Microbiological Methods

Microbiological Methods

Collins and Lyne’s

Eighth Edition

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A member of the Hodder Headline GroupLONDON

Microbiological MethodsEighth Edition

Collins and Lyne’s

C. H. Collins, MBE, MA, DSc, FRCPath, FIBiol, FIBMS

The Ashes, Hadlow, Kent, UK

Patricia M. Lyne, CBiol, MIBiol

The Ashes, Hadlow, Kent, UK

J. M. Grange, MD, MSc

Visiting Professor, Centre for Infectious Diseases and International Health, Royal Free and University College Medical School,

Windeyer Institute for Medical Sciences, London, UK

J. O. Falkinham III, PhD

Professor, Fralin Biotechnology Center, VirginiaPolytechnic Institute and State University, Blacksburg, Virginia, USA

First published in 1964 by Butterworth-HeinemannReprinted 1966Second edition 1967Reprinted 1970Third edition 1970Fourth edition 1976Reprinted with additions 1979Fifth edition 1984Revised reprint 1985Reprinted 1987Sixth edition 1989Revised reprint 1991Seventh edition 1995Reprinted 1997, 1998

This edition published in 2004 byArnold, a member of the Hodder Headline Group,338 Euston Road, London NW1 3BH

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© 2004 Arnold

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Whilst the advice and information in this book are believed to be true and accurate at the date of going topress, neither the authors nor the publisher can accept any legal responsibility or liability for any errors oromissions that may be made. In particular (but without limiting the generality of the preceding disclaimer)every effort has been made to check drug dosages; however it is still possible that errors have been missed.Furthermore, dosage schedules are constantly being revised and new side-effects recognized. For thesereasons the reader is strongly urged to consult the drug companies’ printed instructions before administeringany of the drugs recommended in this book.

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List of contributors viiPreface to the eighth edition viii

1 Safety in microbiology 12 Quality assurance 123 Laboratory equipment 204 Sterilization, disinfection and the decontamination of

infected material 455 Culture media 616 Cultural methods 817 Identification methods 898 Automated methods 1109 Mycological methods 12910 Estimating microbial numbers 14411 Clinical material 15612 Antimicrobial susceptibility tests 16813 Food poisoning and food-borne disease 18714 Food microbiology: general principles 19415 Meat and poultry 20516 Fresh, preserved and extended shelf-life foods 21417 Fresh fish, shellfish and crustaceans 22918 Milk, dairy produce, eggs and ice-cream 23319 Environmental microbiology 24520 Water microbiology 25121 Key to common aerobic, non-sporing, Gram-negative bacilli 26022 Pseudomonas and Burkholderia and other pseudomonads 26223 Acinetobacter, Alcaligenes, Flavobacterium, Chromobacterium

and Janthinobacterium, and acetic acid bacteria 26924 Vibrio, Plesiomonas and Aeromonas 27425 Key to the enterobacteria 28226 Escherichia, Citrobacter, Klebsiella and Enterobacter 28427 Edwardsiella, Hafnia and Serratia 290

Contents

28 Salmonella and Shigella 29229 Proteus, Providencia and Morganella 30130 Key to some miscellaneous aerobic, non-sporing, Gram-negative

bacilli of medical importance 30331 Brucella, Bordetella and Moraxella 30432 Haemophilus, Gardnerella and Streptobacillus 31033 Campylobacter and Helicobacter 31434 Actinobacillus, Pasteurella, Yersinia, Cardiobacterium and

Francisella 31735 Legionella and Mycoplasma 32236 Staphylococcus and Micrococcus 32937 Streptococcus, Enterococcus, Lactococcus, Aerococcus,

Leuconostoc and Pediococcus 33438 Lactobacillus and Erysipelothrix 34639 Corynebacteria 35040 Listeria and Brochothrix 35641 Neisseria 35942 Bacillus 36243 Gram-negative anaerobic bacilli and cocci 36744 Clostridium 37045 Mycobacterium 37946 Nocardia, Actinomadura, Streptomyces and Rhodococcus 40147 Actinomyces, Propionibacterium, Bifidobacterium and Tropheryma 40448 Bartonella and Mobiluncus 40849 Spirochaetes 41050 Yeasts 41351 Common moulds 41852 Pathogenic moulds 426

Appendix: Regulatory safety issues that affect work with micro-organisms in the UK 435Index 445

vi Contents

L. Berg, BSc, CLS, West Hills Hospital and MedicalCenter, West Hills, California, USA

J. S. Brazier, MSc, PhD, CBiol, MIBiol, FIBMS,Anaerobe Reference Laboratory, NationalPublic Health Service of Wales, UniversityHospital of Wales, Cardiff, UK

E. Y. Bridson, MPhil, PhD, CBiol, FIBiol, FIBMS,Camberley, Surrey, UK

C. K. Campbell, MSc, PhD, Mycology ReferenceLaboratory and National Collection ofPathogenic Fungi, Health Protection Agency,Specialist and Reference MicrobiologyDivision, South West HPA, Bristol, UK

C. H. Collins, MBE, MA, DSc, FRCPath, FIBiol, FIBMS,Hadlow, Kent, UK

Janet E. L. Corry, BSc, MSc, PhD, CBiol, MIBiol, FIST,Division of Farm Animal Food Science,Department of Clinical Veterinary Medicine,University of Bristol, Bristol, UK

Kate Davy, MSc, FIBMS, Mycology ReferenceLaboratory and National Collection ofPathogenic Fungi, Health Protection Agency,Specialist and Reference MicrobiologyDivision, South West HPA, Bristol, UK

T. J. Donovan, PhD, CBiol, FIBiol, FIBMS, Ashford,Kent, UK

J. O. Falkinham III, PhD, Fralin BiotechnologyCenter, Virginia Polytechnic and StateUniversity, Blacksburg, Virginia, USA

J. M. Grange, MD, MSc, Centre for InfectiousDiseases and International Health, RoyalFree and University College Medical School,Windeyer Institute for Medical Sciences,London, UK

Elizabeth Johnson, BSc, PhD, Mycology ReferenceLaboratory and National Collection ofPathogenic Fungi, Health Protection Agency,Specialist and Reference MicrobiologyDivision, South West HPA, Bristol, UK

D. A. Kennedy, MA, PhD, CBiol, MIBiol, FIOSH,Surbiton, Surrey, UK

S. F. Kinghorn-Perry, BSc, PhD, Health and SafetyExecutive, Biological Agents Unit, Bootle,Merseyside, UK

Christine L. Little, BSc, LLM, PhD, MIFST, HealthProtection Agency, EnvironmentalSurveillance Unit, Communicable DiseasesSurveillance Centre, London, UK

Patricia M. Lyne, CBiol, MIBiol, The Ashes,Hadlow, Kent, UK

M. O. Moss, FLS, BSc, PhD, DIC, ARCS, School ofBiomedical and Molecular Sciences,University of Surrey, Guildford, Surrey, UK

I. Ogden, HND, Department of MedicalMicrobiology, University of Aberdeen,Aberdeen, UK

D. N. Petts, MSc, PhD, CBiol, MIBiol, FIBMS,Microbiology Department, BasildonHospital, Basildon, Essex, UK

T. L. Pitt, MPhil, PhD, Laboratory of Health Careand Associated Services, Health ProtectionAgency, London, UK

P. Silley, BSc, PhD, CBiol, FIBiol, Don WhitleyScientific Ltd, Shipley, West Yorkshire, UK

P. Taylor, BTech, MSc, MPhil, FIBMS, Department ofMicrobiology, Royal Brompton andHarefield NHS Trust, London, UK

List of contributors

Forty years have passed since this book was firstpublished and during its seven previous editionsthere have been many changes, not the least beingits evolution from single authorship to multiplecontributors and editors. Nevertheless, no singlecontributor is responsible for any one of the chap-ters. The system of peer review and revision hasbeen adopted. Although the newer, automated and‘kit’ methods have been accommodated, the basictechniques, essential to good practice in microbiol-ogy, have been retained and in some casesimproved. These are still applicable in areas whereautomation is not yet possible and, we think, nec-essary for both teaching and research. One impor-tant change is the omission of the many formulaefor the preparation of culture media. Very few lab-oratories, anywhere in the world, now make theirown media because excellent, quality-controlledmaterials are now generally available.

Changes in nomenclature are inevitable but insome cases the older terminology, in addition to thelatest, has been retained, on the ‘custom and usage’principle.

We are indebted to our contributors for both theirspecialist knowledge and their general observa-tions, as well as for allowing us to retain the styleand format of the earlier editions, which appears tohave been appreciated by readers and those whouse the volume as a ‘bench book’.

We also acknowledge the help that we havereceived from Topley and Wilson’s Microbiologyand Microbial Infections and Cowan and Steel’sManual for the Identification of Medical Bacteria.

C. H. CollinsPatricia M. Lyne Collins

J. M. GrangeJ. O. Falkinham

Preface to the eighth edition

During the twentieth century some 4000 labora-tory workers became infected with micro-organ-isms in the course of their work and some of themdied. Unfortunately, laboratory-acquired infectionsstill occur, although most of them are clearly pre-ventable (see Collins and Kennedy, 1999).

To avoid becoming infected, therefore, laboratoryworkers should:

• be aware of the potential hazards of thematerials and organisms they handle,especially those containing blood and thosereceived from tropical and subtropicalcountries

• be aware of the routes by which pathogenicmicro-organisms can enter the human bodyand cause infections

• receive proper instruction and thereafterpractise good microbiological technique(GMT), which effectively ‘contains’ micro-organisms so that they do not have access tothose routes.

CLASSIFICATION OF MICRO-ORGANISMS ON THE BASISOF RISK

Micro-organisms vary in their ability to infect indi-viduals and cause disease. Some are harmless, somemay be responsible for disease with mild symp-toms, others can cause serious illnesses, and a fewhave the potential for spreading in the communityand causing serious epidemic disease. Experienceand research into laboratory-acquired infectionsenabled investigators to classify micro-organisms

and viruses into four categories, also known as‘Classes’, ‘Risk Groups’ or ‘Hazard Groups’ (in thisbook the last term is used). These are numbered1–4 on the basis of the hazards that they present tolaboratory workers and to the community shouldthey ‘escape’ from the laboratory. These categoriestake into account the seriousness of the associateddisease, the availability of prophylactic and thera-peutic agents, the routes of infection and thehistory of laboratory infections caused by theorganisms.

A number of classifications have been devisedsince the 1970s. They all agree in principle and aresummarized in Table 1.1, although there are slightdifferences in wording and emphasis. These classi-fications originally applied to laboratory workers,but the increasing use of biological agents andmaterials in industry has led to their application toother workers (termed ‘employees’ in the mostrecent official publications).

Lists of organisms in Hazard Groups 2, 3 and 4have been compiled by a number of states andorganizations. There will inevitably be variationsbetween countries according to their geography,reflecting differences in the incidence of the micro-organisms, their vectors, the associated diseasesconcerned and levels of hygiene. A comparison ofthese variations was made by the EuropeanFederation of Biotechnology (Frommer et al.,1989), but they are under continual revision.Readers should therefore consult their currentnational lists.

In this book, the UK lists (Advisory Committee onDangerous Pathogens or ACDP, 2001) are used.The differences between these and those in the USAreflect the variations noted above. Most of the

1

Safety in microbiology

organisms are in Hazard Groups 1 and 2.Cautions, in bold italic type, are given for those inHazard Group 3. No Hazard Group 4 agents(which are all viruses) are discussed in the text. Inthe USA the lists are published by the Centers forDisease Control and Prevention (CDC, 1999) andthe American Society for Microbiology (Fleming etal., 1995). These publications, and also that ofCollins and Kennedy (1999), also give ‘agent sum-mary statements’ which provide information aboutthe organisms and their histories in relation to lab-oratory-acquired infections.

ROUTES OF INFECTION

There are at least four routes by which micro-organisms may enter the human body.

Through the lungs

Aerosols, i.e. very small droplets (< 0.5–5.0 μm)containing micro-organisms, are frequentlyreleased during normal microbiological manipula-tions such as work with loops and syringes, pipet-ting, centrifuging, blending, mixing andhomogenizing, pouring, and opening culture tubesand dishes. Inhalation of these has probably causedthe largest number of laboratory-acquired infec-tions.

Through the mouth

Biological agents may be ingested during mouthpipetting and if they are transferred to the mouth

by contaminated fingers or articles, such as food,cigarettes and pencils, that have been in contactwith areas or objects contaminated by spills,splashes or settled droplets.

Through the skin

The skin of hands, etc. is rarely free from (oftenminute) cuts and abrasions, through which organ-isms can enter the bloodstream. Infections may alsoarise as a result of accidental inoculation withinfected hypodermic needles, broken glass andother sharp objects.

Through the eyes

Splashes of infected material into the eyes andtransfer of organisms on contaminated fingers tothe eyes are not uncommon routes of infection.

LEVELS OF CONTAINMENT

The Hazard Group number allocated to anorganism indicates the appropriate ContainmentLevel (CL) in the UK or Biosafety Level (BSL) inthe USA, i.e. the necessary accommodation,equipment, techniques and precautions. Thus,there are four of these levels (Table 1.2). In theWorld Health Organization’s (WHO’s) classifica-tion the Basic Laboratory equates with Levels 1and 2, Containment Laboratory with Level 3 andthe Maximum Containment Laboratory withLevel 4.

The bold italic type ‘Caution’ at the head of sec-

2 Safety in microbiology

Table 1.1 Summary of classifications of micro-organisms on the basis of hazard

Hazard Group 1 Hazard Group 2 Hazard Group 3 Hazard Group 4

LOW RISK → → HIGH RISKLow to individual and to Moderate to individual, low High to individual, low to Very high to individual community to community community and to community

tions dealing with Group 3 organisms indicates theappropriate containment level and any additionalprecautions.

Full details are given by the WHO (1993), theCDC (1999), Fleming et al. (1995), the ACDP(2001) and HSAC (2003). A summary is presentedhere and in Table 1.2. More detailed measures aregiven in Table 1.3.

Levels 1 and 2: the Basic laboratories

These are intended for work with organisms inHazard Groups 1 and 2. Ample space should beprovided. Walls, ceilings and floors should besmooth, non-absorbent, easy to clean and disin-fect, and resistant to the chemicals that are likelyto be used. Floors should also be slip resistant.Lighting and heating should be adequate. Handbasins, other than laboratory sinks, are essential.Bench tops should be wide, the correct height forwork at a comfortable sitting position, smooth,easy to clean and disinfect, and resistant to chemi-cals. Adequate storage facilities should be pro-vided.

If agents in Hazard Group 2 are handled, e.g. inclinical laboratories, a microbiological safetycabinet (see Chapter 3) should be provided.Access should be restricted to authorizedindividuals.

Level 3: the Containment laboratory

This is intended for work with organisms inHazard Group 3. All the features of the Basic labo-ratory should be incorporated, plus the following:

• The room should be physically separatedfrom other rooms, with no communication(e.g. by pipe ducts, false ceilings) with otherareas, apart from the door, which should belockable, and transfer grilles for ventilation(see below).

• Ventilation should be one way, achieved byhaving a lower pressure in the Containmentlaboratory than in other, adjacent rooms andareas. Air should be removed to atmosphere(total dump, not recirculated to other parts ofthe building) by an exhaust system coupled toa microbiological safety cabinet (see p. 30) sothat, during working hours, air is continuallyextracted by the cabinet or directly from theroom and airborne particles cannot be movedaround the building. Replacement air entersthrough transfer grilles.

• Internal windows should be provided, e.g. inthe door, with internal mirrors if necessary incase of accidents to the occupants.

• Access should be strictly regulated. Theinternational biohazard warning sign, withappropriate wording, should be fixed to thedoor (Figure 1.1).

Levels of containment 3

Table 1.2 Summary of biosafety/containment levels

Level Facilities Laboratory practice Safety equipment

1 Basic GMT None. Open bench

2 Basic GMT plus protective clothing. Open bench + safety cabinet for Biohazard signs aerosol potential

3 Containment Level 2 + special clothing and Safety cabinet for all activitiescontrolled access

4 Maximum containment Level 3 plus air lock entry, shower Class III safety cabinet, pressure exit, special waste disposal gradient, double-ended autoclave

GMT, good microbiological technique


Table 1.3 Summary of safety measures for work at containment levels 2, 3 and 4*

Level 2 Level 3 Level 4

Sites, buildings, engineering and facilitiesLaboratories separated from other activities in same building If justified by risk Yes Yes, preferably

assessment separate buildingExternal windows Closed during work Lockable Sealed, watertight and

unbreakableInternal windows so that occupants may be observed Not necessary Recommended YesHandbasins Yes In each room In each roomShowers Not necessary Recommended YesSurfaces impermeable to water and cleaning agents All work surfaces All work surfaces and All exposed surfaces

floorsSurfaces impermeable to acids, bases, solvents and disinfectants Recommended Yes YesLaboratories maintained at negative pressure relative to Not necessary Yes With gradientatmosphere

HEPA filtration of intake and extract air Not necessary Extract air Intake and extract airAirlock access to laboratories Not necessary Recommended YesRoom sealable for decontamination Not necessary Yes YesSeparate effluent treatment Not necessary If indicated by risk Yes

assessmentTwo-way intercom system Not necessary Yes YesLaboratories to have own dedicated equipment Not necessary Yes YesMicrobiological safety cabinets If indicated by risk Classes I and/or II Class III

assessmentAutoclaves In building In room Pass-through, double

endedCentrifuges Available In room In room

Operational measuresInitial and annual tests for containment capability Not necessary Yes YesBiohazard sign displayed On all doors On all doors On all doorsAuthorized access Yes Limited Strictly limitedValidated disinfection and disposal methods Yes Yes YesEmergency plans for spillages of infectious material Yes Yes YesSafe storage of biological agents Yes In Level 3 room only In Level 4 room onlyEffective rodent and other vector control Yes Yes YesProtective clothing to be worn Standard overalls Standard overalls Full protectionGloves to be worn If indicated by risk Yes Yes

assessmentMedical supervision By occupational By unit physician By unit physician

health serviceImmunization As for general public As for general public As for general public

and for agents used and for agents usedBase line sera Not necessary Recommended YesAccident reporting Yes Yes YesTwo-person rule Not necessary Recommended YesSupervision of maintenance and service personnel Yes Accompanied by staff Accompanied by staff

member member, after decontamination ofroom

Technical proceduresWork with infectious materials On open bench In Class I or II In Class III micro-

except if aerosol microbiological safety biological safetyrelease is possible cabinent cabinet

Centrifugation In closed containers In sealable safety In sealable safetybuckets buckets

Use of ‘sharps’ As authorized Avoid Banned except whenespecially authorised

*From Collins and Kennedy (1999). Sources: Frommer et al. (1989), WHO (1993), ACDP (1995a). Health Canada (1996), CDC/NIH (1999). See also HSAC (2003).

Level 4: the Maximum Containmentlaboratory

This is required for work with Hazard Group 4materials and is outside the scope of this book.Construction and use generally require governmentlicence or supervision.

PREVENTION OF LABORATORY-ACQUIRED INFECTIONS: A CODEOF PRACTICE

The principles of containment involve good micro-biological technique, with which this book is par-ticularly concerned, and the provision of:

1. primary barriers around the organisms toprevent their dispersal into the laboratory

2. secondary barriers around the worker to actas a safety net should the primary barriers bebreached

3. tertiary barriers to prevent the escape into thecommunity of any organisms that are notcontained by the primary and secondarybarriers.

This code of practice draws very largely on therequirements and recommendations published inother works (CDC, 1999; WHO, 1993, 1997;ACDP, 1995a, 1998, 2001; Fleming et al., 1995;Collins and Kennedy, 1999; HSE, 2002; HSAC,

2003). (See also Approved Codes of Practice in theAppendix to this book.)

Primary barriers

These are techniques and equipment designed tocontain micro-organisms and prevent their directaccess to the worker and their dispersal as aerosols:

• All body fluids and pathological materialshould be regarded as potentially infected.‘High Risk’ specimens, i.e. those that maycontain agents in Hazard Group 3 ororiginate from patients in the AIDS andhepatitis risk categories, should be so labelled(see below).

• Mouth pipetting should be banned in allcircumstances. Pipetting devices (see p. 38)should be provided.

• No article should be placed in or at the mouth,including mouthpieces of rubber tubingattached to pipettes, pens, pencils, labels,smoking materials, fingers, food and drink.

• The use of hypodermic needles should berestricted. Cannulas are safer.

• Sharp glass Pasteur pipettes should bereplaced by soft plastic varieties (see p. 38).

• Cracked and chipped glassware should bereplaced.

• Centrifuge tubes should be filled only towithin 2 cm of the lip. On centrifugation infixed angle centrifuges, the liquid level isperpendicular to the direction of the forces.

• All Hazard Group 3 materials should becentrifuged in sealed centrifuge buckets (seep. 28).

• Bacteriological loops should be completelyclosed, with a diameter not more than 3 mmand a shank not more than 5 cm, and be heldin metal, not glass, handles.

• Homogenizers should be inspected regularlyfor defects that might disperse aerosols (seep. 39). Only the safest models should be used.Glass Griffith’s tubes and tissue homogenizersshould be held in a pad of wadding in agloved hand when operated.

Prevention of laboratory-acquired infections: a code of practice 5

Figure 1.1 International biohazard sign

• All Hazard Group 3 materials should beprocessed in a Containment laboratory (see‘Tertiary barriers’ below) and in amicrobiological safety cabinet (see p. 30)unless exempted by agreed nationalregulations.

• A supply of suitable disinfectant (see p. 50) atthe use dilution should be available at everywork station.

• Benches and work surfaces should bedisinfected regularly and after spillage ofpotentially infected material.

• Discard jars for small objects and re-usablepipettes should be provided at each workstation. They should be emptied,decontaminated and refilled with freshlyprepared disinfectant daily (see p. 56).

• Discard bins or bags supported in bins should beplaced near to each work station. They shouldbe removed and autoclaved daily (see p. 56).

• Broken culture vessels, e.g. after accidents,should be covered with a cloth, Suitabledisinfectant should be poured over the clothand the whole left for 30 min. The debrisshould then be cleared up into a suitablecontainer (tray or dustpan) and autoclaved.The hands should be gloved and stiffcardboard used to clear up the debris.

• Infectious and pathological material to be sentthrough the post or by airmail should bepacked in accordance with government, postoffice and airline regulations, obtainable fromthose authorities. Full instructions are alsogiven on p. 9.

• Specimen containers should be robust andleak proof.

• Discarded infectious material should not leavethe laboratory until it has been sterilized orotherwise made safe (see p. 55).

Secondary barriers

These are intended to protect the worker shouldthe primary barriers fail. They should, however, beobserved as strictly as the latter:

• Proper overalls and/or laboratory coatsshould be worn at all times and fastened.They should be kept apart from outdoor andother clothing. When agents in Hazard Group3 containing, or material containing orsuspected of containing, hepatitis B or theAIDS virus are handled, plastic aprons shouldbe worn over the normal protective clothing.Surgeons’ gloves should also be worn.

• Laboratory protective clothing should beremoved when the worker leaves thelaboratory and not worn in any other areassuch as canteens and rest rooms.

• Hands should be washed after handlinginfectious material and always before leavingthe laboratory.

• Any obvious cuts, scratches and abrasions onexposed parts of the worker’s body should becovered with waterproof dressings.

• There should be medical supervision inlaboratories where pathogens are handled.

• All illnesses should be reported to the medicalor occupational health supervisors. They maybe laboratory associated. Pregnancy shouldalso be reported, because it is inadvisable towork with certain micro-organisms duringpregnancy.

• Any member of the staff who is receivingsteroids or immunosuppressive drugs shouldreport this to the medical supervisor.

• Where possible, and subject to medicaladvice, workers should be immunized againstlikely infections (Wright, 1988; WHO, 1993;Collins and Kennedy, 1999; HSAC, 2003).

• In laboratories where tuberculous materialsare handled the staff should have receivedBCG or have evidence of a positive skinreaction before starting work. They shouldhave a pre-employment chest radiograph(Wright, 1988).

Tertiary barriers

These are intended to offer additional protectionto the worker and to prevent escape into the com-


munity of micro-organisms that are under investi-gation in the laboratory.

They concern the provision of accommodationand facilities at the Biosafety/Containment levelsappropriate for the organisms handled, and ofmicrobiological safety cabinets.

For more information on laboratory design, seeBritish Occupational Hygiene Society (BOHS,1992) and Collins and Kennedy (1999).

PRECAUTIONS AGAINSTINFECTION WITH HEPATITIS B(HBV), THE HUMANIMMUNODEFICIENCY VIRUS (HIV)AND OTHER BLOOD-BORNEPATHOGENS

Laboratory workers who may be exposed to theseviruses in clinical material are advised to follow thespecial precautions described by one or more of thefollowing: ACDP (1990, 1995b, 2001);Occupational Health and Safety Agency (OHSA,1991); WHO (1991); Fleming et al. (1995); Collinsand Kennedy (1997), HSAC (2003).

PRECAUTIONS IN LABORATORIESWORKING WITH RECOMBINANTMICRO-ORGANISMS

It seems generally to be agreed that the potentialrisks associated with recombinant micro-organismsare of the same order as those offered by naturallyoccurring micro-organisms – with reference, ofcourse, to the hazard groups of the hosts andinserts (Organization for Economic Co-operationand Development or OECD, 1986; Frommer et al.,1989; WHO, 1993).

Nevertheless, work of this nature is closely regu-lated. As it is outside the scope of this book, read-ers are referred to the following: Collins and Beale(1992); National Institutes of Health (NIH, 1994);Advisory Committee on Genetic Modification(ACGM, 1997).

PLANT PATHOGENS

Very few, if any, plant pathogens can cause dis-ease in humans. Laboratory precautions and con-tainment are therefore aimed at preventing theescape of these agents to the environment. Thesubject has received little attention, however,except from the European Federation ofBiotechnology (Frommer et al., 1992). In the USA,laboratories must obtain permission from the USDepartment of Agriculture before they work onplant pathogens.

THE SAFE COLLECTION, RECEIPTAND TRANSPORT OFDIAGNOSTIC SPECIMENS AND‘INFECTIOUS SUBSTANCES’

Fears are sometimes expressed that micro-organ-isms might, as a result of poor packaging or acci-dental damage, ‘escape’ from specimens andcultures while they are being transported to orbetween laboratories. There are no recorded cases,however, of illness arising from such an ‘escape’(WHO, 2003).

A distinction is drawn between diagnostic speci-mens and ‘infectious substances’ largely on thebasis of risk assessment.

Diagnostic specimens

A diagnostic specimen is defined (WHO, 2003) as:

. . . any human or animal material, including butnot limited to excreta, blood and its components,tissue or tissue fluids, collected for the purpose ofdiagnosis but excluding live animals.

As a general rule, such specimens do not offermuch risk, but if they are collected during an out-break of an infectious disease of unknown aetiol-ogy they should be regarded as ‘infectioussubstances’ – see below.

The safe collection, receipt and transport of diagnostic specimens and ‘infectious substances’ 7

Specimen containersThese containers should be robust, preferablyscrew capped, and unlikely to leak. Plastic bagsshould be provided to receive the container afterthe specimen has been collected. Request formsshould not be placed in the same plastic bag asthe specimen. Unless the source and laboratoryare in the same premises, robust outer containerswith adequate absorbent material should be pro-vided. Various packages are available com-mercially.

Transport within hospitals and institutionsLeak-proof trays or boxes should be provided forthe transport of diagnostic specimens from wardsto the laboratory. These should be able to with-stand autoclaving or overnight exposure to disin-fectant. Suitable trays and boxes are readilyavailable. Some are known as kitchen ‘tidies’ or‘tidy boxes’, and are sold cheaply by chain stores,hardware and tool shops for cutlery, kitchen uten-sils, shoe-cleaning equipment and handyman’stools. Ordinary hospital ward or surgical trays arealso widely used. Some purpose-made containersare made of stainless steel, others of propylene eth-ylene co-polymer. They should be deep enough forspecimens to be carried upright to avoid leakageand spillage. These trays and boxes should not beused for any other purposes.

Organs in glass jars or plastic buckets, and 24-hurines in 2-litre screw-capped bottles or the stan-dard plastic carboys should be placed in disposableplastic (or paper) bags for transport for bothhygienic and aesthetic reasons.

Surface transport between and to hospitalsDiagnostic specimens are frequently conveyed fromone hospital to another because of the centraliza-tion of laboratory services. The usual means oftransport is by hospital van or hospital car, i.e. thevehicle is under the control of the hospital authori-ties. Similarly, specimens are transported fromlocal authority offices or clinics to laboratories bythe authorities’ own vehicles. Rarely, such material

may be carried by taxi cabs or by messengers onpublic service vehicles.

The UK Health Services Advisory Committee(HSAC, 2003) recommends that secure transportboxes with lids be used. These must be capable ofwithstanding autoclaving and prolonged exposureto disinfectants. Such boxes are in common useamong food inspectors for conveying food, milkand water samples to public analysts and publichealth laboratories. Suitable boxes, used by somelaboratories, included those that can be fitted tothe carriers of motor cycles and scooters, and theplastic ‘Coolkeepers’ or ‘Chilly Bins’ used bymotorists for picnics. The boxes should bemarked quite clearly with the name and addressof the laboratory that owns them. In case they arelost or the vehicle is involved in an accident, theyshould also carry warning labels ‘Danger ofInfection, do not open’ as well as the interna-tional biohazard sign, the meaning of which israrely clear to laypeople. The labels shouldrequest the finder to call the nearest hospital orpolice station. The boxes should be inspecteddaily for evidence of spillage, and should bedecontaminated and then washed out at leastweekly. Again, the drivers or messengers shouldbe warned that the material in the boxes may beinfectious. If the boxes are suitable for the joband are packed with care, the hazards are mini-mal and there should be no need for disinfectantsto be carried.

Some authorities send specimens to laboratoriesby hand on public service vehicles. The same typeof boxes should be used, but it may be advisable tolabel them less explicitly or transport employeesand other passengers may be alarmed.

Inland mail Within state boundaries the usual requirements arethat the specimen is in a leak-proof primary con-tainer, securely cocooned in absorbent materialand packed in a robust outer container, which islabelled ‘Pathological Specimen’ or ‘DiagnosticSpecimen’.

Specimens for mailing across a frontier/border


should be packaged as described below for infec-tious substances

Infectious substances

An infectious substance (WHO, 2003) is:

. . . a substance containing a viable micro-organismsuch as a bacterium, virus, rickettsia, parasite orfungus that is known or reasonably believed tocause disease in humans or animals.

Airmailing infectious substancesUntil recently there have been problems here,because several different organizations are involved(Collins and Kennedy, 1999). These have now beenresolved by international agreement and the WHOhas issued an excellent guidance document (WHO,2003).

The internationally agreed ‘triple packaging sys-tem’ must be used. This must meet the requirements

of the UN class 2 specifications and packaginginstruction PI(602) (UN, 1996) which impose stricttests, including surviving, undamaged, a 9-metredrop on to a hard surface, as well as puncture tests.

1. The material is placed in a watertight, leak-proof receptacle – the ‘primary container’.This is wrapped in enough absorbent materialto absorb all fluid in case of breakage.

2. The primary container is then packed in awatertight ‘secondary container’ with enoughabsorbent material to cushion the primarycontainer.

3. The secondary container is placed in an outershipping package that protects its contentsfrom damage during transit.

An example of a triple package that satisfies therequirements of the UN packaging instruction isshown in Figure 1.2

A certain amount of documentation must accom-pany the package. This is explained in detail by theWHO (2003).

The safe collection, receipt and transport of diagnostic specimens and ‘infectious substances’ 9

Primary culture containerAbsorbent packing material

Plain lid

Screw-on cap

Customs label

Air mail label

Outer container, screw-capped

Secondary container, to besealed with waterproof tape

Description and quantity labelSender name and address

Absorbent, shockproofpacking between thetwo containers

Infectious perishablebiological substances label

Address label(addressee and sender)

Figure 1.2 Packing infectious substances for the overseas post (airmail)

RECEIPT OF SPECIMENS IN THELABORATORY

Specimens from local sources, i.e. within stateboundaries, may be unpacked only by trainedreception staff who wear laboratory protectiveclothing. The specimens should be left in their plas-tic bags pending examination. Specimens fromacross frontiers/borders, which may contain exoticbiological agents, should always be unpacked onlyby trained laboratory staff under appropriate con-tainment conditions.

Instruction in safety

This instruction should be part of the general train-ing given to all microbiological laboratory workersand should be included in what is known as goodmicrobiological technique. In general, methodsthat protect cultures from contamination also pro-tect workers from infection, but this should not betaken for granted. Personal protection can beachieved only by good training and careful work.Programmes for training in safety in microbiologyhave been published (International Association ofMedical Technology or IAMLS, 1991; WHO,1993; Collins and Kennedy, 1999).

Bioterrorism

We consider that it would be inappropriate to sug-gest that ‘routine’ laboratories (the target of thebook) should test samples for agents of bioterror-ism. Such samples should be submitted to anational laboratory that has the necessary expert-ise. States that do not have one should negotiatewith, for example, the UK, the USA and Russia,who have the necessary facilities. See also theAppendix to this book.

For a review of the history, incidence, causes andprevention of laboratory-acquired infection seeFleming et al. (1995) and Collins and Kennedy(1999).

REFERENCES

Advisory Committee on Dangerous Pathogens (ACDP)(1990) HlV, the Causative Agent of AIDS and

Related Conditions. London: HMSO.ACDP (1995a) Categorisation of Pathogens According

to Hazard and Categories of Containment, 2ndedn. London: HMSO.

ACDP (1995b) Protection against Blood-borne

Infections in the Workplace. London: HMSO.ACDP (2001) The management, design and operation

of microbiological containment laboratories.

Advisory Committee on Dangerous Pathogens.Sudbury: HSE Books.

ACDP (2002) Second Supplement to the ACDPguidance on the: Categorisation of Pathogens

According to Hazard and Categories of

Containment. Sudbury: HSE Books.Advisory Committee on Genetic Modification (ACGM)

(1997) Compendium of Guidance on Good

Practice for people Working with Genetically-

modified Micro-organisms. ACGM/HAS/DE NoteNo. 7. London: The Stationery Office.

British Occupational Hygiene Society (1992)Laboratory Design Issues. British Occupational

Hygiene Society Technical Guide No. 10. Leeds:H & Scientific Consultants.

Centers for Disease Control and National Institutes ofHealth (1999) Biosafety in Microbiological and

Biomedical Laboratories, 4th edn, WashingtonDC: CDC.

Collins, C. H. and Beale, A. J. (1992) Safety in

Industrial Microbiology and Biotechnology.Oxford: Butterworth–Heinemann.

Collins, C. H. and Kennedy, D. A. (eds) (1997)Occupational Blood-borne Infections. Wallingford:CAB International.

Collins, C. H. and Kennedy, D. A. (1999) Laboratory-

acquired Infections, 4th edn. Oxford:Butterworth–Heinemann.

Fleming, D. O., Richardson, J. H., Tulis, J. I. andVesley, D. (eds) (1995) Laboratory Safety:

Principles and practice. Washington DC: ASMPress.

Frommer, W.C. and a Working Party on Safety inBiotechnology of the European Federation of


Biotechnology (1989) Safe Biotechnology III. Safetyprecautions for handily microorganisms ofdifferent risk classes. Applied Microbiology and

Biotechnology 30: 541–552.Frommer, W.C. and a Working Party on Safety in

Biotechnology of the European Federation ofBiotechnology (1992) Safe Biotechnology (4).Recommendations for safety levels forbiotechnological operations with microorganismsthat cause diseases in plants. Applied Microbiology

and Biotechnology 38: 139–140.Health and Safety Executive (HSE) (2002) Control of

Substances Hazardous to Health: Approved Codes

of Practice, 4th edn. Sudbury: HSE Books.HSAC, Health Services Advisory Committee (2003)

Safe Working and the Prevention of Infection in

Clinical Laboratories. Sudbury: HSE Books.International Association of Medical Technology,

Stockholm (1991) A Curriculum for Instruction inLaboratory Safety. Medical Technical International

1. Stockholm: IAMLS.National Institutes of Health (1994) Guidelines for

research involving recombinant DNA molecules.Federal Register 5 July, Part IV.

Occupational Health and Safety Agency (OHSA)

(1991) B1ood-borne Pathogens Standard. Federal

Register 56: 64175–64182.Organization for Economic Co-operation and

Development (1986) Recombinant DNA Safety

Considerations. Paris: OECD.United Nations (1996) Committee of Experts on the

Transport of Dangerous Goods. Division 2.

Infectious Substances, 10th edn. Revised NewYork: UN.

World Health Organization (1991) Biosafety

Guidelines for Diagnostic and Research

Laboratories Working with HIV. AIDS Series 9.Geneva: WHO.

WHO (1993) Laboratory Biosafety Manual, 2nd edn.Geneva: WHO.

WHO (1997) Safety in Health-Care Laboratories.

Geneva: WHO.WHO (2003) Guidelines for the Safe Transport of

Infectious Substances and Diagnostic Specimens.

WHO/EMC/97.3. Geneva: WHO.Wright, A. E. (1988) Health care in the laboratory. In:

Collins C. H. (ed.), Safety in Clinical and

Biomedical Laboratories. London: Chapman &Hall.

References 11

2

Quality assurance

The information given in this chapter is intended toprovide users with sufficient information to imple-ment a quality assurance programme. For a com-prehensive treatise of the subject see Snell et al.(1999). Three terms require definition: qualityassurance, quality control and quality assessment.Quality assurance is the total process that guaran-tees the quality of laboratory results and encom-passes both quality control and quality assessment.The quality of a microbiological service is assuredby quality control, i.e. a continual monitoring ofworking practices, equipment and reagents.Quality assessment is a system by which specimensof known but undisclosed content are introducedinto the laboratory and examined by routine proce-dures. Quality control and quality assessment aretherefore complementary activities and one shouldnot be used as a substitute for the other (EuropeanCommittee for Clinical Laboratory Standards orECCLS, 1985). Essentially, with a quality system,we are seeking to ensure that we are doing the rightthings, for the right people, at the right time anddoing them right every time (Crook, 2002).

THE NEED FOR QUALITY

There is increasing reliance on the quality of labo-ratory results and confidence in them is essential tothe organization of the laboratory. Quality controlof laboratory tests cannot be considered in isola-tion: the choice, collection, labelling and transportof specimens are all subject to error and requireregular monitoring for quality and suitability.Additional recognition of the fact that the qualityof microbiology results is only a component within

the chain of patient care (Figure 2.1) is demon-strated by the introduction of policies in the UKthat are aimed at embedding continuous qualityimprovement (CQI) at all levels and across all partsof the National Health Service (NHS) (Heard et al.,2001). The key goal of these policies is to achievechanges in practice, thereby improving patient out-comes. The term ‘clinical governance’ is used todescribe the links between clinical and organiza-tional responsibility for the quality of care. Clinicalgovernance is a system through which NHS organ-izations are accountable for continuously improv-ing the quality of their services and safeguardinghigh standards of care by creating an environmentin which excellence in clinical care will flourish(Campbell et al., 2002).

Cost-effectiveness is increasingly relevant in allareas of microbiology. In the health services, speedof response may affect the outcome of health careand the length and cost of a patient’s stay in hospi-tal, and it is therefore as much an element of qual-ity as the accuracy of the test result.

An important contributory factor to high stan-dards of quality is the competence and motivationof staff. These aspects can be achieved and main-tained by continued education and, above all, bycommunication within and between laboratoriesand with those using the services.

PRACTICAL QUALITY CONTROLWITHIN THE LABORATORY

Practical, realistic and economic quality controlprogrammes should be applied to all procedures,

irrespective of the size of the laboratory. Schedulesshould be based on the known stability of eachitem and its importance to the accuracy of theinvestigation as a whole. It is unnecessary to moni-tor all reagents and methods on every working day:the frequency of monitoring should be in directproportion to the frequency with which errors arediscovered. Nevertheless, to ensure success all thecomponents of a quality programme must be inplace and operating before good quality laboratoryresults can be produced.

Each laboratory must develop its own programmebecause the time spent on quality control will varyaccording to the size of the laboratory and therange of investigations. For its success, a seniormember of staff should supervise the programmethroughout the laboratory. This supervisory rolehas indeed been formalized by the fact that it is arequirement of the UK Clinical PathologyAccreditation (CPA) and the American Society ofClinical Pathologists (ASCP) that a quality man-ager be appointed. This person will act as a focalpoint for all quality issues within the laboratoryand be responsible for ensuring that the qualitymanagement system (QMS) works.

Internal quality control can be divided into fourmain areas: collection of specimens; preventivemonitoring and maintenance of equipment (seeChapter 3); manual procedures; and monitoring ofmedia and reagents.

Collection of specimens

In clinical microbiology, results of studies on spec-imens taken from sites containing indigenous orcolonizing bacteria, e.g. from clinical sites, or thoselikely to be contaminated during collection, areoften difficult to interpret. Paradoxically, speci-mens containing the largest numbers of microbialspecies, with consequent high laboratory cost, areless likely to provide useful results than those yield-ing fewer types of organisms. Guidelines for theevaluation of the quality of specimens and theappropriateness of requests should be establishedby the medical and laboratory staff (Bartlett et al.,1978). The usefulness of rapid reporting of prelim-inary results should also be assessed (Bartlett,1982) (see also Chapter 11).

A revolution is occurring in the detection, identifi-cation and characterization of pathogens(Borriello, 1999), such that the scenario of taking aclinical sample and, within an hour, knowingwhether a pathogen is present and its susceptibilityto antimicrobial agents may soon be a reality. Suchdevelopments, particularly with regard to nearpatient testing, will have important implicationsfor the delivery of health care. Although there areseveral advantages of near patient testing, there arealso disadvantages, not least of which is thereduced ability for internal and external qualityassurance, which in turn may lead to misdiagnosis.To establish widespread use of such methodolo-gies, there must be confidence in the operators aswell as in the kits. Those responsible for health caremust be confident that the correct specimen wasobtained, the kit was used appropriately by prop-erly qualified individuals, the result was interpretedcorrectly, and any machine used in the process hasbeen appropriately maintained and used.

Practical quality control within the laboratory 13

¥˚Sick patient

¥˚Clinician orders test

¥˚Collection of specimen

¥ T̊ransport of specimen

¥˚Clerical work

¥˚Analysis

¥˚Interpretation by pathologist

¥˚Report to clinician

¥˚Interpretation by clinician

¥˚Diagnosis and treatment

Under directlaboratory control

Figure 2.1 Chain of patient care

Preventive monitoring andmaintenance of equipment

This is vitally important because many investiga-tions are conducted with complex automatedequipment. Regular monitoring and maintenanceavoid costly repairs and long periods when theequipment is out of action (‘down time’).Prevention of down time is particularly importantwhen an uninterrupted 24-h service is being pro-vided. Unique preventive maintenance systems,which are adaptable to the needs of an individuallaboratory, have been described (Wilcox et al.,1978). The following components belong to allpreventive maintenance programmes.

Routine service tasksThe tasks needed to keep each item of equipmentcalibrated, running and clean should be defined inthe standard operating procedures manual (seebelow).

Interval of serviceThe frequency with which defined tasks are per-formed is assessed according to the type of equip-ment and its usage. Although regularly usedequipment will require more frequent cleaning andservicing than that used infrequently, minimumstandards of preventive maintenance must bedefined for the latter. In some areas service repre-sentatives will call annually to perform routineservice and calibration work on instruments (e.g.balances and centrifuges) if there are enough ofthem on the same site.

AccountabilityThe concept of accountability must be introduced,and members of staff should be assigned specificresponsibilities for maintenance tasks.

StaffA list of trained people who are readily availablefor trouble-shooting, verification tasks and repairsshould be compiled. Although laboratory staff mayperform minor uncomplicated maintenance work,

advice with respect to complex equipment and pro-cedures must be sought from the manufacturer’sservice department. The cost of a service contractmust be weighed against the possibility of evenmore expensive repair costs and the consequencesof prolonged ‘down time’.

ImplementationEquipment maintenance is not a ‘one-off’ affair.Once tasks have been defined, and staff trained, theprogramme should continue regularly and uninter-rupted (see also Chapter 3).

DocumentationA system of recording is essential and may beadapted to the specific needs of a laboratory, e.g.index cards or a computerized system. Computerrecords are ideal because they may easily beupdated and made available.

Standard operating proceduresmanual

The standard operating procedures (SOP) manualis the most important document for the improve-ment and maintenance of the quality of the service.Such a manual can be in an electronic format andtherefore more readily available and less likely tobe misplaced. An out-of-date manual or one fromanother laboratory may be used as a guide to writ-ing but, as each laboratory is unique, site-specificmanuals must be developed. If there are no writtenmethods and new staff members are taught by‘word of mouth’, techniques will soon ‘drift’. Theheadings standard to any SOP manual are listedand discussed by Fox et al. (1992).

AuthoritativeA senior member of staff who has practical experi-ence of the methods must write the manual.

RealisticThe manual should avoid theoretical ideals andprovide clear, concise and explicit descriptions of

14 Quality assurance

procedures. Unnecessarily complicated procedureswill quickly lead to loss of respect and to the possi-bility of staff introducing unofficial short cuts. Awell-structured manual, organized and indexed,will allow a microbiologist from one laboratory toperform any of the tests described in another.

Up to dateThe updating of manuals is vital and should be acontinuous and formal process. The manual shouldbe revised annually and amended when necessary.An electronic manual is therefore ideal. Only desig-nated individuals should be permitted to modify aprocedure, and no new or modified procedureshould be used until approval has been obtainedfrom the quality manager.

AvailabilityThe manual must be freely available to all relevantpersonnel. It is the responsibility of senior staff toensure that it is used and strictly adhered to. If it isnot in electronic format it need not be a bulkybound item; loose-leaf systems have the advantagethat sections may be separated and kept on relevantbenches. Each section should have a unique andclear code number for identification and for cross-referencing.

Culture media and reagents

Most laboratories now use dehydrated media,which, if purchased from a reputable manufac-turer, will have undergone extensive quality con-trol testing before sale. Repetition of these testsshould be unnecessary but, as dehydrated mediawill have to be reconstituted, heated and possiblysupplemented with additives during preparation, itis essential to control the effect of these processes.Overheating of these additives may reduce theireffectiveness and/or release toxic substances. Theseare good arguments for using media preparators, inwhich temperatures are strictly controlled insteadof autoclaves (see Chapters 3, 4 and 5). Mediamust fulfil four main parameters: sterility; support

of bacterial growth; selective inhibition of growth,in some cases; and appropriate response of bio-chemical indicators.

All batches of media must be tested for sterility.Methods are usually a compromise because thesterility of every plate or tube of medium cannot beensured without incubation of the whole batch,with the concomitant risk of deterioration of itsability to support microbial growth. A representa-tive portion of each new batch should be incubatedovernight to check for sterility before use. Forbatches of 100 ml or less, a 3–5% minimum sam-ple should be tested; for larger batches, 10 plates ortubes taken randomly will suffice (Nicholls, 1999).After overnight incubation, media should be left atroom temperature or in a refrigerator for a further24 h as a check for contamination by psychro-philes. Low-level surface contamination of platesmay not be detected on the controls but is usuallyrecognizable during use by, for example, the obser-vation of growth part way through a streaked pop-ulation.

Selective media, being inhibitory to many organ-isms, pose particular problems: even gross contam-ination may not be detected by the above methodsbut inoculation of a few millilitres of the final prod-uct into 10 times its volume of sterile broth (todilute inhibitory substances) will permit detectionof heavy contamination.

Tests for performance of mediaThe ability of each batch of new medium to sup-port growth and exhibit the required differentialand selective qualities should be determined beforeuse. This may be done by the efficiency of platingtechnique described in Chapter 5.

The organisms used should be stock strains froma recognized culture collection such as the NationalCollection of Type Cultures (NCTC) or AmericanType Cultures (ATCC). As characteristics of bacte-ria may alter when maintained by serial subculture,it is preferable to use stock strains stored at −70°C,freeze-dried or in gelatin discs (see Chapter 6).Selected media are tested with organisms expectedto grow and those expected to be inhibited.Biochemical indicator media are tested with two or

Practical quality control within the laboratory 15

more organisms to demonstrate positive and nega-tive reactions. A practical approach based on aminimum number of strains has been described(Westwell, 1999) (see also Chapters 5 and 6 of thisbook).

ReagentsMost pure chemicals and stains have greater stabil-ity than biological reagents. All reagents must belabelled as to contents, concentration, date ofpreparation and expiry date or shelf-life. Theymust be stored according to manufacturers’instructions and discarded when out of date.Freshly prepared reagents must be subjected toquality control before use; working solutions ofstains are tested weekly and infrequently usedreagents and stains are tested for their requiredpurpose before use. Chemical reagents, e.g.Kovac’s reagent, should be checked for perform-ance against new batches of media. Some chemicalsdeteriorate unless stored correctly, e.g. certainstains are light sensitive. Storage should thereforebe in accordance with the manufacturers’ datasheets.

QUALITY ASSESSMENT

The concepts and statistical methods for the imple-mentation of quality assessment were largely pio-neered in clinical biochemistry laboratories.Schemes in microbiology laboratories are less easilyorganized because of the complexity, expense andlabour intensity of the work. The diversity ofmicrobiological investigations makes it difficult toproduce control materials that cover all areas, andseveral technical difficulties have to be overcome inthe preparation of quality assessment specimens(DeMello and Snell, 1985).

Quality assessment may be organized locallywithin one laboratory (internal quality assessment)(Gray et al., 1995) or on a wider geographical basis(external quality assessment) (World HealthOrganization or WHO, 1994). A senior member ofstaff within the laboratory should organize aninternal scheme to follow which arrangements are

made so that examinations of specimens or samplesare repeated, thereby enabling direct comparisonswith previously obtained results. An obviousadvantage with this approach is that the specimensor samples are real, rather than simulated, ones.False identifications may be given to repeated spec-imens so that staff – unaware that they are qualitycontrol specimens – do not give them special atten-tion. Care should be taken with the use of storedmaterials because deterioration may prevent thereproduction of previous results.

A central laboratory, often on a national basis,usually organizes an external quality assessmentscheme, as, for example, that of the ASCP.Simulated samples of known but undisclosed con-tent are dispatched to participants who examinethem and report their findings to the organizinglaboratory. The organizer compares the resultsfrom all participating laboratories and sends ananalysis to each participant (Figure 2.2), therebyenabling them to gain an insight into their routinecapabilities. If internal quality control proceduresare functioning correctly, external assessmentshould reveal few unpleasant surprises.

Organization

Participation in the UK quality assessment schemeis voluntary and strictly confidential. Con-fidentiality is achieved by allocating each labora-tory a unique identification number known only tothe organizer and participant. This number is used


Organizing laboratory

Prepare QA specimens

Analyse results

Prepare report

Participants

Examine specimens

Report results

Evaluate

Figure 2.2 External quality assessment scheme

in all routine transactions and individual partici-pant’s results are not revealed to other partiesexcept under previously agreed and strictly definedcircumstances.

Difficulties arise in the organization of externalschemes in countries where the size and function ofparticipant laboratories vary widely. There mayalso be differences in the type of laboratory reportissued, particularly in the field of clinical microbi-ology. Thus some laboratories report only theresults of tests whereas others edit and interpret theresults. External quality assessment schemes musttake account of these differences by offering basicand comprehensive options (Gavan, 1974). Otherschemes reflect the philosophy that, although theremay be differences in the types of laboratories, thesame standards should be applied to their assess-ment. Although international schemes do exist,their aims differ from those of national schemesbecause they are principally used to assess selectednational laboratories in developing countries aspart of a wider support programme. Internationalschemes may be used to compare the standards ofmajor laboratories in different countries. Theselaboratories can then use the expertise and knowl-edge gained to initiate their own national schemes(Snell and Hawkins, 1991).

Specimens

External quality assessment specimens shouldresemble, as closely as possible, materials usuallyexamined by the participant laboratories. In someinstances this is easily achieved, as with specimensfor assay of antibodies in serum. In other cases, suf-ficient quantities of natural material may not beavailable and it is necessary to produce artificialspecimens. As external quality assessment shouldtest a laboratory’s routine capabilities, specimensmust to a great extent reflect the routine workload.Thus, as most specimens examined in a clinical lab-oratory do not yield pathogenic organisms, some ofthe simulated specimens should contain only non-pathogens. Commonly isolated organisms must berepresented in quality assessment specimens but, as

most laboratories experience little difficulty in iso-lating and identifying, e.g. Staphylococcus aureus,there is little to be learned from the inclusion of toomany such specimens. Less frequently encounteredorganisms, such as Corynebacterium diphtheriae,must also be distributed because they play animportant role in the education of junior membersof staff.

To gain and maintain credibility, participantsmust have full confidence in the specimens beingdistributed. The homogeneity and stability ofspecimens must be established and controlled toensure their comparability. Specimens designed totest isolation procedures must have the samenumber of organisms in each vial; this is mosteasily achieved by freeze-drying (DeMello andSnell, 1985). Specimens prepared for the detectionof antigen or antibody must react in a normal andpredictable manner in a full range of commonlyused assays. If participants are to evaluate theirperformance, they need to know if their resultsare incorrect. Thus, in general, specimens shouldyield clear-cut results. In reality, this can be quitedifficult, e.g. it is not easy to select strains of bac-teria that are unequivocally susceptible or resist-ant to a range of relevant antibiotics. The use ofstrains giving ‘borderline’ results may, however,highlight the failure of laboratories to use optimalmethods.

Microbial susceptibility tests

Quality control is vital here (see Chapter 12).

Benefits of quality assessment

The success of a quality assessment scheme isjudged ultimately by its usefulness, the extent ofwhich is primarily determined by the attitude of theparticipant. Quality assessment is a managementtool and information gained from the scheme willbe rendered ineffective if not used correctly. Thewhole philosophy of quality assessment is based on

Quality assessment 17

the assumption that the results obtained shouldreflect what is happening in day-to-day practice.For this to be true, quality assessment specimensmust be examined by the same staff and with thesame methods and reagents as specimens in thenormal workload.

Expenditure of extra effort on quality assessmentspecimens, as a conscious attempt to ‘cheat’ or as asubconscious desire to excel in a challenge of abil-ity (La Motte et al., 1977), negates the usefulness ofthe assessment and fails to reveal any unsuspectedproblems. Pressures on laboratory staff to performwell in quality assessment schemes, especially ifsuccess is seen as contributing to the economic via-bility of the laboratory by providing proof of excel-lence to customers, are understandable but shouldbe resisted.

Accreditation

Accreditation is a valuable component of qualityassurance in clinical microbiology (Batstone,1992). The purpose is simple: the audit by anexternal agency of an applicant’s organizationaland quality assurance programme to see that cer-tain defined standards are met. An accreditationscheme should be comprehensive, covering allaspects of the laboratory, including organizationand administration, staff development and educa-tion, facilities and equipment, and policies and pro-cedures. Participation in all relevant qualityassessment schemes is a requirement for accredita-tion. There are several accreditation schemes avail-able and before a laboratory embarks on theprocess consideration should be given to the appro-priateness of the various schemes.

REFERENCES

Bartlett, R. C. (1982) Making optimum use of themicrobiology laboratory. Journal of the American

Medical Association 247: 857–859. Bartlett, R. C., Heard, S. R., Schiller, G. et al. (1978)

Quality assurance in clinical microbiology. In

Horn, S. L. (ed. ) Quality Assurance Practices for

Health Laboratories Washington: American PublicHealth Association, 1978: 871–1005.

Batstone, F. (1992) Medical audit in clinical pathology.Journal of Clinical Pathology 45: 284–287.

Borriello, S. P. (1999) Science, medicine, and the future:Near patient microbiological tests. British Medical

Journal 319: 298–301. Campbell, S. M., Sheaf, R., Sibbald, B. et al. ( 2002)

Implementing clinical governance in Englishprimary care groups/trusts: reconciling qualityimprovement and quality assurance. Quality and

Safety in Health Care 11: 9–14.Crook, M. (2002) Clinical governance and pathology.

Journal of Clinical Pathology 55: 177–179. DeMello, J. V. and Snell, J. J. S. (1985) Preparation of

simulated clinical material for bacteriologicalexamination. Journal of Applied Bacteriology 59:421–436.

European Committee for Clinical Laboratory Standards(1985) Standard for Quality Assurance. Part 1:

Terminology and general principles, Vol 2. Lund:ECCLS: 1–13.

Fox, M. C., Ward, K. and Kirby, J. (1992) First stepsto accreditation. IMLS Gazette August: 412.

Gavan, T. L. (1974) A summary of the bacteriologyportion of the 1972 (CAP) quality evaluationprogram. American Journal of Clinical Pathology

61: 971–979. Gray, J. J., Wreggitt, T. G., McKee, T. A. et al. (1995)

Internal quality assurance in a clinical virologylaboratory. I Internal quality assessment. Journal of

Clinical Pathology 48: 168–173. Heard, S. R., Schiller, G., Shef, R. et al. (2001)

Continuous quality improvement: educatingtowards a culture of clinical governance. Quality in

Health Care 10: 70–78. La Motte, L. C., Guerrant, G.O., Lewis, D.S. et al.

(1977) Comparison of laboratory performancewith blind and mail- distributed proficiency testingsamples. Public Health Report 99: 554–560.

Nicholls, E. (1999) Quality control of culture media.In: Snell, J. J. S., Brown, D. F. J. and Roberts, C.(eds), Quality Assurance Principles and Practice in

the Microbiology Laboratory. London: PublicHealth Laboratory Service, 119–140.


Snell, J. J. S. and Hawkins, J. M. (1991) Qualityassurance – achievements and intended directions.Reviews in Medical Microbiology 3: 28–34.

Snell, J. J. S., Brown, D. F. J. and Roberts. C. (eds)(1999) Quality Assurance Principles and Practice

in the Microbiology Laboratory. London: PublicHealth Laboratory Service.

Westwell, A. (1999) Quality control of bacteriologicalcharacterization tests. In: Snell, J. J. S., Brown, D.F. J. and Roberts, C. (eds) Quality Assurance

Principles and Practice in the Microbiology

Laboratory. London: Public Health LaboratoryService, pp. 141–150.

Wilcox, K. R., Baynes, T.E., Crabbe, J.V. et al. (1978)Laboratory management. In: Horn, S. L. (ed.),Quality Assurance Practices for Health

Laboratories. Washington DC: American PublicHealth Association, pp. 3–126.

WHO (1994) Practice of quality assurance inlaboratory medicine in developing countries. In:Sharma K. B., Agarwell, D. S., Bullock, D. G. et al.

(eds), Health Laboratory Services in Support of

Primary Health Care in Developing Countries.

New Delhi: Regional Publication, South-East AsiaRegional Office, pp. 77–137.

References 19

3

Laboratory equipment

BASIC PRINCIPLES OF EQUIPMENTMANAGEMENT

Without equipment, there can be no microbiologi-cal methods and no practical microbiology.Experience has shown that, to be effective, equip-ment has to be properly managed. Such manage-ment should be an important part of thelaboratory’s overall quality assurance (QA) system.Equipment management is therefore an importanttopic for the microbiologist (see Chapter 2).

LABORATORY EQUIPMENT IN AMODERN LABORATORY

Taking a large modern clinical microbiology labo-ratory as an example, the range of capital equip-ment to be found in use would include thefollowing:

• Microscopes of different types • Centrifuges of different types• Incubators of different types• Water baths and heating blocks• Refrigerators and freezers• Autoclaves• Microbiological safety cabinets• Laminar flow clean air cabinets • Colony counters. • Freeze-drying apparatus• Shakers and mixers• Multi-point inoculators • Micro-organism detection and identification

systems• Automatic pipettors

• Antimicrobial susceptibility test instruments • Disc dispensers• Anaerobic work stations and anaerobic jars• pH meters• Glassware washers• Drying cabinets• Plate pouring and other culture preparation

systems.

In addition to this there are the basic tools andconsumables:

• Inoculating loops, wires and spreaders • Glassware items that are intended to be

recycled, such as slides, bottles, tubes, Petridishes and pipettes

• Pipetting devices and pipette tips• Petri dish and pipette drums• Racks and baskets for tubes and bottles• Disposable plastic items, including Petri

dishes, pipette tips• Specimen and sample containers of glass or

plastic.

IN VITRO DIAGNOSTIC MEDICALDEVICES

By far the largest proportion of the workload of aclinical and public health laboratory is the exami-nation of human samples and the provision ofresults that are intended to help in making clinicaldiagnoses or in clinical management. Accordingly,these laboratories will be using some equipment,together with dedicated consumable items, thatfalls within the coverage of the European Union’s

Directive on in vitro diagnostic medical devices(IVDs) (EU, 1998).

According to the Directive ‘in vitro diagnostic med-ical device’ means any medical device that is areagent, reagent product, calibrator, control mate-rial, kit, instrument, apparatus, equipment or sys-tem, whether used alone or in combination, intendedby the manufacturer to be used in vitro for the exam-ination of specimens, including blood, solely or prin-cipally for the purpose of providing information:

• concerning a physiological or pathologicalstate

• concerning a congenital abnormality• to determine the safety and compatibility with

potential recipients• to monitor therapeutic measures.

The first of these includes the identification of acause of infection. Monitoring therapeutic meas-ures, e.g. an antimicrobial susceptibility test or anantibiotic assay, are everyday activities for clinicalmicrobiology laboratories.

It is important to note that the Directive indicatesthat specimen containers, if specifically intended bytheir manufacturers for the primary containmentand preservation of specimens derived from thehuman body for the purposes of in vitro diagnosticexamination, are considered to be IVDs. However,the Directive emphasizes that items of generallaboratory equipment are not IVDs unless theirmanufacturer specifically intends them to be usedfor in vitro diagnostic examination.

After 7 December 2003, manufacturers of IVDsmust comply with national legislation that imple-ments the requirements of the Directive, i.e. beforethey can be marketed IVDs must meet the relevantessential requirements that are laid down in theDirective. These focus on design and manufactur-ing requirements aimed at ensuring reliable resultsand safety in use.

When satisfied that essential requirements aremet, after 7 December 2003, the manufacturer hasto affix to the product the CE mark of conformity.However, distributors of IVDs may continue to selluntil 7 December 2005 products that are not CEmarked, but which are still in the supply chain.

CE marking involves different levels of regulatorycontrol of different IVDs. The level of controlreflects the risk category of the type of IVD. Themost highly regulated IVDs include tests for mark-ers of HIV-1 and -2, HTLV-I and -II, and hepatitisB virus (HBV), HCV and HDV.

Member states of the EU are required by theDirective to take all necessary steps to ensure thatIVDs may be placed on the market and/or put intoservice only if they comply with the requirementsof the Directive when duly supplied and properlyinstalled, maintained and used in accordance withtheir intended purpose. This raises an additionalimportant point, i.e. users clearly have a role toplay in the proper use of an IVD if it can beexpected to produce reliable results. Indeed, underthe UK’s In Vitro Diagnostic Medical DeviceRegulations (Statutory Instrument, 2000), if a userreassigns the intended use of a CE-marked IVD ormodifies the instructions for use, without the agree-ment of the manufacturer, he or she becomes liablefor any resulting performance failure that arisesfrom such use of the product.

Against this background, with reference to themanufacturer’s intended application, e.g. as statedin advertisements, sales literature and on packagingand labelling, examples of IVDs that might be ofinterest to a clinical microbiology laboratorywould include dedicated equipment for antimicro-bial sensitivity testing, blood culture, HIV andHBV testing, and specimen containers intended forblood, urine and faeces. On the other hand, a labo-ratory centrifuge, microscope, mixing machine,pipette or test tube that is not marketed for a spe-cific application would not fall within the scope ofthe IVD Directive.

Finally, a hospital laboratory may be responsiblefor the management of IVDs that are used outsidethe laboratory by nursing or medical staff to pro-vide on-the-spot results, i.e. in a situation that isknown, among other things, as point of care testing(POCT). Other terms used to describe POCT arenear patient testing, bedside testing, extra-labora-tory testing and disseminated laboratory testing.

Examples that may be relevant to the clinicalmicrobiology laboratory are rapid test systems for

In vitro diagnostic medical devices 21

infectious disease markers and urinalysis. Someissues in the general management of IVDs andpoint of care testing IVDs are dealt with below.

PRINCIPLES OF EQUIPMENTMANAGEMENT

The equipment management principles outlined inthis chapter are based on guidance that has beenissued by the UK Department of Health’s MedicalDevices Agency (MDA) and its predecessor theMedical Devices Directorate (MDD, 1991). Thepurpose is to ensure, in a cost-effective manner,that equipment:

• is suitable for its intended purpose• is understood by the users• is in a safe and serviceable condition• meets current safety and quality requirements• where relevant, has a data output that is

compatible with the laboratory’s existing orenvisaged data management system.

The essential features of equipment managementare:

• justification of need• selection• acceptance upon delivery• training in use• maintenance, repair and modification• replacement (if necessary) and disposal.

Justification of need

It is recommended that the laboratory have a pro-cedure in place that requires the purchaser to makeout a case in support of the need for the equipment.Where the equipment has never been used before inthe laboratory, the case should indicate the job ofwork to be done by the new item and, where areplacement for an existing item of equipment isbeing sought, the need for replacement should bejustified. The justification of need should take intoaccount the current work pattern and likely future

trends, including possible changes in workload andavailability of trained staff.

Selection

Experience has shown that, unless good qualityequipment is purchased in the first place, no subse-quent activity will effect improvement. It is there-fore of the utmost importance that there arearrangements for providing expert advice whenneeded and for carrying out all the necessary pre-purchasing investigations. In the UK, expert adviceis available from the MDA, which organizes anevaluation programme and publishes reports onIVDs and other items used in the laboratory. Thisprovided independent third party assessment ofperformance, sometimes carried out as compara-tive evaluation between different products with thesame intended purpose. The MDA operated a vol-untary adverse incident reporting programme andpublished safety notices that are of interest to thoseinvolved in the purchase of laboratory equipment.On 1 April 2003, however, the MDA merged withthe Medicines Control Agency to form theMedicines and Health Care Products RegulatoryAgency, and it is not clear what kind of advice thisnew body will provide.

In any case, before new equipment is purchased, itis always advisable to obtain the personal advice ofmicrobiologists who have had experience in its use.It is bad policy to rely entirely on advertisements,catalogues, extravagant claims of representatives,and the opinions of purchasing officers who aremainly concerned with balancing budgets. The bestis not always the most expensive, but it is rarely thecheapest.

A detailed specification should be drawn up forthe equipment. This should itemize the perform-ance requirements with reference to its intendedimmediate use. Although care should be taken notto specify a performance better than needed, thelonger-term use of the equipment in the light ofpossible future test requirements should be takeninto account. Especially when the purchase ofexpensive capital that is available from more than

22 Laboratory equipment

one supplier is contemplated, there is a need for anobjective demonstration of how the specification ismet by each of them. The emphasis should be onsafety and quality, the latter being defined as fitnessfor purpose. Furthermore, the equipment shouldhave been designed specifically for use in microbio-logical laboratories. That designed for other disci-plines or other purposes is rarely satisfactory.

Although nowadays it is unlikely that laboratoryequipment offered for sale will not meet the currentsafety requirements, especially if it is manufacturedfor the European or American markets, neverthe-less compliance with safety standards should beinvestigated. Fortunately, if an item of equipmentfrom a reputable manufacturer carries the EuropeanCE marking or claims of compliance with a rele-vant standard are made, a reasonable presumptioncan be made that the relevant safety standards arelikely to be met. Nevertheless, it is advisable toinclude safety checks as part of the acceptance pro-cedure (see below). A list of British Standards (BSand BS EN) for laboratory equipment is given inthe Addendum to this chapter (see p. 43).

Although the EU Directive applies only to stateswithin the EU and the MDA operates only in theUK, the principle of both could be adopted, withadvantage, in other countries.

Other factors to consider when selecting equip-ment are the availability, throughout the plannedlife of the equipment, of adequate maintenance andrepair facilities, availability of spare parts and con-sumables, and training for staff. The overall cost ofequipment is a highly significant factor when mak-ing a purchasing decision. In assessing the cost ofequipment, the whole life figure should be consid-ered rather than simply the initial capital expendi-ture. Over the lifetime of the equipment, recurringrevenue costs can often amount to more than theinitial purchase price. Indeed, manufacturers oftenrely on the recurring sale of consumables for theirprofits. Other revenue costs include those for main-tenance and repair, spare parts, installation andtraining.

It is recommended that, wherever possible, theselection process should be a team effort, the aimbeing that the final purchasing decision is a consen-

sus following consultation with the intended usersand with full consideration being given to theirviews.

Given that they will have to work with the equip-ment that is chosen, perhaps for a significant partof a working day, if there is a choice of more thanone supplier at the end of the line, it is probablybest that the deciding vote should go to the users.Some fields of investigation that might be pursuedare set out below.

Acceptance procedure

A formal acceptance procedure is needed to ensurethat the entry of all equipment into service is prop-erly controlled. This procedure will include the fol-lowing:

• an initial inspection with safety checks forelectrical equipment

• incorporation into the equipmentmanagement record system

• formal commissioning.

Initial inspectionOn receipt of a new acquisition, an initial inspec-tion should be carried out to ensure that the equip-ment is complete with all the accessories and users’operating manuals, and that it is undamaged and inproper working order.

RecordsIt is recommended that a record be maintainedand kept at all times with the equipment. Thisrecord should contain brief descriptions of allinspections, maintenance, repair and any modifi-cation. The first entry should be of the acceptanceof the equipment into service. The record shouldbe used to note minor faults that are found duringnormal use so that they can be brought to theattention of the service technician at the nextscheduled planned preventive maintenance visit(see below). Periods of time that the equipment isout of service because of breakdown should alsobe noted in the record.

Principles of equipment management 23

CommissioningSome types of equipment, e.g. autoclaves and bio-logical safety cabinets, will require, after installa-tion, a formal commissioning procedure againstobjective test criteria to demonstrate that they arefunctioning safely and effectively before routineuse. This will need to be recorded.

Staff training

No new laboratory equipment should be allowedto enter service until potential users have had ade-quate training in its use, user servicing and emer-gency action in the event of malfunction. It isrecommended that those unfamiliar with the equip-ment should be forbidden to operate it unlesssupervised or until they are considered competentin its use.

The content of the training and who delivers thetraining will depend on the complexity of theequipment, but as a general rule it is recommendedthat, if a supplier offers a training course for a newitem of equipment, this should be taken up. It isimportant that the members of staff who attend thecourse will be the ones who will actually use it.Furthermore, it should be recognized that trainedstaff will move on to other posts and their placesmay be taken by those who may not have seen oroperated the equipment. Reliance should not beplaced on them being able to pick up how to oper-ate equipment from established operators; thisapproach often perpetuates bad habits and danger-ous short-cuts. It is important that replacementstaff receive full and systematic training accordingto a syllabus. Those for centrifuges and microbio-logical safety cabinets are given on pp. 29 and 35,respectively.

This is particularly important where user servic-ing is concerned because experience has shown thatoperational malfunctions and dangerous occur-rences, such as fires or explosions, have sometimesoccurred immediately after faulty user servicing(see below).

Maintenance, repair and modification

If equipment is to remain safe, serviceable and reli-able throughout its working life, planned con-trolled maintenance is essential. In addition,arrangements should be in place to ensure thatemergency repairs and any modifications that haveto be carried out to improve the performance orsafety of equipment are carried out promptly andeffectively.

DecontaminationEquipment that has to be inspected, maintained,repaired, modified or scrapped (see below) shouldbe in a condition that makes it safe to be handledby all those who may come into contact with it dur-ing transit or subsequent handling (MDA, 1998).Before any such work is done on microbiology lab-oratory equipment, to reduce the risk of transmis-sion of infection, or fear of infection, it must becleaned and decontaminated in accordance with themanufacturer’s specific instructions, if provided. Inthe absence of specific instructions for decontami-nation, the manufacturer should be consulted aboutcompatibility with intended agents to ensure, as faras possible, that they are compatible with the equip-ment and do not damage it (see underMicrobiological safety cabinets and Chapter 4).

If the equipment is to be sent out of the laboratoryfor the work to be done, e.g. it is to be sent to themanufacturer’s factory, or the work is to be carriedout on site by staff other than those employed bythe laboratory, such as by a visiting engineer, adecontamination certificate should be issued underthe authority of the head of the laboratory.

MaintenanceThere are two levels of maintenance to consider:

1. Basic scheduled maintenance, i.e. userservicing, which the manufacturer intendsshould be carried out by the actual user of theequipment; sometimes this amounts tonothing more than the laboratory equivalentof good housekeeping, e.g. cleaning andsimple adjustments,


2. Planned preventive maintenance (PPM),which the manufacturer intends should becarried out to ensure long-term reliableperformance in accordance with hisspecification. This often involvescomprehensive inspection, changingcomponents and subassemblies that requireregular replacement, calibration, performancetests and a final safety check. This amounts tothe laboratory equivalent of car servicing.

Responsibility for day-to-day supervision of userservicing should rest with the head of the labora-tory who may wish to delegate this function to asenior member of the staff. It is essential that thework is done only by trained staff strictly in accor-dance with a written schedule, and that completeand up-to-date records are kept. A daily checklistwill usually suffice.

Although contract PPM will usually be availablefrom the manufacturer or an appointed agent, itmay also be available from a third-party commer-cial source or though an in-house maintenancescheme, e.g. as is sometimes operated by hospitalengineering departments in the UK. Setting asideeconomic considerations, the head of the laboratorywill need to ensure that whoever carries out the PPMcan demonstrate that they have readily available:

• sufficient technical information and spareparts

• sufficient trained staff, including supervisors• necessary tools and test equipment.

RepairIf user servicing and PPM are being carried outproperly and in accordance with the manufac-turer’s schedule, breakdowns should be minimized.However, random failures can occur even withwell-maintained equipment, and the head of thelaboratory should ensure that arrangements are inplace so that repairs can be effected speedily. Inaddition to the requirements detailed for the provi-sion of PPM, whoever is given the responsibility ofattending to a request for a repair should be able toprovide a guarantee that they can respond withinan agreed timescale.

ModificationUncontrolled modification of laboratory equipmentcan adversely affect performance and can be dan-gerous. Modification should be carried out only ifformal authorization has been given by the head ofthe laboratory or a senior member of staff to whomauthority has been delegated. It is preferable thatmodification be carried out by the manufacturer inorder to ensure that essential safety features are notimpaired. Manufacturers under contract for theprovision of PPM will sometimes offer modifica-tions that have been developed to improve thesafety or performance of their equipment. One ofthe disadvantages of third-party contract or in-house provision of PPM is that the availability ofsuch modifications may not become apparent.

Replacement and disposal

In due course, equipment may have to be replacedfor one or more of the following reasons. The fol-lowing can happen to the equipment:

• It may be worn out beyond economic repair.• It may be damaged beyond economic repair.• It may be unreliable.• Results produced are clinically obsolete or the

methodology for producing them istechnologically obsolete.

• Spare parts are no longer available.• Consumables are no longer available.• More cost-effective equipment is available.

It is good practice to keep all existing equipmentunder review so that an item of equipment that willneed replacement can be identified in good time,thereby facilitating the necessary financial and sys-tematic procurement arrangements for replace-ment. In particular, the head of the laboratoryshould resist any drive that would lead to the hur-ried or unstructured purchase of replacementequipment at the end of a financial year because offear that ‘the money will be lost otherwise’. Suchactivity has often been associated with poor valuefor money or the purchase of equipment that is notreally needed.

Principles of equipment management 25

However unwanted equipment is disposed of,even if it is destined for the scrapyard, it shouldnever pass out of the control of the laboratorywithout it being cleaned and decontaminated andaccompanied by a signed decontamination certifi-cate.

MANAGEMENT OF IVDS

Purchasers of IVDs will need to be aware that theessential requirements of the IVD Directive are thatproducts must achieve the claims that are made forthem by manufacturers in terms of analytical sensi-tivity, diagnostic sensitivity, analytical specificity,diagnostic specificity, accuracy, repeatability andreproducibility, including control of known rele-vant interference and limits of detection.Furthermore, the manufacturer must be able todemonstrate with data that the performance claimshave been met. The Directive also states that theproduct must not compromise the health or safetyof users.

In short, this means that a customer who pur-chases a CE-marked product from a reputablemanufacturer should be able to have a high degreeof confidence that it is capable of working properlyif used as specified, and will be safe to use.However, it is important to emphasize that theDirective does not require a manufacturer todemonstrate clinical utility as a condition of plac-ing an IVD on the market. This means that theonus is on the purchaser to assess whether theproduct is suitable for the use to which the resultswill be put in the clinical management of patients.

Another aspect to consider is that the IVDDirective exists to remove technical barriers totrade within the EU. Thus, it can be expected thatmanufacturers from across the expanding EU willbe offering IVDs that, although they may have beendeveloped originally for a specific home market,will be widely exported. This may necessitate trans-lations of the original instructions. Good clearinstructions have a crucial role in the safe and effec-tive use of IVDs. In the light of these points, thelaboratory is advised to ensure (MDA, 2002a) that:

• The users of IVDs and the clinicians who willuse the results produced by them should beconsulted when making purchasing decisions.

• Performance claims made by manufacturersand arrangements for provision of trainingand maintenance and the compatibility of acandidate system’s output with thelaboratory’s data management system shouldbe considered at the selection stage.

• Before an IVD is introduced into routine use,it should be subjected to a thoroughevaluation in the hands of the laboratorystaff, as a means of validation.

• The evaluation should include aninvestigation to establish whether existingspecimen collection devices, e.g. blood sampletubes and bacteriological swabs, can adverselyaffect the results produced by the IVD.

• The instructions for use must beunderstandable by all the intended users – thisis especially important when the instructionsthat have been issued are translations.

• Adequate levels of staff training aremaintained.

• Arrangements are in place for maintenanceand repair.

Point of care testing IVDs

Although today’s POCT IVDs are generally morereliable and less prone to error than those of previ-ous generations, the successful implementation ofPOCT service is still dependent on the effectiveorganization and management of staff.

Users of IVDs for POCT should have a soundunderstanding of the relevant analytical principles,the importance of QA and the interpretation of testresults. It is therefore important that the users ofPOCT IVDs have access to clear guidance on theseissues. In the light of these points, the laboratorythat is responsible for providing the clinical micro-biology service for the place where a POCT IVD isto be used is advised (MDA, 2002b) to ensure thatit:


• is involved at the earliest opportunity in allaspects of the provision of a POCT serviceinvolving a microbiological test

• oversees all training, updating and monitoringof all staff involved in the POCT service, andshould be prepared to act as an initial‘trouble-shooter’ for all problems involvingPOCT IVDs

• oversees the preparation of all standardoperating procedures involving the POCTservice

• oversees the integration of the POCT serviceinto its overall quality system.

CAPITAL EQUIPMENT

Equipment for which there are British Standardsare marked with an asterisk(*) (see the Addendumto this chapter on p. 43).

Microscopes

Laboratory workers should, of course, be familiarwith the general mechanical and optical principlesof microscopes but a detailed knowledge of eitheris unnecessary and, apart from superficial cleaning,maintenance should be left to the manufacturers,who will arrange for periodic visits by their techni-cians. Most manufacturers publish handbooks con-taining useful explanations and information.

Wide-field compensating eyepieces should be fit-ted; they are less tiring than other eyepieces and aremore convenient for spectacle wearers. For low-power work, × 5 and × 10 objectives are most usefuland, for high-power (oil immersion) microscopy,× 50 and × 100 objectives are desirable.

Careful attention to critical illumination, centringand the position of diaphragms is essential for ade-quate microscopy.

Low-power microscopesLow-power, binocular, dissecting microscopes arebetter than hand lenses for examining colony mor-

phology. Experience in their use may save muchtime and expense in pursuing unnecessary tests (seep. 85). They also facilitate the subculture of smallcolonies from crowded Petri dishes, thus avoidingmixed cultures.

Incubators

Incubators are available in various sizes. In general,it is best to obtain the largest possible models thatcan be accommodated, although this may createspace problems in laboratories where several differ-ent incubation temperatures are employed. Thesmaller incubators suffer wider fluctuations in tem-perature when their doors are opened than thelarger models, and most laboratory workers findthat incubator space, like refrigerator space, is sub-ject to ‘Parkinson’s law’.

Although incubators rarely develop faults, it isadvisable, before choosing one, to ascertain thatservice facilities are available. The circuits are notcomplex but their repair requires expert technicalknowledge. Transporting incubators back to themanufacturers is most inconvenient.

In medical and veterinary laboratories, incubatorsare usually operated only at 35–37°C. The food orindustrial laboratory usually requires incubatorsoperated at 15–20°C, 28–32°C and 55°C. For tem-peratures above ambient there are no problems,but lower temperatures may need a cooling as wellas a heating device.

CO2 incubatorsIncubation in an atmosphere of 5–8% CO2 ispreferable for the cultivation of many bacteria ofmedical importance. Incubators that provide auto-matic control of CO2 and humidity are now avail-able.

Water used to maintain humidity may becomecontaminated with fungi, especially aspergilli,causing problems with cultures that require pro-longed incubation.

Capital equipment 27

Cooled incubatorsFor incubation at temperatures below the ambient,incubators must be fitted with modified refrigera-tion systems with heating and cooling controls.These need to be correctly balanced.

Automatic temperature changesThese are necessary when cultures are to be incu-bated at different temperatures for varying lengthsof time, as in the examination of water by mem-brane filtration, and when it is not convenient tomove them from one incubator to another. Twothermostats are required, wired in parallel, and atime switch wired to control the thermostat set atthe higher temperature. These are now built in tosome models.

Portable incubatorsThese are useful in field work, e.g. environmentalmicrobiology and water examination (see p. 258).

Incubator roomsThese should be installed by specialist suppliers.Metal shelving is preferable to wood: if the humid-ity in these rooms is high the growth of mouldfungi may be encouraged by wood. Solid shelvingshould be avoided and space left at the rear of theshelves to allow for the circulation of air.

If, on the other hand, low humidity is a problemcultures may be prevented from drying out by plac-ing them along with pieces of wet filter paper inplastic boxes such as those sold for the storage offood.

Centrifuges*

Over the years there have been a number of acci-dents involving centrifuges. Some of these involvedthe ejection into the laboratory of centrifuge buck-ets and other parts. Although some of these acci-dents were caused by faulty components, otherswere the result of misuse (Kennedy, 1988). Thehigh kinetic energy of parts ejected from a cen-trifuge poses a serious risk of injury. In addition, if

an accident occurs while bodily fluids are beingcentrifuged, an infection risk arises from micro-bially contaminated environmental surfaces andaerosols (Collins and Kennedy, 1999).

Mechanical, electrical and microbiological safetyare important considerations in the purchase anduse of centrifuges. They should conform tonational and international standards, e.g. thoselisted in the Addendum (see p. 43), and also theInternational Federation of Clinical Chemistry(IFCC, 1990) and the International ElectricalCommission (IEC, 1990).

Although modern centrifuges are designed tomeet high safety standards (including those of BSEN, 1995, see Addendum) and are less likely tolend themselves to misuse, it is strongly recom-mended that all potential users of a centrifuge aretrained in its safe use before they are permitted tostart using it. It is important that users receivetraining in every type of centrifuge that they will beusing, including operations involving sealed buck-ets if these are to be used. A model set of instruc-tions and precautions for the safe use of centrifugesare offered below.

The ordinary laboratory centrifuge is capable ofexerting a force of up to 3000 g and this is the forcenecessary to deposit bacteria within a reasonabletime.

For general microbiology, a centrifuge capable ofholding 15-ml and 50-ml buckets and working at amaximum speed of 4000 rev./min is adequate. Theswing-out head is safer than the angle head becauseit is less likely to distribute aerosols if uncappedtubes are used (Collins and Kennedy, 1999).

For maximum microbiological safety, sealedbuckets should be fitted. These confine aerosols ifbreakage occurs and are safer than sealed rotors.Several companies supply sealed buckets (‘safetycups’). Windshields offer no protection. Thereshould be an electrically operated safety catch sothat the lid cannot be opened when the rotors arespinning.

Centrifuge buckets are usually made of stainlesssteel and are fitted with rubber buffers. They arepaired and their weight is engraved on them. Theyare always used in pairs, opposite to one another,


and it is convenient to paint each pair with differ-ent coloured patches so as to facilitate recognition.If the buckets fit in the centrifuge head on trun-nions, these are also paired.

Centrifuge tubes to fit the buckets are made ofglass, plastic, nylon or spun aluminium. Conicaland round-bottomed tubes are made. Although theconical tubes concentrate the deposit into a smallbutton, they break much more readily than theround-bottomed tubes. When the supernatantfluid, not the deposit, is required, round-bottomedtubes should be used.

Instructions for using the centrifuge1. Select two centrifuge tubes of identical lengths

and thickness. Place liquid to be centrifugedin one tube and water in the other to withinabout 2 cm of the top.

2. Place the tubes in paired centrifuge bucketsand place the buckets on the pans of thecentrifuge balance. This can be made byboring holes large enough to take the bucketsin small blocks of hard wood, and fittingthem on the pans of a simple balance.

3. Balance the tubes and buckets by adding 70%alcohol to the lightest bucket. Use a Pasteurpipette and allow the alcohol to run downbetween the tube and the bucket.

4. Place the paired buckets and tubes indiametrically opposite positions in thecentrifuge head.

5. Close the centrifuge lid and make sure thatthe speed control is at zero before switchingon the current. (Many machines are fittedwith a ‘no-volt’ release to prevent the machinestarting unless this is done.)

6. Move the speed control slowly until the speedindicator shows the required number ofrevolutions per minute.

Precautions1. Make sure that the rubber buffers are in the

buckets, otherwise the tubes will break.2. Check the balancing carefully. Improperly

balanced tubes will cause ‘head wobble’ andspin-off accidents, and wear out the bearings.

3. Check that the balanced tubes are reallyopposite one another in multi-bucketmachines.

4. Check that the buckets are properly locatedon the centrifuge head.

5. Never start or stop the machine with a jerk.6. Observe the manufacturer’s instructions about

the speed limits for the various loads.7. Load and open sealed centrifuge buckets in a

microbiological safety cabinet.

MaintenanceThe manufacturers will, by contract, arrange peri-odic visits for inspection and maintenance in theinterests of safety and efficiency.

Water baths

The contents of a test tube placed in a water bathare raised to the required temperature much morerapidly than in an incubator. These instruments aretherefore used for short-term incubation. If thelevel of water in the bath comes to one-half to two-thirds of the way up that of the column of liquid inthe tube, convection currents are caused, whichkeep the contents of the tube well mixed and hastenreactions such as agglutination.

All modern water baths are equipped with electri-cal stirrers and in some the heater, thermostat andstirrer are in one unit, easily detached from thebath for use elsewhere or for servicing. Water-baths must also be lagged to prevent heat lossthrough the walls. A bath that has not been laggedby the manufacturers may be insulated with slabsof expanded polystyrene.

Water-baths should be fitted with lids in order toprevent heat loss and evaporation. These lids mustslope so that condensation water does not drip onthe contents. To avoid chalky deposits on tubesand internal surfaces, only distilled water should beused, except, of course, in those baths that operateat or around ambient temperature, which are con-nected to the cold water supply and fitted with aconstant level and overflow device.


This kind of bath offers the same problems as thelow-temperature incubators unless the water is sup-plied at a much lower temperature than that requiredin the water bath. In premises that have an indirectcold water supply from a tank in the roof, the watertemperature in summer may be near or even abovethe required water bath temperature, giving the ther-mostat no leeway to operate. In this case, unless itis possible to make direct connection with the risingmain, the water supply to the bath must be passedthrough a refrigerating coil. Alternatively, the waterbath may be placed in a refrigerator and its elec-tricity supply adapted from the refrigerator light orbrought in through a purpose-made hole in the cab-inet. The problem is best overcome by fitting labo-ratory refrigeration units designed for this purpose.

Metal block heatersThese give better temperature control than waterbaths and do not dry out, but they can be used onlyfor tubes and thin glass bottles that must fit neatlyinto the holes in the steel or aluminium blocks.

It is advisable to use these blocks for work withtubes containing Group 3 agents that might leakinto and contaminate the water in water baths.

Refrigerators and freezers

Although domestic appliances are usually satisfac-tory for laboratory work they may pose a hazard ifthey have unsealed electrical contacts and if chemi-cals with flammable or explosive vapours arestored in them. If there is a need to store such mate-rial in a domestic-type refrigerator, it should be asmall amount in a sound container with a leak-proof closure and preferably packaged inside alarger container.

Ideally all sources of ignition, e.g. thermostat con-tacts and light switch mechanisms, should be out-side the refrigeration chamber. Intrinsically saferefrigerators are available commercially but theyare expensive.

Refrigerators and freezers that are not intendedfor the storage of flammable liquids should belabelled as such (Kennedy, 1988).

Sterilization equipment

For details about this equipment, see Chapter 4.

Microbiological safety cabinets*

Microbiological safety cabinets (MSCs), which arealso known as biological safety cabinets (BSCs), areintended to capture and retain infected airborneparticles released in the course of certain manipula-tions and to protect the laboratory worker frominfection that may arise from inhaling them.

There are three kinds: Classes I, II and III. InEurope Class I and Class II cabinets are used indiagnostic, Level 2 and Containment, Level 3 labo-ratories for work with Hazard Group 3 organisms.Class III cabinets are used for Hazard Group 4viruses. Some other countries use only Class II andClass III cabinets.

A Class I cabinet is shown in Figure 3.1. Theoperator sits at the cabinet, works with the handsinside, and sees what he or she is doing through theglass screen. Any aerosols released from the cul-tures are retained because a current of air passes inat the front of the cabinet. This sweeps the aerosolsup through the filters, which remove all or most ofthe organisms. The clean air then passes throughthe fan, which maintains the airflow, and is


Figure 3.1 Class I microbiological safety cabinet

exhausted to atmosphere, where any particles ororganisms that have not been retained on the filterare so diluted that they are no longer likely to causeinfection if inhaled. An airflow of between 0.7 and1.0 m/s must be maintained through the front ofthe cabinet, and modern cabinets have airflow indi-cators and warning devices. The filters must bechanged when the airflow falls below this level.

The Class II cabinet (Figure 3.2) is more compli-cated and is sometimes called a laminar flow cabi-net, but as this term is also used for clean aircabinets that do not protect the worker it should beavoided (see p. 37). In the Class II cabinet about70% of the air is recirculated through filters, sothat the working area is bathed in clean (almoststerile) air. This entrains any aerosols produced inthe course of the work, which are removed by thefilters. Some of the air (about 30%) is exhausted toatmosphere and is replaced by a ‘curtain’ of roomair, which enters at the working face. This preventsthe escape of any particles or aerosols released inthe cabinet.

There are other types of Class II cabinets, withdifferent airflows and exhaust systems, which mayalso be used for toxic chemicals and volatile chem-icals. Manufacturers should be consulted.

Class III cabinets are totally enclosed and are

tested under pressure to ensure that no particlescan leak from them into the room. The operatorworks with gloves that are an integral part of thecabinet. Air enters through a filter and is exhaustedto atmosphere through one or two more filters(Figure 3.3).

Purchasing standards for safety cabinetsSafety cabinets should comply with national stan-dards, e.g. British Standards Institution (seeAddendum, p. 43) and the (US) NationalSanitation Foundation (NSF, 1992).

Use of safety cabinetsThese cabinets are intended to protect the workerfrom airborne infection. They will not protect theworker from spillage and the consequences of poortechniques. The cabinet should not be loaded withunnecessary equipment or it will not do its jobproperly. Work should be done in the middle to therear of the cabinet, not near the front, and theworker should avoid bringing hands and arms outof the cabinet while working. After each set ofmanipulations and before withdrawing the hands,the worker should wait for 2–3 min to allow anyaerosols to be swept into the filters. The hands andarms may be contaminated and should be washedimmediately after ceasing work. Bunsen burner


Figure 3.2 Class II microbiological safety cabinet

Figure 3.3 Class III microbiological safety cabinet

burners and even micro-incinerators should not beused because they disturb the airflow. Disposableplastic loops (see p. 37) are recommended.

SitingThe efficiency of a safety cabinet depends on cor-rect siting and proper maintenance. Possible sitesfor cabinets in a room are shown in Figure 3.4. SiteA is a poor site, because it is near to the door andairflow into the cabinet will be disturbed every timethe door is opened and someone walks into theroom and past the cabinet. Site B is not much bet-ter, because it is in almost a direct line between thedoor and window, although no one is likely towalk past it. Its left side is also close to the wall andairflow into it on that side may be affected by the‘skin effect’, i.e. the slowing down of air when itpasses parallel and close to a surface. Air passingacross the window may be cooled, and will meetwarm air from the rest of the room at the cabinetface, when turbulence may result.

Site C is better and site D is best of all. If two cab-inets are required in the same room, sites C and Dwould be satisfactory, but they should not be tooclose together, or one may disturb the airflow ofthe other. We remember a laboratory where a ClassI cabinet was placed next to a Class II cabinet.When the former was in use it extracted air fromthe latter and rendered that cabinet quite ineffec-tive.

Care must also be taken in siting any other equip-ment that might generate air currents, e.g. fans andheaters. Mechanical room ventilation may be aproblem if it is efficient, but this can be overcomeby linking it to the electric circuits of the cabinetsso that either, but not both, is extracting air fromthe room at any one time. Alternatively, bafflesmay be fitted to air inlets and outlets to avoid con-flicting air currents near to the cabinet. Tests withsmoke generators (see below) will establish thedirections of air currents in rooms.

None of these considerations needs to apply toClass III cabinets, which are in any case usuallyoperated in more controlled environments.

Testing airflowsThe presence and direction of air currents anddraughts are determined with ‘smoke’. This may begenerated from burning material in a device such asthat used by bee keepers, but is usually a chemicalthat produces a dense, visible vapour. Titaniumtetrachloride is commonly used. If a cottonwool-tipped stick, e.g. a throat swab, is dipped into thisliquid and then waved in the air, a white cloud isformed which responds to quite small air move-ments.

Commercial airflow testers are more convenient.They are small glass tubes, sealed at each end. Bothends are broken off with the gadget provided and arubber bulb fitted to one end. Pressing the bulb topass air through the tube causes it to emit whitesmoke. These methods are suitable for ascertainingair movements indoors.

To measure airflow an anemometer is required.Small vane anemometers, timed by a stopwatch,are useful for occasional work but for serious activ-ities electronic models, with a direct reading scale,


Figure 3.4 Possible sites for safety cabinets in relation to crossdraughts from door and window and movement of staff. A is bad;B is poor; C is better; D is best

are essential. The electronic vane type has a diame-ter of about 10 cm. It has a satisfactory time con-stant and responds rapidly enough to show thechanges in velocity that are constantly occurringwhen air is passing into a Class I safety cabinet.Hot wire or thermistor anemometers may be usedbut they may show very rapid fluctuations andneed damping. Both can be connected to recorders.

It is necessary to measure the airflow into a ClassI cabinet in at least five places in the plane of theworking face (Figure 3.5). At all points this shouldbe between 0.7 and 1.0 m3/s. No individual meas-urement should differ from the mean by more than20%. If there is such a difference there will be tur-bulence within the cabinet. Usually some piece ofequipment, inside or outside the cabinet, or theoperator, is influencing the airflow.

Although airflows into Class I cabinets are usuallymuch the same at all points on the working face,this is not true for Class II cabinets, where the flowis greater at the bottom than at the top. The aver-age inward flow can be calculated by measuring thevelocity of air leaving the exhaust and the area ofthe exhaust vent. From this the volume per minuteis found and this is also the amount entering thecabinet. The average velocity is obtained by divid-ing this volume by the area of the working face. Arough and ready way, suitable for day-to-day use,requires a sheet of plywood or metal that can be fit-ted over the working face. In the centre of this anaperture is made which is 2 × 2.5 cm. The inward

velocity of air through this is measured and theaverage velocity over the whole face calculatedfrom this figure and the area of the working face.The inflow at the working face should be not lessthan 0.4 m/s. A check should also be made withsmoke to ensure that air is in fact entering the cab-inet all the way round its perimeter and not just atthe lower edge.

The downward velocity of air in a Class II cabinetshould be measured with an anemometer at 18points in the horizontal plane 10 cm above the topedge of the working face (Figure 3.6). The meandownflow should be between 0.25 and 0.5 m/s andno individual reading should differ from the meanby more than 20%.

It is usual, when testing airflows with ananemometer, to observe or record the readings ateach position for several minutes, because of possi-ble fluctuations.

To measure the airflow through a Class III cabinetthe gloves should be removed and readings taken ateach glove port. Measurements should also betaken at the inlet filter face when the gloves areattached.


Figure 3.5 Testing airflow into a Class I safety cabinet. Anemo-meter readings should be taken at five places, marked with an X,in the plane of the working face with no-one working at thecabinet

Figure 3.6 Testing the vertical airflow in a Class II safety cabinet. Anemometer readings should be taken at points markedX on an imaginary grid 6 inches within the cabinet walls and justabove the level of the bottom of the glass window

DecontaminationAs safety cabinets are used to contain aerosols,which may be released, during work with HazardGroup 3 micro-organisms, the inside surfaces andthe filters will become contaminated. The workingsurface and the walls may be decontaminated on aday-to-day basis by swabbing them with disinfec-tant. Glutaraldehyde is probably the best disinfec-tant for this purpose because phenolics may leavesticky residues and hypochlorites may, in time, cor-rode the metal (Caution – see below).

Glutaraldehyde vapour is a cause of occupa-tional asthma, irritation of the eyes, nose andthroat. It is also a skin sensitizer and may causeallergic dermatitis. In the UK the Health andSafety Executive sets a maximum exposure limitof 0.05 ppm.

For thorough decontamination, however, andbefore filters are changed, formaldehyde (Caution– see below) should be used and precautions shouldbe taken before use. The installation should bechecked to ensure that none of the gas can escapeinto the room or other rooms. The front closure(night door) of the cabinet should seal properly onto the carcase, or masking tape should be availableto seal it. Any service holes in the carcase should besealed and the high efficiency particulate air (HEPA)filter seating examined to ensure that there are noleaks. If the filters are to be changed and the primaryor roughing filter is accessible from inside the cabi-net, it should be removed and left in the workingarea. The supply filter on a Class III cabinet shouldbe sealed with plastic film.

Formaldehyde is severely irritating to the eyes andthe respiratory tract. Inhalation may cause lungoedema. It is possibly carcinogenic to humans. TheInternational Labour Organization advises anoccupational exposure limit of 0.3 ppm. Mixturesof 7.75% (v/v) formaldehyde and dry air are explo-sive: the ignition point is 430°C.

Formaldehyde is generated by:

• boiling formalin, which is a 40% solution ofthe gas in water

• heating paraformaldehyde, which is its solidpolymer.

Boiling formalinThe volume used is important. Too little will beineffective; too much leads to deposits of the poly-mer, which is persistent and may contribute to thenatural blocking of the filters. The British Standard(BS, 1992 – see Addendum, p. 43) specifies a con-centration of 50 mg/m3. To achieve this 60 mlformalin, mixed with 60 ml water, is required foreach cubic metre of cabinet volume (this volume isspecified by the manufacturer).

The formalin may be boiled in several ways. Somecabinets have built-in devices in which the liquid isdripped on to a hot-plate. Other manufacturerssupply small boilers. A laboratory hot-plate, con-nected to a timing clock, is adequate. The correctamount of formalin and water is placed in a flask.

Heating paraformaldehydeTablets (1 g) are available from chemical manufac-turers. Three or four tablets are adequate for anaverage size cabinet. They may be heated on anelectric frying pan connected to a time clock, orplaced in a dish and heated by an electric hairdrier.

Exposure timeIt is convenient to start decontamination in the lateafternoon and let the gas act overnight. In themorning the fan should be switched on and thefront closure ‘cracked’ open very slightly to allowair to enter and purge the cabinet. Some new cabi-nets have a hole in the front closure, fitted with astopper that can be removed for this purpose. WithClass III cabinets the plastic film is taken off thesupply filter to allow air to enter the cabinet andpurge it of formaldehyde. After several minutes thegloves may be removed. The fan is then allowed torun for about 30 min, which should remove allformaldehyde.

Summary of decontamination procedureThe prefilter should be removed from inside thecabinet (where possible) and placed on the work-ing surface. It is usually pushed or clipped intoplace and is easily removed, but as it is likely to be


contaminated gloves should be worn. Formalin60 ml plus water 60 ml, per cubic metre of cabinetvolume, should be placed in its container on theheater in the cabinet or the reservoir. The frontclosure is then put in place and sealed if necessary.The heater is switched on and the formalin boiledaway. After switching off the heater the cabinet isleft closed overnight. The next morning the cabi-net fan is switched on, and the front closure isopened very slightly to allow air to pass in andpurge the cabinet of formaldehyde. After severalminutes the front closure is removed and the cabi-net fan allowed to run for about 30 min. Anyobvious moisture remaining on the cabinet wallsand floor may then be wiped away and the filterschanged.

Changing the filters and maintenanceThe cabinet must be decontaminated before filtersare changed and any work is done on the motorand fans. If these are to be done by an outside con-tractor, e.g. the manufacturer’s service engineer,the front closure should be sealed on again after theinitial purging of the gas and a notice – Cabinetdecontaminated but not to be used – placed on itawaiting the engineer’s arrival. He or she will alsorequire a certificate stating that the cabinet hasbeen decontaminated.

The primary, or prefilters, should be changedwhen the cabinet airflow approaches its agreedlocal minimum. Used filters should be placed inplastic or tough paper bags, which are then sealedand burned. If the airflow is not restored to at leastthe middle of the range, then arrangements shouldbe made to replace the HEPA filter. This is usuallydone by the service engineer, but may be done bylaboratory staff if they have received instruction.Unskilled operators often place the new filter inupside down or fail to set it securely and evenly inits place. Used filters should be placed in plasticbags, which are then sealed for disposal. They arenot combustible. Some manufacturers accept usedfilters and recover the cases, but this is not usuallya commercial practice – no refund is given. Whenmanufacturers replace HEPA filters they may offera testing service.

Training operatorsWe recommend that all potential users of microbi-ological safety cabinets be trained in the proper useof every type of cabinet that they are likely to use,especially in the following:

• Classification• Appropriate and inappropriate use• Mode of operation and function of all

controls and indicators• Limitations of performance• How to work safely with them • How to decontaminate them after use• Principles of airflow and operator protection

tests (see below).

Testing: further informationThis is beyond the scope of this book and thereader is referred to national standards and otherpublications (Kuhne et al. 1995; Collins andKennedy, 1999; CDC/NIH, 2000).

Although a cabinet may be designed and con-structed satisfactorily and be capable in the factoryof meeting the performance claims made for it,unless it is properly installed in the laboratory itmay not provide adequate operator protection.It was for this reason that BS 5726: Part 3, 1992specified that an operator protection factor test,among other performance tests, must be carriedout after installation. This is a very sensitive testthat actually measures the ability of a cabinet toretain an aerosol. Unfortunately, the EuropeanStandard (designated BS EN 12469:2000 in theUK) that has replaced BS 5726:1992 does notmandate an operator protection factor test (whichit calls a retention test) after installation, butmakes it an option. It is recommended that anoperator protection factor test is included in thecommissioning procedure.

Autoclaves

For information about autoclaves, see Chapter 4.


Anaerobic work stations andanaerobic jars

For information about these, see Chapter 6.

Freeze-drying apparatus

For information about this, see Chapter 6.

Blenders and shakers

These range from instruments suitable for grindingand emulsifying large samples, e.g. food, to glassand plastic devices for homogenizing small piecesof tissue (see ‘Tissue grinders and homogenizers’,below). Most are electrically driven (see BS EN,1996 in the Addendum). They suffer from the dis-advantage of requiring a fresh, sterile cup for eachsample. These cups are expensive and it takes timeto clean and re-sterilize them.

Some of the devices may release aerosols duringoperation and when they are opened. A heavyPerspex or metal cover should be placed over themduring use. This should be decontaminated after use.All blenders, etc. should be opened in a micro-biological safety cabinet because the contents willbe warm and under pressure, and aerosols will bereleased.

The Stomacher Lab-Blender overcomes theseproblems. Samples are emulsified in heavy-dutysterile plastic bags by the action of paddles. This isan efficient machine, capable of processing largenumbers of samples in a short time without pausesfor washing and re-sterilizing. There is little risk ofaerosol dispersal. The bags are automaticallysealed while the machine is working.

Vortex mixers are useful for mixing the contentsof single bottles, e.g. emulsifying sputum, butshould always be operated in a microbiologicalsafety cabinet.

Shaking machinesConventional shaking machines are sold by almostall laboratory suppliers. They are useful for mixingand shaking cultures and for serological tests, butshould be fitted with racks, preferably ofpolypropylene, which hold the bottles or tubesfirmly. These machines should be covered with astout Perspex box when in use to prevent the dis-persal of aerosols from containers that might leak.Alternatively, or in addition, we believe that allbottles that contain infected material and areshaken in any machine should be placed insideindividual self-sealing or heat-sealed plastic bags,in order to minimize the dispersal of infected air-borne particles.

The better models allow bottles, flasks, etc. to berotated at varying speeds as well as for shaking intwo dimensions.

Spiral platers


Micro-organism detection andidentification systems


Antimicrobial susceptibility testinstruments

For information about these, see Chapters 8 and12.

Disc dispensers

These allow antimicrobial sensitivity and otherpaper discs to be easily and rapidly dispensed,singly or in sets on media in Petri dishes.


Glassware washing machines

These are useful in large laboratories that useenough of any one size of tube or bottle to give aneconomic load.

Glassware drying cabinets

A busy wash-up room requires a drying cabinetwith wire-rack shelves and a 3-kW electric heaterin the base, operated through a three-heat con-trol. An extractor fan on the top is a refinement;otherwise the sides near the top should belouvered.

Laminar flow, clean air work stations

These cabinets are designed to protect the workfrom the environment and are most useful for asep-tic distribution of certain media and plate pouring.A stream of sterile (filtered) air is directed over theworking area, either horizontally into the room orvertically downwards when it is usually re-circu-lated. They are particularly useful for preventingcontamination when distributing sterile fluids andhand pouring into Petri dishes.

Laminar flow clean air work stations are NOTmicrobiological safety cabinets and must NOTused for manipulations with micro-organisms ortissue culture cells. The effluent air, which may becontaminated, is blown into the face of the opera-tor.

BENCH EQUIPMENT, GLASSWAREAND PLASTICS

Equipment for which there are British Standardsare marked with an asterisk(*) (see Addendum,p. 43).

Bunsen burner burners and loopincinerators

The usual Bunsen burner with a bypass is satisfac-tory for most work, but for material that may spat-ter or that is highly infectious a hooded Bunsenburner should be used. There are several versionsof these, but we recommend the Kampff andBactiburner types, which enclose the flame in aborosilicate tube.

‘Electric Bunsen burners’ or electric incineratorburners are available (e.g. the Bacti-Cinerator,made by Oxford Labware, St Louis, MO, USA). Aloop or inoculating wire (see below) is inserted intothe heated ceramic tube and after about 6 secondsorganic material is incinerated and sterilized at atemperature of about 815°C. Any spatter is con-tained within the heated tube. These devices aresuitable for use in Class I and Class II MSCs, pro-vided that the cabinets are free of any flammablematerial.

Inoculating loops and wires

These are usually made of 25 SWG Nicrome wire,although this is more springy than platinum irid-ium. They should be short (not more than 5 cmlong) in order to minimize vibration and thereforeinvoluntary discharge of contents. Loops should besmall (not more than 3 mm in diameter). Largeloops are also inclined to empty spontaneously andscatter infected airborne particles. They should becompletely closed, otherwise they will not holdfluid cultures. This can be achieved by twisting theend of the wire round the shank, or by taking apiece of wire 12 cm long, bending the centre rounda nail or rod of appropriate diameter, and twistingthe ends together in a drill chuck. Ready-madeloops of this kind are available.

Loops and wires should not be fused into glassrods because these may shatter in a Bunsen burnerflame. Aluminium holders are sold by most labora-tory suppliers.

Plastic disposable loops are excellent. There are

Bench equipment, glassware and plastics 37

two sizes – 1 and 10 μl – and both are useful. Theyare sold sealed in packs of about 25, sterilizedready for use. Pointed plastic rods, for use in placeof ‘straight wire’, for subculturing are now avail-able but some are too coarse for picking up verysmall colonies.

Spreaders

Many workers prefer these to loops for inoculatingPetri dishes with more than one loopful of material.To make them, cut a glass rod of 3–4 mm diameterinto 180-mm lengths and round off the cut ends ina flame. Hold each length horizontally across abat’s wing flame so that it is heated and bendsunder its own weight, approximately 36 mm fromone end, and an L shape is obtained. Plastic spread-ers are on the market, sterilized ready for use.

Microscope slides and cover glasses

Unless permanent preparations are required, thecheaper microscope slides are satisfactory. Slideswith ground and polished edges are much moreexpensive. Most microscope slides are sold inboxes of 100 slides. They should not be washedand reused but discarded.

Cover glasses are sold in several thicknesses andsizes. Those of thickness grade No. 1, 16 mmsquare, are the most convenient. They are sold inboxes containing about 100 glasses. Plastic coverglasses are available.

Pasteur pipettes*

Glass Pasteur pipettes are probably the most dan-gerous pieces of laboratory equipment in unskilledhands. They are used, with rubber teats, to transferliquid cultures, serum dilutions, etc.

Very few laboratories make their own Pasteurpipettes nowadays; they are obtainable, sterile andready for use, from most laboratory suppliers and

are best purchased in bulk. Long and short formsare available and most are made of 6–7 mm diam-eter glass tubing. Some are supplied.

Safer Pasteur pipettes with integral teats, made oflow-density polypropylene, are now available andare supplied ready sterilized.

Pasteur pipettes are used once only. Attempts torecycle them may result in cuts and pricks tofingers. There is a British Standard for glass dis-posable Pasteur pipettes (BS 5732:1985 – seeAddendum).

Graduated pipettes

Straight-sided blow-out pipettes, 1–10 ml capacityare used. They must be plugged with cottonwool atthe suction end to prevent bacteria entering fromthe pipettor or teat and contaminating the materialin the pipette. These plugs must be tight enough tostay where they are during pipetting, but not sotight that they cannot be removed during cleaning.About 25 mm of non-absorbent cottonwool ispushed into the end with a piece of wire. The endsare then passed through a Bunsen burner flame totidy them. Wisps of cottonwool that get betweenthe glass and the pipettor or teat may permit air toenter and the contents to leak.

Pipettes are sterilized in the hot air oven in square-section aluminium containers, similar to those usedfor Pasteur pipettes (square-section containers thatdo not roll have supplanted the time-honouredround-section boxes). A wad of glass wool at thebottom of the container prevents damage to the tips.

Disposable 1-ml and 10-ml pipettes are available.Some firms supply them already plugged, wrappedand sterilized.

Pipetting aids

‘Mouth pipetting’ is, properly, banned in allmicrobiological laboratories that handlepathogens or potential pathogens.


Rubber teats and pipetting devices provide alter-natives to mouth pipetting.

Rubber teatsChoose teats with a capacity greater than that ofthe pipettes for which they are intended, i.e. a 1-mlteat for Pasteur pipettes, a 2-ml teat for a 1-mlpipette; otherwise the teat must be used fully com-pressed, which is tiring. Most beginners compressthe teat completely, then suck up the liquid and tryto hold it at the mark while transferring it. This isunsatisfactory and leads to spilling and inaccuracy.Compress the teat just enough to suck the liquid alittle way past the mark on the pipette. Withdrawthe pipette from the liquid, press the teat slightly tobring the fluid to the mark and then release it. Thecorrect volume is now held in the pipette withouttiring the thumb and without risking loss. To dis-charge the pipette, press the teat slowly and gently,and then release it in the same way. Violent opera-tion usually fails to eject all the liquid; bubbles aresucked back and aerosols are formed.

‘Pipettors’A large number of devices that are more sophisti-cated than simple rubber teats are now available.Broadly speaking there are four kinds:

1. Rubber bulbs with valves that control suctionand dispensing

2. Syringe-like machines that hold pipettes morerigidly than rubber bulbs and have a plungeroperated by a rack and pinion or a lever

3. Electrically operated pumps fitted withflexible tubes in which pipettes can be inserted

4. Mechanical plunger devices that take smallplastic pipette tips and are capable ofrepeatedly delivering very small volumes withgreat accuracy.

It is extremely difficult to give advice on the rela-tive merits of the various devices. That which suitsone operator, or is best for one purpose, may notbe suitable for others. The choice should be madeby the operators, not by managers or administra-tors who will not use them. None of those in cate-gories 1, 2 and 4 above is expensive, and it should

be possible for several different models to be avail-able. What is necessary is some system of instruc-tion in their use and in their maintenance. Noneshould be expected to last forever.

In the USA, a number of manufacturers offercleaning and calibration services at a reasonablecost.

Tissue grinders and homogenizers

Hand-held devices are used for work with smallpieces of tissue or other material. They are avail-able in several sizes. A heavy glass tube is con-stricted near to its closed end and a pestle, usuallymade of glass covered with polyfluorotetraethylene(PTFE) or of stainless steel, is ground into the con-striction. With the pestle in place, the tissue andsome fluid are put into the tube. The pestle isrotated by hand. The tissue is ground through theconstriction and the emulsion collects in the bot-tom of the tube.

These grinders present some hazards. The tubes,even though they are made of borosilicate glass,may break and disperse infected material. Theyshould be used inside a microbiological safety cab-inet and be held in the gloved hand in a wad ofabsorbent material.

Recently, single-use, plastic, disposable tissuegrinders have become available. Although they areless likely to break than the glass models, they dohave a minor disadvantage that small shards ofsharp-edged plastic end up in the ground tissue,although these are easily identifiable.

Small pieces of soft tissue, e.g. curettings, may beemulsified by shaking in a screw-capped bottle witha few glass beads and 1 or 2 ml broth on a vortexmixer.

Glassware and plastics*

For ordinary bacteriological work, soda-glass tubesand bottles are satisfactory. In assay work, themore expensive borosilicate glass might be justi-


fied. An important consideration is whether glass-ware should be washed or discarded. Purchasingcheaper glassware in bulk and using plastic dispos-able Petri dishes and culture tubes may, in some cir-cumstances, be more economical than employinglabour to clean them. Plastics fall into two cate-gories:

1. Disposable items, such as Petri dishes,specimen containers and plastic loops, whichare destroyed when autoclaved and cannot besterilized by ordinary laboratory methods (butsee Chapter 4)

2. Recoverable material, which must be sterilizedin the autoclave.

Non-autoclavable plasticsThese include polyethylene, styrene, acrylonitrile,polystyrene and rigid polyvinyl chloride.

Autoclavable plasticsThese withstand a temperature of 121°C andinclude polypropylene, polycarbonate, nylon,PTFE (polytetrafluororethylene or Teflon), polyal-lomer, TPX (methylpentene polymer), Viken andvinyl tubing.

It is best to purchase plastic apparatus from spe-cialist firms who will give advice on the suitabilityof their products for specific purposes. Most recov-erable plastics used in microbiology can be washedin the same way as glassware. Some plastics softenduring autoclaving and may become distortedunless packed carefully.

Petri dishes*

Disposable plastic Petri dishes (see BS 1990 in theAddendum) are now used in most laboratories indeveloped countries. They are supplied alreadysterilized and packed in batches in polythene bags.They can be stored indefinitely, are cheap whenpurchased in bulk, and are well made and easy tohandle. Two kinds are available: vented andunvented. Vented dishes have one or two nibs thatraise the top slightly from the bottom and are to be

preferred for anaerobic and carbon dioxide cul-tures.

Glass Petri dishes are, however, still popular insome areas. In general, two qualities are obtain-able. The thin blown dishes, usually made ofborosilicate glass, are pleasant to handle, their topsand bottoms are flat and they stack safely. They donot become etched through continued use but arefragile, must be washed with care and are expen-sive. The thick pressed glass dishes are often con-vex, cannot always be stacked safely, and are easilyetched and scratched with use and washing. On theother hand, they are cheap and not fragile.Aluminium, stainless steel and plastic ‘lids’ areavailable, which doubles the number of glassdishes.

Glass Petri dishes are usually placed in aluminiumdrums and sterilized by hot air.

Test tubes and bottles, plugs, capsand stoppers

Culture media may be distributed in either testtubes or small bottles. Apart from personal choice,test tubes are easier to handle in busy laboratoriesand take up less space in storage receptacles andincubators, although bottles are more convenientin the smaller workroom where media are kept forlonger periods before use. Media in test tubes maydry up during storage. Screw-capped test tubes areavailable.

The most convenient sizes of test tube are:127 × 12.5 mm, holding 4 ml of medium;152 × 16 mm, holding 5–10 ml; 152 × 19 mm,holding 10–15 ml; and 178 × 25 mm, holding20 ml. Rimless test tubes of heavy quality are madefor bacteriological work. The lipped, thin, glasschemical test tubes are useless and hazardous.

Cottonwool plugs have been used for many yearsto stopper test-tube cultures, but have largely beenreplaced by metal or plastic caps, or in some labo-ratories by soft, synthetic, sponge bungs.

Aluminium test-tube caps were introduced someyears ago but they have a limited tolerance and, in


spite of alleged standard specifications of test tubes,a laboratory very soon accumulates many tubesthat will not fit the caps. Caps that are too loose areuseless. These caps are cheap, last a long time,available in many colours, and save a great deal oftime and labour. Those held in place by a smallspring have a wider tolerance and fit most tubes.Polypropylene caps in several sizes and colours andwhich stand up to repeated autoclaving can also beobtained. Rubber stoppers of the orthodox shapeare useless because they are blown out of the tubesin the autoclave, although synthetic sponge rubberstops remain in situ. Temporary closures can bemade from kitchen aluminium foil.

Aluminium capped test tubes are sterilized in thehot air oven in baskets. Polypropylene cappedtubes must be autoclaved.

Several sizes of small culture bottles are made formicrobiological work. Some of these bottles are ofstrong construction and are intended for reuse.Others, although tough enough for safe handling,are disposable. The most useful sizes are: thoseholding 7, 14 and 28 ml; and the ‘UniversalContainer’, 28 ml, which has a larger neck than theothers and is also used as a specimen container.These bottles usually have aluminium screw capswith rubber liners. The liners should be made ofblack rubber; some red rubbers are thought to giveoff bactericidal substances. Polypropylene caps arealso used; they need no liners but in our experiencethey may loosen spontaneously during long incuba-tion or storage. The medium dries up. They are sat-isfactory in the short term

All of these bottles can be sterilized byautoclaving.

Media storage bottles*

‘Medical flats’ or ‘rounds’ with screw caps aremade in sizes from 60 ml upwards. The most con-venient sizes are 110 ml, holding 50–100 ml ofmedium, and 560 ml, for storing 250–500 ml. Theflat bottles are easiest to handle and store but theround ones are more robust.

Specimen containers

The screw-capped glass or plastic ‘UniversalContainers’, which hold about 28 ml, are the mostpopular. There are many other containers, mostlymade of plastic. There are too many different con-tainers, many of plastic: some are satisfactory, oth-ers not; some leak easily and others do not stand upto handling by patients. Only screw-capped con-tainers with more than one-and-a-half threads ontheir necks should be used. Those with ‘push-in’ or‘pop-up’ stoppers are dangerous. They generateaerosols when opened. Waxed paper pots shouldnot be used because they invariably leak. For largerspecimens, there is a variety of strong screw-cappedjars.

Before any containers are purchased in bulk, it isadvisable to test samples by filling them withcoloured water and standing them upside down onblotting paper for several days after screwing thecaps on moderately well. Patients and nurses maynot screw caps on as tightly as possible. Similarbottles should be sent through the post, wrapped inabsorbent material in accordance with postal regu-lations (see p. 8). Leakage will be evident by thestaining of the blotting paper or wrapping. Moresevere tests include filling the containers withcoloured water and centrifuging them upside downon a wad of blotting paper. Specimen containerproblems and tests are reviewed by Collins andKennedy (1999).

Sample jars and containers

Containers for food samples, water, milk, etc.should conform to local or national requirements.In general, large screw-capped jars are suitable butare rather heavy if many samples are taken, andshould not be used in food factories. Plastic con-tainers may be used because leakage and spillageare not such a problem as with pathological mate-rials. Strong plastic bags are useful, particularly ifthey are of the self-sealing type. Otherwise ‘quick-ties’ may be used.


Racks and baskets

Test-tube and culture bottle racks should be madeof polypropylene or of metal covered withpolypropylene or nylon so that they can be auto-claved. These racks also minimize breakage, whichis not uncommon when metal racks are used.Wooden racks are unhygienic. Some metal racksmay rust and should not be used in water baths.

Aluminium trays for holding from 10 to 100 bot-tles, according to size, are widely employed in theUK. They can be autoclaved and are easily takenapart for cleaning.

The traditional wire baskets are unsafe for hold-ing test tubes. They contribute to breakage hazardsand do not retain spilled fluids. For non-infectivework, those baskets in which the wire is coveredwith polypropylene or nylon are satisfactory, butautoclavable plastic boxes of various sizes are saferfor use with cultures.

Other bench equipment

A hand magnifier, forceps and a knife or scalpelshould be provided, and also a supply of tissues formopping up spilled material and general cleaning.Swab sticks, usually made of wood and about6 inches (15 cm) long, are useful for handling somespecimens, and wooden throat spatulas are usefulfor food samples. These can be sterilized in largetest tubes or in the aluminium boxes used forPasteur pipettes.

Some form of bench ‘tidy’ or rack is desirable tokeep loops and other small articles together.

Discard jars and disinfectant pots are consideredin Chapter 4.

REFERENCES

British Standards (BS and BS EN) are listed sepa-rately in the Addendum (see below).Centers for Disease Control and National Institutes of

Health (CDC/NIH) (2000) Primary Containment

for Biohazards Selection, Installation and Use of

Biological Safety Cabinets. Washington:Government Printing Office.

Collins, C.H. and Kennedy, D.A. (1999) Laboratory

Acquired Infections, 4th edn. Oxford:Butterworth–Heinemann.

European Union (1998) Directive 98/79/EC of theEuropean Parliament and of the Council of 27October 1998 on in vitro diagnostic medicaldevices. Official Journal of the European

Communities L331, 7.12.98, pp. 1–37.International Electrical Commission (1990) 1010 Part

1. Safety Requirements – Electrical Equipment for

Measuring and Control and Laboratory Use.

Geneva: IEC. International Federation of Clinical Chemistry (1990)

Guidelines for the Selection of Safe Laboratory

Centrifuges and their Safe Use. Copenhagen: IFCC. Kennedy, D. A. (1988) Equipment-related hazards. In:

Collins, C. H. (ed.), Safety in Clinical and

Biomedical Laboratories. London: Chapman &Hall Medical, pp. 11–46.

Kuhne, R. W. Chatigny, M. A., Stainbrook, B. W. et al.

(1995) Primary barriers and personal protectiveequipment in biomedical laboratories. In: Fleming,D. O., Richardson, J. H., Tulis, J. I. et al. (eds),Laboratory Safety: Principles and Practices, 2ndedn. Washington: ASM Press.

Medical Devices Agency (MDA) (1998) Medical deviceand equipment management, for hospital andcommunity-based organisations. Device Bulletin,MDA DB 9801, January 1998.

MDA (2002a) Management of in vitro diagnosticmedical devices. Device Bulletin, MDA DB 2002(02), March 2002.

MDA (2002b) Management and use of IVD point ofcare test devices. Device Bulletin, MDA DB 2002(03), March 2002.

Medical Devices Directorate (MDD) (1991) Managementof medical equipment and devices, Health

Equipment Information 98: November 1990.National Sanitation Foundation (1992) Standard 49.

Class II (Laminar Flow) Biohazard Cabinetry. AnnArbor: NSF.

SI (2000) In Vitro Diagnostic Medical Device Regulations2000, Statutory Instrument 2000 No. 1315.


ADDENDUM: BRITISH STANDARDSFOR LABORATORY EQUIPMENT

Autoclaves, sterilizers and disinfectors

BS EN 61010-1:1993. Safety requirements for electricalequipment for measurement, control andlaboratory use. General requirements.

BS EN 61010-2-041:1997. Safety requirements forelectrical equipment for measurement, controland laboratory use. Particular requirementsfor autoclaves using steam of the treatmentof medical materials and for laboratory processes.

BS EN 61010-2-042:1997. Safety requirements forelectrical equipment for measurement, control andlaboratory use. Particular requirements forautoclaves and sterilizers using toxic gas for thetreatment of medical materials and for laboratoryprocesses.

BS EN 61010-2-043:1998. Safety requirements forelectrical equipment for measurement, control andlaboratory use. Particular requirements for dryheat sterilizers using either hot air or hot inert gasfor the treatment of medical materials and forlaboratory processes.

BS EN 61010-2-045:2001. Safety requirements forelectrical equipment for measurement, control andlaboratory use. Particular requirements for washerdisinfectors used in medical, pharmaceutical,veterinary and laboratory fields.

BS 2646-2:1990 [1996]. Autoclaves for sterilizationin laboratories. Guide to planning and installation.

BS 2646-4:1991 [1998]. Autoclaves for sterilization inlaboratories. Guide to maintenance.

BS 2646-1:1993 [2000]. Autoclaves for sterilization inlaboratories. Specification for design, construction,safety and performance.

BS 2646-3:1993 [2000]. Autoclaves for sterilizationin laboratories. Guide to safe use and maintenance.

BS 2646-5:1993 [2000]. Autoclaves for sterilization inlaboratories. Methods of test for function andperformance.

Cell disruptors

BS EN 12884:1999. Biotechnology. Performancecriteria for cell disruptors.

Centrifuges

BS EN 61010-2-020:1995. Safety requirements forelectrical equipment for measurement, control andlaboratory use. Particular requirements forlaboratory centrifuges.

BS EN 12884:1994. Biotechnology. Performancecriteria for centrifuges.

Drying ovens

BS 2648:1955 [2000]. Performance requirements forelectrically-heated laboratory drying ovens.

Glassware

BS EN ISO 4796-1:2001. Laboratory glassware. Screw-neck bottles.

BS EN ISO 4796-2:2001. Laboratory glassware.Conical-neck bottles.

BS EN ISO 4796-3:2001. Laboratory glassware.Aspirator bottles.

BS ISO 4798:1997. Laboratory glassware. Filter funnels.BS ISO 4800:1998. Laboratory glassware. Separating

funnels and dropping bottles.BS 5732:1985 [1997]. Specification for glass disposable

Pasteur pipettes.

Microbiologicial safety cabinets

BS EN 12469:2000. Biotechnology. Performancecriteria for microbiological safety cabinets.

BS 5726:Part 1:1992. Microbiological safety cabinets.Part 1. Specification for design, construction andperformance prior to installation.

Addendum: British Standards for laboratory equipment 43

BS 5726:Part 2:1992. Microbiological safety cabinets.Part 2. Recommendations for information to beexchanged between purchaser, vendor and installerand recommendations for installation.

BS 5726:Part 3:1992. Microbiological safety cabinets.Part 3. Specification for performance afterinstallation.

BS 5726:Part 4:1992. Microbiological safety cabinets.Part 4. Recommendations for selection use andmaintenance.

Mixers and stirrers

BS EN 61010-2-051:1996. Safety requirements forelectrical equipment for measurement, control andlaboratory use. Particular requirements for laboratoryequipment for mechanical mixing and stirring.

pH meters

BS 3145:1978 [1999]. Specification for laboratory pHmeters.

Plastics ware

BS 5404-1: 1976 [1994]. Plastics laboratory ware.Beakers.

BS 5404-2:1977 [1994]. Plastics laboratory ware.Graduated measuring cylinders.

BS 5404-3:1977 [1994]. Plastics laboratory ware. Filterfunnels.

BS 5404-4:1977 [1994]. Plastics laboratory ware. Washbottles.

BS 611-2:1990. Specification for plastics Petri dishesfor single use.


DEFINITION OF TERMS

• Sterilization implies the complete destructionof all micro-organisms, including spores. It isaccomplished by physical methods.

• Disinfection implies the destruction ofvegetative organisms that might cause diseaseor, in the context of the food industries, thatmight cause spoilage. It usually employschemicals and does not necessarily kill spores.

These two terms are not synonymous.

• Decontamination is the preferred term inmicrobiological laboratories for renderingmaterials safe for use or disposal.

STERILIZATION

The methods commonly used in microbiologicallaboratories are:

• red heat (flaming)• dry heat (hot air)• steam under pressure (autoclaving)• steam not under pressure (tyndallization)• filtration• inspissation.

Incineration is also a method of sterilization butas it is applied, usually outside the laboratory, forthe ultimate disposal of laboratory waste, it is con-sidered separately (see p. 59).

Red heat

Instruments such as inoculating wires and loops aresterilized by holding them in a Bunsen flame untilthey are red hot. Hooded Bunsens (see p. 37) arerecommended for sterilizing inoculating wires con-taminated with highly infectious material (e.g.tuberculous sputum) to avoid the risk of splutteringcontaminated particles over the surrounding areas.

Micro-incinerators, electrically operated andintended to replace Bunsens, are becoming increas-ingly popular.

Dry heat

This is applied in electrically heated ovens, alsoknown as dry-heat sterilizers, which are thermo-statically controlled and fitted with a large circulat-ing fans to ensure even temperatures in all parts ofthe load. Modern equipment has electronic con-trols which can be set to raise the temperature tothe required level, hold it there for a prearrangedtime and then switch off the current. A solenoidlock is incorporated in some models to prevent theoven being opened before the cycle is complete.This safeguards sterility and protects the staff fromaccidental burns. There is a British Standard (BS2648) for these appliances (see p. 43).

Materials that can be sterilized by this methodinclude glass Petri dishes, flasks, pipettes andmetal objects. Various metal canisters and drumsthat conveniently hold glassware during steriliza-tion and keep it sterile during storage are availablefrom laboratory suppliers. Some laboratory

4

Sterilization, disinfection and thedecontamination of infected material

workers still prefer the time-honoured method ofwrapping pipettes, etc. individually in brown(Kraft) paper.

LoadingAir is not a good conductor of heat so oven loadsmust be loosely arranged, with plenty of space toallow the hot air to circulate.

Holding times and temperaturesWhen calculating processing times for hot air ster-ilizing equipment, there are three time periods thatmust be considered:

1. The heating-up period, which is the timetaken for the entire load to reach thesterilization temperature; this may take about1 h.

2. The holding periods at different sterilizationtemperatures recommended in the UK are160°C for 2 h or 180°C for 30 min (BritishPharmacopoeia, 1993; Russell, 1999).

3. The cooling-down period, which is carriedout gradually to prevent glassware fromcracking as a result of a too rapid fall intemperature; this period may take up to 2 h.

Control of hot air sterilizersTests for electrically operated fan ovens aredescribed in the British Standard (BSI, 1998).

Hot air sterilization equipment should be cali-brated with thermocouples when the apparatus isfirst installed and checked with thermocouplesafterwards when necessary.

Ordinary routine control can be effected simplywith commercial chemical indicator tubes.

Steam under pressure: the autoclave

The temperature of saturated steam at atmosphericpressure is approximately 100°C. Temperatureincreases with pressure, e.g. at 1 bar (about15 lb/in2 – some older autoclaves use Imperialunits) it is 121°C. Bacteria are killed by autoclavingat this temperature for 15–20 min. Air has an

important influence on the efficiency of autoclav-ing. The above relationship holds well only if no airis present. If about 50% of the air remains in theautoclave the temperature of the steam/air mixturewill be only 112°C. If air is present in the autoclaveload, it will also adversely affect penetration by thesteam.

All the air that surrounds and permeates the loadmust first be removed before sterilization can com-mence. In some autoclaves this is done by vacuumpumps.

Loads in autoclavesAs successful autoclaving depends on the removalof all the air from the chamber and the load, thematerials to be sterilized should be packed loosely.‘Clean’ articles may be placed in wire baskets, butcontaminated material (e.g. discarded cultures)should be in solid-bottomed containers not morethan 20 cm deep (see ‘Disposal of infected waste’below). Large air spaces should be left around eachcontainer and none should be covered. If large vol-umes of fluid are to be sterilized, then the timeshould be lengthened to ensure that this reaches theappropriate temperature.

Types of autoclaveOnly autoclaves designed for laboratory work andcapable of dealing with a ‘mixed load’ should beused. ‘Porous load’ and ‘bottled fluid sterilizers’ arerarely satisfactory for laboratory work. There aretwo varieties of laboratory autoclave:

1. pressure cooker types2. gravity displacement models with automatic

air and condensate discharge.

Laboratory bench autoclavesThese, as domestic pressure cookers, are still in usein many parts of the world. The more modern typehas a metal chamber with a strong metal lid thatcan be fastened and sealed with a rubber gasket. Ithas an air and steam discharge tap, pressure gaugeand safety valve. There is an electric immersionheater in the bottom of the chamber (Figure 4.1).

46 Sterilization, disinfection and the decontamination of infected material

Operating instructionsThere must be sufficient water inside the chamber.The autoclave is loaded and the lid is fasteneddown with the discharge tap open. The safety valveis then adjusted to the required temperature andthe heat is turned on.

When the water boils, the steam will issue from thedischarge tap and carry the air from the chamberwith it. The steam and air should be allowed toescape freely until all the air has been removed. Thismay be tested by attaching one end of a length ofrubber tubing to the discharge tap and inserting theother end into a bucket or similar large container ofwater. Steam condenses in the water and the air risesas bubbles to the surface; when all of the air has beenremoved from the chamber, bubbling in the bucketwill cease. When this stage has been reached, theair–steam discharge tap is closed and the rubber tub-ing removed. The steam pressure then rises in thechamber until the desired pressure and temperatureare reached and steam issues from the safety valve.

When the load has reached the required tempera-ture (see ‘Testing autoclaves’ below), the pressure isheld for 15 min. At the end of the sterilizing period,the heater is turned off and the autoclave is allowedto cool.

The air and steam discharge tap is opened very slowlyafter the pressure gauge has reached zero (atmosphericpressure). If the tap is opened too soon, while the auto-clave is still under pressure, any fluid inside (liquid

media, etc.) will boil explosively and bottles contain-ing liquids may even burst. The contents are allowedto cool. Depending on the nature of the materials beingsterilized, the cooling (or ‘run-down’) period neededmay be several hours for large bottles of agar to coolto 80°C, when they are safe to handle.

Gravity displacement autoclavesThese may be relatively simple in their constructionand operation, as shown diagrammatically inFigure 4.2, or extremely sophisticated pieces ofengineering in which air and, finally, steam areremoved by vacuum pumps and in which the wholesterilization cycle may be programmed.

The jacket surrounding the autoclave consists ofan outer wall enclosing a narrow space around thechamber, which is filled with steam under pressureto keep the chamber wall warm. The steam entersthe jacket from the mains supply, which is at highpressure, through a valve that reduces this pressureto the working level. The working pressure is meas-ured on a separate pressure gauge fitted to thejacket. This jacket also has a separate drain for airand condensate to pass through.

The steam enters the chamber from the samesource that supplies steam to the jacket. It is intro-duced in such a way that it is deflected upwardsand fills the chamber from the top downwards,

Sterilization 47

TrivetWaterHeater

Lid

Air/steam discharge valve

Pressure gauge

Safety valve

Chamber

Figure 4.1 ‘Pressure cooker’ laboratory autoclave

Combined pressureand vacuum gauge

Cotton woolfilter

SafetyvalveSafety

valvePressuregauge

Jacket

To jacket

ValveBaffle

Chamber

To chamber

Valve

To vacuum pumpor steam ejector

Mai

ns s

team

supp

ly

Valve

Near-to-steamtraps

Strainers

Valve

Non-return valve

Thermometer

Door

Figure 4.2 Gravity displacement autoclave

thus forcing the air and condensate to flow out ofthe drain at the base of the chamber by gravity dis-placement. The drain is fitted with strainers to pre-vent blockage by debris. The drain discharges intoa closed container (not shown in the diagram) sothat there is a complete air break that preventsbackflow. There is also a filter to ensure thataerosols are not released into the room.

The automatic steam trap or ‘near-to-steam’ trapis designed to ensure that only saturated steam isretained inside the chamber, and that air and con-densate, which are at a lower temperature than sat-urated steam, are automatically discharged. It iscalled a ‘near-to-steam’ trap because it opens if thetemperature falls to about 2°C below that of satu-rated steam and closes within 2°C or nearer to thesaturated steam temperature. The trap operates bythe expansion and contraction of a metal bellowsthat operates a valve.

There may be a thermometer probe in the drainbut, as this registers the temperature of steam atthat point and not that in the load within the cham-ber, this may be misleading, e.g. the temperature inthe drain may reach 121°C whereas that in the loadis only 50°C (Collins and Kennedy, 1999).

In modern autoclaves flexible thermocoupleprobes are fitted in the chamber so that tempera-tures in various parts of the load may be recorded.In older models the thin thermocouple leads maybe safely taken in through the door seals.

There are usually interlocking devices that pre-vent the opening of the door before the tempera-ture in the chamber has fallen to 80°C. This doesnot imply that the temperature in the load has alsofallen to a safe level. In large, sealed bottles it maystill be over 100°C, when the contents will be at ahigh pressure. Sudden cooling may cause the bot-tles to explode. The autoclave should not beopened until the temperature in the load has fallento 80°C or below. This may take a very long time,and in some autoclaves there are locks that permitthe door to be opened only fractionally to cool theload further before it is finally released. This is agood reason for not using these autoclaves for thepreparation and melting of culture media (seeChapter 5).

Operation of a gravity displacementautoclave

If the autoclave is jacketed, the jacket must first bebrought to the operating temperature. The chamberis loaded, the door is closed and the steam valve isopened, allowing steam to enter the top of the cham-ber. Air and condensate flow out through the drainat the bottom (Figure 4.2). When the drain ther-mometer reaches the required temperature a furtherperiod must be allowed for the load to reach the tem-perature. This should be determined initially and peri-odically for each autoclave as described below. Unlessthis is done the load is unlikely to be sterilized. Theautoclave cycle is then continued for the holding time.When it is completed the steam valves are closed andthe autoclave allowed to cool until the temperaturedial reads less than 80°C. Not until then is the auto-clave safe to open. It should first be ‘cracked’ oropened very slightly and left in that position for sev-eral minutes to allow steam to escape and the load tocool further (see ‘Protection of the operator’ below).

Time/temperature cyclesFor most purposes the time/temperature cycles inTable 4.1 will ensure sterilization of a properlypacked load (see p. 46) (MDA, 1993/1996).

These are the holding times at temperature (HTAT),determined as described below. The usual HTAT inmicrobiology laboratories is 15 min at 121°C.

Material containing, or suspected of containing,the agents of spongiform encephalopathies should,however, be autoclaved for 18 min or for six con-secutive cycles of 3 min at 134°C (Advisory Com-mittee on Dangerous Pathogens or ACDP, 1994).

Table 4.1 Time/temperature cycles

Testing autoclavesThe time/temperature cycles should be tested under‘worst load’ conditions, e.g. a container filled with

Temperature (°C) Holding period (min)

121–124 15134–138 3


5 ml screw-capped bottles. This should be placed inthe centre of the chamber and, if space is available,other loaded containers may be placed around it.Thermocouple leads are placed in the middle of theload and at other places. The cycle is then startedand timed.

There are three periods:

1. Warming up, until the temperature in themiddle of the load is 121°C.

2. Sterilization, i.e. HTAT, in which thetemperature in the load is maintained at121°C for 15 min.

3. Cooling down, after the steam valve has beenclosed and the temperature in the load hasfallen to 80°C.

If the autoclave is to be operated manually thesetimes should be noted and displayed. Automatedautoclaves may be programmed.

MonitoringDuring daily use or from time to time the recordingthermometers may be supplemented with indica-tors. There are three types. Two give immediateresults, the other is retrospective:

1. Bowie-Dick autoclave tape (Bowie et al.,1963): this is tape impregnated with achemical and placed in the load. The colourchanges if there is adequate steampenetration. These tapes should be reservedfor vacuum-type autoclaves.

2. Chemical indicators: these are usually insealed tubes or sachets, and change colour ifthe correct time and temperature combinationhas been achieved.

3. Biological indicators. Spores of two organismsare used: Bacillus stearothermophilus (NCTC1007, ATCC 7935) and Clostridiumsporogenes (NTCC 8596, ATCC 7955). Theyare used as suspensions or absorbed oncarriers such as filter paper strips. Laboratorypreparations may be unreliable because theheat resistance of the spores depends on theculture media used. It is best to purchasecommercial products. Strips are placed atvarious locations in the load and, after

autoclaving, are added to tubes of broth,which are then incubated. Turbidity indicatesunsuccessful processing.

There are British Standards for the design, instal-lation, maintenance and testing of laboratory auto-claves (BSI, 1997a, 1997b, 1996, 1998, 2000a,2000b, 2000c – see Addendum to Chapter 3).

For further information on autoclaving seeKennedy (1988), Gardner-Abbate (1998) andRussell et al. (1999).

Protection of the operatorSerious accidents, including burns and scalds tothe face and hands have occurred when autoclaveshave been opened, even when the temperaturegauge is read below 80°C and the door lock hasallowed the door to be opened. Liquids in bottlesmay still be over 100°C and under considerablepressure. The bottles may explode on contact withair at room temperature.

When autoclaves are being unloaded operatorsshould wear full-face visors of the kind that coverthe skin under the chin and throat. They shouldalso wear thermal protective gloves (see Kennedy,1988).

Steam at 100°C (‘tyndallization’)

This process, named after the Irish physicist cumbacteriologist John Tyndall, employs a Koch orArnold steamer, which is a metal box in the bottomof which water is boiled by a gas burner, electricheater or steam coil. The articles to be processedare placed so that they rest on a perforated rackjust above the water level. The lid is conical so thatthe condensation runs down the sides instead ofdripping on the contents. A small hole in the top ofthe lid allows air and steam to escape.

This method is used to sterilize culture media thatmight be spoiled by exposure to higher tempera-tures, e.g. media containing easily hydrolysed car-bohydrates or gelatin. These are steamed for30–45 min on each of 3 successive days. On thefirst occasion, vegetative bacteria are killed; any

Sterilization 49

spores that survive will germinate in the nutrientmedium overnight, producing vegetative forms thatare killed by the second or third steaming.

Filtration

Micro-organisms may be removed from liquids bypassing them through filters with very small poresthat trap bacteria but, in general, not mycoplasmasor viruses. The method is used for sterilizing serumfor laboratory use, antibiotic solutions and specialculture media that would be damaged by heat. It isalso used for separating the soluble products ofbacterial growth (e.g. toxins) in fluid culture media.

Filters of historical interest, now rarely used,include the Berkefeld (made of Kieselguhr), theChamberland (unglazed porcelain), the Seitz(asbestos) and sintered glass. Details of these maybe found in older bacteriology textbooks. Theyhave been superseded by membrane filters.

Membrane filtersThese filters are made from cellulose esters (cellu-lose acetate, cellulose nitrate, collodion, etc.). Arange of pore sizes is available. Bacterial filters havea pore size of less than 0.75 μm. The membranesand the devices that hold them for use may be ster-ilized by autoclaving.

For use, a sterile membrane is mounted, usingaseptic precautions, on a perforated platform, usu-ally made of stainless steel, which is sealed togetherbetween the upper and lower funnels. Filtration isachieved by applying either a positive pressure tothe entrance side of the filter or a negative pressureto the exit side.

Small filter units for filtering small volumes offluid (e.g. 1–5 ml) are available. The fluid passesthrough the filter by gravitational force in a cen-trifuge or is forced through a small filter from asyringe.

Membrane filters and filter holders to suit differ-ent purposes are obtainable and several manufac-turers publish useful booklets or leaflets about theirproducts.

CHEMICAL DISINFECTION

Some disinfectants present health hazards (Table4.2). It is advisable to wear eye and hand protec-tion when making dilutions.

Many different chemicals may be used and they arecollectively described as disinfectants or (micro)bio-cides. The former term is used in this book. Someare ordinary reagents and others are special formu-lations, marketed under trade names. Disinfectantsshould not be used to ‘sterilize’ materials or whenphysical methods are available. In some circum-stances, e.g. in food establishments, cleaning withdetergents is better. The effects of time, tempera-ture, pH, and the chemical and physical nature ofthe article to be disinfected and of the organic mat-ter present are often not fully appreciated.

Types and laboratory usesof disinfectants

There is an approximate spectrum of susceptibilityof micro-organisms to disinfectants. The mostsusceptible are vegetative bacteria, fungi and lipid-containing viruses. Mycobacteria and non-lipid-containing viruses are less susceptible and sporesare generally resistant.

Consideration should be given to the toxicity ofdisinfectants and any harmful effects that they mayhave on the skin, eyes and respiratory tract.

Only those disinfectants that have a laboratoryapplication are considered here. For further infor-mation and other applications, see Gardner-Abbate(1998), Russell et al. (1999) and Ayliffe et al. (1999).

The most commonly used disinfectants in labora-tory work are clear phenolics and hypochlorites.Aldehydes have a more limited application, and alco-hol and alcohol mixtures are less popular but deservegreater attention. Iodophors and quaternary ammo-nium compounds (QACs) are more popular in theUSA than in the UK, whereas mercurial compoundsare the least used. The properties of these disinfec-tants are summarized below and in Table 4.2. Othersubstances, such as ethylene oxide and propiolac-tone, are used commercially in the preparation of


Table 4.2 Properties of some disinfectants

Active against Inactivated by Toxicity

Fungi Bacteria Myco- Spores Lipid Non- Protein Natural Synthetic Hard Deter- Skin Eyes LungsG + G − bacteria viruses lipid materials materials water gent

viruses

Phenolics + + + + + + + + + + + − + v + + + + + + C + + −Hypochlorites + + + + + + + + + + + + + + + + + + C + + + Alcohols − + + + + + + + + + − + v + + + + − − + −Formaldehyde + + + + + + + + + + + + + + +a + + + + + + − + + + Glutaraldehyde + + + + + + + + + + + + + + +b + + NA + + + − + + +Iodophors + + + + + + + + + + + + + + + + + + + + + A + + −QACs + + + + + + − − − − + + + + + + + + + + + A(C) + + −

From Collins and Kennedy (1999)

+ + +, good; + +, fair; +, slight; −, nil; v, depends on virus; a > 40°C; b > 20°C; C, cationic; A, anionic; NA, not applicable; QACs, quarternary ammonium compounds.

sterile equipment for hospital and laboratory use.They are not normally used in laboratories but areused in some hospitals for medical equipment.Commercial equipment is available for this purpose.

Clear phenolicsThese compounds are effective against vegetativebacteria (including mycobacteria) and fungi. Theyare inactive against spores and non-lipid-containingviruses. Most phenolics are active in the presence ofconsiderable amounts of protein but are inactivatedto some extent by rubber, wood and plastics. Theyare not compatible with cationic detergents.Laboratory uses include discard jars and disinfec-tion of surfaces. Clear phenolics should be used atthe highest concentration recommended by the man-ufacturers for ‘dirty situations’, i.e. where they willencounter relatively large amounts of organic mat-ter. This is usually 2–5%, as opposed to 1% for‘clean’ situations where they will not encounter muchprotein. Dilutions should be prepared daily anddiluted phenolics should not be stored for labora-tory use for more than 24 h, although many dilutedclear phenolics may be effective for more than 7 days.

Skin and eyes should be protected.

HypochloritesThe activity is caused by chlorine, which is veryeffective against vegetative bacteria (excludingmycobacteria), spores and fungi. Hypochlorites areconsiderably inactivated by protein and to someextent by natural non-protein material and plastics,and they are not compatible with cationic deter-gents. Their uses include discard jars and surfacedisinfection, but as they corrode some metal care isnecessary. They should not be used on the metalparts of centrifuges and other machines that aresubjected to stress when in use.

The hypochlorites sold for industrial and labora-tory use in the UK contain 100 000 ppm availablechlorine. They should be diluted as follows:

• Reasonably clean surfaces 1 : 100 giving1000 ppm

• Pipette and discard jars 1 : 40 giving 2500 ppm• Blood spillage 1 : 10 giving 10 000 ppm

Some household hypochlorites (e.g. those used forbabies’ feeding bottles) contain 10 000 ppm andshould be diluted accordingly. Household‘bleaches’ in the UK and the USA contain50 000 ppm available chlorine and dilutions of1 : 20 and 1 : 5 are appropriate.

Hypochlorites decay rapidly in use, although theproducts as supplied are stable. Diluted solutionsshould be replaced after 24 h. The colouring matteradded to some commercial hypochlorites is intendedto identify them: it is not an indicator of activity.

Hypochlorites may cause irritation of skin, eyesand lungs.

Sodium dichloroisocyanate (NaDCC) is a solidchlorine-releasing agent. The tablets are useful forpreparing bench discard jars and the powder fordealing with spillages, especially of blood.

AldehydesFormaldehyde (gas) and glutaraldehyde (liquid) aregood disinfectants. They are active against vegeta-tive bacteria (including mycobacteria), spores andfungi. They are active in the presence of proteinand not very much inactivated by natural or syn-thetic materials, or detergents.

Formaldehyde is not very active at temperaturesbelow 20°C and requires a relative humidity of atleast 70%. It is not supplied as a gas, but as a solidpolymer, paraformaldehyde, and a liquid, formalin,which contains 37–40% formaldehyde. Both formsare heated to liberate the gas, which is used for disin-fecting enclosed spaces such as safety cabinets androoms. Formalin diluted 1 : 10, to give a solutioncontaining 4% formaldehyde, is used for disinfectingsurfaces and, in some circumstances, cultures. Solid,formaldehyde-releasing compounds are now on themarket and these may have laboratory applicationsalthough they have not yet been evaluated for thispurpose. Formaldehyde is used mainly for decontam-inating safety cabinets (see p. 34) and rooms.

Some glutaraldehyde formulations need an activa-tor, which is supplied with the bulk liquid. Most acti-vators contain a dye so that the user can be sure thatthe disinfectant has been activated. Effectiveness andstability after activation vary with the product andthe manufacturer’s literature should be consulted.


Aldehydes are toxic. Formaldehyde is particu-larly unpleasant because it affects the eyes andcauses respiratory distress. Special precautions arerequired (see below).

Glutaraldehyde is moderately toxic and is alsoan irritant, especially to eyes, skin and the upperrespiratory tract.

Alcohol and alcohol mixturesEthanol and propanol, at concentrations of about70–80% in water, are effective, albeit slowly,against vegetative bacteria. They are not effectiveagainst spores or fungi. Protein and other mate-rial or detergents do not especially inactivatethem.

Effectiveness is enhanced by the addition offormaldehyde, e.g. a mixture of 10% formalin in70% alcohol, or hypochlorite to give 2000 ppm ofavailable chlorine.

Alcohols and alcohol mixtures are useful fordisinfecting surfaces and, with the exception ofalcohol–hypochlorite mixtures, for balancingcentrifuge buckets.

They are relatively harmless to skin but may causeeye irritation.

Quaternary ammonium compoundsThese are cationic detergents known as QACs orquats, and are effective against vegetative bacteriaand some fungi, but not against mycobacteria orspores. They are inactivated by protein, by a vari-ety of natural and plastic materials, and by anionicdetergents and soap. Their laboratory uses aretherefore limited, although they have the distinctadvantages of being stable and not corroding met-als. They are usually employed at 1–2% dilutionfor cleaning surfaces, and are very popular in foodhygiene laboratories because of their detergentnature.

QACs are not toxic and are harmless to the skinand eyes.

IodophorsAs with chlorine compounds these iodines areeffective against vegetative bacteria (includingmycobacteria), spores, fungi, and both lipid-con-

taining and non-lipid-containing viruses. They arerapidly inactivated by protein and to a certainextent by natural and plastic substances, and theyare not compatible with anionic detergents. For usein discard jars and for disinfecting surfaces theyshould be diluted to give 75–150 ppm iodine but,for hand-washing or as a sporicide, diluted in 50%alcohol to give 1600 ppm iodine. As sold,iodophors usually contain a detergent and theyhave a built-in indicator: they are active as long asthey remain brown or yellow. They stain the skinand surfaces but stains may be removed withsodium thiosulphate solution.

Iodophors are relatively harmless to skinalthough some eye irritation may be experienced.

Mercurial compoundsActivity against vegetative bacteria is poor andmercurials are not effective against spores. They dohave an action on viruses at concentrations of1 : 500 to 1 : 1000 and a limited use, as saturatedsolutions, for safely making microscopic prepara-tions of mycobacteria.

Their limited usefulness and highly poisonousnature make mercurials unsuitable for general lab-oratory use.

Precautions in the use of disinfectants

As indicated above, some disinfectants haveundesirable effects on the skin, eyes and respira-tory tract. Disposable gloves and safety specta-cles, goggles or a visor should be worn by anyonewho is handling strong disinfectants, e.g. whenpouring from stock and preparing dilutions foruse.

Testing disinfectants

Several countries and organizations have ‘official’tests for disinfectants. They are used by manufac-turers and have no place in clinical or public health

Chemical disinfection 53

laboratories that might use them only occasionally,because reproducibility is then a problem. Some,e.g. the Rideal–Walker and the Chick–Martin tests,have fallen into disrepute because they have beenused to compare unlikes. Those that use Salmonellatyphi, even strains that are claimed to be avirulent,should be abandoned altogether. Details of current‘official’ tests will not, therefore, be given here –only the principle.

Suspensions containing known numbers ofcolony-forming units (cfu)/millilitre of Staphylo-coccus aureus and/or Pseudomonas aeruginosa areprepared and added to various dilutions of the testproduct. After predetermined contact times, aknown volume of the mixture is removed to a neu-tralizing fluid that deactivates the disinfectant. Thenumber of surviving colony-forming units is thencounted. A satisfactory disinfectant might beexpected to reduce the number of colony-formingunits by 5 log units within an hour.

Laboratory testsAlthough the standard or official tests are best leftto manufacturers, there is one, the ‘in use’ test, thatis simple and useful.

The ‘in-use’ testSamples of liquid disinfectants are taken from suchsources as laboratory discard jars, floor-mop buck-ets, mop wringings, disinfectant liquids in whichcleaning materials or lavatory brushes are stored,disinfectants in central sterile supply departments,used instrument containers and stock solutions ofdiluted disinfectants. The object is to determinewhether the fluids contain living bacteria, and inwhat numbers. The test (Maurer, 1972) isdescribed here in detail for use in any laboratory,because meaningful results can be obtained only inthe light of local circumstances.

1. Take a 1-ml sample of the disinfectantsolution from each pot or bucket withseparate sterile pipettes.

2. Add to each sample 9 ml diluent in a sterileuniversal container or a 25-ml screw-cappedbottle and mix well. Select the diluent

according to the group to which thedisinfectant belongs (Table 4.3).

3. Within 4 h use separate Pasteur pipettes towithdraw small volumes of thedisinfectant/diluent and to place 10 drops,separately, on the surface of each of two,well-dried, nutrient agar plates.

4. Incubate one plate at 32 or 37°C for 3 daysand the other at room temperature for 7 days.The optimum temperature for mostpathogenic bacteria is 37°C but those thathave been damaged by disinfectants oftenrecover more readily at 32°C.

5. After incubation, examine the plates.

The growth of bacterial colonies on one or both ofa pair of plates is evidence of the survival of bacte-ria in the particular pot from which the sample istaken. One or two colonies on a plate may be ignored– a disinfectant is not a sterilant and the presence ofa few live bacteria in a pot is to be expected.

The growth of five or more colonies on oneplate should arouse suspicion that all is not well.The relationship between the number of colonieson the plate and the number of live bacteria in thepot may easily be calculated, because the disinfec-tant sample is diluted 1 in 10 and the 50-droppipette delivers 50 drops/ml. If five colonies aregrown from ten drops of disinfectant/diluent, thenfive live bacteria were present in one drop of dis-infectant and 250 live bacteria were present in1 ml disinfectant.


Table 4.3 ‘In-use’ test neutralizing diluents

Diluent Disinfectant group

Nutrient broth AlcoholsAldehydesHypochloritesPhenolics

Nutrient broth + Hypochlorites +Tween 80, 3% w/v detergent

IodophorsPhenolics + detergentQACs

QACs, quarternary ammonium compounds

DECONTAMINATION ANDDISPOSAL OF INFECTED MATERIALS

It is a cardinal rule that no infected material shallleave the laboratory.

This principle (Collins et al., 1974) still holdswell. Laboratory waste that contains micro-organ-isms is technically clinical waste (Health ServicesAdvisory Committee or HSAC, 1999). It is clearlythe responsibility of the laboratory management toensure that no waste containing viable micro-organisms leaves the laboratory premises, when itmay offer hazards to other people. The wasteshould therefore be made safe on site before finaldisposal. The best way of doing this is to autoclaveit. This procedure offers no problems in properlyequipped and well-managed laboratories.

Disinfectants alone should not be used. We havealways believed that disinfection is a first-linedefence, and for, discarded bench equipment, forexample, it is a temporary measure, to be followedas soon as possible by autoclaving.

Table 4.4, adapted from Collins and Kennedy(1993), lists items that should be regarded as infec-tious and should therefore be autoclaved. (If incin-eration is contemplated, see p. 59.)

Containers for discarded infectedmaterial

In the laboratory there should be five importanttypes of receptacles for discarded infected materials:

1. Colour-coded discard bins or plastic bags forspecimens and cultures

2. Discard jars to receive slides, Pasteur pipettesand small disposable items

3. Pipette jars for graduated (recoverable) pipettes4. Colour-coded plastic bags for combustibles

such as specimen boxes and wrappers thatmight be contaminated

5. Colour-coded ‘sharps’ containers forhypodermic needles and syringes.

Decontamination and disposal of infected materials 55

Table 4.4 Proposed classification of clinical and biomedical laboratory waste

Disposables other than sharpsSpecimens or their remains (in their containers) submitted for tests: containing blood, faeces, sputum, urine,

secretions, exudates, transudates, other normal or morbid fluids but not tissuesAll cultures made from these specimens, directly or indirectlyAll other stocks of micro-organisms that are no longer requiredUsed diagnostic kits (which may contain glass, plastics, chemicals and biologicals)Used disposable transfer loops, rods, plastic Pasteur pipettesDisposable cuvettes and containers used in chemical analysesBiologicals, standards and quality control materialsFood samples submitted for examination in outbreaks of food poisoningPaper towels and tissues used to wipe benches and equipment and to dry handsDisposable gloves and gowns

SharpsHypodermic needles (with syringes attached if custom so requires)Disposable knives, scalpels, blades, scissors, forceps, probesGlass Pasteur pipettes; slides and cover glassesBroken glass, ampoules and vials

Tissues and animal carcasesBedding from animal cages

Adapted from Collins and Kennedy (1993)

Recommended colours in the UK (HSAC, 1999)are:

• Yellow: for incineration• Light blue or transparent with blue

inscription: for autoclaving (but maysubsequently be incinerated)

• Black: normal household waste – localauthority refuse collection

• White or clear plastic: soiled linen (e.g.laboratory overalls).

Discard bins and bags

Discard bins should have solid bottoms that shouldnot leak, otherwise contaminated materials mayescape. To overcome steam penetration problems,these containers should be shallow, not more than20 cm deep and as wide as will fit loosely into theautoclave. They should never be completely filled.Suitable plastic (polypropylene) containers areavailable commercially, although not specificallydesigned for this purpose. Some authorities preferstainless steel bins.

Plastic bags are popular but only those made forthis purpose should be used. They should be sup-ported in buckets or discard bins. Even the mostreliable may burst if roughly handled. It is possiblethat, under new EU legislation, rigid containers willsupersede plastic bags.

For safe transmission to the preparation room,the bins may require lids and the bags may be fas-tened with wire ties.

Discard bins and bags should be colour coded, i.e.marked in a distinctive way so that all workers rec-ognize them as containing infected material.

Discard jars

The jars or pots of disinfectant that sit on thelaboratory bench and into which used slides,

Pasteur pipettes and other rubbish are dumpedhave a long history of neglect and abuse. In all toomany laboratories these jars are filled infrequentlywith unknown dilutions of disinfectants, are over-loaded with protein and articles that float, and areinfrequently emptied. The contents are rarely prop-erly disinfected.

Choice of containerOld jam jars and instant coffee jars are not suitable.Glass jars are easily broken and broken glass is anunnecessary laboratory hazard, especially if it is likelyto be contaminated. Discard jars should be robustand autoclavable and the most serviceable articles are1-litre polypropylene beakers or screw-cappedpolypropylene jars. These are deep enough to holdsubmerged most of the things that are likely to be dis-carded, are quite unbreakable and survive many auto-clave cycles. They go dark brown in time, but thisdoes not affect their use. Screw-capped polypropy-lene jars are better because they can be capped afteruse and inverted to ensure that the contents are allwetted by the disinfectants, and air bubbles that mightprotect objects from the fluid are removed.

Correct dilutionA 1-litre discard jar should hold 750 ml of diluteddisinfectant and leave space for displacement with-out overflow or the risk of spillage when it is moved.A mark should be made at 750 ml on each jar, prefer-ably with paint (grease pencil and felt-pen marks areless permanent). The correct volume of neat disin-fectant to be added to water to make up this volumefor ‘dirty situations’ can be calculated from the man-ufacturers’ instructions. This volume is then markedon a small measuring jug, e.g. of enamelled iron, ora plastic dispenser is locked to deliver it from a bulkcontainer. The disinfectant is added to the beakerand water added to the 750-ml mark.

Sensible useLaboratory supervisors should ensure that inap-propriate articles are not placed in discard jars.There is a reasonable limit to the amount of paperor tissues that such a jar will hold, and articles thatfloat are unsuitable for disinfectant jars, unless


these can be capped and inverted from time to timeto wet all the contents.

Large volumes of liquids should never be addedto dilute disinfectants. Discard jars, containing theusual volume of neat disinfectant, can be pro-vided for fluids such as centrifuge supernatants,which should be poured in through a funnel thatfits into the top of the beaker. This preventssplashing and aerosol dispersal. At the end of theday water can be added to the 750-ml mark andthe mixture left overnight. Material containinglarge amounts of protein should not be added todisinfectants but should be autoclaved or inciner-ated.

Regular emptyingNo material should be left in disinfectant in discardjars for more than 24 h, or surviving bacteria maygrow. All discard jars should therefore be emptiedonce daily, but whether this is at the end of the dayor the following morning is a matter for localchoice. Even jars that have received little or nothingduring that time should be emptied.

‘Dry discard jars’

Instead of jars containing disinfectants there is aplace in some laboratories for the plastic containersused for discarded disposable syringes and theirneedles. These will accommodate Pasteur pipettes,slides, etc. and can be autoclaved or incinerated.

Pipette jars

Jars for recoverable pipettes should be made ofpolypropylene or rubber. These are safer thanglass. The jars should be tall enough to allowpipettes to be completely submerged without thedisinfectant overflowing. A compatible detergentshould be added to the disinfectant to facilitatecleaning the pipettes at a later stage. Tall jars areinconvenient for short people, who tend to placethem on the floor. This is hazardous. The square-

based rubber jars may be inclined in a box or on arack that is convenient and safer.

Plastic bags for combustibles

These should be colour coded (yellow in the UK,see p. 56).

Treatment and disposal procedures

There are three practical methods for the treatmentof contaminated, discarded laboratory materialsand waste:

1. Autoclaving2. Chemical disinfection3. Incineration.

The choice is determined by the nature of the mate-rial: if it is disposable or recoverable; if the latter, ifit is affected by heat. With certain exceptions noneof the methods excludes the others. Disinfectionalone is advisable only for graduated, recoverablepipettes. It will be seen from Figure 4.2 that incin-eration alone is advised only if the incinerator isunder the control of the laboratory staff.

Organization of treatmentThe design features of a preparation room for deal-ing with discarded laboratory materials shouldinclude autoclaves, a sluice, a waste disposal unitplumbed to the public sewer, deep sinks, glasswarewashing machines, drying ovens, sterilizing ovensand large benches.

These should be arranged to preclude any possi-ble mixing of contaminated and decontaminatedmaterials. The designers should therefore work to aflow, or critical pathway chart, provided by a pro-fessional microbiologist.

Such a chart is shown in Figure 4.3. The contam-inated materials arrive in colour-coded containersonto a bench or into an area designated and usedfor that purpose only. They are then sorted accord-ing to their colour codes and dispatched to the

Decontamination and disposal of infected materials 57

incinerator or loaded into the autoclave. Nothingbypasses this area. After autoclaving, the contain-ers are taken to the sorting bench where the con-tents are separated:

• Waste for incineration, which is put intocolour-coded containers

• Waste for the rubbish tip, which is also putinto different colour-coded containers

• Waste suitable for the sluice or waste disposalunit

• Recoverable material which is passed to thenext section or room for washing and re-sterilizing. This room should have a separateautoclave. Contaminated waste and materialsfor re-use or re-issue should not be processedin the same autoclave.

Procedures for various items

Before being autoclaved the lids of discard binsshould be removed and then included in the auto-clave load in such a way that they do not interferewith steam penetration. Plastic bags should havethe ties removed and the bags opened fully in thebins or buckets that support them.

Contaminated glasswareAfter autoclaving, culture media may be pouredaway or scraped out, and the tubes and bottles, etc.washed by hand or mechanically with a suitable

detergent. The washing liquid or powder used willdepend on the hardness of the water supply and themethod of washing. The advice of several labora-tory detergent manufacturers should be sought.

Busy laboratories require glassware washingmachines. Before purchasing one of thesemachines, it is best to consider several and to askother laboratories which models they have foundsatisfactory. A prerequisite is a good supply of dis-tilled or deionized water.

If re-usable articles are washed by hand doublesinks, for washing and then rinsing, are necessary,plus plastic or stainless steel bowls for final rinsingin distilled or deionized water. Distilled water fromstripper stills, off the steam line, is rarely satisfac-tory for bacteriological work.

Rubber liners should be removed from screw capsand the liners and caps washed separately andreassembled. Colanders or sieves made ofpolypropylene are useful for this procedure.

New glassware, except that made of borosilicateor similar material, may require neutralization.When fluids are autoclaved in new soda-glass tubesor bottles, alkali may be released and alter the pH.Soaking for several hours in 2–3% hydrochloricacid is usually sufficient, but it is advisable to test asample by filling with neutral water plus a fewdrops of suitable indicator and autoclaving.

Discard jarsAfter standing overnight to allow the disinfectantto act, the contents of the jars should be poured


Figure 4.3 Design of preparation (utility) rooms. Flow chart for the disposal of infected laboratory waste and re-usable materials

Landfill

Sortingarea

Wastereception

area

Waste disposalunitIncinerator

Autoclave

SterilizingRe-issue

Barrier

Washing

LABORATORIES

carefully through a polypropylene colander andflushed down the sluice sink. The colander and itscontents are then placed in a discard bin and auto-claved. Rubber gloves should be worn for theseoperations. The empty discard jars should be auto-claved before they are returned to the laboratoryfor further use. There may be residual contamina-tion.

Re-usable pipettesAfter total immersion overnight in disinfectant (e.g.hypochlorite, 2500 ppm available chlorine – ananionic detergent may be added), the pipettesshould be removed with gloved hands.

Before they are washed, the cottonwool plugsmust be removed. This can be carried out by insert-ing the tip into a piece of rubber tubing attached toa water tap. Difficult plugs can be removed with asmall crochet hook. Several excellent pipette wash-ing machines are manufactured that rely on waterpressure and/or a siphoning action, but the finalrinse should be in distilled or deionized water.

24-hour urinesAlthough rare in microbiology laboratories otherpathology departments may send them for disposalon the grounds that they may contain pathogens.Ideally they should be processed in the departmentconcerned as follows.

Sufficient disinfectant, e.g. hypochlorite, shouldbe added to the urine to give the use dilution. Afterstanding overnight the urine should be pouredcarefully down the sink or sluice to join similarmaterial in the public sewer. The containers, whichare usually plastic, may then be placed in colour-coded bags for incineration.

Incineration

The problems with this method of disposal ofinfected waste that has not been autoclaved are inensuring that the waste actually reaches the incin-erator, and that if it does it is effectively sterilizedand none escapes, either as unburned material orup the flue. Incinerators are rarely under the con-

trol of laboratory staff. Sometimes they are noteven under the control of the staff of the hospital orinstitution, but are some distance away, and con-taminated and infectious material has to be sent tothem on the public highway. Some older incinera-tors are inefficient.

Unburned material may be found among the ashand, from its appearance, it may be deduced that itmay not have been heated enough to kill micro-organisms. We have recovered unconsumed animaldebris, including fur and feathers and entrails, froma laboratory incinerator. The updraught of air maycarry micro-organisms up the flue and into theatmosphere if the load is too large or badly distrib-uted.

In the UK there are strict regulations about theincineration of clinical waste, which includesinfected laboratory waste (see Collins andKennedy, 1999) and inefficient incinerators arebeing phased out. The ACDP (1995) states that allwaste must be made safe before disposal orremoval to the incinerator but the HSAC (1991)permits it to be transported to an incinerator pro-vided that ‘it is securely packaged’ and the inciner-ator is ‘under the supervision of an operator who isfully conversant with appropriate safe workingprocedures for handling contaminated waste’. Asinfected laboratory waste, especially discarded cul-tures, is the most hazardous of all clinical waste,and as all reputable microbiological laboratoriespossess autoclaves, this practice should be resistedand the material made safe by autoclaving it beforeit leaves the laboratory (Collins et al., 1974; Collinsand Kennedy, 1993, 1999; Collins, 1994; ACDP,1995).

There is a British Standards Institution Code ofPractice (BS 3316) for the incineration of hospitalwaste.

REFERENCES

Advisory Committee on Dangerous Pathogens (ACDP)(1994) Precautions for Work with Human and

Animal Transmissable Spongiform

Encephalopathies. London: HMSO.

References 59

Advisory Committee on Dangerous Pathogens (1995)Categorization of Pathogens according to Hazard

and Categories of Containment. London: HMSO. Ayliffe, G. A. J., Babb, J. R. and Taylor, L (1999)

Hospital Acquired Infections, 3rd edn. Oxford,Butterworth-Heinemann.

Bowie, J. W., Kelsey, J. C. and Thompson, G. R.(1963) The Bowie and Dick autoclave tape test.Lancet i: 586–587.

British Pharmacopoeia (1993). London: HMSO.British Standards Institution (BSI) (1987) BS 3316 Part

4. Code of Practice for the design, specification andcommissioning of incineration plant for thedestruction of hospital waste. London: BritishStandards Institution.

British Standards Institution (BSI) (1998) BS EN61010-2-043:1998. Safety requirements forelectrical equipment for measurement, control andlaboratory use. Particular requirements for dryheat sterilizers using either hot air or hot inert gasfor the treatment of medical materials and forlaboratory processes. London: British StandardsInstitution.

Collins, C. H. (1994) Infected laboratory waste. Letters

in Applied Microbiology 19: 61–62.Collins, C. H. and Kennedy, D. A. (1993) Treatment

and Disposal of Clinical Waste. Leeds: ScienceReviews.

Collins, C. H. and Kennedy, D. A. (1999) Laboratory

Acquired Infections, 4th edn. Oxford: Butterworth-Heinemann.

Collins, C. H., Hartley, E. G. and Pilsworth, R. (1974)

The Prevention of Laboratory-Acquired Infections.PHLS Monograph No. 6. London: HMSO.

Gardner-Abbate, S. (1998) Introduction to Sterilization

and Disinfection, 3rd edn. London: ChurchillLivingstone.

Health Services Advisory Committee (HSAC) (1991)Safe Working and the Prevention of Infection in

Clinical Laboratories. London: HMSO.HSAC (1999) Safe Disposal of Clinical Waste. London:

HMSO.Kennedy, D. A. (1988) Equipment-related hazards. In:

Collins, C. H. (ed.), Safety in Clinical and

Biomedical Laboratories. London: Chapman &Hall, pp. 11–46.

Maurer, I. M. (1972) The management of laboratorydiscard jars. In: Shapton, D. A. and Board, R. G.(eds), Safety in Microbiology Society for AppliedBacteriology Technical Series No. 6, London:Academic Press, pp. 53–59.

Medical Devices Agency (MDA) (1993/1996)Sterilization, Disinfection and Cleaning of Medical

Equipment. London: HMSO.Russell, A. D. (1999) Microbial susceptibility and

resistance to chemical and physical agents. In:Collier, L., Balows, S. and Sussman, M. (eds),Topley and Wilson’s Microbiology and Microbial

Infections, 9th edn, Vol. 2, Systematic

Bacteriology. London: Arnold, Chapter 7.Russell, A. D., Hugo, W. B. and Ayliffe, G. A. J. (1999)

Principles and Practice of Disinfection,

Preservation and Sterilization, 3rd edn. Oxford:Blackwell Scientific.


In developed countries it is unusual for medicalmicrobiology laboratories to make their own cul-ture media from raw materials. Furthermore, eventhe reconstitution of dehydrated media takes placeless and less in the individual laboratory as bottles,tubes and plates of ready-to-use media becomeavailable from central suppliers and/or commercialsources.

Although this loss of skill in the preparation ofmedia may be regretted, the growing weight of invitro diagnostics legislation makes independent lab-oratory manufacture a legal minefield. For every per-son making a product, two more staff will be requiredto check activity, labels, instructions for use, shelf-life limits and safety precautions in use. One effectof centralization of supplies is an inevitable reduc-tion in the variety of media formulations available.Commercial manufacturers annually review their listof media and monitor the sales of each medium.They may decide to cease manufacture when salesfall below specified figures. Central non-commercialsuppliers will operate from a limited list of consen-sus decisions about the variety of media that can besupplied to the receiving laboratories. This meansthat the days have long gone since Sir Graham Wilsononce stated that a UK microbiologist would ratherborrow a colleague’s toothbrush than use his or herparticular culture medium.

Nevertheless, there will always be culture mediafor special purposes that are not available commer-cially. Formulae and methods for those that arementioned in this book are therefore included (seep. 72). Laboratory workers who do decide to maketheir own media can find the formulae, etc. in thecommercial catalogues and handbooks and in oldereditions of this book.

All culture media must now display labels describ-ing the formulation, with the amounts of the indi-vidual constituents present, normally expressed asgrams per litre. The labels of the same mediumfrom different suppliers will appear to be identicalbut each label will also state that some variation informulation may be made to meet performancestandards. This caveat allows formula manipula-tion to accommodate variation in the raw materialsused to achieve standard performance results.Some culture media have commercially protectednames and, although such media may have thesame formulation between different manufactur-ers; others cannot use the protected name.

Further details about the formulation and per-formance of culture media may be found in the fol-lowing: MacFaddin (1985), Baird et al. (1987),Barrow and Feltham (1993) and Bridson (1994).Also the current editions of BBL, Difco, LabM andOxoid manuals and catalogues are useful sourcesof information.

IN VITRO CULTIVATION OFBACTERIA

The work of Pasteur (Gieson, 1955) and his col-leagues in France, together with that of RobertKoch (Brock, 1988) and his team in Germany, pro-vided the foundation of in vitro cultivation ofmicro-organisms in the second half of the nine-teenth century. Cultivation was essential because,apart from the morphology of individual cells,these extremely small units of life could not be sep-arately studied to determine their metabolism and

5

Culture media

biochemistry, and to classify their separate identi-ties, without using millions of cells – a situationthat, despite advances in molecular biology, stilllargely prevails today. It was this essential ground-work that provided the proof of the causation ofinfectious disease.

All the organisms of importance in medical micro-biology are described as chemo-organotrophsbecause they obtain energy from organic mole-cules. The constituents of early culture media fortheir cultivation were undefined, complex, organicmixtures. This is another characteristic of medicalmicrobiology that remains to this day. Pasteurstarted with simple organic solutions and thenmoved on to urine and blood, finally using live ani-mals. Koch started with a meat stew (meat extract)for the isolation of his first pathogenic organism –Bacillus anthracis. He did not consider this to be auniversal culture medium and he also experimentedwith blood, serum and egg. Koch and his col-leagues in Berlin then went on to isolate and iden-tify the bacterial causes of all the major infectiousdiseases in Europe. This work was completed overa period of 20 years – 1880–1900.

This great achievement came about because oftwo important developments. First, Koch created asolid culture medium format from his meat extractsolution, the ‘Poured Plate’, using at first gelatinand later agar as the solidifying agent. He estab-lished the principle of one cell forming one colonyof identical cells, which was a great improvementover Pasteur’s dilution to extinction method usingfluid cultures. Second, his colleague Loeffler sup-plemented Koch’s meat extract with peptone(hydrolysed protein). The full significance of thissupplement only became apparent in the closingyears of the twentieth century.

The major work in microbiology in the first halfof the twentieth century was the study of themetabolism in prokaryote cells: the catabolic chem-ical pathways producing energy and the anabolicpathways of biosynthesis consuming it. At the sametime, study of the dynamics of bacterial growthrevealed the lag, exponential growth, and the sta-tionary and decline phases of growth that are typi-cal in any closed system of bacterial cultivation. In

the second half of the twentieth century, antibioticswere developed and the DNA/RNA genetic codewas cracked to reveal the complexities of themolecular control of life.

THE COMMON COMPONENTS OFCULTURE MEDIA FOR MEDICALMICROBIOLOGY

An examination of the formulae of culture mediacommonly shows a few key nutrient ingredients,and several other components added for the selec-tion and/or identification of separate groups orspecies of bacteria.

All life is dependent on the presence of water andall the nutrients from which micro-organisms syn-thesize cell material and obtain energy must be dis-solved in water. Simple chemical analysis ofbacterial cells reveals 11 macroelements – C, H, O,N, S, P, K, Na, Ca, Mg, Fe – and many trace ele-ments that are often found as impurities in themacroelements, e.g. Mn, Mb, Zn, Cu, Co, Ni, V, B,Se, Si, W. This simple analysis is not very helpful inthe construction of specific culture media and it isbetter to divide the constituents into one or morefunctional parts.

Amino-nitrogen nutrientsProtein hydrolysates (peptones) infusions orextracts: these nutrients often contain sufficientenergy-rich molecules and trace elements tobecome the sole ingredient necessary for thegrowth of common medical bacteria.

Energy sourceThis is usually glucose but other easily used carbo-hydrates may be substituted. Peptones can alsoprovide carbon energy, especially those derivedfrom plants.

Growth factorsSome more fastidious organisms appear to requireheat-labile supplements to stimulate growth, e.g.blood, serum and vitamin complexes. Further work

62 Culture media

has shown that, with Bordetella, Neisseria,Campylobacter and Legionella species, the essen-tial role of blood is to act as a protective agentagainst toxic oxygen radicals rather than act as asupplementary nutrient.

Buffer saltsSoluble sodium or magnesium phosphate, acetateor citrate salts are commonly added to culturemedia containing carbohydrates, to maintain pHstability when fermentative organisms are growing.These buffer salts, however, can chelate essentialmetals, especially Fe2+ ions (Munro, 1968). Somegrowth factors are toxic (e.g. fatty acids) and mustbe added in a form or composition (e.g. with albu-min or as a detergent) to permit growth and sur-vival.

Mineral salts and metalsSupplements of salts and metals are normallyrestricted to synthetic (defined) culture media.Undefined media should provide an adequate sup-ply unless extra is required to overcome a chelatingsubstance in the formulation or an excess of NaClis added to select halophilic organisms.

Selective agentsToxic chemicals, antibiotics and inhibitory dyeshave been used separately or in combination in cul-ture media. It is essential that the selective agentsare at the correct strength for the particularmedium formulation and the selected organism.There is an element of compromise for all selectivemedia, i.e. not all unwanted organisms will beinhibited and not all desired organisms will grow(Miller and Banwart, 1965; Bridson, 1978). Theactive concentration of an antibacterial agent (e.g.Hg2+, antibiotic) may be influenced by the compo-nents of the medium (e.g. by albumin).

Indicator dyesDyes such as phenol red, neutral red and bromocre-sol purple are added to culture media to indicatechanges in pH value, during and after growth.Some dyes are colourless (e.g. triphenyltetrazoliumchloride) and act as electron acceptors, and their

reduced forms (e.g. triphenylformazan) are pig-mented (red) and insoluble, resulting in dye deposi-tion around colonies in agar medium or red colourin broths as an indication of growth. Fermentativecarbohydrates are normally added to the formula-tion. The dyes used can be toxic to sensitive orstressed cells and this fact needs to be kept in mind,if growth is absent.

Gelling agentsAgar is the most common gelling agent used in cul-ture media because it has natural advantages overgelatin and alginate. It is not an inert constituent ofculture media; it can contribute metals, minerals,sulphate and pyruvate. Agar can also bind waterand inhibit growth of organisms (see ‘Pouredplates’, p. 69).

DEFINED (SYNTHETIC) VERSUSUNDEFINED (COMPLEX) CULTUREMEDIA

The great success of Koch and his colleagues in thelast 20 years of the nineteenth century was basedon totally undefined and (as we now know) highlycomplex soups and stews made in the laboratorykitchen. With the exception of Mycobacteriumtuberculosis, a wide variety of pathogenic organ-isms were isolated with the same nutrientbroth/agar medium first described by Koch andLoeffler. With small modifications, this formulahas continued to serve bacteriologists to the presentday. There was a period, however, when microbi-ologists became ashamed of their apparent culinaryart. In the 1950s, the biochemical neighbours ofbacteriologists began to surge ahead with newknowledge of energy-yielding and synthetic path-ways of metabolism, which they combined withrapid, automated, microanalytical techniques.Bacteriologists became aware that, if Koch orPasteur were to enter the average clinical bacteriol-ogy laboratory, it would soon feel like home tothem. Thus began a 25-year search for defined(synthetic) culture media to replace the soup and

Defined (synthetic) versus undefined (complex) culture media 63

stew. There were two sound reasons for the devel-opment of defined media at that time:

1. Existing (largely manufactured by individuallaboratories) undefined media could givewidely variable results between and withinlaboratories.

2. Increasing exploration of microbialmetabolism, using defined media, suggestedthat reproducible results could come onlyfrom synthetic culture media.

Over the next 25 years, active research wasreported in a large number of published papers,which mostly demonstrated hope rather than real-ity. Nutrient agar/broth can grow practically all thegenera of clinically important organisms and withone or two supplements will grow most of the oth-ers. This includes all the species and subvariants ofthe genera. None of the synthetic media formula-tions published could meet this standard. Mostworkers started with the Synthetic Mineral-SaltsMedium of Davis and Mingioli (1950). This con-tained:

Carbon source 2 gK2HPO4 7 gKH2PO4 3 gMgSO4⋅7H2O 0.1 g(NH4)2SO4 1 gNa citrate⋅3H2 O 0.5 gWater 1 litre(Agar) (15 g)pH 7.0

This formula allowed the growth of Escherichiacoli from a generous inoculum. To grow moredemanding organisms supplementation with aminoacids, nucleotides, vitamins, trace metals, etc. wasnecessary The list of supplements increased consid-erably for organisms such as pathogenic neisserias.One such defined medium for Neisseria gonor-rhoeae required 10 multi-component stock solu-tions plus 5 separated components, a total of 50components. Catlin’s (1973) paper describing thismedium showed that reproducible defined mediacannot be prepared by weighing all the dried ingre-dients into water, boiling to dissolve them and

finally autoclaving the mixture. A very large,unpredictable and highly complex interaction ofthe components would result, which would not berepeatable with every batch of the same medium.Furthermore, even the stock solutions had to beprotected from photo- and chemo-oxidation.Experience showed that simpler defined media,designed around a less demanding genus, wouldseldom show growth of other genera and oftenwould not grow all the species of the single genus,without further supplementation.

The search for defined (synthetic) media thatcould be used in general clinical bacteriologyslowly ebbed away in the mid-1970s. There weretwo major reasons for abandoning the search:

1. By the mid-1970s most bacteriologicallaboratories were purchasing commerciallyprepared, dehydrated and ready-to-use media.The gross disparity of quality in culture mediabetween and within laboratories had gone.

2. In 1966, Ellner et al. published the results oftheir work on Columbia agar. These workershad postulated that, if a one-peptone mediumshowed good results with many organisms,then more than one peptone would be better.They finally incorporated four differentpeptones and formulated a general-purposemedium for medical microbiology, which hasstill not been eclipsed in performance. Theexplanation for its astonishing success lies inthe complex mixture of polypeptidesproduced from different proteolytic enzymesdigesting different proteins. Subsequently, itwas realized that the Columbia formula couldrecover growth from small inocula of stressedorganisms.

STRESSED ORGANISMS ANDRESUSCITATION

In the early days of clinical bacteriology, when sur-geons were delighted to see ‘laudable’ pus fromtheir patients, it was considered that all pathogenicorganisms were aggressive, vigorous cells. The idea

64 Culture media

that such organisms might be stressed or need spe-cial nurture would have seemed ludicrous to bacte-riologists in those by-gone days.

Preservation of food by drying, salting, heating toscalding temperature, chilling with ice or makingacid with vinegar and/or the use of spices evolvedempirically, long before the existence of micro-organisms was known. Pasteur rationalized the heat-ing process now called ‘pasteurization’ and he wasable to explain why the new ‘autoclave’ canning offood worked to overcome putrefaction on storageat room temperature. It is unlikely that the foodpreservation steps described above could destroy allthe viable micro-organisms in food. However, thosethat just survived were probably damaged to a degreethat fermentation or putrefaction was long delayed.Such damaged or stressed organisms may also notsurvive a period of acid digestion in the stomachs ofthose eating the food. Those micro-organisms thatdid escape alive into the small intestine would meetother defence mechanisms. However, in an imper-fect world not all consumers have the same immunesystems and food poisoning is still a hazard today.Food microbiologists, aware that their productscould contain damaged (stressed) micro-organisms,were the first to recognize that special steps neededto be taken to recover them. Quantitative counts oforganisms in food samples, before and after pro-cessing, were essential facts required to make judge-ments about the duration and temperatures requiredto process foods safely.

The recovery of stressed cells by theprocess of resuscitation

The traditional test to detect sublethal injury inbacteria caused by inhibitory agents (chemical,physical or biological) is recognition of failure togrow on a selective medium that normally supportsits growth, although growth occurs in the absenceof the inhibitory agent (Mossel and Ratto, 1970;Hackney et al., 1979). Although this selective/non-selective medium test can be helpful to detectinjured cells, it is not entirely reliable. Damaged

cells may not be able to grow at all until the recov-ery (repair) process has taken place (Walker, 1966;Dukan et al., 1997). In the 1960s it was recognizedthat the addition of pyruvate or catalase to culturemedia yielded higher colony counts. Slowly, overthe next 30 years, a full appreciation of the effectsof oxidation/reduction in culture media and theireffects on microbial growth was made. Oxidationhas been considered as the cause of ‘viable but non-culturable’ (VBNC) organisms (Bloomfield et al.,1998)but this hypothesis has been contested (Barerand Harwood, 1999).

An unexpected but essential factor in the recoveryof stressed bacteria was the discovery of a widevariation in the length of lag phases of growth asso-ciated with the numbers of organisms present in theinoculum. Stephens et al. (1997) used an auto-mated growth analysis system to measure timesand growth of heat-stressed salmonellas in a resus-citation broth, in which the inocula varied fromone cell to 107 cells. Single stressed cell inoculapro-duced lag phases in different recovery broths,which varied from 6 to 36 h. The lag phases of non-heat-injured single cells varied from 6 to 8 h in thesame recovery broths. At 100-cell inocula, bothheat-injured and non-heat-injured cells producedsimilar lag phases of 6–8 h. There is some protec-tive action when numbers of stressed cells attemptrecovery of growth that is not available to the sin-gle stressed cell. Reducing agents are beneficialbecause, in later work, Stephens (1999) demon-strated that the most inhibitory broths had higherlevels of peroxide present and that the addition ofreducing agents to the inhibitory media overcamethe long lag phase with single stressed cells.

Is there a single optimum recovery medium forpre-enrichment or resuscitation of bacteria thathave been sublethally injured by various methods?Experience has shown that complex undefinedmedia are preferable to chemically defined mediaor buffer salt solutions. Key factors seem to be thepresence of polypeptides and a reduced Ehmedium. A review of the literature made in 1977listed the recovery agents that can be added tomedia to assist the recovery of stressed organisms(Mossel and Corry, 1977):

Stressed organisms and resuscitation 65

1. Neutralizing substances to counter the effectsof damaging residual chemicals, e.g. β-lactamases, sulphydral compounds, wettingagents

2. Lysozyme to help germination of ‘dead’spores

3. Catalase or whole blood to remove peroxides4. Pyruvate and other citric acid cycle

intermediates to improve recovery frommembrane damage

5. Magnesium to stabilize RNA and cellmembranes

6. General recovery agents: oxygen radicalscavengers, whole blood, activated charcoal.

SELECTIVE AND ELECTIVECULTURE MEDIA

The concept of selective enrichment of micro-organisms was developed early in the history ofculture media. Bile and bile salts were found toselect Salmonella typhi in particular and coliformbacteria in general. Conjugated bile salts (com-bined with taurine or glycine) are less toxic thanfree bile acids (deoxycholic and cholic acids). Thetoxic activity of selective agents has to be accu-rately determined. The amount of agent added tothe formulation is calculated and checked by test-ing each new batch with a standard reference batchof the same formulation.

Chemical selective agents

Typical chemical selective agents are:

1. Inorganic salts: azide, bismuth, lithium,selenite, tellurite, thallium

2. Dyes: brilliant green, malachite green, crystalviolet

3. Surface active agents: bile, cetrimide, laurylsulphate, Tergitol (Bridson, 1990) and Tween80

4. Others: dichloran, irgasan, phenylethylalcohol.

Chemical selective agents are relatively crude inaction and in most circumstances a line must bedrawn in a trial to define an optimum concentra-tion to be used. An ideal concentration that willdestroy 100% of unwanted organisms and allow100% of the desired organisms to survive is unob-tainable. From a limited range of concentrations(predetermined by experiment) that can allow apredominance of the desired organism(s) and max-imum suppression of the unwanted organisms, thebest concentration is selected for use with the cho-sen medium. Further details on chemical selectiveagents can be found in the Oxoid publication(Bridson, 1994).

Antibiotic selective agents

Fleming carried out the first demonstration of theselective action of antibiotics in culture media in1929, using his crude penicillin preparation to iso-late Haemophilus species. This work could not berepeated until commercial supplies of penicillinbecame available in the 1950s. Lacey (1954)returned to Fleming’s technique using penicillin toisolate H. pertussis. By that date, however, the inci-dence of penicillin resistance was sufficiently com-mon to require other selective agents to be usedwith it.

Although not the first of the antibiotic-selectivemedia to be used, the medium of Thayer andMartin (1966) for the isolation of Neisseria specieswas the most widely known early example. Thesemedia opened the door for opportunities to isolate‘new’ and ‘difficult’ organisms. A host of exoticorganisms could now be found and cultivated inany general microbiology laboratory, e.g.Campylobacter (Helicobacter), Legionella,Gardnerella, Listeria, Mycoplasma and Yersiniaspecies, and Clostridium difficile.

Early work with these media, using single antibi-otics, was promising, e.g. novobiocin for salmonel-las and neomycin for anaerobic organisms, butmixtures of two or more antibiotics gave betterlong-term prospects against antibiotic-resistant

66 Culture media

contaminating organisms. Although there are verymany antibiotics, their basic mechanisms of actionare few:

• alteration of cell membranes• inhibition of protein synthesis• inhibition of nucleic acid synthesis • inhibition of cell wall synthesis• anti-metabolic activity or competitive

antagonism.

By incorporating at least two of these five inhibitoryactions in the antibiotic mixture, the likelihood ofresistance developing is much reduced.

Commercial media suppliers were requested tosupply antibiotic selective media. Only two antibi-otics, chloramphenicol and kanamycin, were suffi-ciently stable to be incorporated into dehydratedculture media. However, the hazards of inhalingantibiotic-containing dust by customers and manu-facturing staff were considered to be too great.Incorporating labile antibiotics into preparedmedia meant that the individual activities of theantibiotics would fall from the day of preparationuntil the media were used. The best solution was tohave separate vials of sterile, lyophilized antibioticmixtures, ready for rehydration and adding to pre-scribed volumes of pre-sterilized, cooled media. Itwas Skirrow’s medium for the isolation ofCampylobacter species that started the commercialproduction of lyophilized antibiotic supplements in1977 (Skirrow, 1977). In a relatively short periodof time, campylobacters arose from obscurity tobecome identified as the most common cause offood poisoning in the UK. Since that time, another60–70 antibiotic mixtures have been made avail-able to microbiological laboratories.

Elective media

A selective medium contains a surfeit of nutrientsand inhibitory agents are added to restrict growthof undesired organisms and make it selective for aparticular genus/species. By contrast, an electivemedium is designed to have minimal nutritionalingredients, enabling only a limited group of micro-

organisms to grow. Elective media are not used inmedical microbiology because they are ineffectivefor resuscitation of stressed organisms. Reuter(1985) has described elective media for lactic acidbacteria and gives examples of selection and elec-tion.

THE SIGNIFICANCE OF COMPLEXPOLYPEPTIDES IN UNDEFINEDCULTURE MEDIA

It was Loeffler’s contribution of peptone to Koch’ssystem of cultivating bacteria that ensured 120years later that the original nutrient broth/agarmedium formulation has scarcely changed.Throughout most of this time, the role of peptonewas seen to be as a nutritional supplement but itscomplex structure of peptides was little appreciateduntil the work of Ziska (see Bridson 1994) waspublished in 1967–68. This, combined with thework of Ellner et al. (1966), raised a possibility thatpolypeptides might have a special role in the recov-ery of stressed organisms. Columbia Agar, with itswide spectrum of peptides ranging in size from 50to 2 amino acids in length plus numerous freeamino acids, was superior in performance to cul-ture media containing one peptone only. The suc-cess of undefined polypeptides over definedmixtures of amino acids may be attributed to oneor more of the following hypotheses.

Polypeptides may act as redox agents

It is known that larger numbers of stressed organ-isms are easier to recover than single or few cells.This protective effect may be a reduced Eh in theimmediate cell environment. Polypeptides may havea similar protective effect in lowering the Eh of themedium. Wright (1933) demonstrated that ‘toxic’(oxidized) peptones could be reduced by heatingwith meat particles. He found that such reduced pep-tones would then recover small numbers of fastidi-ous strains of Streptococcus pneumoniae.

The significance of complex polypeptides in undefined culture media 67

Use of peptides may be more efficientthan amino acids

The formation of proteins in the living cell involvesassembly of specific amino acids into strings andthen folding of the amino acid sequence into sec-ondary, tertiary or quarternary structures. It is thethree-dimensional structure of the sequence,assisted by protein chaperones, that determineswhether the protein will have a structural or a cat-alytic function. Protein formation is a heavy bur-den for the stressed microbial cell; the creation andpolymerization of amino acids consume 75% ofthe available cell energy (Stouthamer, 1973). It alsorequires a fully functional cascade of enzymes thatmay not be present in the stressed cell. For thestressed cell, a supply of appropriate polypeptidesmay enable the cell to overcome these constraints.

Transport of polypeptides

Transport of amino acids and peptides into the cellcosts energy. It requires one proton to transport asingle amino acid or a peptide. Peptide transporthas greater energy advantages over amino acidtransport.

How large polypeptides (greater than a tetrapep-tide) cross cell membranes is still a matter of con-jecture. Chaperone molecules could capture,transport and, if required, refold large peptides(Clarke, 1996). Stressed cells produce increasedamounts of chaperone molecules as heat shock pro-teins (Hsp), as well as other SOS proteins, andthese may have a critical transport role (Martin andHartl, 1997).

Polypeptide mixtures and ribozymes

Existing technology cannot reveal the complexchemical pathways involved in the resuscitation ofa single cell. All currently described microbial bio-chemical pathways are based on mean value analy-ses of millions of cells. The fact remains, however,

that a severely disabled cell has to create a largevariety of enzymes and structural proteins toenable it to commence normal metabolism anddivision.

There are theories that RNA developed beforeDNA in the evolutionary pathway. These theoriesare based on the fact that nucleotide subunits ofDNA are modified RNA molecules, and that manyenzymes require coenzymes that are RNA mole-cules or close relatives. In 1982, RNA moleculeswith catalytic powers (ribozymes) were discoveredin protozoa and bacteria. Ribozymes are hybridmolecules, part peptide and part ‘gene’. Moleculescan be selected by the ‘fitness’ of the peptide armand then amplified with the attached ‘gene’. Theyare used to cut strands of DNA and they can bereplicated in a synthesizer. When replicated using asynthesizer that allows ‘errors’ to occur in copying,the mutant enzymes can show increases in DNAcutting efficiency of 100%. This discovery fitted thethree main pillars of Darwinian evolution:

• a mechanism to introduce mutations inmolecules

• a selection or ‘fitness’ pressure that favourssome mutations over others

• an amplification mechanism that encouragesfavoured mutants to multiply.

Exposing stressed cells to millions of polypeptidevariations would allow cells to select polypeptidesby ‘fitness’ of combination with the cell ribozymes.These RNA–polypeptide molecules may be used ina non-ribosomal pathway as ‘starters’ for the syn-thesis of key enzymic and structural proteins.

These speculative but intriguing hypotheses aremore fully discussed in Bridson (1999).

STORAGE AND PROTECTION OFPREPARED CULTURE MEDIA FROMENVIRONMENTAL FACTORS

It is a not uncommon paradox that much scienceand care can be expended in producing high-qual-ity media, only to have the products stored under

68 Culture media

conditions in which they will deteriorate. Mediasupplied from commercial or central sources nowhave precise instructions about shelf-lives, storagetemperature limits and light protection factors.Microbiological laboratories are expected to showevidence that they conform to these storage and useinstructions. Like good wine, culture media solu-tions are best kept cool (2–8°C), not frozen, and inthe dark (photo-oxidation spoils both wine andbroth). All bottles should carry labels identifyingthe contents and show the preparation date, shelf-life, batch number and expiry date. Media shouldbe used in strict date order and outdated materialdiscarded. There are also a few elementary obser-vations that can be carried out. Bottles withreduced clarity and darkening of colour (especiallythe upper layer of the liquid) should be rejected andthe pH value checked for acid drift.

Great damage can be caused to culture media inthe autoclave. If powdered culture media is addedto the bottles, water simply poured on top to theprescribed volume and the bottles placed in theautoclave for sterilization, the problem can be seenwhen the autoclave is opened. There is a dark-brown concentrate of nutrients at the bottom of thebottle. When shaken, the colour becomes a uni-form straw colour but the damage has been done.Superheating biological concentrates causes inter-active complexity of the amino acids, phosphatesand free metal ions. It reduces the nutritive value ofthe medium and reduces the shelf-life of the prod-uct. All prepared culture media are dynamic com-plexes of ingredients that can only deteriorate onstorage. The best storage conditions are those thatreduce the rate of deterioration to the minimum.Stirred media preparators are preferable to staticautoclaves.

PROTECTION OF PREPAREDPLATES

The most labile prepared culture medium that iswidely used in microbiological laboratories is thenutrient gel slab residing in the Petri dish. What

causes the rapid fall off in microbiological per-formance? Chemo- and photo-oxidation have arole but the most serious effect is the loss of surfacemoisture. Modern agars at 1.2% w/v hold water ina firm gel. The agarose polymer, however, has anatural contraction rate which squeezes water outof the gel; this is called syneresis. If the coveredplate is left on the open bench, this water evapo-rates and the surface ‘skin’ of agar becomes lessporous. The greater the water loss, the thicker the‘skin’ of inhibitory agar on the surface. The twoforces of syneresis and evaporation work togetherand can result in a thin layer of very hard agar atthe surface of the dish. The microbial inoculum onthe surface of the agar quickly uses the availablenutrients in its immediate locality. It must nowwait while nutrients diffuse up through the agarthickness, or from the periphery of the inoculum toreplace nutrients taken up by the microcolony.Anything that slows down this flow of nutrientsresults in attenuated growth but the agar ‘skin’ is apoor diffuser of solute. All bacteria suffer in thissituation and usually produce smaller colonies, butmore serious is the fact that stressed cells are par-ticularly vulnerable and may fail to multiply at all.

How much water must be lost before there is asignificant effect on bacterial growth? Tests carriedout in Oxoid laboratories many years ago, beforestressed cells were fully appreciated, showed that ifprepared plates lost more than 5% of their grossweight (Petri dish plus agar layer) on storage, whencompared with their fresh weight, then a reductionin colony size of unstressed bacteria could bedetected. This is quite a small amount of water butit is removed from the highly critical surfacebetween organism and agar.

Armed with this knowledge, is it possible to com-pare the performance of current commercially pre-pared plates with those made in the individuallaboratory 30 years or so ago? It would be tempt-ing to say that current plates are much superior tothose in the past. Although 7-day-old plates madein the past were poorer than present-day, commer-cially prepared plates of the same age, in thosedays, 7-day-old plates were not used in most labo-ratories. Plates were often poured in the morning

Protection of prepared plates 69

and used the same day. These freshly preparedplates would probably be comparable in perform-ance with most commercially/centrally pouredplates distributed today.

Packaging is a very important factor but it is notthe sole answer. It is possible to see moisture bead-ing the lids and packaging of packs of preparedplates. This problem is caused by changes in tem-perature and great care is now paid to keeping thetemperature of the plates as steady as possible withpolystyrene packs. Unfortunately, once agar haslost water it cannot reabsorb it unless it is raised tothe melting temperature of agar (80°C.) It is possi-ble to improve the growth of micro-organisms byplacing the inoculated dishes in closed containers,together with moisture-saturated air but little ornone of the water can rehydrate the agar.

The most successful way to keep moisture in agarplates is to place a sterile plastic film over the sur-face of the agar. This will prolong the serviceablelife of the plate by minimizing the water loss fromthe surface. A simple test to demonstrate this is toincubate two sterile nutrient agar plates at 35°C for48 hours. Take them out of the incubator and, usinga sterile scalpel blade, cut around the meniscus ofagar of one plate. Replace it in the lid and slap theplate smartly on the bench. The agar slab will fallinto the lid. Inoculate both plates lightly with a sus-pension of Staphylococcus aureus (Oxford strain)NTCC 6571. After 16 hours’ incubation, the con-ventionally exposed agar surface will show poorergrowth than the upside-down surface, which hasbeen protected by the plastic dish. The poor growthrepresents a reduction in water activity and evenmoderately high water activity (aw) reduction cantotally inhibit the growth of stressed cells.

QUALITY CONTROL AND THEPERFORMANCE TESTING OFCULTURE MEDIA

Total quality management (TQM) of culture mediais discussed on pages 134–146 of Bridson (1994),but clinical laboratories will require only the end-user control tests. These will be to check storage

facilities and shelf-lives of culture media in the lab-oratory or to compare one supplier’s media withanother. Only quantitative tests should be used forstatistical purposes but a great deal of essentialmicrobiological detail is obtained from colonialqualitative characteristics, e.g. colour, translu-cence, surface reflectance of light, pigment diffu-sion, size variation, capsule formation, etc. For theidentification of certain organisms, such character-istics may be quite critical and the lack or variationof these could fail a particular batch or anothersupplier’s product, and constitute a failure to meetthe user’s quality standards.

When clinical laboratories made their own culturemedia, they tested them with laboratory stock cul-tures and with clinical specimens taken frompatients. When commercial suppliers commencedmanufacturing media for use in clinical diagnosis,they could either have requested that an outside hos-pital laboratory tested samples with clinical speci-mens or have begged for clinical material from localhospital laboratories. Both practices were carriedout and both were equally difficult to control.Increasingly stringent safety rules in industry madethe importation of clinical material into manufac-turing premises very difficult. At this time the expan-sion of food microbiology led to demands from thatindustry that all culture media made for them shouldbe tested with specified standard lyophilized strainsfrom national collection centres, e.g. ATCC(American Type Culture Collection) or NCTC(National Collection of Type Cultures) in particular.

As food industry requirements included bothspoilage and pathogenic organisms, media manu-facturers turned their backs on clinical isolates andused these stable and reproducible organisms forall their testing. Unfortunately, this change openeda gulf between the manufacturer and the medicallaboratory. The manufacturer could supply muchdetail about the quantitative recovery of standardorganisms, but the clinical customer did not isolatestandard organisms from patients. The manufac-turer’s task was to nurture very unhappy organ-isms, taken from a hostile environment of immuneand antibiotic attacks, possibly in quite small num-bers, i.e. a few highly stressed cells. The difference

70 Culture media

between these testing situations was exacerbatedby the use of selective media. Bartl (1985) discussedthis problem where he compared ‘wild’ versus ‘cul-ture collection’ strains on selective culture media.He accepted, however, that uncertainties arosefrom using random non-reproducible ‘wild’ strains.A better solution was required. The answer seemsto be to stress standard strains under strictly con-trolled conditions, e.g. exposing them to higher orlower temperatures for fixed periods of time, suchas 20 min at 52°C and 30 min at −20°C or anyvariations between these extremes of temperature.There is still much work required to standardizethe stress conditions for a wide variety of commer-cially available lyophilized cultures and to applythe appropriately stressed organisms to test a vari-ety of culture media.

Meanwhile, various schedules of testing methodsfor culture media are in use. Appendix A of the‘QA and QC of microbiological culture media’(National Committee for Clinical LaboratoryStandards or NCCLS, 1996) can be recommended.

• The NCCLS QA Standard for CommerciallyPrepared Microbiological Culture Media(NCCLS, 1996) describes the appropriatecontrol organisms and test procedures thatare to be used by manufacturers of culturemedia in the USA. If these standards are metcustomer laboratories in the USA are exemptfrom further testing. Two culture mediaformulations have failed to meet NCCLSstandards (MacFaddin, 1985): CampylobacterAgar and selective media for pathogenicNeisseria species. When using these media,the customer laboratory must carry outappropriate quality control tests.

• The British Standard BS 12322:1999 (BSI,1999) is a concise description and definitionof terms used, performance evaluationcriteria, control strains used, quality criteriaand information to be supplied by themanufacturer.

• DD ENV ISO 110133-1:2000: (DD, 2000) isa draft publication of extensive terminologywith dozens of headings but the small print

underneath each heading is not as helpful asCEN/TC 275 (CEN, 2001).

• The CEN/TC 275 N489 (2001) on practicalguidelines on performance testing of culturemedia is much more detailed than the twostandards above, although both should bestudied. The tables of commonly used culturemedia with test organisms, culture conditionsand expected reactions are particularly useful.

• BS 6068-4.12:1998 (ISO 11731) (BSI, 1998)is concerned with water quality and thedetection and enumeration of Legionellaspecies in particular. It recommends usingBuffered Charcoal Yeast-Extract AgarMedium (BCYE) with GVPC (glycine–vancomycin–polymyxin–cyclohexamide). Aspecific warning is given against using stockstrains of L. pneumophila to check thegrowth properties of BCYE–GVPC medium.Known positive water samples containing‘wild’ strains of Legionella are preferable.

Corry et al. (1995) have updated their 1987 pub-lication on culture media for food microbiology.Each medium is fully described with a brief historyplus physical properties, shelf-life, inoculationmethod, reading results and interpretation, qualityassessment (productivity and selectivity) withappropriate references.

A closing comment should be made about culturemedia incubation temperature. It is a sad and per-sistent error that all European standards specify37°C as the mean incubation temperature, ratherthan the US standard of 35°C. Walk-in incubatorsnormally operate at ± 2°C of the stated mean tem-perature. Stressed organisms will recover and growbetter between 33 and 37°C than they will between35 and 39°C.

IS THERE A FUTURE FORCONVENTIONAL CULTUREMEDIA?

This is a question that has been regularly askedover the last 30 years, as novel microbial detection

Is there a future for conventional culture media? 71

and identification methods have come and gone.One weakness with many ‘alternative’ tests hasbeen that, with the exception of methods based onnucleic acid amplification, high concentrations oforganisms are required before they can be applied.Culture media and all their problems are stillrequired before the novel method can be used. Thenovel part is then often very expensive comparedwith conventional culture media microbiology.

Rapid diagnostic tests for the presence of patho-genic organisms in patients can be justified, in spiteof high costs, in certain circumstances:

• Where growth of the suspected organism isvery slow or uncertain in culture media

• Where very early diagnosis or differentiationof agents causing identical symptoms isrequired to allow life-saving procedures to beapplied, e.g. meningitis

• Where rapid screening of potential patients isrequired for barrier nursing or isolation fromwards

• Where identification of very specific antigen-variant types of the pathogen is urgently sought.

Other situations could be listed if the costs wereof less consequence. Unfortunately, health servicesconsume money with the same avidity that ‘blackholes’ consume matter. In the ‘rob Peter to payPaul’ situation that is common in most health serv-ices, a best value solution is always sought.

There are good reasons why conventional meth-ods of culturing micro-organisms will continue fora substantial period of time:

• Unsuspected or ‘novel’ micro-organisms thatemerge (e.g. legionellas).

• The methods used are very economical and itcan be envisaged that smaller culture, media-packaged formats will be produced on high-speed machines, thus improving the economyof the methods.

• There are large parts of the world where, eventoday, microbial diagnostic services are sadlylacking. Even simple pathology services wouldbe a great advantage to the deprivedpopulations.

• Most importantly, only broad-spectrumculture media are capable of nurturing andcultivating quite unexpected organisms.Until it is possible accurately to interprettissue SOS signals, the pyrexial, hypotensiveand very unwell patient requires an openmind and non-discriminate investigations,including conventional microbiologicaltesting.

As science and medicine progress at an accelerat-ing rate, it would be foolish to attempt to predictwhen a totally new and reliable system of identify-ing significant microbial infection in a patient willappear. The agar plate is about 120 years old and,despite many forecasts of its imminent demise, it iscurrently manufactured in larger numbers thanever. It still appears to be in its log phase of growth.Experience proves that technology does notbecome obsolete until it is superseded by somethingthat is clearly superior. Microbiologists await thearrival of superior microbial diagnostic methodswith great interest.

LABORATORY-PREPARED MEDIA

Most of the media mentioned in this book areavailable commercially but some are not and theformulae for these are given below. A few othersare in appropriate chapters.

Arginine broth

This is useful in identifying some streptococci andGram-negative rods:

Tryptone 5 g Yeast extract 5 g Dipotassium hydrogen phosphate 2 g L-Arginine monohydrochloride 3 g Glucose 0.5 g Water 1000 ml

72 Culture media

Dissolve by heating, adjust to pH 7.0; dispense in5- to 10-ml amounts and autoclave at 115°C for10 min.

For arginine breakdown by lactobacilli, use MRS(p. 347) broth in which the ammonium citrate isreplaced with 0.3% arginine hydrochloride.

Carbohydrate fermentation andoxidation tests

Formerly known as ‘peptone water sugars’, someof these media are now more nutritious and do notrequire the addition of serum except for very fas-tidious organisms. Liquid, semisolid and solidbasal media are available commercially and con-tain a suitable indicator, e.g. Andrade (pH 5–8),phenol red (pH 6.8–8.4) or bromothymol blue (pH5.2–6.8).

Some sterile carbohydrate solutions (glucose,maltose, sucrose, lactose, dulcitol, mannitol,salicin) are available commercially. Prepare othersas a 10% solution in water and sterilize by filtra-tion. Add 10 ml to each 100 ml of the reconstitutedsterile basal medium and tube aseptically. Do notheat carbohydrate media. Durham’s (fermentation)tubes need to be added only to the glucose tubes;gas from other substrates is not diagnostically sig-nificant.

These media are not suitable for some organisms;variations are given below.

Ammonium salt ‘sugars’Pseudomonads and spore bearers produce alkalifrom peptone water and the indicator may notchange colour. Use this basal synthetic mediuminstead.

Ammonium dihydrogen phosphate 1.0 g Potassium chloride 0.2 g Magnesium sulphate 0.2 g Agar 10.0 g Water 1000 ml

Dissolve by heating, add 4 ml 0.2% bromothymolblue and a final 1% sterile (filtered) carbohydrate

solution (0.1% aesculin, 0.2% starch – these aresterilized by steaming).

‘Anaerobic sugars’Peptone water or serum peptone water sugar medianeed to have a low oxygen tension for testing thereactions of anaerobes, even under anaerobic con-ditions. Add a clean wire nail to each tube beforesterilization. Do not add indicator until aftergrowth is seen.

Baird-Parker’s carbohydrate mediumFor the sugar reactions of staphylococci and micro-cocci use Baird-Parker (1966) formula:

Yeast extract 1 g Ammonium dihydrogen phosphate 1 g Potassium chloride 0.2 g Magnesium sulphate 0.2 g Agar 12 g Water 1000 ml

Steam to dissolve and adjust to pH 7.0. Add20 ml of 2% bromocresol purple and bottle in95-ml amounts. Sterilize at 115°C for 10 min.For use, melt and add 5 ml 10% sterile carbohy-drate.

Lactobacilli fermentation mediumFor sugar reactions of lactobacilli, make MRS base(see p. 347) without Lab-Lemco and glucose andadjust to pH 6.2–6.5. Bottle in 100-ml amountsand autoclave at 115°C for 15 min. Melt 100 ml,add 10 ml 10% sterile (filtered) carbohydrate solu-tion and 2 ml 0.2% chlorophenol red. Dispenseaseptically.

Robinson’s serum water sugarsSome organisms will not grow in peptone watersugars. Robinson’s serum water medium is supe-rior to that of Hiss:

Peptone 5 g Disodium hydrogen phosphate 1 g Water 1000 ml

Steam for 15 min, adjust to pH 7.4 and add250 ml horse serum. Steam for 20 min, add 10 ml

Laboratory-prepared media 73

Andrade indicator and 1% of appropriate sugar(0.4% starch).

Unheated serum contains diastase and may con-tain a small amount of fermentable carbohydrate,so a buffer is desirable, particularly in starch fer-mentation tests.

Gluconate broth

For differentiation among enterobacteria:

Yeast extract 1.0 g Peptone 1.5 g Dipotassium hydrogen phosphate 1.0 g Potassium gluconate 40.0 g (Or sodium gluconate 37.25 g)Water 1000 ml

Dissolve by heating, adjust to pH 7.0, filter if nec-essary, dispense in 5- to 10-ml amounts and auto-clave at 115°C for 10 min.

Hugh and Leifson’s medium

Peptone 2.0 g Sodium chloride 5.0 g Dipotassium hydrogen phosphate 0.3 g Agar 3.0 g Water 1000 ml

Heat to dissolve, adjust to pH 7.1 and add 15 ml0.2% bromothymol blue and sterile (filtered) glu-cose to give a final 1% concentration. Dispenseaseptically in narrow (< 1 cm) tubes in 8- to 10-mlamounts.

Baird-Parker (1966) modification forstaphylococci and micrococci

Tryptone 10 g Yeast extract 1 g Glucose 10 g Agar 2 g Water 1000 ml

Steam to dissolve, adjust to pH 7.2. Add 20 ml0.2% bromocresol purple, tube in 10-ml amountsin narrow (12 mm) tubes and sterilize at 115°C for10 min.

Donovan’s (1966) medium

Originally devised to differentiate klebsiellas, this isuseful for screening lactose fermenting Gram-nega-tive rods:

Tryptone 10 g Sodium chloride 5 g Triphenyltetrazolium chloride 0.5 g Ferrous ammonium sulphate 0.2 g 1% aqueous bromothymol blue 3 ml Inositol 10 g Water 1000 ml

Dissolve by heating and adjust to pH 7.2.Distribute in 4-ml amounts and autoclave at 115°Cfor 15 min. Allow to set as butts.

Dorset’s egg medium

Wash six fresh eggs in soap and water, and breakinto a sterilized basin. Beat with a sterile fork andstrain through sterile cotton gauze into a sterilemeasuring cylinder. To three parts of egg add onepart nutrient broth. Mix, tube or bottle, and inspis-sate in a sloped position for 45 min at 80–85°C.Glycerol 5% may be added if desired. (Griffith’segg medium uses saline instead of broth and is use-ful for storing stock cultures.)

Egg yolk salt broth and agar

For lecithinase tests:

Nutrient broth or blood agar base 10 ml Egg yolk emulsion 1 ml NaCl (5%) 0.2 ml

74 Culture media

EMJ medium for leptospires

The formula is given by Waitkins (1985) but acommercial preparation is available.

Selective EMJH/5FU medium(Waitkins, 1985)

Dissolve 1 g 5-fluorouracil (5FU) in about 50 mldistilled water containing 1 mol/l NaOH by gentleheat (do not exceed 56°C). Adjust to pH 7.4–7.6with 1 mol/l HCl, make up to 100 ml with distilledand sterilize through a membrane filter (0.45 μm).Dispense in 1-ml lots and store at –4°C.

Thaw 1 ml at 56°C and add it to 100 ml of the EMJMedium (final concentration of 5FU is 100 μg/ml).

Modified alkaline peptone water

This is used for isolating Vibrio cholerae:

Sodium chloride 10.0 gMagnesium chloride hexahydrate 4.0 gPotassium chloride 4.0 gTryptone peptone 10.0 g

Adjust to pH 8.6, distribute and sterilize by auto-claving (Roberts et al., 1995).

N medium (Collins, 1962)

This is used in the differentiation of mycobacteria:

NaCl 1.0 gMgSO4·7H2O 0.2 gKH2HPO4 0.5 gNa2HPO4·12H2O 3.0 g(NH4)2SO4 10.0 gGlucose 10.0 g

Phenylalanine agar

DL-Phenylalanine 2 g Yeast extract 3 g

Na2HPO 1 g NaCl 5 g Agar 20 g Distilled water 1000 ml

Dissolve by heat; sterilize at 115°C for 10 minand tube as slopes.

Phenolphthalein phosphate agar

For the phosphatase test: add 1 ml of a 1% solutionof phenolphthalein phosphate to 100 ml of nutrientagar or commercial blood agar base and pour plates.

Phenolphthalein sulphate agar

For the arylsulphatase test: dissolve 0.64 g potas-sium phenolphthalein sulphate in 100 ml water(0.01 mol/l). Dispense one of the MiddlebrookBroth media in 2.7-ml amounts and add 0.3 ml ofthe phenolphthalein sulphate solution to each tube.

SPS (Sulphite Polymyxin Sulphadiazineagar)

Peptone (from casein) 15.0 gYeast extract 10.0 gFerric citrate 0.5 gSodium sulphite 0.5 gSodium sulphadiazine 0.12 gAgar 13.9 gpH at 25ºC 7.0

From Angelotti et al. (1962).

Tyrosine agar: xanthine agar

To 100 ml melted nutrient agar, add 5 g tyrosineor 4 g xanthine and steam for 30 min. Mix well tosuspend amino acid and pour plates.

Laboratory-prepared media 75

‘Sloppy agar’ (Craigie tube)

Broth containing 0.1–0.2% agar will permit themigration of motile organisms but will not allowconvection currents. This permits motile organismsto be separated from non-motile bacteria in theCraigie tube.

Dispense sloppy agar into 12-ml amounts inscrew-capped bottles. To each bottle add a piece ofglass tubing of dimensions 50 × 5 or 6 mm. Theremust be sufficient clearance between the top of thetube and the surface of the medium so that a menis-cus bridge does not form, and the only connectionbetween the fluid and the inner tube is through thebottom.

Starch agar

Prepare a 10% solution of soluble starch inwater and steam for 1 h. Add 20 ml of this solu-tion to 100 ml melted nutrient agar and pourplates.

Diluents

Saline and Ringer’s solutionsPhysiological saline, which has the same osmoticpressure as micro-organisms, is a 0.85% solutionof sodium chloride in water. Ringer’s solution,which is ionically balanced so that the toxic effectsof the anions neutralize one another, is better thansaline for making bacterial suspensions. It is used atone-quarter the original strength.

Sodium chloride (AnalaR) 2.15 g Potassium chloride (AnalaR) 0.075 g Calcium chloride, anhydrous (AnalaR) 0.12 g Sodium thiosulphate pentahydrate 0.5 g Distilled water 1000 ml

The pH should be 6.6.Both may be purchased as tablets.

Calgon Ringer’sAlginate wool, used in surface swab counts, dis-solves in this solution:

Sodium chloride 2.15 g Potassium chloride 0.075 g Calcium chloride 0.12 g Sodium hydrogen carbonate 0.05 g Sodium hexametaphosphate 10.00 g pH 7.0

It is conveniently purchased as tablets.

Thiosulphate Ringer’sThis may be preferred for dilutions of rinses wherethere may be residual chlorine. It is convenientlymanufactured in tablet form.

Maximum recovery diluent (MRD)The safest and least lethal of diluents is 0.1% pep-tone water.

Buffered peptone waterThis is a useful pre-enrichment medium for theenrichment of salmonellas from foods.

Peptone 10.0 g Sodium chloride 5.0 g Disodium hydrogen phosphate 3.5 g Potassium dihydrogen phosphate 1.5 gDistilled water 1000 ml

The pH should be 7.2.

Phosphate-buffered salineThis is a useful general diluent at pH 7.3:

Sodium chloride 8.0 g Potassium dihydrogen phosphate 0.34 g Dipotassium hydrogen phosphate 1.21 g Water 1000 ml

76 Culture media

Adjustment of pH

The pH of reconstituted and laboratory-preparedmedia should be checked with a pH meter but, forpractical purposes, devices such as the BDHLovibond Comparator are good enough.

Pipette 10 ml of the medium into each of two 152× 16 mm test tubes. To one add 0.5 ml 0.04% phe-nol red solution. Place the tubes in a LovibondComparator with the blank tube (without indica-tor) behind the phenol red colour disc, which mustbe used with the appropriate screen. Rotate the discuntil the colours seen through the apertures match.The pH of the medium can be read in the scaleaperture. Rotate the disc until the required pH fig-ure is seen, add 0.05 mol/l sodium hydroxide solu-tion or hydrochloric acid to the medium plusindicator tube and mix until the required pH isobtained.

From the amount added, calculate the volume of1 or 5 mol/l alkali or acid that must be added to thebulk of the medium. After adding, check the pHagain.

For example, 10 ml of medium at pH 6.4 requires0.6 ml 0.05 mol/l sodium hydroxide solution togive the required pH of 7.2. Then the bulk mediumwill require:

[0.6 × 100]/20 = 3.0 ml 1 mol/l NaOH/litre

Discs of other pH ranges are available.All final readings of pH must be made with the

medium at room temperature because hot mediumwill give a false reaction with some indicators.Note the volume of acid and alkali used and com-pare with records of previous batches. Major dif-ferences in pH or buffering capacity may indicatean error in calculation or changes in the content ofraw materials.

GENERAL PRECAUTIONS

Ensure that all equipment, including autoclaves,preparators and distributors, is regularly servicedand maintained in good condition.

Do not overheat media or quality will beimpaired. Do not autoclave concentrates beforethey are dissolved by prior heating or they will setat the bottom of the vessel. Do not hold partlyprocessed media overnight.

Screw caps, aluminium or propylene closures arebetter than cottonwool plugs.

CONTAMINATION

This is a major cause of media loss and may becaused by inadequate heat processing or failure tosterilize the distributing apparatus. The appearanceof a few colonies on plated media after storage mayindicate environmental fall-out. This may bereduced or even eliminated by using automatedplate pouring machines and/or by pouring media inlaminar-flow clean air cabinets.

ISOLATION AND SELECTIVEMEDIA: EFFICIENCY OF PLATINGTECHNIQUE

New batches of culture media may vary consider-ably and should be tested in the following way.

Prepare serial tenfold dilutions, e.g. 10−2–10−7, ofcultures of various organisms that will grow on orbe inhibited by the medium. For example, whentesting DCA use S. sonnei, S. typhi, S.typhimurium, several other salmonellas and E. coli.Use at least four well-dried plates of the testmedium for each organism and at least two plateseach of a known satisfactory control medium and anon-selective medium, e.g. nutrient agar. Do Milesand Misra drop counts with the serial dilutions ofthe organisms on these plates so that each plate isused for several dilutions (Figure 5.1) (see p. 148).

Count the colonies, tabulate the results and com-pare the performances of the various media. Thesemay suggest that in a new batch of a specialmedium it is necessary to alter the proportion ofcertain ingredients.

Stock cultures will, however, frequently grow on

Isolation and selective media: efficiency of plating technique 77

media that will not support the growth of ‘dam-aged’ organisms from natural materials. It is best,therefore, to use dilutions or suspensions of suchmaterial.

With some culture media, these drop counts donot give satisfactory results because of reduced sur-face tension. An alternative procedure is recom-mended.

Make cardboard masks to fit over the tops ofPetri dishes and cut a square 25 × 25 mm in thecentre of each. Place a mask over a test plate anddrop one drop of the suspension through thesquare on the medium. Spread the drop over thearea limited by the mask. This method has theadditional advantage of making colony counts eas-ier to perform but more plates must, of course, betested.

In addition to these EOP tests, ordinary platingmethods should be used to compare colony sizeand appearance.

IDENTIFICATION MEDIA

Maintain stock cultures (see p. 86) of organismsknown to give positive or negative reactions withthe identification tests in routine use. The sameorganism may often be used for a number of tests.When a new batch of any identification medium isanticipated, subculture the appropriate stock strainso that a young, active culture is available. Test thenew medium with this culture.

Keep stock cultures of the organisms likely to beencountered, preferably from a type culture collec-tion and maintained freeze-dried until required.

Subculture these cultures to test new batches of cul-ture media.Caution: bacteria in Hazard Group 3 should notbe used unless the tests can be conducted in Level3 laboratories.

REFERENCES

Angelotti, R., Hall, H. E., Foter, M. J. et al. (1962)Quantitation of Clostridium perfringens in foods.Applied Microbiology 10: 193–199.

Baird, R. M., Corry, J. E. L. and Curtis, G. D. W. (eds)(1987) Pharmacopoeia of culture media for foodmicrobiology. International Journal of Food

Microbiology 5: 187–229.Baird-Parker, A. C. (1966) An improved diagnostic and

selective medium for isolating coagulase-positivestaphylococci. Journal of Applied Bacteriology 25:12–19.

Barer, M. R. and Harwood, C. R. (1999) Bacterialviability and culturability. Advances in Microbial

Physiology 41: 93–137.Barrow, G. I. and Feltham, R. K. A. (eds) (1993)

Cowan & Steel’s Manual for the Identification of

Medical Bacteria, 3rd edn. Cambridge: CambridgeUniversity Press.

Bartl V. (1985) Choice of test strains. International

Journal of Food Microbiology 2: 99–102.Bloomfield, S. F., Stewart, G. S., Dodd, C. C. et al.

(1998) The viable but non-culturable phenomenonexplained? Microbiology 144: 1–3.

Bridson, E. Y. (1978) Problemen by de standaardisatievan selective stoffen voor isolatie media voorEnterobacteriaceae. In: Van Monster tot Resultaat.

Wageningen: Nederlands Society for Microbiology,pp. 58–67.

Bridson, E. Y. (1990) Media in Microbiology. Review

of Medical Microbiology 1: 1–9.Bridson, E. Y. (1994) The Development, Manufacture

and Control of Microbiological Culture Media.

Basingstoke: Oxoid Ltd. Bridson, E. Y. (1999) Quantal microbiology: the

interactive effect of chaos and complexity onstressed cell recovery. PhD Thesis, University of theWest of England.

78 Culture media

No colonies

19 colonies

Two colonies

Uncountable(more than 200 colonies)

10−5

10−410−3

10−6

Figure 5.1 Miles and Misra count

British Standards Institution (BSI) (1998) BritishStandard 6068-4.12:1998 (ISO 11731:1998) Waterquality – Part 4: Microbiological methods – Section4.12: Detection and enumeration of Legionella.London: BSI.

BSI (1999) British Standard BS EN 12322:1999. Invitro diagnostic medical devices – Culture mediafor microbiology – Performance criteria for culturemedia. London: BSI.

Brock, T. (1988) Robert Koch: A life in medicine and

bacteriology. Madison, WI: Science TechnicalPublications.

Catlin, B. W. (1973) Nutritional profiles of Neisseria

gonorrhoeae, N. meningitidis and N. lactamica inchemically defined media and the use of growthrequirements for gonococcal typing. Journal of

Infectious Disease 128: 178–181.CEN (2001) CEN/TC 275 N489:2001. Microbiology

of food and animal feeding stuffs – Guidelines on

preparation and production of culture media – Part

2: Practical guidelines on performance testing of

culture media. Brussels: EEC.Clarke, A. R. (1996) Molecular chaperones in protein

folding and translocation. Current Opinions in

Structural Biology 6: 43–60.Collins, C. H. (1962) The classification of ‘anonymous’

acid fast bacilli from human source. Tubercle 43:293–298.

Corry, J. E. L. , Curtis, G. W. D. and Baird, R. M.(eds) (1995) Culture Media for FoodMicrobiology. In: Progress in Industrial

Microbiology, Vol. 34. Oxford: Elsevier Science.

Davis, B. D. and Mingioli, E. S. (1950) Mutants ofEscherichia coli requiring methionine or vitaminB12. Journal of Applied Bacteriology 60: 17–21.

DD (2000) DD ENV ISO 11133-1:2000. Microbiology

of food and animal feeding stuffs – Guidelines on

quality assurance and performance testing of culture

media. Part 1: General guidelines. Brussels: EEC.Donovan, T. J. (1966) A Klebsiella screening medium.

Journal of Medical Laboratory Technology 23:11194–11196.

Dukan, S., Levi, Y. and Touati, D. (1997) Recovery ofculturability of an HOCl-stressed population ofEscherichia coli after incubation in phosphate

buffer: resuscitation or regrowth? Applied and

Environmental Microbiology 63: 4204–3209.Ellner, P. D., Stossel, C. I., Drakeford, E. et al. (1966)

A new culture medium for medical microbiology.American Journal of Clinical Pathology 45:502–507.

Gieson, G. L. (1955) The Private Science of Louis

Pasteur. Princeton: Princeton University Press. Hackney, C. R., Ray, B., Speck, M. L. (1979) Repair

detection procedure for enumeration of faecalcoliforms and enterococci from seafoods andmarine environments. Applied and Environmental

Microbiology 37: 947–953.Lacey, B. W. (1954) A new selective medium for

Haemophilus pertussis containing diamidine,sodium fluoride and penicillin. Journal of Hygiene

52: 273–303.MacFaddin, J. F. (1985) Media for the

Isolation–Cultivation–Identification–Maintenance

of Medical Bacteria, Vol. 1. Baltimore, MA:Williams & Williams.

Martin, J. and Hartl, F. U. (1997) Chaperone-assistedprotein folding. Current Opinions in Structural

Biology 7: 41–52. Millar, V. R. and Banwart, G. J. (1965) Effect of

various concentrations of brilliant green and bilesalts on salmonella and other organisms. Applied

Microbiology 13: 77–80.Mossel, D. A. A. and Corry, J. E. L. (1977) Detection

and enumeration of sublethally injured pathogenicand index bacteria in foods and water processedfor safety. Alimenta 16: 19–34.

Mossel, D. A. A. and Ratto, M. A. (1970) Rapiddetection of sublethally impaired cells ofEnterobacteriaceae in dried foods. Applied

Microbiology 20: 273–275.Munro, A. L. S. (1968) Measurement and control of

pH values. In: Norris JR, Ribbons DW. (eds),Methods in Microbiology, Vol. 2. London:Academic Press, pp. 39–89.

National Committee for Clinical Laboratory Standards(NCCLS) (1996) Quality Assurance for

Commercially Prepared Microbiological Culture

Media, 2nd edn. Approved Standard. 1996. CCLS M22-A2, Vol. 16, No. 16. Villanova, PA:NCCLS.

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Reuter, G .(1985) Elective and selective media for lacticacid bacteria. International Journal of Food

Microbiology 2: 55–68.Roberts, D., Hooper, W. and Greenwood, M. (eds)

(1995) Practical Food Microbiology. London:Public Health Laboratory Service, p. 155.

Skirrow, M. B. (1977) Campylobacter enteritis – a‘new’ disease. British Medical Journal ii:9–11.

Stephens, P. J. (1999) Recovery of stressed salmonellas.PhD Thesis, University of Exeter.

Stephens, P. J., Joynson, J. A., Davies, K. W. et al.(1977). The use of an automated growth analyserto measure recovery times of single heat-injuredsalmonella cells. Journal of Applied Microbiology

83: 445–455.Stouthamer, A. H. (1973) The search for a correlation

between theoretical and experimental growth yields.In: Quayle JR (ed.), International Review of

Biochemistry and Microbiological Biochemistry, Vol.21. Baltimore, MA: University Park Press, pp. 1–47.

Thayer, J. D. and Martin, J. E. (1966) Improvedmedium selective for cultivation of Neisseria

gonorrhoea and N. meningitidis. Public Health

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Walker, G. C. (1966) The SOS response of Escherichia

coli. In: Neidhardt FC (ed.), Escherichia coli and

Salmonella, Vol. 1. Washington DC: ASM Press,pp. 1400–1416.

Willis, A. T. and Hobbs, B. (1959) A medium for theidentification of clostridia producing opalescence inegg yolk emulsions. Journal of Pathology and

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reduction of peptone in the preparation of mediafor the pneumococcus and other organisms. Journal

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80 Culture media

GENERAL BACTERIAL FLORA

Specimens and samples for microbiological exami-nation may contain mixtures of many differentmicro-organisms. To observe the general bacterialflora of a sample of any material, use non-selectivemedia. No one medium or any one temperaturewill support the growth of all possible organisms. Itis best to use several different media, each favour-ing a different group of organisms, and to incubateat various temperatures aerobically and anaerobi-cally. The pH of the medium used should approxi-mate that of the material under examination.

For bacteria, examples are nutrient agar, glucosetryptone agar and blood agar and, for moulds andyeasts, malt agar and Sabouraud’s agar.

Methods for estimating bacterial numbers aregiven in Chapter 10, and the dilution methodsemployed for that purpose can be used to makeinocula for the following techniques. Three meth-ods are given here for plating material. Theydepend on separating clumps or aggregates of bac-teria so that each will grow into a separate colony(but see Chapter 10). Colonies resulting from thesemanipulations may then be subcultured for furtherexamination.

Pour plate method

Melt several tubes each containing 15 ml medium.Place in water bath at 45–50°C to cool. Emulsifythe material to be examined in 0.1% peptone waterand prepare 10−1, 10−2 and 10−3 dilutions in the samediluent (see p. 146). Add 1 ml of each dilution to15 ml of the melted agar, mix by rotating the tube

between the palms of the hands, and pour into aPetri dish. Make replicate pour plates of each dilu-tion for incubation at different temperatures.Allow the medium to set, invert the plates and incu-bate. This method gives a better distribution ofcolonies than that used for the plate count (seep. 146), but cannot be used for that purposebecause some of the inoculum, diluted in the agar,remains in the test tube.

Spreader method

Dry plates of a suitable medium. Make dilutions ofthe emulsified material as described above. Placeabout 0.05 ml (1 drop) of dilution in the centre ofa plate and spread it over the medium by pushingthe glass spreader backwards and forwards whilerotating the plate. Replace the Petri dish lid andleave for 1–2 h to dry before inverting and incubat-ing as above. Plates that are wet may yield conflu-ent growth in spots because of growth of cells inthe liquid on the surface.

Looping-out method

Make dilutions of material as described above.Usually the neat emulsion and a 10−1 dilution willsuffice. Place one loopful of material on themedium near the rim of the plate and spread it overthe segment (Figure 6.1a). Flame the loop andspread from area A over area B with parallelstreaks, taking care not to let the streaks overlap.Flame the loop and repeat with area C, and so on.

6

Cultural methods

Each looping out dilutes the inoculum. Invert theplates and incubate.

When looping-out methods are used, two loopsare useful: one is cooling while the other is beingused.

These surface plate methods are said to give betterisolation of anaerobes than pour plates.

Spiral or rotary plating

Spiral platers are used extensively in the food,pharmaceuticals, cosmetics and water industries tocount bacteria. The benefits of spiral plating com-pared with conventional methods include minimalset-up time, the elimination of serial dilutions, upto 69% labour saving, a reduction in consumablecosts and laboratory waste with up to a two-thirdsreduction in incubator space requirements.Incubated plates can then be counted manually orautomatically using image analysis systems withthe requisite software.

Although the early spiral platers were purelymechanical the current generation of instrumentsuse a microprocessor to control all aspects of thedeposition of liquid sample on to the surface of arotating agar plate. The stylus arm dispenses liquidsamples in an Archimedes spiral, either uniformlyacross the plate, or as a continuous decreasing vol-ume producing the equivalent of a thousandfolddilution across a single plate. Pre-programmedoptions allow liquid sample to be dispensed in avariety of ways, using up from 10 to 400 μl of

sample. One impressive feature of the WASP sys-tem (Don Whitley Scientific Ltd, UK) is the abilityto load sample, inoculate a plate and then clean thestylus with a single keystroke. This instrument alsohas an automatic cleaning cycle, which uses sanitiz-ing solution from an integral reservoir and sterilewater contained in autoclavable vessels. To ensurethat less experienced users can operate the instru-ment with the minimum risk of inaccurate results,the user-selectable parameters can be chosen andthen locked. The microprocessor continually mon-itors the travel and relative positions of the syringe,carriage and turntable, and these can be interro-gated by service engineers. The results are thencompared with factory settings, generating valu-able instrument calibration data, essential fortoday’s quality standards.

Hand-operated platers (with electrically driventurntable) are also available. Place an open plate onthe turntable. Touch the rotating plate with thecharged loop at the centre of the medium and drawit slowly towards the circumference. A spiral ofbacterial growth will be obtained after incubationwhich will show discrete colonies (see Figure 6.1b).

Multipoint inoculators

These are convenient tools when many replicatecultures are needed. They are fully or semi-auto-matic and spot inoculate large numbers of Petridish cultures. A number of pins take up the inocu-lum from a single reservoir or different inoculafrom the wells in, for example, a microtitre plateand spot inoculate the medium on one or a succes-sion of Petri dishes. Multipoint inoculators haveheads for up to 36 pins for 90-mm dishes and 96for larger dishes. A variety of these instruments isavailable commercially. They are particularly use-ful for combined antibiotic susceptibility, mini-mum inhibitory concentration tests, the qualitycontrol of culture media, the assessment of bacter-ial loads of large numbers of samples (e.g. urines,foods) and in some identification procedures, e.g.of staphylococci and enterobacteria.

82 Cultural methods

B

C

D

(a) (b)

A B

C

D

E

(a)

Figure 6.1 (a) ‘Spreading’ or ‘looping-out’ on a plate; (b) rotaryplating

The UK Public Health Laboratory Service haspublished an excellent guide to multipoint inocula-tion methods (Faiers et al., 1991).

Shake tube cultures

These are useful for observing colony formation indeep agar cultures, especially of anaerobic ormicroaerophilic organisms.

Dispense media, e.g. glucose agar or thioglycol-late agar, in 15- to 20-ml amounts in bottles ortubes 20–25 mm in diameter. Melt and cool toapproximately 45°C. Add about 0.1 ml inoculumto one tube, mix by rotating between the palms,remove one loopful to inoculate a second tube, andso on. Allow to set and incubate. Submergedcolonies will develop and will be distributed as fol-lows: obligate aerobes grow only at the top of themedium and obligate anaerobes only near the bot-tom; microaerophiles grow near but not at the top;and facultative organisms grow uniformly through-out the medium. Banding of growth in response ofoxygen concentration may be observed by reducingthe agar concentration in the medium to 0.5–1.0%(‘sloppy agar’).

Stab cultures

These can be used to observe motility, gas produc-tion and gelatin liquefaction. Dispense the mediumin 5-ml amounts in tubes or bottles of 12.5 mmdiameter and inoculate by stabbing a loaded wiredown the centre of the agar.

Subcultures

Place the plate culture under a low-power binocu-lar microscope and select the colony to be exam-ined. With a flamed straight wire (not a loop) touchthe colony. There is no need to dig or scrape; toovigorous handling may result in contamination

with adjacent colonies or with those beneath theselected colony.

With the charged wire, touch the medium onanother plate, flame the wire and then use a loop tospread the culture to obtain individual colonies.This should give a pure culture but the proceduremay need repeating.

To inoculate tubes or slopes, follow the same pro-cedure. Hold the charged wire in one hand and thetube in the other in an almost horizontal position.Remove the cap or plug of the tube by grasping itwith the little finger of the hand holding the wire.Pass the mouth of the tube through a Bunsen flameto kill any organism that might fall into the culture,and inoculate the medium by drawing the wirealong the surface of a slope or touching the surfaceof a liquid. Replace the cap or plug and flame thewire. Several tubes may be inoculated in successionwithout recharging the wire.

ANAEROBIC CULTURE

Anaerobic jars

Modern anaerobic jars are made of metal or trans-parent polycarbonate, are vented by Schradervalves and use ‘cold’ catalysts. They are thereforesafer than earlier models; internal temperaturesand pressures are lower. More catalyst is used andthe escape of small particles of catalyst, which canignite hydrogen–air mixtures, is prevented.Nevertheless, the catalyst should be kept dry, driedafter use and replaced frequently.

For ordinary clinical laboratory work the com-mercial sachets are a convenient source of hydro-gen and hydrogen–carbon dioxide mixtures. Arecent development is a sachet that achieves reli-able anaerobic conditions without generatinghydrogen. The final atmosphere contains less than1% oxygen, supplemented with carbon dioxide.

Place the plates ‘upside down’, i.e. with themedium at the bottom of the vessel. If they areincubated the right way up, the medium sometimesfalls into the lid when the pressure is reduced. Dry

Anaerobic culture 83

the catalyst capsule by flaming and replace thelid.

Some workers prefer to use pure hydrogen or amixture of 90% hydrogen and 10% carbon diox-ide. A non-explosive mixture of 10% hydrogen,10% carbon dioxide and 80% nitrogen is safer (seeKennedy, 1988), but may be difficult to obtain insome countries. There is a common misconceptionabout storing these cylinders horizontally: theymay safely be stored vertically.

For the evacuation–replacement jar techniquewith a mixture containing 90% or more hydrogenattach a vacuum pump to the jar across amanometer and remove air to about −300 mmHg.Replace the vacuum with the gas mixture, discon-nect both tubes and allow the secondary vacuumto develop as the catalyst does its work. Leave for10 min and reconnect the manometer to checkthat there is a secondary vacuum of about–100 mmHg. This indicates that the seals of thejar are gas tight and that the catalyst is workingproperly. Replace the partial vacuum with gas toatmospheric pressure.

Note that if the gas mixture contains only 10%hydrogen the jar must be evacuated to at least–610 mmHg so that enough oxygen is removed forthe catalyst and hydrogen to remove the remainder.

Indicators of anaerobiosis

‘Redox’ indicators based on methylene blue andresazurin are available commercially. There is asimple test to demonstrate that anaerobiosis isachieved during incubation: inoculate a bloodagar plate with Clostridium perfringens and placeon it a 5-μg metronidazole disc. This bacterium isnot very exacting in its requirement for anaerobio-sis and will grow in suboptimal conditions. A zoneof inhibition around the growth indicates thatconditions were sufficiently reduced for the drugto work and that anaerobiosis is adequate. Thereis little to commend the use of Pseudomonasaeruginosa as a ‘negative’ control. Even if a ‘posi-tive’ control such as C. tetani fails to grow, it does

not necessarily suggest inadequate anaerobic con-ditions.

Anaerobic cabinets

Although the development of anaerobic jars provedto be a major advance, it was not until the adventof anaerobic workstations that the microbiologistcould easily process, culture and examine sampleswithout exposing them to atmospheric oxygen. It iswell established that anaerobe isolation rates aresignificantly increased through the use of anaerobicworkstations. Wren (1977), working at theLondon Hospital, showed an increase in isolationrate of anaerobes from 9.7% when using anaerobicjars opened at both 24 and 48 hours for examina-tion to 28.1% with jars left for 48 hours beforebeing opened, and to 35.7% when using an anaer-obic workstation. Wren concluded that ‘uninter-rupted anaerobic incubation for 48 h substantiallyincreases the yield of anaerobic isolates’. Ten yearslater a study at Newcastle General Hospital bySissons et al. (1987) showed an isolation rate of43.6% using anaerobic jars, which was increasedto 56.4% with an anaerobic workstation. The prin-cipal reason for these differences is straight-forward: anaerobic workstations provide animmediate and sustained oxygen-free atmospherethroughout the sample manipulation and incuba-tion period. Furthermore, anaerobic jars are fre-quently not anaerobic as a result of either apoisoned catalyst or the poor technique used whensetting up the jar. It must of course be mentionedthat fundamental to good anaerobic microbiologyis the requirement to ensure that samples are notexposed to oxygen after sampling. Oxygen expo-sure will of course reduce culturability of the moststringent of anaerobes in the sample.

Culture under carbon dioxide

Some microaerophiles grow best when the oxygentension is reduced. Capnophiles require carbon

84 Cultural methods

dioxide. For some purposes, candle jars are ade-quate, providing approximately 2.5% CO2. Placethe cultures in a can or jar with a lighted candle ornight-light. Replace the lid. The light will go outwhen most of the oxygen has been removed and bereplaced with CO2.

Alternatively, place 25 ml 0.1 mol/l HC1 in a 100-ml beaker in the container and add a few marblechips before replacing the lid.

Commercial CO2 sachets, hydrogen–CO2 sachetsor CO2 from a cylinder may be used with an anaer-obic jar. Remove air from the jar down to–76 mmHg and replace with CO2 from a footballbladder. This is the best method for capnophiles,which require 10–20% CO2 to initiate growth, but,if extensive work of this nature is carried out, aCO2 incubator is invaluable. A nitrogen–CO2 mix-ture can be used instead of pure CO2.

Many clinical laboratories now purchase ‘CO2

incubators’ that automatically maintain a predeter-mined level of CO2 and humidity (see p. 27).

Incubation of cultures

Incubators are discussed in Chapter 2. In most clin-ical laboratories the usual temperature to be main-tained is 35–37°C. A dish of water should be placedon the floor of an incubator (especially if air is cir-culated to prevent the drying of media in Petridishes). For culturing at 20–22°C incubators fittedwith cooling coils may be necessary. Alternatively,cultures may be left on the bench, but a cupboard isbetter because draughts can be avoided. A maximum

and minimum thermometer is necessary. Ambienttemperatures often fluctuate widely when windowsare open or heating is switched off at night.

Psychrophiles are usually incubated at 4–7°C.Again, incubators need a cooling coil, but a domes-tic refrigerator may be used, provided that it isnot opened too often. Thermophiles require55–60°C.

Incubate Petri dish cultures upside down, i.e.medium uppermost (except in anaerobic vessels),otherwise condensation collects on the surface ofthe medium and prevents the formation of isolatedcolonies.

If it is necessary to incubate Petri dish cultures forseveral days, seal them with adhesive tape or placethem in plastic bags or plastic food containers.

COLONY AND CULTURALAPPEARANCES

Although colony recognition is often now consid-ered to be ‘old-fashioned’ much time and labourmay be saved in the identification of bacteria ifworkers familiarize themselves with colonialappearances on specific media. These appearancesand the terms used to describe them are illustratedin Figure 6.2.

Examine colonies and growths on slopes with ahand lens or, better, plate microscope. Observe pig-ment formation both on top of and under the sur-face of the colony and note whether pigmentdiffuses into the medium. In broth cultures, surfacegrowth may occur in the form of a pellicle.

Colony and cultural appearances 85

Figure 6.2 Description of colonies

Odours

Some cultures develop characteristic odours, butcaution is required before smelling them.Laboratory workers have acquired infections, e.g.with brucellas, as a result of sniffing cultures(Collins and Kennedy, 1999).

Nevertheless, odours may be helpful in noting thepresence of some bacteria. Examples are the ‘grapy’odour of P. aeruginosa and the ‘fruity’ odour ofAlcaligenes faecalis, likened to green apples. Someother organisms with quite distinct odours are:Proteus and Providencia spp. (similar to Proteusspp., but somewhat sweeter), Serratia odorifera(usually described as musty, or compared withmouldy potatoes), Streptococcus milleri (likecaramel), Klebsiella spp. (a fishy, amine odour),Enterobacter spp. (resembling mouldy oranges),Nocardia (earthy) and Citrobacter spp. (faecal andquite unpleasant). Some of these odours aredescribed by Murray et al. (1999).

AUXANOGRAMS

These are used to investigate the nutritionalrequirements of bacteria and fungi, e.g. whichamino acid or vitamin substance is required by‘exacting strains’ or which carbohydrates theorganisms can use as carbon sources.

Make a minimal inoculum suspension of theorganism to be tested in phosphate-buffered saline.Centrifuge and re-suspend in fresh phosphate-buffered saline. Wash again in the same way andadd 1 ml to several 15-ml amounts of mineral saltsmedium (see p. 64) with indicator. Pour into plates.On each plate place a filter-paper disc of 5-mmdiameter and on the filter paper place one loopfulof a 5% solution of the appropriate carbohydrate.Incubate and observe growth.

When testing for amino acid or vitamin require-ments in this way, use the basal medium plus glu-cose. Several discs, each containing a differentamino acid, can be used on a single plate.

Commercially prepared discs and auxanogramkits are available.

STOCK CULTURES

Most laboratories need stock cultures of ‘standardstrains’ of micro-organisms for testing culturemedia and controlling certain tests, and so thatworkers may become familiar with cultural appear-ances and other properties. These must be subcul-tured periodically to keep them alive, with theconsequent risk of contamination; more impor-tantly, in laboratories engaged on assay work, thisis for the involuntary selection of mutants. It isbecause of mutations in bacterial cultures that theso-called ‘standard strains’ or ‘type cultures’ sooften vary in behaviour from one laboratory toanother.

To overcome these difficulties, type culture collec-tions of bacteria, fungi, yeasts and other micro-organisms of medical and industrial importance aremaintained by government and other laboratories.In these establishments, the cultures are preservedin a freeze-dried state, and in the course of mainte-nance of the stocks they are tested to ensure thatthey conform to the official descriptions of thestandard or type strains.

In the UK these collections, under the aegis of theUK National Culture Collection (UKNCC), are dis-tributed among a number of institutions. Theaddresses of the various collections may be foundon the website (www.ukncc.co.uk). In the USA theAmerican Type Culture Collection (ATCC) is atPO Box 1549, Manassas, VA20108, USA.

Strains that are to be used occasionally, or rarely,should be obtained when required and not main-tained in the laboratory. Strains that are in con-stant use may be kept for short periods by serialsubculture, for longer periods by one of the dryingmethods and indefinitely by freeze-drying. Whenstocks of dried or freeze-dried cultures need renew-ing, however, it is advisable to start again with aculture from one of the National Collection ratherthan to perpetuate one’s own stock.

86 Cultural methods

www.ukncc.co.uk

Serial subculture

The principle is to subculture as infrequently aspossible in order to keep the culture alive, and toarrest growth as early as possible in the logarithmicphase so as to avoid the appearance and possibleselection of mutants. Useful media for keepingstock cultures of bacteria are Dorset egg, fastidiousanaerobe medium, Robertson’s cooked meat andlitmus milk. Fungi may be maintained onSabouraud’s medium. These media must be inscrew-capped bottles. It is difficult to keep culturesof some of the very fastidious organisms, e.g. gono-cocci, and it is advisable to store them above liquidnitrogen (see below).

Inoculate two tubes, incubate until growth is justobvious and active, e.g. 12–18 h for enterobacteria,other Gram-negative rods and micrococci, and 2 or3 days for lactobacilli. Cool the cultures and storethem in a refrigerator for 1–2 months.

Keep one of these cultures, A, as the stock, anduse the other, B, for laboratory purposes.

Stock A1 → A2 → A3 → A4

↓ ↓ ↓ ↓Use B B B B

PRESERVATION OF CULTURES

Drying cultures

Two methods are satisfactory. To dry by Stamp’smethod, make a small amount of 10% gelatin innutrient broth and add 0.25% ascorbic acid. Add athick suspension of the organisms before themedium gels (to obtain a final density of about 1010

organisms/ml). Immerse paper sheets in wax, drainand allow to set. Drop the suspension on the waxedsurface. Dry in a vacuum desiccator over phospho-rous pentoxide. Store the discs that form in screw-capped bottles in a refrigerator. Petri dishessmeared with a 20% solution of silicone in lightpetroleum and dried in a hot-air oven can be usedinstead of waxed paper.

Rayson’s method is slightly different. Use 10%serum broth or gelatin broth and drop on discs ofabout 10-mm diameter cut from Cellophane andsterilized in Petri dishes. Dry in a vacuum desicca-tor over phosphorous pentoxide. Store in screw-capped bottles in a refrigerator.

To reconstitute, remove one disc with sterile for-ceps into a tube of broth and incubate.

Freezing

Make thick suspensions of bacteria in 0.5 ml steriletap water or skim milk in screw-capped bottles andplace in a deep freeze at −40°C or, better, −70°C.Many organisms remain viable for long periods inthis way.

Make heavy suspensions in 30% glycerol in 1%peptone water (preserving medium) and distributein small vials. Freeze at −70°C (−20°C may suffice).There are commercial systems (e.g. Prolab, UK) inwhich suspensions are added to vials containingprepared beads. The excess fluid is removed andthe vial frozen. One bead is removed to culturemedium as required.

Suspensions may also be stored in ampoules in thevapour phase above liquid nitrogen in a Dewar ves-sel. Note that this should not be in the liquid nitro-gen itself. If the liquid finds its way into animperfectly sealed ampoule it will expand rapidlyto its gaseous phase when the ampoule is removedfrom the Dewar vessel, causing it to explode anddisperse its contents. Also, if an ampoule breaks theliquid nitrogen can become contaminated with themicro-organisms.

Freeze-drying

Suspensions of bacteria in a mixture of one part30% glucose in nutrient broth and three parts com-mercial horse serum are frozen and then dried byevaporation under vacuum. This preserves the bac-teria indefinitely and is known as lyophilization.Yeasts, cryptococci, nocardias and streptomycetes

Preservation of cultures 87

can be lyophilized by making a thick suspension insterile skimmed milk fortified with 5% sucrose.This simple protective medium can be sterilized byautoclaving in convenient portions, e.g. 3 ml in5-ml screw-capped bottles.

Several machines are available. The simplest con-sists of a metal chamber in which solid carbondioxide and ethanol are mixed. The bacterial sus-pension is placed in 0.2-ml amounts in ampoules orsmall tubes together with a label, and these are heldin the freezing mixture, which is at about −78°C,until they freeze. They are then attached to themanifold of a vacuum pump capable of reducingthe pressure to less than 0.01 mmHg. Between thepump and the manifold, there is a metal container,usually part of the freezing container, in which amixture of solid carbon dioxide and phosphorouspentoxide condenses the moisture withdrawn fromthe tubes. The tubes or ampoules are then sealedwith a small blowpipe.

Simultaneous freezing and drying is carried out incentrifugal freeze-dryers in which bubbling of thesuspension is prevented by centrifugal action.

REFERENCES

Collins, C. H. and Kennedy, D. A. (1999) Laboratory

Acquired Infections, 4th edn. Oxford: Butterworth-Heinemann.

Faiers, M., George, R., Jolly, J. and Wheat, P (1991)Multipoint Methods in the Clinical Laboratory.London: Public Health Laboratory Service.

Kennedy, D. A. (1988) Equipment-related hazards. In:Collins C. H. (ed.), Safety in Clinical and

Biomedical Laboratories. London: Chapman &Hall, pp. 11–46.

Murray, P. R., Baron, J. O., Pfaller, M. A. et al. (1999)Manual of Clinical Microbiology, 7th edn.Washington: ASM Press.

Sisson P. R., Ingham H. R. and Byrne P. O. (1987)Wise anaerobic station: an evaluation. Journal of

Clinical Pathology 40: 286–291.Wren, M. W. D. (1977) The culture of clinical

specimens for anaerobic bacteria. Journal of

Medical Microbiology 10: 195–201.

88 Cultural methods

In general, the identification of micro-organismsrequires both microscopic examination and cul-tural tests. It may also require serological tests.Pure cultures are essential and methods for obtain-ing them are described in Chapter 6. It may be nec-essary, however, to plate out subculturesrepeatedly to ensure that only one kind of organismis present. General-purpose media should be used,provided that they support the growth of theorganism in question. Colonies taken from selec-tive media may contain organisms, the growth ofwhich has been suppressed.

Cultures for identification should first be exam-ined microscopically. Stained films may revealmixed cultures.

MICROSCOPY

Preparation of films for microscopy

It is not easy to observe the morphology of mostbacteria in wet, unstained preparations, even withlight, dark-field or phase contrast microscopy. It isbetter to stain thin films of the organisms, preparedas follows.

Place a very small drop of saline or water in thecentre of a 76 × 25 mm glass microslide. Remove asmall amount of bacterial growth with an inoculat-ing wire or loop, emulsify the organisms in the liq-uid, spreading it to occupy about 1 or 2 cm2. For allstains it is important to be able to examine singlecells, not cells in clumps. Cells in clumps may notdisplay the correct stain reaction. This is why aminimal amount of material is transferred to the

slide surface. Allow to dry in air or by waving highover a Bunsen burner flame, taking care that theslide becomes no warmer than can be borne on theback of the hand. Pass the slide, film side down,once only through the Bunsen flame to ‘fix’ it. Thiscoagulates bacterial protein and makes the film lesslikely to float off during staining. It may not kill allthe organisms, however, and the film should still beregarded as a source of infection. Methods forstaining are given below. After staining, drain, blotwith fresh clean filter paper and dry by gentle heatover a Bunsen flame.

Most laboratories now buy stains in solutionready for use or as concentrated solutions, ratherthan as dry substances. The formulae of some are,however, given below. Make sure that any stainsolution used is free of particulate matter. Filter ifnecessary.

In the USA only stains certified by the BiologicalStains Commission should be used. In the UK thereis no comparable system and stains prepared byone of the specialist organizations should be pur-chased.

A large number of stains, mostly basic anilinedyes, have been used and described. For practicalpurposes, however, one or two simple stains, theGram method and an acid-fast stain are all that arerequired, plus one stain for fungi. A few otherstains are described here, including methods forstaining capsules and spores.

Methylene blue stainStain for 1 min with a 0.5% aqueous solution ofmethylene blue or Loeffler’s methylene blue: mix30 ml saturated aqueous methylene blue solutionwith 100 ml 0.1% potassium hydroxide. Prolonged

7

Identification methods

storage, with occasional shaking, yields ‘poly-chrome’ methylene blue for M’Fadyean’s reactionfor Bacillus anthracis in blood films.

Fuchsin stainStain for 30 s with the following: dissolve 1 g basicfuchsin in 100 ml 95% ethanol. Stand for 24 h.Filter and add 900 ml water. Or use carbol-fuchsin(see ‘Acid-fast stain’) diluted 1 : 10 with phenolsaline.

Gram stainThere are many modifications of this. This isJensen’s version:

1. Dissolve 0.5 g methyl violet in 100 mldistilled water.

2. Dissolve 2 g potassium iodide in 20 mldistilled water. Add 1 g finely ground iodineand stand overnight. Make up to 300 mlwhen dissolved.

3. Dissolve 1 g safranin or 1 g neutral red in100 ml distilled water.

Stain with methyl violet solution for 20 s. Washoff and replace with iodine solution. Leave for 1min. Wash off iodine solution with 95% alcohol oracetone, leaving on for a few seconds only. Washwith water. Counterstain with fuchsin or safraninfor 30 s. Wash and dry.

Some practice is required with this stain toachieve the correct degree of decolourization.Acetone decolourizes much more rapidly thanalcohol.

The Hucker method is commonly used inAmerican laboratories.

1. Dissolve 2 g crystal violet in 20 ml 95%ethanol. Dissolve 0.8 g ammonium oxalate in80 ml distilled water. Mix these twosolutions, stand for 24 h and then filter.

2. Dissolve 2 g potassium iodide and 1 g iodinein 300 ml distilled water using the methoddescribed for Jensen’s Gram stain.

3. Grind 0.25 g safranin in a mortar with 10 ml95% ethanol. Wash into a flask and make upto 100 ml with distilled water.

Stain with the crystal violet solution for 1 min.Wash with tap water. Stain with the iodine solutionfor 1 min. Decolourize with 95% ethanol until nomore stain comes away. Wash with tap water. Stainwith the safranin solution for 2 min. Wash and dry.

Acid-fast stainThe Ziehl–Neelsen method employs carbol fuchsin,acid–alcohol and a blue or green counterstain.Colour-blind workers should use picric acid coun-terstain.

1. Basic fuchsin 5 g Crystalline phenol (caution) 25 g 95% alcohol 50 ml Distilled water 500 ml

Dissolve the fuchsin and phenol in the alcoholover a warm water bath, then add the water. Filterbefore use.

2. 95% ethyl alcohol 970 ml Conc. hydrochloric acid 30 ml

3. 0.5% methylene blue or malachite green, or0.75% picric acid in distilled water.

Pour carbol–fuchsin on the slide and heat care-fully until steam rises. Stain for 3–5 min but do notallow to dry. Wash well with water, decolourizewith acid–alcohol for 10–20 s, changing twice, andcounterstain for a few minutes with methyleneblue, malachite green or picric acid.

Some workers prefer the original decolourizingprocedure with 20% sulphuric acid followed by95% alcohol.

When staining nocardias or leprosy bacilli, use1% sulphuric acid and no alcohol.

Cold staining methods are popular with someworkers. For the Muller–Chermack method add 1drop Tergitol 7 (sodium heptadecyl sulphate) to25 ml carbol–fuchsin and proceed as for theZiehl–Neelsen stain.

For Kinyoun’s method make the carbol–fuchsinas follows:

Basic fuchsin 4 g Melted phenol (caution) 8 g 95% ethanol 20 ml Distilled water 100 ml

90 Identification methods

Dissolve the fuchsin in the alcohol. Shake gentlywhile adding the water. Then add the phenol.Proceed as for Ziehl–Neelsen stain but do not heat.Decolourize with 1% sulphuric acid in water.

Fluorescent stain for acid-fast bacilliThere are several of these. We have found thisauramine–phenol stain adequate:

1. Phenol crystals (caution) 3 g Auramine 0.3 g Distilled water 100 ml

2. Conc. HCl 0.5 ml NaCl 0.5 g Ethanol 75 ml

3. Potassium permanganate 0.1% in distilled water.

Prepare and fix films in the usual way. Stain withauramine–phenol for 4 min. Wash in water.Decolourize with acid–alcohol for 4 min. Washwith potassium permanganate solution.Permanganate bleaches the colour, so be carefulnot to decolourize too much.

Staining corynebacteriaTo show the barred or beaded appearance and themetachromatic granules of these organisms,Laybourn’s modification of Albert’s stain may beused:

1. Dissolve 0.2 g malachite green and 0.15 gtoluidine blue in a mixture of 100 ml water,1 ml glacial acetic acid and 2 ml 95%alcohol.

2. Dissolve 3 g of potassium iodide in50–100 ml distilled water. Add 2 g finelyground iodine and leave overnight. Make upto 300 ml.

Stain with solution (1) for 4 min. Wash, blot anddry, and stain with solution (2) for 1 min. Thegranules stain black and the barred cytoplasm lightand dark green.

Spore stainingMake a thick film, and stain for 3 min with hot car-bol–fuchsin (Ziehl–Neelsen). Wash and flood with30% aqueous ferric chloride for 2 min.

Decolourize with 5% sodium sulphite solution.Wash and counterstain with 1% malachite green.

Flemming’s technique substitutes nigrosin for thecounterstain. Spread nigrosin over the film with theedge of another slide.

Spores are stained red. Cells are green, or withFlemming’s method are transparent on a grey back-ground.

Capsule stainingPlace a small drop of India ink on a slide. Mix intoit a small loopful of bacterial culture or suspension.Place a cover glass over the drop avoiding air bub-bles and press firmly between blotting paper.Examine with a high-power lens. (Dispose of blot-ting paper in disinfectant.)

For dry preparations mix one loopful of India inkwith one loopful of a suspension of organisms in5% glucose solution at one end of a slide. Spreadthe mixture with the end of another slide, allowdrying and pour a few drops of methyl alcohol overthe film to fix. Stain for a few seconds with methylviolet (Jensen’s Gram solution No. 1). The organ-isms appear stained blue with capsules showing ashaloes.

Giemsa stain may be used. Fix the films inabsolute methanol for 3 min. Pour on Giemsa stainand leave until it has almost dried up. Wash rapidlyin water and then in phosphate buffer (0.001 mol/l,pH 7.0). Blot and dry. Capsules are stained pinkand bacterial cells blue.

Flagella stainsThis is a modification of Ryu’s method (Kodaka etal., 1982).

1. Tannic acid powder 10 g Phenol 5% in water (caution) 50 mlAluminium potassium sulphate⋅

12H2O, saturated solution 50 ml 2. Crystal violet 12 g

Ethanol 100 ml

Mix 10 parts of (1) with 1 part of (2) and stand for3 days before use.

Use a young culture and dilute it with water untilit is barely turbid. Place a small drop on a very

Microscopy 91

clean, flamed slide. Allow it to spread and dry inair. Stain for 15 min, wash and dry.

Diene’s stain for mycoplasmas

Methylene blue 2.5 g Azure II 1.25 g Sodium bicarbonate 0.15 g Maltose 10 g Benzoic acid 0.2 g

Dissolve in 100 ml distilled water.

CULTURE PROCEDURES

There are three approaches to the identification ofbacteria: ‘traditional’, ‘kit’ and ‘automated’. Asmany of the traditional methods have now beenreplaced in most clinical laboratories by paperdiscs, strips and batteries of tests in kits, these willbe considered first. Automated methods for identi-fication are described in Chapter 8.

Controls

As indicated in Chapter 2, quality control is impor-tant. Stock cultures of known organisms shouldtherefore be included in test procedures. Suitableorganisms, with their NCTC and ATCC cataloguenumbers, are listed in Table 7.1. They may be storedand resuscitated for use as described on p. 87.

Kits for bacteriology

These methods offer certain advantages, e.g. thesaving of time and labour but, before an arbitrarychoice is made between conventional tests and thenewer methods, and between the several productsavailable, the following factors should be con-sidered.

1. There are no ‘universal kits’. Some organismscannot be identified by these methods.

2. Caution is required in interpretation. A kitresult may suggest unlikely organisms, e.g.Yersinia pestis, Brucella melitensis,


Table 7.1 Bacterial strains for use in control tests inidentification procedures

Strain NCTC ATCC

Acinetobactercalcoaceticus 7 844 15 308lwoffi 5 866 15 309

Aeromonas hydrophila 8 049 7 966Alkaligenes faecalis 415 19 018Bacillus

cereus 10 876 7 464subtilis 6 633 10 400

Clostridiumhistolyticum 19 401 503perfringens 13 124 8 237

Edwardsiella tarda 10 396 19 547Enterobacter

aerogenes 13 048 10 006cloacae 10 005 –

Enterococcus faecalis 29 212 –Escherichia coli 25 922 10 418Mycobacterium

fortuitum 10 349 6 841kansasii 10 268 14 471phlei 8 151 19 249terrae 10 856 15 755

Nocardiabraziliensis 11 274 19 296otitidiscaviarum 1 934 14 629

Proteusmirabilis 10 975 –rettgeri 7 475 –

Pseudomonas aeruginosa 27 853 10 662Serratia marcescens 13 880 10 218Staphylococcus

epidermidis 12 228 –aureus 25 923 6 571

Streptococcusagalactiae 13 813 8 181milleri 10 708 –pneumoniae 6 303 –salivarius 8 618 7 073

Pseudomonas mallei, and ill-consideredreporting of these could cause havoc.

3. Kits may not employ media suitable forgrowth of novel micro-organisms. If onechoses to use different media, be sure that theresults and interpretation of the results areconsistent with another method for measuringthe same characteristic or activity.

4. Conventional, paper strip and disc methodsenable the user to make his or her own choiceof tests. Kit methods do not; they give 10–20test results whether the user needs them or not.

5. Identification of organisms by conventional,paper strip or disc methods usually requiresthe judgement of the user. With kit methods,the results are often interpreted by numericalcharts or computers.

6. If final identification depends on serology, afew screening tests are usually adequate. Ifidentification depends on biochemical tests,the more tests used the more reliable will bethe results. Kits may then be the methods ofchoice, especially with unusual organisms andin epidemiological investigations when the kitmanufacturers’ services are particularly useful.

7. Some techniques may be more hazardous thanothers. Those that involve pipetting increasethe risk of dispersing aerosols and ofenvironmental contamination. Thoseemploying syringe and needle work increasethe risk of self-inoculation.

8. Some methods are much more expensive peridentification than others.

9. Kits devised for identifying bacteria ofmedical importance may not give the correctanswers if they are used to identify organismsof industrial significance

It follows that choices should be made accordingto the nature of the investigations, the professionalknowledge and skill of the workers, and the size ofthe laboratory budget. The relative merits of theindividual products mentioned below are notassessed here but assistance may be obtained fromthe survey of an advisory group (Bennett andJoynson, 1986) in which 10 commercial kits were

tested with over 1000 bacterial strains. Kit manu-facturers will supply other references on request.

CONVENTIONAL TESTS

Only those tests mentioned in this book areincluded here.

Aesculin hydrolysis

Inoculate aesculin medium or Edwards’ mediumand incubate overnight. Organisms that hydrolyseaesculin blacken the medium.

Controls: positive: Serratia marcescens; negative:Edwardsiella tarda.

Ammonia test

Incubate culture in nutrient or peptone broth for 5days. Wet a small piece of filter paper with Nesslerreagent and place it in the upper part of the culturetube. Warm the tube in a water bath at 50–60°C.The filter paper turns brown or black if ammonia ispresent.

Arginine hydrolysis

Incubate the culture in arginine broth for 24–48 hand add a few drops of Nessler reagent. A browncolour indicates hydrolysis. (For lactobacilli, seep. 73.)

Controls: positive: Enterobacter cloacae; negative:Proteus rettgeri.

CAMP test

This test is named after the initials of its originators(Christie, Atkins and Munch-Petersen, 1944).

Conventional tests 93

Inoculate a plate of sheep blood agar (preferablylayered over nutrient agar) with a single streak ofStaphylococcus aureus that produces β-toxin(NCTC 7428) across its diameter. Streak the testorganisms at right angles to this but do not touchthe staphylococcus streak. Incubate aerobicallyovernight at 37°C and examine immediately afterremoval from the incubator. A positive CAMPreaction is shown by a clear zone where the inoculameet.

Carbohydrate fermentation andoxidation

Liquid, semisolid and solid media are described onp. 73. They contain a fermentable carbohydrate,alcohol or glucoside, and an indicator to show theproduction of acid. To demonstrate gas produc-tion, a small inverted tube (Durham’s tube, gastube) is placed in the fluid media. In solid media(stab tube, see p. 83), gas production is obviousfrom the bubbles and disruption of the medium.Normally, gas formation is recorded only in theglucose tube and any that occurs in tubes of othercarbohydrates results from the fermentation ofglucose formed during the first part of thereaction.

• For most bacteria, use peptone or broth-basedmedia.

• For Lactobacillus spp., use MRS base (seep. 347) without glucose or meat extract andadjust to pH 6.2–6.5. Add chlorphenol redindicator.

• For Bacillus spp. and Pseudomonas spp., useammonium salt ‘sugar’ media (see p. 73).These organisms produce ammonia frompeptones and this may mask acid production.

• For Neisseria spp. and other fastidiousorganisms use solid or semisolid media.Neisserias do not like liquid media. Enrichwith Fildes’ extract (5%) or with rabbit serum(10%) Horse serum may give false resultsbecause it contains fermentablecarbohydrates. Robinson’s buffered serum

sugar medium (see p. 73) overcomes thisproblem and is the medium of choice forCorynebacterium spp.

Treat all anaerobes as fastidious organisms. Theindicator may be decolourized during incubation,so add more after incubation. A sterile iron nailadded to liquid media may improve the anaerobicconditions.

Tests may be carried out on solid medium con-taining indicator. Inoculate the medium heavily,spreading it all over the surface and place carbohy-drate discs on the surface. Acid production is indi-cated by a change of colour in the medium aroundthe disc. We do not recommend placing more thanfour discs on one plate.

Casein hydrolysis

Use skimmed milk agar and observe clearingaround colonies of casein-hydrolysing organisms.The milk should be dialysed so that acid produc-tion by lactose fermenters does not interfere.To detect false clearing caused by this, pour a10% solution of mercuric chloride (caution) in20% hydrochloric acid over this medium. Ifthe cleared area disappears, casein was nothydrolysed.

Controls: positive: Neisseria braziliensis; negative:N. otitidiscaviarum.

Catalase test

There are several methods:

1. Emulsify some of the culture in 0.5 ml of a1% solution of Tween 80 in a screw-cappedbottle. Add 0.5 ml 20-vol (6%) hydrogenperoxide (caution) and replace the cap.Effervescence indicates the presence ofcatalase. Do not do this test on an uncoveredslide because the effervescence creates


aerosols. Cultures on low-carbohydratemedium give the most reliable results.

2. Add a mixture of equal volumes 1% Tween80 and 20-vol (6%) hydrogen peroxide to thegrowth on an agar slope. Observeeffervescence after 5 min.

3. Test mycobacteria by Wayne’s method (seep. 389). Both for mycobacteria and for otherbacteria, for bubbles to form the hydrogenperoxide must enter cells because catalase isan intracellular enzyme.

4. Test minute colonies that grow on nutrientagar by the blue slide test. Place one dropof a mixture of equal parts methylene bluestain and 20-vol (6%) hydrogen peroxide ona slide. Place a cover slip over the colonies tobe tested and press down firmly to makean impression smear. Remove the cover slipand place it on the methylene blue–peroxidemixture. Clear bubbles, appearing within30 s, indicate catalase activity.

Controls: positive: Staphylococcus epidermidis;negative: Enterobacter faecalis.

Citrate utilization

Inoculate solid medium (Simmons) or fluid medium(Koser) with a straight wire. Heavy inocula maygive false-positive results. Incubate at 30–35°C andobserve growth.

Controls: positive: P. rettgeri; negative: S.epidermidis.

Coagulase test

Staphylococcus aureus and a few other organismscoagulate plasma (see p. 330).

Controls: positive: S. aureus; negative: S.epidermidis.

Decarboxylase tests

Falkow’s method may be used for most Gram-neg-ative rods but Moeller’s method gives the bestresults with Klebsiella spp. and Enterobacter spp.Commercial Falkow medium allows only lysinedecarboxylase tests, whereas commercial Moellermedia permit lysine, ornithine and arginine tests tobe used.

Inoculate Falkow medium and incubate for 24 hat 37°C. The indicator (bromocresol purple)changes from blue to yellow as a result of fermen-tation of glucose. If it remains yellow, the test is neg-ative; if it then changes to purple, the test is positive.

When using Moeller medium, include a controltube that contains no amino acid. After inocula-tion, seal with liquid paraffin to ensure anaerobicconditions and incubate at 37°C for 3–5 days.There are two indicators: bromothymol blue andcresol red. The colour changes to yellow if glucoseis fermented. Decarboxylation is indicated by apurple colour. The control tube should remainyellow.

Controls. Arginine: positive: E. cloacae; negative: E.aerogenes. Lysine: positive, Serratia marcescens;negative: P. rettgeri. Ornithine: positive: S.marcescens; negative: P. rettgeri.

DNase test

Streak or spot the organisms heavily on DNaseagar. Incubate overnight and flood the plate with1 mol/l hydrochloric acid, which precipitatesunchanged nucleic acid. A clear halo around theinoculum indicates a positive reaction.

Controls: positive: S. marcescens; negative: E.aerogenes.

Gelatin liquefaction

1. Inoculate nutrient gelatin stabs, incubate atroom temperature for 7 days and observe


digestion. For organisms that grow only attemperatures when gelatin is fluid, include anuninoculated control and after incubationplace both tubes in a refrigerator overnight.The control tube should solidify. This methodis not entirely reliable.

2. Inoculate nutrient broth. Place a denaturedgelatin charcoal disc in the medium andincubate. Gelatin liquefaction is indicated bythe release of charcoal granules, which fall tothe bottom of the tube.

3. For a rapid test, use 1 ml 0.01 mol/l calciumchloride in saline. Inoculate heavily (wholegrowth from slope or Petri dish). Add a gelatincharcoal disc and place in a water bath at37°C. Examine at 15-min intervals for 3 h.

4. Inoculate gelatin agar medium. Incubateovernight at 37°C and then flood the platewith saturated ammonium sulphate solution.Haloes appear around colonies of organismsproducing gelatinase.

Controls: positive: Aeromonas hydrophila; negative:E. coli.

Gluconate oxidation

Inoculate gluconate broth (see p. 74) and incubatefor 48 h. Add an equal volume of Benedict’sreagent and place in a boiling water bath for 10min. An orange or brown precipitate indicates glu-conate oxidation.

Controls: positive: E. cloacae; negative: P. rettgeri.

Hippurate hydrolysis

Inoculate hippurate broth, incubate overnight andadd excess 5% ferric chloride. A brown precipitateindicates hydrolysis.

Controls: positive: Streptococcus agalactiae;negative: S. salivarius.

Hugh and Leifson test (oxidationfermentation test)

This is also known as the ‘oxferm’ test. Someorganisms metabolize glucose oxidatively, i.e. oxy-gen is the ultimate hydrogen acceptor and culturemust therefore be aerobic. Others ferment glucose,when the hydrogen acceptor is another substance;this is independent of oxygen and cultures may beaerobic or anaerobic.

Heat two tubes of medium (see p. 74) in boilingwater for 10 min to drive off oxygen, cool andinoculate; incubate one aerobically and the othereither anaerobically or seal the surface of themedium with 2 cm melted Vaseline or agar to giveanaerobic conditions.

• Oxidative metabolism: acid in aerobic tubeonly.

• Fermentative metabolism: acid in both tubes.

For testing staphylococci and micrococci, use theBaird-Parker modification (see p. 73).

Controls: oxidation: Acinetobacter calcoaceticus;fermentation: E. coli; no action: Alkaligenes faecalis.

Hydrogen sulphide production

1. Inoculate a tube of nutrient broth. Place astrip of filter paper impregnated with a leadacetate indicator in the top of the tube,holding it in place with a cottonwool plug.Incubate and examine for blackening of thepaper.

2. Inoculate one of the iron or lead acetatemedia. Incubate and observe blackening.

TSI (Triple Sugar Iron) agar and similar media donot give satisfactory results with sucrose-ferment-ing organisms. The indicator paper (lead acetate) isthe most sensitive method and the ferrous chloridemedia the least sensitive method, but the latter isprobably the method of choice for identifying sal-monellas and for differentiation in other groups


where the amount of hydrogen sulphide producedvaries with the species.

Controls: positive: E. tarda; negative: P. rettgeri.

Indole formation

Grow the organisms for 2–5 days in peptone ortryptone broth.

1. Ehrlich’s methodDissolve 4 g p-dimethylaminobenzaldehyde in amixture of 80 ml concentrated hydrochloric acidand 380 ml ethanol (do not use industrial spirit –this gives a brown instead of a yellow solution). Tothe broth culture add a few drops of xylene andshake gently. Add a few drops of the reagent. Arose-pink colour indicates indole.

2. Kovac’s methodDissolve 5 g p-dimethylaminobenzaldehyde in amixture of 75 ml amyl alcohol and 25 ml concen-trated sulphuric acid. Add a few drops of thisreagent to the broth culture. A rose-pink colourindicates indole.

3. Spot test (Miller and Wright, 1982)Dissolve 1 g p-dimethylaminocinnamaldehyde(DMAC) (caution) in 100 ml 10% hydrochloricacid. Moisten a filter paper with it and smear on acolony from blood or nutrient agar. A blue-greencolour is positive and pink negative. This test maybe applied directly to colonies but is unreliable inthe presence of carbohydrates, e.g. on MacConkeyor CLED media.

Controls: positive: P. rettgeri; negative: S.marcescens.

Lecithinase activity

1. Inoculate egg yolk, salt broth and agar (seep. 74) and incubate for 3 days. Lecithinase-producing colonies are surrounded by zonesof opacity.

2. Inoculate egg yolk salt broth (see p. 74) andincubate for 3 days. Lecithinase producersmake the broth opalescent. Some organisms(e.g. Bacillus cereus) give a thick turbidity in13 h.

Controls: positive: B. cereus; negative: B. subtilis.

Levan production

Inoculate nutrient agar containing 5% sucrose.Levan producers give large mucoid colonies afterincubation for 24–48 h.

Controls: positive: Streptococcus salivarius; negative,S. milleri.

Lipolytic activity

Inoculate tributyrin agar. Incubate for 48 h at25–30°C. A clear zone develops around coloniesof fat-splitting organisms. These media can beused for counting lipolytic bacteria in dairyproducts.

Controls: positive: S. epidermidis; negative: Proteusmirabilis.

Malonate test

Inoculate one of the malonate broth media andincubate overnight. Growth and a deep-blue colourindicate malonate utilization.

Controls: positive: E. cloacae; negative: P. rettgeri.

Motility

Hanging drop methodPlace a very small drop of liquid bacterial culture inthe centre of a 16-mm square No. 1 cover glass,


with the aid of a small (2-mm) inoculating loop.Place a small drop of water at each corner of thecover glass. Invert, over the cover glass, amicroslide with a central depression – a ‘well slide’.The cover glass will adhere to the slide and whenthe slide is inverted the hanging drop is suspendedin the well.

Instead of using ‘well slides’ a ring of Vaselinemay be made on a slide. This is supplied in col-lapsible tubes or squeezed from a hypodermicsyringe.

Bring the edge of the hanging drop, or the air bub-bles in the water seal, into focus with the 16-mmlens before turning to the high-power dry objectiveto observe motility.

Bacterial motility must be distinguished frombrownian movement. There is usually little diffi-culty with actively motile organisms, but feeblymotile bacteria may require prolonged observationof individual cells.

Careful examination of hanging drops mayindicate whether a motile organism has polarflagella – a darting zigzag movement – or per-itrichate flagella – a less vigorous and more vibra-tory movement.

To examine anaerobic organisms for motility growthem in a suitable liquid medium. Touch the culturewith a capillary tube 60- to 70-mm long and boreof about 0.5–1 mm. Some culture will enter the tube.Seal both ends of the tube in the Bunsen flame andmount on Plasticine on the microscope stage.Examine as for hanging drops.

Craigie tube methodCraigie tubes (see p. 76) can be used instead ofhanging drop cultures. Inoculate the medium in theinner tube. Incubate and subculture daily from theouter tube. Only motile organisms can growthrough the sloppy agar.

Nagler test

This tests for lecithinase activity (see p. 97) but theword has come to mean the half antitoxin plate test

for Clostridium perfringens. Egg yolk agar contain-ing Fildes’ enrichment or Willis and Hobbsmedium is satisfactory.

Control: C. perfringens – no activity in presence ofits antitoxin.

Nitrate reductase test

1. Inoculate nitrate broth medium and incubateovernight.

2. Grow the organisms in a suitable broth. Adda few drops of 1% sodium nitrate solutionand incubate for 4 h.

Acidify with a few drops of 1 mol/l hydrochloricacid and add 0.5 ml each of a 0.2% solution of sul-phanilamide and 0.1% N-naphthylethylenedi-amine hydrochloride (caution) (these two reagentsshould be kept in a refrigerator and freshly pre-pared monthly). A pink colour denotes nitratereductase activity. As some organisms furtherreduce nitrite, however, if no colour is producedadd a very small amount of zinc dust. Any nitratepresent will be reduced to nitrite and produce apink colour, i.e. a pink colour in this part of the testindicates no nitrate reductase activity and nocolour indicates that nitrates have been completelyreduced. This latter point is important because theabsence of nitrite does not necessarily mean thatthere is no nitrate reductase. Rather, the absence ofnitrite could be the result of rapid reduction ofnitrite by nitrite reductase. Assimilative nitratereductase may not be produced in the presence ofammonia or amino nitrogen, and dissimilatorynitrate reductase may not be produced under aero-bic conditions.

Controls: positive: S. marcescens; negative:Acinetobacter lwoffii.

ONPG (o-nitrophenylgalactoside) test

Lactose is fermented only when β-galactosidaseand permease are present. Deficiency of the latter


gives late fermentation. True non-lactose fer-menters do not possess β-galactosidase.

Place an ONPG disc in 0.1 ml 0.85% sodiumchloride (physiological saline) in which a colony ofthe test organism has been emulsified. Incubate for6 h. If β-galactosidase is present, a yellow colour isformed as the result of o-nitrophenol liberation. Ifthere is no colour, incubate overnight to detectlactose fermenters.

Controls: positive: S. marcescens; negative: P.rettgeri.

Optochin test

Pneumococci are sensitive but streptococci areresistant to optochin (ethylhydrocupreinehydrochloride).

Streak the organisms on blood agar and place anoptochin disc on the surface. Incubate overnightand examine the zone around the disc.

Controls: positive: S. pneumoniae; negative: S.milleri.

Oxidase test (cytochromeoxidase test)

1. Soak small pieces of filter paper in 1%aqueous tetramethyl-p-phenylenediaminedihydrochloride or oxalate (which keepsbetter). Some filter papers give a blue colourand these must not be used. Dry or use wet.Scrape some of a fresh young culture with aclean platinum wire or a glass rod (dirty orNichrome wire gives false positives) and rubon the filter paper. A blue colour within 10 sis a positive oxidase test. Old cultures areunreliable. Tellurite inhibits oxidase as dofermentable carbohydrates. Organisms thathave produced acid from a carbohydrateshould be subcultured to a sugar-freemedium.

2. Incubate cultures on nutrient agar slopes for24–48 h at the optimum temperature for thestrain concerned. Add a few drops each offreshly prepared 1% aqueous p-amino-dimethylaniline oxalate and 1% α-naphtholin ethanol. Allow the mixture to run over thegrowth. A deep blue colour is a positivereaction.

Controls: positive: Pseudomonas aeruginosa;negative: A. lwoffii.

Oxidative or fermentative metabolismof glucose

See Hugh and Leifson test above.

Phenylalanine test (PPA or PPD test)

Inoculate phenylalanine agar and incubateovernight. Pour a few drops of 10% ferric chlo-ride solution over the growth. A green colour indi-cates deamination of phenylalanine tophenylpyruvic acid. Among the enterobacteriaonly strains of Proteus and Providencia have thisproperty.

Controls: positive: P. rettgeri; negative: E. cloacae.

Phosphatase test

Some bacteria, e.g. S. aureus, can split ester phos-phates. Inoculate a plate of phenolphthalein phos-phate agar (see p. 75) and incubate overnight.Expose the culture to ammonia vapour (caution).Colonies of phosphatase producers turn pink.Phosphate may prevent the synthesis of phos-phatase.

Controls: positive: S. aureus; negative: S.epidermidis.


Proteolysis

Inoculate cooked meat medium and incubate for7–10 days. Proteolysis is indicated by blackening ofthe meat or digestion (volume diminishes).Tyrosine crystals may appear.

Controls: positive: C. histolyticum; negative:Cl. perfringens.

Starch hydrolysis

Inoculate starch agar (see p. 76). Incubate for 3–5days, then flood with dilute iodine solution.Hydrolysis is indicated by clear zones aroundthe growth. Unchanged starch gives a bluecolour.

Controls: positive: B. subtilis; negative: E. coli.

Sulphatase test

Some organisms (e.g. certain species of myco-bacteria) can split ester sulphates. Grow theorganisms in media (Middlebrook 7H9 formycobacteria) containing 0.001 mol/l potassiumphenolphthalein disulphate for 14 days. Add a fewdrops of ammonia solution (caution). A pinkcolour indicates the presence of free phenolph-thalein. Sulphate may prevent the synthesis of sul-phatase.

Controls: positive: Mycobacterium fortuitum;negative: M. phlei (3-day test only).

Tellurite reduction

Some mycobacteria reduce tellurite to telluriummetal (see p. 389).

Controls: positive: M. fortuitum; negative: M. terrae.

Tween hydrolysis

Some mycobacteria can hydrolyse Tween (polysor-bate) 80, releasing fatty acids that change thecolour of an indicator. This test is used mostly withmycobacteria (see p. 389).

Controls: positive: M. kansasii; negative:M. fortuitum.

Tyrosine decomposition

Inoculate parallel streaks on a plate of tyrosineagar (see p. 75) and incubate for 3–4 weeks.Examine periodically under a low-power micro-scope for the disappearance of crystals around thebacterial growth.

Controls: positive: N. braziliensis; negative:N. otitidiscaviarum.

Urease test

Inoculate one of the urea media heavily and incu-bate for 3–12 h. The fluid media give more rapidresults if incubated in a water bath. If urease ispresent, the urea is split to form ammonia, whichchanges the colour of the indicator from yellow topink.

Controls: positive: P. rettgeri; negative: S.marcescens.

Voges Proskauer reaction (VP)

This tests for the formation of acetyl methylcarbinol (acetoin) from glucose. This is oxidized bythe reagent to diacetyl, which produces a redcolour with guanidine residues in the media.

Inoculate glucose phosphate broth and incubatefor 5 d at 30°C. A very heavy inoculum and 6 hincubation may suffice. Test by one of the follow-ing methods.


1. Add 3 ml 5% alcoholic α-naphthol solutionand 3 ml 40% potassium hydroxide solution(Barritt’s method).

2. Add a ‘knife point’ of creatinine and 5 ml40% potassium hydroxide solution(O’Meara’s method).

3. Add 5 ml of a mixture of 1 g copper sulphate(blue) dissolved in 40 ml saturated sodiumhydroxide solution plus 960 ml 10%potassium hydroxide solution (APHAmethod).

A bright pink or eosin red colour appearing in5 min is a positive reaction. For testing Bacillusspp., add 1% NaCl to the medium.

Controls: positive: S. marcescens; negative:P. rettgeri.

Xanthine decomposition

Inoculate parallel streaks on a plate of xanthineagar (see p. 75) and incubate for 3–4 weeks.Examine periodically under a low-power micro-scope for the disappearance of xanthine crystalsaround the bacterial growth.

Controls: positive: N. otitidiscaviarum; negative:N. braziliensis.

AGGLUTINATION TESTS

These tests are performed in small test tubes (75 ×9 mm). Dilutions are made in physiological salinewith graduated pipettes controlled by a rubber teator (for a single-row test), with Pasteur pipettesmarked with a grease pencil at approximately0.5 ml. Automatic pipettes or pipettors are usefulfor doing large numbers of tests.

Standard antigen suspensions and agglutinatingsera can be obtained commercially. Antigen sus-pensions are used mainly in the serological diagno-sis of enteric fever, which may be caused by severalrelated organisms, and of brucellosis.

Standard agglutinating sera are used to identifyunknown organisms.

Testing unknown sera against standardH and O antigen suspensions

To test a single suspensionPrepare a 10−1 dilution of serum by adding 0.2 mlto 1.8 ml saline. Set up a row of seven small tubes.Add 0.5 ml saline to tubes 2–7 and 0.5 ml of the10−1 serum to tubes 1 and 2. Rinse the pipette bysucking in and blowing out saline several times.Mix the contents of tube 2 and transfer 0.5 ml totube 3. Rinse the pipette. Continue with doublingdilutions but discard 0.5 ml from tube 6 instead ofadding it to tube 7. Rinse the pipette between eachdilution. The dilutions are now 1 : 10 to 1 : 320.

Add 0.5 ml of standard suspension to each tube.The last tube, containing no serum, tests the stabil-ity of the suspension. The dilutions are now 1 : 20to 1 : 640.

To test with several suspensionsIn some investigations, e.g. in enteric fevers, a num-ber of suspensions will be used. Set up six largetubes (152 × 16 mm). To tube 1 add 9 ml saline, totubes 2–6 add 5 ml saline and to tube 1 add 1 mlserum. Mix and transfer 5 ml from tube 1 to tube2, and continue in this way, rinsing the pipettebetween each dilution.

Set up a row of seven small tubes (75 × 9 mm) foreach suspension to be tested. Transfer 0.5 ml fromeach large tube to the corresponding small tube.Work from right to left, i.e. weakest to strongestdilution to avoid unnecessary rinsing of the pipette.The final dilutions are now 1 : 20 to 1 : 640.

Incubation temperature and timesIncubate tests with O suspensions in a water bathat 37°C for 4 h, then allow to stand overnight inrefrigerator.

Incubate H tests for 2 h in a water bath at50–52°C (not 55–56°C, as the antibody may bepartly destroyed). The level of water in the bath

Agglutination tests 101

should be adjusted so that only about half of theliquid in the tubes is below the surface of the water.This encourages convection in the tubes, mixingthe contents.

Brucella agglutinationsTo avoid false-negative results resulting from theprozone phenomenon, double the dilutions for atleast three more tubes, i.e. use 1 : 20 to 1 : 5120.

Reading agglutinationsExamine each tube separately from right to left, i.e.beginning with the negative control. Wipe dry anduse a hand lens. If the tubes are scratched, dip themin xylene. The titre of the serum is that dilution inwhich agglutination is easily visible with a low-power magnifier.

Testing unknown organisms againstknown sera

Preparation of O suspensionsThe organism must be in the smooth phase. Growon agar slopes for 24 h. Wash off in phenol–salineand allow lumps to settle. Remove the suspensionand dilute it so that there are approximately 1 ×109 bacteria/ml by the opacity tube method (seep. 145). Heat at 60°C for 1 h. If this antigen is to bestored, add 0.25% chloroform.

If a K antigen is suspected, heat the suspension at100°C in a water bath for 1 h (although the B anti-gen is thermostable).

Preparation of H suspensionsCheck that the organism is motile and grow it innutrient broth for 18 h, or in glucose broth for4–6 h. Do not use glucose broth for the overnightculture because the bacteria grow too rapidly. Addformalin to give a final concentration of 1.0% andleave for 30 min to kill the organisms. Heat at50–55°C for 30 min (this step may be omitted if thesuspension is to be used at once). Dilute to approx-imately 1 × 109 organisms/ml by the opacity tubemethod.

Agglutination testsUse the same technique as that described under‘Testing unknown sera . . .’ but as most sera areissued with a titre of at least 1 : 250 (and labelledaccordingly) it is not necessary to dilute beyond1 : 640. In practice, as standard sera are highly spe-cific and an organism must be tested against severalsera, it is usually convenient to screen by addingone drop of serum to about 1 ml suspension in a75 × 9 mm tube. Only those sera that give aggluti-nation are then taken to titre.

Slide agglutinationThis is the normal procedure for screening with Osera.

Place a loopful of saline on a slide and, next to it,a loopful of serum. With a straight wire, pick acolony and emulsify in the saline. If it is sticky orgranular, or autoagglutinates, the test cannot bedone. If the suspension is smooth, mix the serum inwith the wire. If agglutination occurs it will berapid and obvious. Dubious slide agglutinationshould be discounted.

Only O sera should be used. Growth on solidmedium is used for slide agglutination and this isnot optimum for the formation of flagella. False-negative results may be obtained with H sera unlessthere is fluid on the slope.

Suspensions of organisms in the R phase areagglutinated by 1 : 500 aqueous acriflavine solu-tion.

FLUORESCENT ANTIBODYTECHNIQUES

The principle of fluorescent antibody techniques isthat proteins, including serum antibodies, may belabelled with fluorescent dyes by chemical combi-nation without alteration or interference with thebiological or immunological properties of the pro-teins. These proteins may then be seen in micro-scopic preparations by fluorescence microscopy.

The preparation is illuminated by ultraviolet orultraviolet blue light. Any fluorescence emitted by


the specimen passes through a barrier filter abovethe object. This filter transmits only the visible flu-orescence emission. Microscopes suitable for thispurpose are now readily available, and it is a sim-ple matter to convert standard microscopes for flu-orescence work. Fluorescent dyes are used insteadof ordinary dyes because they are detectable inmuch smaller concentrations. They are available ina form that simplifies the conjugation procedureconsiderably. The fluorochromes in frequent cur-rent use are fluorescein isothiocyanate (FITC) andLissamine rhodamine B (RB200). Of these, FITC isthe most commonly used. It gives an apple-greenfluorescence.

Fluorescence labelling is often used in microbiol-ogy and immunology along with or instead of tra-ditional serological tests. Immune serum globulinconjugated with fluorochrome is usually employedto locate the corresponding antigen in microbiolog-ical investigations.

Reagents, accompanied by technical instructions,are available commercially.

TYPING MICRO-ORGANISMS

Reasons for typing micro-organisms

The principal reason for typing or fingerprintingmicro-organisms is to provide information to per-mit the tracing of epidemics of infection or toidentify the source of an outbreak (Maslow et al.,1993), e.g. fingerprints of isolates from a clusterof patients can be used to determine whether theyshare the same or different clones of a particularspecies. If isolates from different individuals sharethe same fingerprint or type, it suggests that theywere infected from a single source. In outbreaksof disease that are thought to be food related,typing can be used to identify the source of theinfectious micro-organism. Also, fingerprintingmethods (e.g. multilocus enzyme electrophoresisor MLEE) can be used to describe the populationgenetics of micro-organisms (Selander et al.,1986, 1987).

Typing targets

Typing or fingerprinting methods fall into two cat-egories based on the target molecule. One categoryuses phenotypic features of the micro-organismsuch as cell surface antigens, e.g. proteins or poly-saccharides (i.e. serotyping), profiles of enzymaticactivities (biotyping), profiles of enzyme polymor-phisms (MLEE), or patterns of susceptibility tobacteriophage (i.e. phage typing) or antibiotics (i.e.antibiograms). Unfortunately, expression of thesecharacteristics can be influenced by growth condi-tions and they are thus subject to variation. It isalso common that some strains are untypable forreasons other than the absence of the particulartyping target, e.g. if a typing method employsagglutination with antisera, cells that sponta-neously clump cannot be typed.

In the second category of typing methods DNA isthe target. Repeated sequences, e.g. a transposablegenetic element such as an insertion sequence (IS)or a reiterated sequence, are commonly employedas targets. DNA-based fingerprinting can also beperformed by comparison of large genomic frag-ments generated by cleavage with restrictionendonucleases and separated by pulsed-field gelelectrophoresis (PFGE). As all cells contain DNA,growth conditions do not necessarily influence theresults, unless they result in cells resistant to lysisand release of DNA. It is also important to remem-ber that DNA-fingerprinting methods involving theuse of restriction endonucleases (i.e. restrictionfragment length polymorphism or RFLP) can beinfluenced by the presence of DNA-modifyingenzymes which can prevent cleavage of specificDNA sequences.

Requirements of targets for typing orfingerprinting

Excellent descriptions of the principles of typing orfingerprinting (Maslow et al., 1993) and interpret-ing typing data (Tenover et al., 1995) have beenpublished. First, it should be pointed out that

Typing micro-organisms 103

typing or fingerprinting should always be validatedwith a large number of isolates. Further, in cases ofoutbreak investigations, a large number of isolatesshould be tested. The results from small samplescan be influenced by chance and thus may notreflect the entire population. If the objective of thetyping is to identify clusters (implying a commonsource of infection), small samples may omit mem-bers of clusters and the extent of clustering wouldbe underestimated (Murray, 2002).

A primary requirement of a target is that all iso-lates must express or have that target. There cannotbe isolates that lack the particular target (i.e. unty-pable). There must be a diversity of patterns or pro-files exhibited by the population. However, oneshould be careful to choose a target with sufficientdiversity to provide meaningful results, e.g. if therewere little variation in the target among isolates,then all isolates would be thought to belong to alimited number of different types. Use of such a tar-get might lead to a false clustering of isolates orlinkage to a possible source. Such targets might beuseful as species markers rather than as typing tar-gets. At the other end of the spectrum, it is possibleto use a target that displays too much variationwithin the studied population. Typing or finger-printing with such a target would result in a falseabsence of clustering or linkage to a possiblesource. This would be the case if a transposablegenetic element were subject to a very high rate oftransposition. In this case, almost all isolates willbe thought to belong to individual groups, to theextreme of one isolate per group.

Another way of stating the requirement that therebe a diversity of typing or fingerprinting patterns isthat a useful target provides a high level of discrim-ination. A discrimination index can be calculatedusing Simpson’s index of diversity, originally devel-oped for the description of species diversity withinthe habitat or an organism (Simpson, 1949). Usingthat index, it was possible to compare the discrim-inatory power of different methods used for typingisolates of Candida albicans (Hunter and Gaston,1988). Clearly, the number of types directly influ-ences the discrimination index. A greater numberof different types are associated with a higher dis-

criminatory index. Fortunately, this method takesinto account the reduction in discriminatory indexwhen many isolates belong to a single group, e.g.there is a non-random distribution of sites of inser-tion of IS6110 in Mycobacterium tuberculosiswhich is the result of the existence of sites(‘hotspots’) favoured for integration (McHugh andGillespie, 1998).

Typing methods should be simple, rapid, inexpen-sive and within the technical skill of personnel.Typing methods, as with other methods employedin the laboratory, should include quality controland quality assessment using reference strains. Formethods involving electrophoretic separation ofeither proteins (e.g. MLEE) or DNA fragments (e.g.RFLP, PFGE or arbitrary primed polymerase chainreaction), the criteria for determining whether anindividual band’s mobility is the same or differentin two isolates should be established by repeatedmeasurement, e.g. it is not uncommon to find thatmobility of identical bands in different gels variesby about ± 5%. Thus, if the mobilities of bands intwo different isolates are within 5%, they will becounted as identical in the analysis. Finally, thereshould be a straightforward method of analysisthat provides meaningful information to users (e.g.clinicians or public health personnel). Basically, theobject is to determine whether isolates belong to asingle clone. If not members of the same clone, therelatedness of clones must be determined. Onemethod of estimating the relatedness between iso-lates is to calculate the Dice (1945) coefficient ofsimilarity. That value equals twice the number ofshared bands divided by the total number of bandsin both isolates (Dice, 1945).

Serotyping

Serotyping is one of the oldest methods for typingmicro-organisms and has had wide use in tracingthe sources of food-associated infections. Antiseraare raised against different isolates of a particularspecies and used to agglutinate cells. Targets forserotyping can be cell surface polysaccharides or


proteins. Provided that there is sufficient variationin the targets and a large number of isolates areemployed, a panel of antisera can be identified thatcan distinguish between categories (i.e. serotypes)of isolates. To prevent cross-reactions, sera can bemixed with isolates of different types to leave onlyantibodies reactive against targets that are uniqueto the type used to generate the antiserum(adsorbed sera). The widespread applicability ofserotyping is limited by the need to produce andstore antisera. Growth conditions can lead to anabsence or modification of the targets of the antis-era (false type). If evidence of the presence of a par-ticular target is based on agglutination, cells ofisolates that can spontaneously agglutinate cannotbe typed.

Phage typing

Phage typing followed the discovery that bacteriawere susceptible to infection by a variety of differ-ent viruses (e.g. bacteriophage) and those virusesrecognized different cell surface molecules (i.e.receptors) for attachment. For phage typing, lawnsof individual isolates are spotted with virus-con-taining lysates and susceptibility (i.e. lysis of thelawn) scored for each virus. As is the case for alltyping methods, there must be a large number ofdifferent viruses recognizing different receptors andsufficient variation in the cell surface receptors(present or absent) to allow sufficient discrimina-tion between isolates. As is the case for serotyping,growth conditions can lead to an absence of a par-ticular receptor and assignment of an isolate to afalse type.

Multilocus enzyme electrophoresis

Multilocus enzyme electrophoresis relies on thevariation in amino acid composition of cellularproteins (Selander et al., 1986). It has been used tocharacterize the populations of E. coli (Whittam etal., 1983), Legionella pneumophila (Selander et al.,

1985), and a variety of other bacteria (Selander etal., 1987). Populations of micro-organisms containmutant individuals expressing an active enzyme theamino acid composition of which is different. Insome instances the difference in amino acid resultsin a change in electrophoretic mobility. The mobil-ity of enzymes is measured by electrophoretic sepa-ration and detection of in situ enzyme activity (e.g.production of an insoluble coloured product). Thediscriminatory power of MLEE is dependent on theexistence of mutant variants with different mobili-ties in a population. Further, measuring the mobil-ity of a large number of different enzymes increasesthe discrimination index. MLEE requires thatgrowth conditions be chosen to ensure expressionof a wide variety of different enzymes, the activitiesof which can be detected in gels (Selander et al.,1986). Although time-consuming, labour intensiveand requiring sophisticated equipment and techni-cal skill, MLEE is a powerful method for typingisolates.

Pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis enables the separa-tion of large fragments of DNA by gel elec-trophoresis (Schwartz et al., 1983; Carle et al.,1986). The fragments are generated by cleavage ofgenomic DNA with restriction endonucleases thatrecognize few sequences (e.g. 8- to 12-base recogni-tion sequences) and generate fragments of typicallybetween 100 and 1000 kb (Allardet-Servent et al.,1989). Methods for isolation and purification oflarge DNA-restriction fragments are presented inSelden and Chory (1987). Variations in size and,hence, mobility of restriction fragments are basedon mutations leading to acquisition or loss of arecognition site for the enzyme. As such large-sizedfragments of DNA would be subject to fragmenta-tion by shearing in solution, cells are immobilizedin agarose blocks and followed sequentially by celllysis and restriction endonuclease digestion ofreleased DNA to reduce shear forces. The agaroseblocks are inserted into wells of agarose and PFGE

Typing micro-organisms 105

performed. PFGE can be used for typing virtuallyevery microbial species. Unfortunately, in spite ofits wide applicability, the number of required num-ber of steps makes PFGE time-consuming. FurtherPFGE requires expensive equipment and sophisti-cated technical assistance.

It is possible to calculate the expected average sizeand number of fragments generated by a specificrestriction endonuclease and the guanine + cytosinepercentage (%G + C) of the micro-organism.Choosing restriction enzymes that produce a largenumber of fragments results in greater discrimina-tion. Care should be taken to ensure that differ-ences in patterns are not the result of the presenceof a modifying methylase, which prevents cleavageof a recognition site. A guide for interpretation ofPFGE patterns has been published (Tenover et al.,1995). Criteria used to determine whether bandsare identical or different must be established byrepeated measurements of digests of a limited num-ber of strains. In addition to simple visual inspec-tion of the fragment patterns, Dice coefficients ofsimilarity can be calculated and phylogenetic treesconstructed (Fitch and Margoliash, 1967;Felsenstein, 1985). There are also a number ofcommercial software programs that can scan gelsand calculate relatedness between isolates.

Restriction fragment lengthpolymorphism

A variety of targets has been chosen for RFLPanalysis including insertion sequences and repeatedsequences. For RFLP analysis, DNA is recoveredfrom a variety of isolates and subjected to restric-tion endonuclease digestion. The restriction frag-ments are separated by gel electrophoresis and alabelled nucleic acid probe is used to detect bandscontaining the target sequence by hybridization.Methods for DNA isolation – restriction endonu-clease digestion, gel electrophoresis, blotting (i.e.southern transfer) and hybridization – are alldescribed in detail in Sambrook et al. (1989). Incases where fingerprinting is widely used, e.g. in

tuberculosis epidemiology, a standard methodol-ogy has been published (Van Embden et al., 1993).

The common feature of useful targets is that theyare present in multiple copies in the genome (e.g.> 10). One way of detecting a genetic element ofhigh copy number is to subject genomic DNA tocleavage with a variety of restriction endonucleasesand separate the fragments by gel electrophoresis.Often, repeated sequences appear as more intensebands when stained with ethidium bromide andthey can be cloned for sequencing. As is the case forPFGE, the variation in bands is caused by muta-tions in restriction endonuclease recognitionsequences flanking or within a repeated genetic ele-ment. Variation is also a consequence of the trans-position of an insertion sequence.

As with PFGE, the target for RFLP analysis isDNA. Few isolates are found to be untypable andthis is because of the inability to recover sufficientamounts of DNA. RFLP and PFGE are bothamenable to quality control and quality assuranceprogrammes. The criteria for interpretation ofRFLP patterns is no different from those used forPFGE, as is the method of analysis and presenta-tion of data. As a result of the need for separatesteps of electrophoresis, blotting and hybridization,RFLP analysis requires time, is rather expensiveand demands technical sophistication.

Polymerase chain reactionfingerprinting methods

PCR amplification of sequences betweeninsertion sequencesIt is possible to amplify DNA between insertionsequences or repetitive genetic elements using thepolymerase chain reaction (PCR). To be successful,there must be a high number of target sequences inthe genome (i.e. > 10 and preferably 20). Oneexample of a repeated sequence that is highly con-served among many different bacteria is the repeti-tive extragenic palindromic (REP) sequence(Versalovic et al., 1991). Primers are chosen toamplify the target sequence (or related target


sequences), and amplification of the sequencebetween targets occurs by a primer binding to oneelement and the second primer binding to a secondelement. If the two targets are not too far apart toprevent successful PCR amplification (e.g. < 5 kb),amplified products will be produced. One advan-tage of this approach is that the number of stepsnecessary for fingerprinting is reduced and therequired level of DNA purity is reduced. One dis-advantage of this approach is that it is less discrim-inatory than RFLP analysis. In a comparison ofIS1245 fingerprinting in Mycobacterium avium,fewer amplified fragments were produced com-pared with the number of bands seen in RFLP(Picardeau and Vincent, 1996). PCR-based finger-printing based on consensus primers for the REPsequence has been used for epidemiological studiesof a wide range of bacteria, including Citrobacterdiversus (Woods et al., 1992), Neisseria gonor-rhoeae (Poh et al., 1996) and Acinetobacter spp.(Snelling et al., 1996). A commercial kit for per-forming REP-PCR is available (Bacterial BarCodes,Inc., Houston, TX).

Arbitrary primed PCR and random amplifiedpolymorphic DNA Micro-organisms can be typed using the PCR-based technique that employs single primers toamplify sequences in DNA (Welsh andMcClelland, 1990; Williams et al., 1990). Themethod is called either arbitrary primed PCR (AP-PCR) or random amplified polymorphic DNA(RAPD). The advantage of this approach is thatprimers can be screened without knowledge of thegenome’s characteristics and thus the technique canbe employed with a wide variety of micro-organ-isms. Screening chooses single primers that repro-ducibly yield multiple amplified products. Detailedmethods that also provide examples of the applica-tion of AP-PCR to fingerprinting of a variety ofmicro-organisms have been published (Williams etal., 1993). Using isolates of E. coli, it was shownthat AP-PCR was more sensitive than MLEE in dis-tinguishing isolates (Wang et al., 1993). Data fromAP-PCR (i.e. band patterns) can be treated asbands from PFGE or RFLP analysis and the data

can be used to estimate nucleotide sequence diver-gence (Clark and Lanigan, 1993). As is characteris-tic of all PCR-based techniques, extreme care mustbe taken to avoid amplification of contaminatingDNA and amplification of non-target, but relatedsequences.

Conclusion

One of the distinct advantages of PFGE and RFLPanalysis is that gels can be scanned, phylogenetictrees constructed and coefficients of relatedness cal-culated by computer. Such data can be available toclinicians in a hospital or other investigators at dis-tant locations.

It is possible to select the level of discriminationdesired in any epidemiological study. Care must betaken to avoid choosing a method that provides toolittle discrimination leading to false grouping ofisolates. In contrast, a fingerprinting method with ahigh discrimination index may lead to separationof isolates that truly belong to the same group.Using MLEE data, it is assumed that isolates withthe same mobility for a particular enzyme share thesame allele (i.e. DNA sequence). However, this isnot necessarily the case; the isolates may have dif-ferent alleles. Therefore, it is not surprising thatAP-PCR can be more discriminatory than MLEEfor isolates of E. coli (Wang et al., 1993). Oneshould not conclude that the most discriminatorymethod is the ‘best’ for typing. One method, e.g.MLEE, may provide evidence of large relatedgroups that can be found world wide (Whittam etal., 1983). Such patterns would be missed by amore discriminatory method.

REFERENCES

Allardet-Servent, A. N., Bouziges, M. J., Carles-Nurit,G. et al. (1989) Use of low-frequency-cleavagerestriction endonucleases for DNA analysis inepidemiological investigations of nosocomialbacterial infections. Journal of Clinical

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Clark, A. G. and Lanigan, C. M. S. (1993) Prospectsfor estimating nucleotide divergence with RAPDs.Molecular and Biological Evolution 10:1096–1111.

Dice, L. R. (1945) Measures of the amount ofecological association between species. Ecology 26:297–302.

Felsenstein, J. (1985) Confidence limits on phylogenies:an approach using the bootstrap. Evolution 39:783–791.

Fitch, W. M. and Margoliash, E. (1967) Constructionof phylogenetic trees. Science 155: 279–284.

Hunter, P. R. and Gaston, M. A. (1988) Numericalindex of the discriminatory ability of typingsystems: an application of Simpson’s index ofdiversity. Journal of Clinical Microbiology 26:2465-2466.

Kodaka, H., Armfield, A. Y., Lombard, G. and Dowell,V. R. (1982) Practical procedure for demonstratingbacterial flagella. Journal of Clinical Microbiology

16: 948.McHugh, T. D. and Gillespie, S. H. (1998) Non-

random association of IS6110 and Mycobacterium

tuberculosis: implications for molecularepidemiological studies. Journal of Clinical

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(1993) Molecular epidemiology: application ofcontemporary techniques to the typing ofmicroorganisms. Clinical Infectious Disease 17:153–164.

Miller, J. M. and Wright, J. W. (1982) Spot indole test:

evaluation of four methods. Journal of Clinical

Pathology 15: 589–592.Murray, M. (2002) Sampling bias in the molecular

epidemiology of tuberculosis. Emerging Infectious

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Mycobacterium avium isolates by PCR. Journal of

Clinical Microbiology 34: 389–392.Poh C. L., Ramachandran, V. and Tapsall, J. W.

(1996) Genetic diversity of Neisseria gonorrhoeae

IB-2 and IB-6 isolates revealed by whole-cellrepetitive element sequence-based PCR. Journal of

Clinical Microbiology 34: 292–295.Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989)

Molecular Cloning: A laboratory manual, 2nd edn.New York: Cold Spring Harbor Laboratory Press.

Schwartz, D. C., Saffran, W. J. Welsh, W. J. et al.(1983) New techniques for purifying large DNAsand studying their properties and packaging. Cold

Spring Harbor Symposium on Quantitative

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(1985) Genetic structure of populations ofLegionella pneumophila. Journal of Bacteriology

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(1986) Methods of multilocus enzymeelectrophoresis for bacterial population geneticsand systematics. Applied and Environmental

Microbiology 51: 873–884.Selander, R. K., Musser, J. M., Caugant, D. A. et al.

(1987) Population genetics of pathogenic bacteria.Microbiology and Pathology 3: 1–7.

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Simpson, E. H. (1949). Measurement of diversity.Nature 163: 688.

Snelling, A. M., Gerner-Smidt, P., Hawkey, P. M. et al.

(1996) Validation of use of whole-cell repetitiveextragenic palindromic sequence-based PCR (REP-PCR) for typing strains belonging to theAcinetobacter calcoaceticus–Acinetobacter


baumanii complex and application of the methodto the investigation of a hospital outbreak Journal

of Clinical Microbiology 34: 1193–1202.Tenover, F. C., Arbeit, R. D., Goering, R. V. et al.

(1995) Interpreting chromosomal DNA restrictionpatterns produced by pulsed-field gelelectrophoresis: criteria for bacterial strain typing.Journal of Clinical Microbiology 33: 233–2239.

Van Embden, J. D. A., Cave, M. D., Crawford, J. T. et

al. (1993) Strain identification of Mycobacterium

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Versalovic, J., Koeuth, T., McCabe, E. R. et al. (1991)Distribution of repeated DNA sequences ineubacteria applied to fingerprinting of bacterialgenomes. Nucleic Acid Research 19: 6823–6824.

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(1983) Multilocus genetic structure in naturalpopulations of Escherichia coli. Proceedings of the

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(1993) Genetic analysis using random amplifiedpolymorphic DNA markers. Methods in

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References 109

In medical, agricultural and industrial microbiol-ogy, there is a continued need for ‘rapid methods’that will shorten the time between receipt of a spec-imen or sample and the issue of a report. This hasled to the development of instrumentation, usuallyknown as automation, which has the additionaladvantage that it does not require constant atten-tion, can proceed overnight and has thereforeearned the title ‘walk-away’. Such automation maybe described as the operation of an instrument bymechanical, electrical or electronic proceduresalone or in combination, thereby removing theneed for direct human action.

The equipment described here can be truly con-sidered as automation and is widely available.Clearly, not all such equipment can be included.Nor can details be given of use and operation; thesemay be found in the manufacturers’ instructionmanuals.

IDENTIFICATION ANDANTIMICROBIAL SUSCEPTIBILITY

A pure culture is a prerequisite. Thereafter, the userhas a wide choice. Most systems incorporateantimicrobial agent testing options.

VITEK

Initially introduced as the automated system in1976 by BioMerieux (France), this is based on thedetection of microbial growth in microwells withinplastic cards. The original cards were designed for

the identification of urine isolates and used themost probable number (MPN) system to determinesignificant bacteriuria. Additional cards becameavailable for the identification of pure cultures andfor antimicrobial susceptibility testing. VITEK is acompletely automated instrument that offers rapidresults with an average 2- to 6-hour, same-day,turnaround time. The system allows for random orbatch processing and has a built-in quality controlmodule with a complete data management system.There are five VITEK models available, all of whichprovide the same analysis with rapid results andonly differ in the number of cards they process: 32,60, 120, 240 or 480.

Results from the identification cards are inter-preted automatically. For antimicrobial susceptibil-ity testing, over 40 antimicrobial agents areavailable and the results from each test include aninterpolated minimum inhibitory concentration(MIC) as well as the National Committee ofClinical Laboratory Standards (NCCLS) break-points of susceptible, intermediate and resistant(NCCLS, 2000).

The VITEK Test Card is the size of a playing cardand made up of 30 or 45 microwells, containingeither identification substrates or antibiotics.VITEK offers a wide range of cards for the identifi-cation and susceptibility testing of most organismsencountered in the laboratory. The Test Card is asealed container, so no aerosols are produced,thereby reducing the likelihood of personal con-tamination. Disposable waste is apparentlyreduced by more than 80% over microtitremethods. The current range of tests is summarizedin Table 8.1.

The VITEK is an integrated modular system con-

8

Automated methods

sisting of a filler–sealer unit, reader–incubator,computer and printer, and it may be interfacedwith other laboratory information managementsystems (LIMSs). The inoculum is prepared from apredetermined medium and, after adjustment toeither MacFarlane 1 or 2 standard, the suspensionis automatically transferred to the Test Card duringthe vacuum cycle of the filling module. The cardsare then incubated in trays at 35°C. The opticaldensity is monitored hourly; the first reading estab-lishes a baseline, after which the light reductioncaused by growth or biochemical reaction isrecorded.

The VITEK has been introduced as a fully auto-mated system that performs identification and sus-ceptibility testing analyses from a standardizedinoculum. The system combines both complemen-tary and highly advanced skills to transform theantibiotic susceptibility test into a performancetool that detects and helps in the fight against bac-terial resistance.

VITEK2 is targeted at the clinical market andclaims to reduce time to identification and antibi-otic susceptibility results. It offers a more extensiveanalysis menu than the original VITEK, also bene-fiting from a miniaturized card-format consumableitem (10 cm × 6 cm × 0.5 cm), which consists of 64wells for increased analytical capabilities.

Identification is based on fluorescent technologythat provides broad profiles for the reliable identifi-cation of the most clinically relevant organisms.

Data analysis uses algorithms to look at a rangeof parameters and test conditions to ensure accu-rate test results; it is combined with an AdvancedEXPERT Rules System for quality assurance pur-poses.

After primary isolation a suspension of the organ-ism is prepared in a tube of saline and verified witha densitometer. The inoculum tube is then placedinto a rack. The sample identification number isentered into the Smart Carrier via a barcode or key-pad and electronically linked to the supplied bar-code on each test code. Identification (ID) andantimicrobial susceptibility testing (AST) TestCards can be mixed and matched in the cassette tomeet specific laboratory needs. All informationentered at the bench is transported to the instru-ment in a memory chip attached to the cassette.This provides positive text tracking from the benchto the report. From start to finish, all processingsteps are completely autonomous, standardized,controlled and checked.

The optical system reads all 64 wells every 15min. This kinetic monitoring provides an extendeddatabase for analysis and interpretation allowingVITEK2 to provide rapid results.

As AST becomes increasingly requested and com-plex as a result of emerging and low-level microbialresistances, the EXPERT system provides variouslevels of security, validations and in-depth analysisof results to predict the clinical outcome of therapymost accurately.

The EXPERT System allows for:

1. Biological validation of the quality of theresults from a technical point of view.

2. Result interpretation to facilitate correctionfor improved clinical outcomes.

3. Systematic addition of recommendations thatthe laboratory wishes to communicate tophysicians. These may be selected fromfootnotes pertinent to the national standardsto which the laboratory is working, e.g. theNCCLS (2000), the British Society for

Identification and antimicrobial susceptibility 111

Table 8.1 VITEK: range of tests

Product card Organisms identified

GNI + Card V1311 Enterobacteria, vibrios, P. aeruginosa and other non-fermenters

GPI Card V1305 Streptococcus, Staphylococcus,Enterococcus, Listeria andCorynebacterium spp.

YBC Card V1303 Most clinically relevant yeastsANI Card V1309 > 80 anaerobesNHI Card V1308 Neisseria, Haemophilus spp. and

other fastidious organismsUID-3 V1102 Nine urinary pathogensUID-1 V1106 Nine urinary pathogens

Antimicrobial Chemotherapy (BSAC: Phillipset al., 1999) or specific recommendationsdefined by the user’s laboratory.

VITEK have introduced DataTracas, an informa-tion management system that oversees the test datagenerated by the VITEK or VITEK2. The informa-tion is gathered and formatted into a variety ofreports. In addition data can be exported for analy-sis into other PC-based software programs.Addition of the CAR program allows the VITEKand VITEK2 user to compose rules easily, listingspecific conditions which, when met, suppress thereporting of toxic, inappropriate or more costlyantimicrobial agents on the patient report.

The quality control module provides the labora-tory with the ability to store the results of allVITEK cards, thereby allowing the quality of con-trol performance to be tracked. Cumulative qualitycontrol reports, which list any results with theexceptions flagged or exceptions only, are easilygenerated.

The Bi-directional Computer Interface (BCI)package allows VITEK to receive patient informa-tion directly from the LIMS and transmit microbi-ology test data directly to it. This minimizeskeystrokes and data transcription errors whileautomatically uploading test data into the LIMS.

The system has been evaluated by many workers.Jorgensen et al. (2000) showed that VITEK2 pro-vided rapid, reliable susceptibility category deter-minations with a range of challenge and clinicalisolates of Streptococcus pneumoniae. The meantime for generation of susceptibility results is 8.1 h.Hansen et al. (2002) used VITEK GN1+ and GNS-GA cards to identify and perform susceptibilitytesting on positive blood cultures from theBACTEC system. They concluded that the directVITEK method could correctly report identifica-tions and susceptibility patterns within 6 h, makingsame-day reporting possible for almost two-thirdsof bacteraemic episodes with Gram-negativebacilli.

Joyanes et al. (2001) evaluated VITEK2 for iden-tification and susceptibility testing of Pseudomonasaeruginosa, Acinetobacter baumannii and

Stenotrophomonas maltophilia, and concludedthat it allowed rapid identification of the S. mal-tophilia strains and most of the P. aeruginosa andA. baumannii isolates. It also performed reliablesusceptibility testing of many of the antimicrobialagents used against these organisms.

In a multicentre evaluation of the VITEK2Advanced Expert System for interpretive reading ofantimicrobial resistance tests, Livermore et al.(2002) demonstrated the capacity of the system todetect and interpret resistance mechanisms with ahigh level of accuracy and standardization. Only64 of 963 (6.6%) interpretations at 10 Europeancentres were discrepant, with half of these concern-ing S. pneumoniae isolates that were intermediatelyresistant to penicillin by previous methods. In asimilar evaluation of the Advanced Expert System,Cantón et al. (2001) showed it to be a reliable toolfor the detection of extended spectrum β-lacta-mases or inhibitor-resistant TEM β-lactamasesproduced by Enterobacteriaceae.

Sensititre

The Sensitive System (Trek Diagnostic Systems,Inc.) is made up of a number of ‘mix-and-match’components and provides walk-away automationfor identification and susceptibility testing. Themodular system is made up of an auto-inoculator,plate reader, the fully automated AutomatedReading and Incubation System (ARIS) and theSensititre Automated Management System (SAMS)software.

The Sensititre system uses fluorescence-basedtechnology. Substrates are linked to a fluorophoresuch as 4-methylumbelliferone (4MU) or 7-aminomethylcoumarin (7AMC). The substrate flu-orophore is normally non-fluorescent but, in thepresence of specific enzymes, the substrates arecleaved from the fluorophore, when the unbound4MU or 7AMC fluoresces under ultraviolet (UV)light.

The test media contain specifically designedprobes for a number of reactions. Carbohydrateuse is detected by monitoring the pH shift with a

112 Automated methods

fluorescent indicator. When the pH in the test wellis alkaline, the indicator is fluorescent but, as thecarbohydrate is oxidized or fermented, producingacid, the fluorescence decreases, indicating a posi-tive reaction. Carbon-source use, and decarboxy-lase and urease reactions also depend onmonitoring pH changes with fluorescence indica-tors. Bacterial enzyme tests detect the productionof various enzymes, including peptidase, pyranosi-dase, phosphatase and glucuronidase. Enzymes aredetected by their ability to cleave a quenched fluo-rophore, resulting in fluorescence, e.g. aesculin, afluorescent glycoside, breaks down to the non-fluo-rescent aescletin and glucose; non-fluorescencetherefore indicates a positive result. Tryptophandeamination by the catabolic enzyme tryptophaseresults in a coupled reaction in which the formationof a dark colour suppresses the fluorescent signaland thus indicates a positive result.

A real benefit of a fluorescence endpoint is thatemission may be detected with greater sensitivityand a reaction can therefore be observed much ear-lier than is possible with colour changes or turbid-ity readings. Fluorescence also overcomes some ofthe problems associated with conventionalapproaches such as loss of resolution caused bypigment formation in cultures, scanty or variablegrowth, and the use of opaque or pigmented sup-plements.

The system is based on the standard 96-wellmicrotitre tray and consists of an auto-inoculator,plate reader and data handler. Inocula are pre-pared, with the aid of the built-in nephelometer, bytransferring colonies from the isolation media tosterile distilled water to equal a 0.5 McFarlanestandard. The inoculator dispenses 50 μl into eachtest well. The test plate is read after incubation.The light source is a broad-band xenon lamp(360 nm) which generates microsecond pulses ofhigh-peak-power light. This passes through inter-ference filters and a beam-splitting cube with wave-length-selective coatings to lenses that focus thelight on a test well and the detector. The detector isa photomultiplier tube that transmits raw fluores-cence data to a computer. Approximately 30 s arerequired to read a plate and the biocode generated

is matched to the Sensititre database. There may beadditional test prompts and a reincubate promptalso occurs at 5 h if there is no, or a low, probabil-ity of identification. The result of each test isprinted and the quality of each identification isdetermined on the basis of calculated probabilityvalues. ARIS has a 64-plate capacity, which allowsthe user to load any MIC, breakpoint or identifica-tion plates for a combination of 192 possible testsin a single instrument.

Stager and Davis (1992) reviewed the perform-ance reports of the Sensititre and stated that thelimitations were the requirement for off-line incu-bation and the necessity for reincubation of somepanels. The development of systems for the rapididentification of Gram-positive bacteria wouldextend its usefulness.

One of the real developments in automatedinstrumentation has been the introduction of datamanagement software. Sensititre is no exceptionand has introduced the SAMS software, which pro-vides for a wide range of data-tracking and report-ing capabilities. Extensive survey facilities allow avariety of customizable and automatically format-ted reports, including epidemiology, statistics andworkload data, to be produced. The QC Moduleautomatically checks quality control ranges, show-ing Pass or Fail on reports. The Expert System flagswhen predefined NCCLS standards are violated.Automated Backup allows scheduling of automaticnightly data back-up and the Automated Interfaceallows scheduling of automatic initiation of results.

Biolog

A number of system options are available althoughit is the OmniLog ID system that is fully auto-mated. It uses Biolog’s proprietary carbon sourceuse test methodology in a convenient 96-wellformat. When an organism is introduced to thewide variety of pre-selected carbon sources, itrapidly produces a characteristic pattern or ‘finger-print’, which is compared with an extensive data-base. Unlike other systems, the Biolog results are


based on 95 reactions from 6 to 8 different classesof carbon sources. The Biolog technology candetect mixed or contaminated cultures, and is alsoable to identify environmental and fastidiousorganisms. The sensitivity and precision of thetechnology have allowed Biolog to build extensivedatabases that include environmental organisms,veterinary organisms, plant pathogens and humanpathogens.

A unique feature of the OmniLog System is user-defined incubation temperature. The OmniLog IDSystem was designed to identify a wide variety oforganisms, which must be incubated at their opti-mal temperature to obtain an accurate identifica-tion. Other automated systems that incubate andread identification test panels do so at one set tem-perature, usually 37°C. As such they are not alwaysable to identify bacteria such as those isolatedwhen doing environmental monitoring. For thoseorganisms that may require special incubationenvironments (e.g. elevated CO2 or extreme tem-peratures), the system allows the user to incubatethe MicroPlates ‘off line’ before placing them intothe Reader for interpretation.

The OmniLog System simultaneously incubates,reads and interprets the Biolog MicroPlates (Table8.2). It continuously processes samples but allowsthe user complete access at any time during a sam-ple run. Samples can be loaded when ready andremoved when complete without disturbing othersamples still in process. Inside the Reader there are25 trays. Each tray holds 2 MicroPlates, giving theReader a total capacity to incubate and read 50 iso-lates.

Before the user inoculates the appropriateMicroPlates, they log the MicroPlate information

into the OmniLog software. By simply followingthe software’s instructions, the user then opens thedoor of the OmniLog Reader and places eachMicroPlate in the appropriate tray slot indicated bythe software. Once all the MicroPlates are loadedand the door of the Reader is closed, the OmniLogsoftware takes over the responsibility for incubat-ing, reading, saving and printing the results.

The OmniLog ID System begins reading theMicroPlates 4 h after they have been placed intothe Reader. The pattern is compared with the iden-tification database and an ID is called if enoughpositive reactions have developed. This result isthen displayed on the Read menu screen, stored inthe computer’s memory, and an identificationreport is printed out. If no result is obtained after6 h, the instrument automatically continues toincubate the MicroPlate and begins reading againafter 16 h and up to 24 h.

The OmniLog ID System uses Windows-basedsoftware. The status of all the samples can beobserved by simply looking at the Read menuscreen of the OmniLog ID System software. All ofthe information for a specific MicroPlate is con-tained on a single line of this screen. Once the sys-tem has determined a final identification for aspecific MicroPlate, it indicates this status with acheck mark icon. A clock icon is used for thosesamples that are still incubating because an identi-fication result has not yet been determined.

The ‘Plus’ version of the OmniLog adds the addi-tional capability of identifying yeasts, anaerobesand filamentous fungi (Table 8.3).


Table 8.2 The OmniLog System Microbe Identification Process

Step 1 Isolate a pure culture on Biolog media Step 2 Do a Gram stain and determine testing

protocolStep 3 Prepare inoculum at specified cell densityStep 4 Inoculate MicroPlates and place in ReaderStep 5 Obtain ID results from printer

Table 8.3 Omnilog and Omnilog Plus: range of tests

OmniLog OmniLog Plus

Gram-negative bacteria ✔ ✔

500 speciesGram-positive bacteria ✔ ✔

500 speciesYeasts 250 species ✔

Anaerobic bacteria 350 species ✔

Filamentous fungi 500 species ✔

In today’s regulated environments control andvalidation of the microbial identification systemand control are two crucial elements of regulatorycompliance. In conjunction with validationexperts, Biolog developed a comprehensive valida-tion package for the OmniLog System. TheValidation Protocol Manual provides the frame-work for performing the Installation Qualificationand the Operation Qualification for each of the sys-tem elements. The Validation Package also includestest strains that can be run on the OmniLog to pro-vide validation of the system performance. Biologalso provides optional software features to controloperator access and to control access for buildingor modifying data files. This new capability isindicative of how Biolog is consistently respondingto its customers’ ongoing needs.

The operator can use one of the Biolog databasesor compile a user-defined file. An unknown bio-chemical profile may then be compared with eitherthe databases or a combination of the two. Otherfeatures of the software include on-line informa-tion about any species in the library, cluster analy-sis programs in the form of dendrograms and two-or three-dimensional plots to demonstrate therelatedness of strains or species, and the separationof the Gram-negative database into clinical andenvironmental sectors.

It is the last of these that gives some distinction tothe Biolog because it identified a niche in the mar-ket that other systems had failed to fill. It includesnon-clinical isolates as a feature of its initial data-base, thereby addressing the particular needs ofenvironmental and research workers. This is bestillustrated by considering the ES Microplate whichis designed for characterizing and/or identifyingdifferent strains of Escherichia coli and Salmonellaspp., for characterizing mutant strains and forquality control tests on E. coli and S. typhimuriumstrains carrying recombinant plasmids.

Phoenix

The Phoenix System is a new, automated, shortincubation system for the rapid identification and

susceptibility testing of clinically relevant bacteria.Phoenix can test up to 100 ID and AST combina-tion panels at a time. The Phoenix system uses anoptimized colorimetric redox indicator for ASTand a variety of colorimetric and fluorimetric indi-cators for bacterial ID. A sealed and self-inoculat-ing moulded polystyrene tray with 136 microwellsserves as the Phoenix ID/AST Combination dispos-able.

The Phoenix System includes an inoculationstation for panel set-up/ inoculation and aninstrument with an Incubator/Reader carouselmodule. The carousel houses four horizontaltiers of 26 panel carriers to accommodate atier-specific Normalizer and 25 PhoenixPanels.

The Phoenix Panel uses up to 51 microwells foridentification and up to 85 microwells for suscepti-bility testing of 16 antimicrobial agents at five con-centrations, or more than 25 agents at threeconcentrations per agent.

An optimized Mueller–Hinton broth base with aredox indicator and an optimized Inoculum Fluid isused for AST and ID inoculum preparations,respectively.

A bacterial inoculum concentration, approxi-mately equivalent to a 0.5 McFarland Standard,is required for the identification of eitherGram-negative or Gram-positive bacteria.Susceptibility testing is performed with an inocu-lum concentration of 3–7 × 105 colony-formingunits (cfu)/ml with a colorimetric redox indica-tor. Kinetic measurements of bioreactivitywithin individual microwells via red, green,blue and fluorescence readings are collectedand comparatively analysed with the Phoenixdatabase.

The average time to results is 6 h for Gram-posi-tive and 6–12 h for Gram-negative organisms. Theinstrument will provide interpreted test results withan integrated expert system (BDXpert). The systemwill offer an optional workstation (Epicenter) tocomplement Phoenix results with data manage-ment features, including epidemiology, user-defined reporting and Advanced BDXpertcapability.


Midi microbial identification system

The Midi (Microbial ID Inc., USA) is a fully auto-mated, computerized gas chromatography (GC)system that can separate, distinguish and analysemore than 300 C9–C20 fatty acid methyl esters. Thevalue of this type of analysis is that the fatty acidcomposition is a stable genetic trait, which is highlyconserved within a taxonomic group.

The system software includes operational proce-dures, automatic peak naming, data storage andcomparison of unknown profiles with the database,using pattern recognition algorithms. The databasecontains more than 100 000 strain profiles, includ-ing representatives of Enterobacteriaceae andPseudomonas, Staphylococcus and Bacillus spp. Italso includes mycobacteria, anaerobes and yeasts.

Representative isolates must be subcultured indefined media before preparation of the methylester, after which the whole process is automated.The system is calibrated by a mixture of straight-chain fatty acids. This is necessary after analysis of10 samples and corrects for any changes in sampleinjection volume and variability in gas flow rates.Calibration samples can be set up automatically.The autosampler will take up to 2 days of analyti-cal capacity and may therefore be considered astruly walk-away.

Gas chromatography analysis is a well-estab-lished tool in the anaerobe laboratory and exten-sive work has been done in this field, notably at theAnaerobe Laboratory, Virginia PolytechnicInstitute and State University. McAllister et al.(1991) reported that the Midi system correctlyidentified 97% of the anaerobes tested.

The Midi database consists of more than 100 000analyses of strains obtained from experts and fromculture collections. The cultures were collectedfrom around the world to avoid potential geo-graphical bias. Where possible, 20 or more strainsof a species or subspecies were analysed to makethe entry. When fatty-acid profile subgroups werefound within a taxon, more strains were obtainedto delineate each group.

The system will analyse about 45 samples per day(with a dual tower, 90 samples per day can be

analysed). A single technician can extract about 75samples per day and so the operator time per sam-ple averages at about 6 min.

Assessment of identification systems

Stager and Davis (1992) reviewed five studies inwhich the accuracy of either two or three of the sys-tems was compared: percentage accuracy variedfrom as low as 35% to as high as 99.2%. This typeof study is of somewhat limited value, however,because the companies are continually improvingtheir systems, particularly with respect to the qual-ity of the databases, which are, of course, funda-mental to correct identification. This is best seen inthe studies of Kelly et al. (1984), Stevens et al.(1984) and Truant et al. (1989), who all presentedevidence that variation in biotypes of individualspecies from different geographical areas may inpart be responsible for performance variability.This needs to be considered both by the manufac-turers and by the users, especially by the latterwhen they are considering a purchase.

BLOOD CULTUREINSTRUMENTATION

BACTEC

The BACTEC series of instruments (BectonDickinson, USA) is generally associated with bloodcultures but has other uses. Samples of blood,spinal, synovial, pleural and other normally sterilebody fluids are injected into the BACTEC vials.Growth of organisms is detected by the productionof CO2 from culture media. As the first of the bloodculture systems, it is worth considering the devel-opment of this product line. Users should be awareof the fact that cross-contamination could occurbetween samples, should there be inadequatesterilization of the sample needles that are used tocollect samples.


BACTEC 460

This early BACTEC system used the makers’ cul-ture medium containing 14C-labelled substrate. Theinstrument takes 60 vials, which are inoculatedwith 5 ml of the test fluid. It then automaticallymeasures the radioactivity in the headspace gas for14CO2 each hour and prints the result. If the activityexceeds a preset threshold the blood culture is con-sidered to be positive.

BACTEC NR-660, NR730 and NR-860

The NR series replaced the radioactive substrate byemploying infrared (IR) spectrometry to detect theCO2. Infrared light is absorbed by the CO2 in thetest cell and the amount passing through to thedetector is registered. The amplifier converts thismeasurement of conductivity to voltage, which isconverted to a read-out value. There is an inverserelationship between the amount of CO2 generatedand the amount of IR light detected. This isaccounted for in the calculations that generate thegrowth value (GV).

During the test a pair of needles penetrates thevial septum and the headspace gas is drawnthrough one of them into the detector. The otherneedle is connected to an external gas cylinder sothat the appropriate gas for aerobiosis or anaero-biosis can be replaced. The various models in theNR series use different IR systems. In the NR-860a positive culture is flagged if the GV, or the differ-ence between two consecutive measurements,exceeds a predetermined threshold. The vial head-space is also measured and when that exceeds adefault setting the sample is flagged as potentiallypositive. Vial testing is automatic and an auto-mated tray transport mechanism shuttles the traysbetween the incubator and the IR sensor unit. Abarcode scanner reads the sample label. The NR-860 can store 480 samples in eight drawers. Thetwo bottom drawers, containing the most recentaerobic cultures, are mounted on an orbital shaker.

The working capacity is 48 new cultures per daywith a 5-day test protocol. When positive samplesare identified they are removed for further investi-gation.

BACTEC 460/TB for mycobacteria

The BACTEC 460 instrument was modified to the460/TB for the detection and susceptibility testingof mycobacteria. In this the cover was replaced byas hood with a forced, recirculating air supply andHEPA (high efficiency particulate air) filters toavoid dispersion of any aerosols into the laboratoryatmosphere. This instrument, subsequently modi-fied to detect CO2 by IR spectrometry, instead ofradioactivity, has proved to be very useful in theearly detection of tubercle bacilli in sputum cul-tures and of antimicrobial resistance of mycobacte-ria.

BACTEC 9000 Series

This 9000 series instruments are fluorescence-based, walk-away detection systems reputedlyoffering increased sensitivity; as they are non-inva-sive, they offer a high level of built-in microbiolog-ical safety. As no venting is required the samplesmay be collected in Vacutainer blood-collectingsets. A dye in the sensor reacts with CO2, therebymodulating the amount of light that is absorbed bya fluorescent material in the sensor. The photode-tectors measure the fluorescence, which is relatedto the amount of CO2 released in the culture. Oninitiation of a test the machine carries out a diag-nostic routine, after which it starts continuous andautomated on-line testing. A test cycle of all racksis continually being monitored every 10 min andpositive cultures are flagged by a light on the frontof the instrument and displayed on the monitor.

The BACTEC 9240 can monitor 240 culturevials, arranged in six racks. The vials are incubatedat 35°C with agitation. The working capacity is 24new culture sets per day with a 5-day test protocol.

Blood culture instrumentation 117

Also available is the BACTEC 9120, which moni-tors 120 culture vials. The BACTEC 9050 has beenintroduced for laboratories that process fewer than150 blood culture sets each month. The compactsize of the BACTEC 9050 minimizes space require-ments, yet it still features fully automated, non-invasive, continuous fluorescent monitoring ofgrowth, but occupies only 4.5 square feet of benchspace (less than half a square metre). Software andoperating systems are totally built in, so there is noneed for an external computer to take up morespace and complicate operations. The instrument isdesigned for laboratories processing up to fiveblood cultures per day. The technician simplyopens the door of the instrument, presses the ‘VialEntry’ key, scans the vial barcode at the fixed bar-code reader and places the vial into the assignedposition. Once the door is shut, processing auto-matically begins.

In addition to the ability of networking up to 20BACTEC instruments for large or group laborato-ries, Becton Dickinson have introduced BACTECVISION Software, a data control centre for theBACTEC 9000 series of blood culture instruments.

The software allows for extensive data manage-ment, total reporting and sorting flexibility in aWindows environment.

Other BACTEC 9000 Data ManagementSystems

• BACTEC Level I: with bar code icon interfaceto increase work flow efficiency. Monitorsinstrument status, tracks and reports inprotocol specimens.

• BACTEC Level II: includes Level I plusconnectivity to all interfaces written to ASTMstandards. Provides bidirectional capability todownload patient demographics directly tocore computer, saving technician time andspeeding reporting of results. Monitorsinstrument status, tracks and reports in-protocol specimens, and records patient andspecimen demographics.

Note: BACTEC VISION is a Level III System andincludes all items in Levels I and II.

BacT/Alert

The BacT/Alert system (Biomerieux) was introducedin 1990–91 as a fully automated system and there-fore as an alternative to semi-automated radiomet-ric and IR systems. Like the BACTEC, it uses theproduction of CO2 and employs a novel colorimet-ric sensor in the base of the aerobic and anaerobicblood culture bottles. The sensor changes colour asCO2 is produced; the rate of change is detected by areflectometer and the data are passed to a computerwhose algorithm can distinguish between the con-stant CO2 production by blood cells and the accel-erating CO2 production from a positive bloodculture. The sensor is covered by an ion-exclusionmembrane, which is permeable to CO2 but not tofree hydrogen ions, media components or wholeblood. Indicator molecules in the water-impregnatedsensor are dark green in their alkaline state, chang-ing progressively to yellow as the pH decreases. CO2

from a positive culture passes through the mem-brane, giving the following reaction:

CO2 + H2O → HCO3− + H+

The free hydrogen ions react with the indicatormolecules causing the sensor to change colour. Thisis detected by a red light-emitting diode and meas-ured by a solid-state detector.

Aerobic and anaerobic culture bottles are avail-able and are maintained at negative pressure forease of inoculation. The recommended adult sam-ple is 5–10 ml per bottle. It is necessary to vent theaerobic bottles occasionally. Cultures taken outsidenormal laboratory hours may be incubated exter-nally and entered into the system on the followingday. The system holds up to 240 bottles, each withits own detector. Up to four data-detection unitsmay be interfaced with the same computer, giving amaximum throughput for one system of 960 bot-tles at any one time. A single detection unit has 10blocks of 24 wells, each block operating independ-ently, thereby allowing easy removal and routineservicing. A Reflectance Standard Kit is suppliedwith each system, which allows the user to cali-brate and test for quality control. The PC analysesall data, interpreting both positive and negative


growth curves. It also allows complete patient filesto be kept and will interface with existing in-housesystems.

The system has been evaluated against the radio-metric BACTEC 460 and shown to give compara-ble results (Thorpe et al., 1990). Wilson et al.(1992) also concluded that the BacT/Alert andBACTEC 660/730 non-radiometric systems werecomparable for recovering clinically significantmicro-organisms from adult patients with bacter-aemia or fungaemia. The BacT/Alert, however,detected microbial growth earlier than theBACTEC and gave significantly fewer false-positiveresults. In addition to the standard Bact/Alert sys-tem, MB/BacT has been introduced as an easier,safer and more cost-effective method for detectionof mycobacteria than conventional or radiometricmethods. It is a fully automated system for contin-uous non-invasive monitoring of non-blood speci-mens and, based on the unique colorimetrictechnology, it offers the same benefits in terms ofspeed, accuracy, ease of use and cost-effectiveness.

A further development is BacT/ALERT 3D whichis a test system that combines blood, body fluid andmycobacteria specimen testing in one instrument.Its flexible modular design consists of one to sixIncubator Modules, each capable of accommodat-ing 240 bottles, and is directed by a touchscreen-activated Control Module. This allows for atext-free user interface for more direct randomrapid loading and unloading of the test samples.The control Module supervises the reading of theIncubator Module’s sensors and controls the deci-sion-making algorithms, to determine whether ornot a given bottle is positive.

Bact/VIEW is a complete data management pro-gram for use with BacT/ALERT and MB/BacT. Ithas an easy to learn and operate touchscreen inter-face. Collecting patient data, launching routinetasks, loading and unloading bottles, and customiz-ing reports are very straightforward matters. Thereis no need to memorize commands or routines. Asingle computer system with BacT/VIEW can con-trol up to nine microbiological units. In additionBacT/VIEW may be connected directly to an LIMSusing BacT/LINK.

ESP

ESP (Trek Diagnostic Systems) is a fully automatedculture system for routine blood culture, mycobac-teria detection and Mycobacterium tuberculosissusceptibility testing on a single instrument plat-form. It is a closed system design, thereby allowingsafe handling of all specimens throughout the labo-ratory.

The instrument design can be configured to allowfor a system that efficiently accommodates a rangeof test volumes: 1920 test sites may be monitoredby one PC, thereby reducing costs and improvingefficiency.

ESP detects a wide range of micro-organisms bymeasuring both consumption and production of allgases. The technology does not rely on forced pro-duction of CO2 for micro-organism detection. Thenon-invasive measurement of pressure changes inthe headspace of the culture bottle provides aneffective and rapid detection of microbial growth.Aerosol-free venting of the culture bottle occursautomatically before entry and after removal of apositive specimen. A hydrophobic membrane in theconnector prevents aerosols from escaping into thelaboratory environment.

Proprietary media support the growth of thewidest variety of micro-organisms and are effectivefor all specimen types without any additives.Included essential growth factors allow a muchsmaller amount of blood to be drawn. TheAmerican Food and Drugs Administration (FDA)has cleared the use of volumes as low as 0.1 ml toaccommodate difficult draws. Paediatric bottles aretherefore not necessary with ESP. The optimizedblood : broth dilution ratio neutralizes effects ofantibiotics and other inhibitory substances, mean-ing that resins and fan media are not required.

A non-radiometric M. tuberculosis detection sys-tem is provided for. The ESP Culture System IIcombines a liquid culture medium, a growth sup-plement and, for potentially contaminated speci-mens, an antibiotic supplement, with a detectionsystem that automatically incubates and continu-ally monitors culture bottles inoculated with speci-mens suspected of containing mycobacteria. ESP

Blood culture instrumentation 119

detects mycobacterial growth by automaticallymonitoring (every 24 min) the rate of consumptionof oxygen within the headspace of the culture bot-tle and reports that growth response with a visiblepositive sign.

An ESP Myco Susceptibility Kit consisting ofthree first-line anti-tuberculosis drugs – rifampin,isoniazid and ethambutol – is available. The totalESP susceptibility system is made up of specific lotsof ESP media that are qualified to be used with eachESP Myco Susceptibility Kit. The primary drugs areinjected into supplemented ESP Myco bottles. Aninoculum of the M. tuberculosis isolate to be testedcan be prepared from colonies grown on solidmedia or from a seed bottle. The inoculum is addedto the drug-containing bottles and to a drug-freecontrol bottle, used as a positive control. The pres-ence of growth is determined automatically by theESP instrument. If the organism is susceptible to adrug, microbial growth will not be detected or willbe significantly delayed compared with the drug-free control bottle. If the organism is resistant to adrug, microbial growth will be detected within 3days of the control bottle.

Safety has been addressed by the use of a dispos-able plastic connector with recessed probe thateliminates risk and liability of injury from ventingprobes and provides safe, non-invasive venting.There is a 0.2-μm hydrophobic membrane withinthe connector which prevents the escape of danger-ous aerosols into the laboratory. The use of a non-radiometric mycobacteria broth eliminatespotential hazards of radioactive components andeliminates the requirement for radioactive disposalor licensing fees.

As with all the current generation of instrumenta-tion, a data management system has been devel-oped to extend the functionality of the culturesystem. It is a fully featured Windows NT-baseddata management system using the MicrosoftAccess database. Custom reports can easily be inte-grated with standard reports, providing a wealth ofinformation about epidemiology, quality of results,workplace efficiency, and cost containment. Aninterface allows download of patient demographicsduring specimen entry and automatic transfer of

test results to the LIMS. ESP data can be retrievedfrom any computer on the network using a com-patible version of Microsoft Access.

URINE EXAMINATION

There have been many attempts to automate urineanalysis, none of which has had significant com-mercial success. Zaman et al. (2001) considered theUF-100 urine flow cytometer (Merck, Eurolab) andconcluded that the technology does not accuratelypredict the outcome of urine cultures, and that thissystem is therefore unsuitable for the safe screeningof urine samples for urinary tract infections.

Trek Diagnostic Systems, Inc. have introduced theCellenium System which is claimed to provide thefirst rapid, fully automated approach to urine screen-ing. Using proprietary advanced robotic operation,measured amounts of each urine sample are dis-pensed into a Cellenium cassette and fluorescentnucleic acid stains are added. The cassette is auto-matically coupled with a Cellenium membrane.Vacuum filtration results in a monolayer of stainedmicro-organisms on the membrane, which are exam-ined using computerized fluorescent microscopyimaging. Results are equivalent to the colony-form-ing units per millilitre of a standard culture.

This approach can eliminate the culturing of neg-ative samples and can detect and enumerate bacte-ria in urine, as well as providing Gram staininformation. In positive specimens, bacteria arenoted as Gram-positive cocci, Gram-negative rodsor Gram-positive rods. It can also be used to detectyeast and white blood cells, and provides Gramstain morphological information.

The instrument is able to detect, enumerate, andclassify pathogenic micro-organisms in urinewithin 30 min, thus eliminating the requirement toculture negative specimens. It consequently pro-vides the ability to report negative results a day ear-lier than traditional culture methods.

Patient specimen data may be entered either bybarcode or by manual entry through the instru-ment’s input station. An intuitive Windows NT-based software guides the loading of reagents and


allows laboratory personnel to monitor the testprocess at any time. The proprietary reagents aresupplied in a ready-to-use form to eliminate anyrequirement for special preparation. The instru-ment is capable of continuous operation (24 h/day)analysing up to 70 specimens per h.

Reporting can be carried out in several ways,including a hard copy printout, and a graphic mapthat classifies specimens as positive, negative orinconclusive, or through an interface to the LIMS.

IMPEDANCE INSTRUMENTATION

Four systems are generally available: Bactometer(BioMerieux, France), Malthus (Radiometer,Denmark), RABIT (Don Whitley, UK) and Bactrac(Sy-Lab, Austria). All operate on the same princi-ple.

Impedance may be defined as resistance to flow ofan alternating current as it passes through a con-ductor (for detailed theory, see Eden and Eden,1984; Kell and Davey, 1990). When two metalelectrodes are immersed in a conducting fluid thesystem behaves either as a resistor and capacitor inseries or as a conductor and capacitor in parallel(Kell and Davey, 1990). When it is in series combi-nation, the application of an alternating sinusoidalpotential will produce a current that is dependenton the impedance, Z, of the system which, in turn,is a function of its resistance, R, capacitance, C,and applied frequency, F. Thus:

Z = �R2 + ��12

��πFC�2�

Any increase in conductance, defined here as thereciprocal of resistance, or capacitance results in adecrease in impedance and an increase in current.The AC equivalent of conductance is admittance,defined as the reciprocal and the units of measure-ment are siemens (S).

Microbial metabolism usually results in anincrease in both conductance and capacitance,causing a decrease in impedance and a consequentincrease in admittance. The concepts of impedance,conductance, capacitance and resistance are only

different ways of monitoring the test system andare interrelated. An important factor, however, isthat all are dependent on the frequency of the alter-nating current and all are complex quantities, inthat they contain both real and imaginary parts(Kell and Davey, 1990). In practical terms the usermust be aware that the electrical response is fre-quency dependent, has a conductive and capacita-tive component, and is temperature dependent.Temperature control in any impedance system is ofcritical importance. A temperature increase of 1°Cwill result in an average increase of 0.9% in capac-itance and 1.8% in conductance (Eden and Eden,1984), and from this it can be calculated that a lessthan 100 millidegree temperature drift can result ina false detection. It is interesting to note that threeof the four systems mentioned above have adopteda different method of temperature control:Bactometer, a hot-air oven; Malthus, a water bath;and RABIT and BacTrac a solid block heating sys-tem.

These systems provide a measurement of netchanges in conductivity of the culture medium.Tests are monitored continually and, when the rateof change in conductivity exceeds the user-definedpre-set criterion, the system reports growth. Thetime required to reach the point of detection is the‘time to detection’ (TTD) and is a function of thesize of the initial microbial population, its growthkinetics and the properties of the culture medium.For a given protocol, the TTD is inversely propor-tional to the initial microbial load of the sample. Atthe point of detection it is generally considered thatthere will be approximately 106 cfu/ml of the testorganism present in the sample. This will varyaccording to the type of organism, growth media,etc., but will be constant for any one organismgrowing under defined test conditions.

The attributes of the principal systems are out-lined in Table 8.4, which shows that there are dif-ferences in the measuring modes. The Bactometerhas the option of measuring either conductance orcapacitance, the Malthus measures conductanceand the principal signal component in the RABITis conductance, although there is a also a capaci-tance component. The benefits of capacitance are

Impedance instrumentation 121

best exemplified in attempts to detect organisms inwhich normal metabolism results in no netincrease in medium conductivity. This does notpreclude the Malthus and RABIT from such appli-cations, however, because both can utilize the indi-rect technique (Owens et al., 1989) in which anelectrical signal is generated by CO2 production.The technique also has the advantage of being fullycompatible with samples and test media that havea high salt content, which is not the case with theBactometer. The indirect technique also allows theuser a greater choice of culture media becausethese do not have to be optimized for electricalresponse but simply for growth of the targetorganism.

ATP MEASUREMENT

Stewart and Williams (1992) have reviewed themechanism of bioluminescence assay, mediated bythe luciferin/luciferase reaction, for adenosinetriphosphate (ATP). The luciferin/luciferase com-plex reacts specifically with the ATP in living cellsand emits a light signal proportional to the amountof ATP present. There is a linear relationshipbetween the ATP and the plate count (Stannardand Wood, 1983). The ATP in the sample origi-nates from both living cells and other sources (the

somatic ATP), and the two must be differentiated ifthe technique is to be used in microbiological test-ing (although it is not necessary for rapid hygienemonitoring, as in the food industry). The twocommonly used methods are separation of theorganisms from the sample before extractingthe ATP, and prior destruction of the somaticATP.

Bactofoss

Bactofoss (Foss Electric, Denmark) is a unique,fully automated instrument that combines the prin-ciples mentioned above to remove sample ‘noise’.All functions are monitored by the central process-ing unit and messages are presented on the screen.Bactofoss is a simple push-button instrumentwhich provides a result within 5 min.

The test sample is taken up automatically anddeposited in a temperature-controlled funnel whereseveral filtrations and pre-treatments removesomatic ATP. The micro-organisms are left on afilter paper which is then positioned in an extrac-tion chamber. Extraction reagent is added toextract microbial ATP, the amount of which isdetermined by measurement of luminescence afterthe addition of luciferin and luciferase. Allreagents, etc. are supplied by the manufacturer and


Table 8.4 Comparison of the three principal impedance systems/ Bactometer, Malthus and RABIT

Bactometer Malthus 2000 RABIT

Test capacity 512 480 512Modular system Yes No YesTemperature options per system 8 1 16 maxCell volume 2 ml max 2 and 5 ml 2–10 mlCell format Block of 16 Single SingleReusable/disposable Disposable Reusable and disposable ReusableMedia availability Yes Yes YesMeasuring mode Conductance or Conductance Impedance

capacitanceIndirect mode No Yes YesFull access to cell throughout test period No No YesTemperature control Hot-air oven Water-bath Dry heating block

the instrument can perform 20 analyses per hour.Limond and Griffiths (1991) have shown that it isapplicable to meat and milk samples, with detec-tion sensitivities of 3 × 104 – 3 × 108 cfu/g and 1 ×104 – 1 × 108 cfu/ml for milk.

Direct epifluorescence techniques

The direct epifluorescence technique (DEFT) wasoriginally developed for counting bacteria in rawmilk (Pettipher et al., 1980, 1989). It takes lessthan 30 min and uses membrane filtration and epi-fluorescence microscopy. Pre-treatment of samplesmay be necessary to facilitate rapid filtration anddistribution of bacteria on the membrane. A suit-able fluorescent stain shows organisms that are eas-ily distinguished from debris by an epifluorescencemicroscope, which is linked to an automatic count-ing system.

The direct epifluorescence technique has beenused in manual or semi-automatic modes for sev-eral years, but the new Cobra system (France) isfully automated.

Bactoscan

Bactoscan (Foss Electric, Denmark) is fully auto-mated and employs the DEFT principle, but is ded-icated to the quality control of milk by makingdirect bacterial counts.

A 2.5-ml sample of milk is treated with lysingfluid to dissolve protein and somatic cells. The bac-teria are then separated from the milk by gradientcentrifugation. The bacterial suspension is mixedwith a protease enzyme to dissolve particulate pro-tein and is then stained with acridine orange. Thestained organisms are differentiated from debris onthe basis of dye uptake and are counted by a con-tinuously functioning epifluorescence microscopewith the results displayed on screen. Eighty samplesper hour can be screened and the system is flushedwith rinsing fluid between each sample. When test-ing is finished the machine is automatically cleaned

The protease solution must be prepared daily butthe other reagents need be made up only everyother day. The start-up procedure takes about 30min and involves a calibration routine, lens check-ing and setting the discrimination level of thecounting module. Milk samples are placed in spe-cial racks, heated to 40°C and loaded on the instru-ment. The sample identification can be keyed inmanually or entered by a bar code reader. Resultsare displayed on a screen. Bactoscan counts corre-late well with standard plate counts and may there-fore be converted to equivalent colony-formingunits per millilitre.

FLOW CYTOMETRY

Flow cytometry, widely used in research, has nowbeen developed for routine industrial use. The tech-nique allows cell-by-cell analysis of the test samplesand, coupled with a range of fluorescent labels,provides a rapid and automated method for detect-ing microbes and examining their metabolic state.The sample is injected into a ‘sheath’ fluid thatpasses under the objective lens via a hydrodynamicfocusing flow cell. The sheath fluid passes continu-ously through the flow cell, thereby focusing thesample stream into a narrow, linear flow. The sam-ple then passes through a light beam that causes thelabelled cells to emit fluorescent pulses. Each pulseis detected and subsequent analysis allows pulses tobe recognized as separate counts and graded interms of fluorescence intensities.

Systems currently available include the Chemunex(AES Laboratoire) range of products, which cannow detect at the single cell level. The results maybe printed as hard copy or transferred to a com-puter. The labelling and counting take less than30 min per sample. Other systems from companiessuch as Coulter Electronics and Becton Dickinsonare available.

The ability of flow cytometry to analyse and sortcells into defined populations on the basis of cellsize, density and discriminatory labelling is apowerful new microbiological tool.

Flow cytometry 123

PYROLYSIS MASS SPECTROMETRY

Pyrolysis mass spectrometry (PMS) has consider-able potential for the identification, classificationand typing of bacteria (Magee et al., 1989;Freeman et al., 1990; Sisson et al., 1992). In theRAPyD-400 benchtop automated PMS (HorizonInstruments, UK) samples are spread on V-shapedNi–Fe pyrolysis foils held in pyrolysis tubes.Samples are then heated by Curie-point techniquesin a vacuum, which causes pyrolysis in a controlledand reproducible manner. The gas produced ispassed through a molecular beam and analysed bya rapid scanning quadruple mass spectrometer toproduce a fingerprint of the original sample over amass range of 12–400 Da. Data are analysed byonboard multivariate statistical routines, providingprincipal component analysis, discriminant func-tion analysis, cluster analysis and factor spectra.The system is entirely automated, with sampleloading, extraction, indexing to next sample anddata collection controlled by a computer. Routineservicing and maintenance are minimal. Routineanalysis takes approximately 90 s per sample witha batch of 300 samples.

It is important that the organisms are originallycultured on media that do not impose stress andconsequent alteration of phenotype expression,which may obscure the relatedness of isolates. Itmay be desirable to facilitate expression of certainphenotypes before analysis, thereby allowing thetechnique to differentiate between unrelatedstrains, e.g. differentiation between toxigenic andnon-toxigenic strains (Sisson et al., 1992).Experience in UK public health laboratories sug-gests that speed, low running costs and versatilityof PMS makes it suitable for the initial screening inoutbreaks of infection.

MOLECULAR MICROBIOLOGY

Advances in molecular microbiology have added todiagnostic techniques, mainly with DNA probes orthe polymerase chain reaction (PCR).

Probe-based technology

A DNA probe is a piece of single-stranded DNAthat can recognize and consequently hybridize witha complementary DNA sequence. The probe alsocarries a label that ‘lights up’ the hybrid. Probes canbe prepared from total cellular DNA, short-chainoligonucleotides or cloned DNA fragments andtheir specificity measured. There are many assayformats, although all follow the same principlewhereby the strands of the DNA double helix of thetarget organism are separated by heating or alkali,and immobilized on a membrane. Immobilizationprevents the complementary strands re-hybridizingand allows access for the probe.

After challenge with the probe the membrane iswashed to remove any unbound probe and theresulting hybridization is visualized. The dot–blotassay immobilizes target DNA on a membrane andvisualizes hybridization as a coloured spot.Colony–blot assays take up cells from a plate on toa sterile membrane, thereby forming a replica ofthat plate. The cells are then lysed to release theDNA, which is immobilized and challenged by theprobe. Liquid hybridization formats use a probethat carries a chemifluorescent label, which is addeddirectly to lysed cells. Adding a proprietary reagentbreaks down non-hybridized probe. The amount offluorescence measured is directly proportional tothe number of target organisms. Paddle and beadformats carry the probe on their solid surfaces.After hybridization the unbound probe is washedoff and detection is visualized. Several commercialkits are available for this type of examination (e.g.Gen-Probe Inc., San Diego, CA).

DNA fingerprinting

This is considered elsewhere in the book (seep. 103) because it is not yet fully automated.

Otherwise known as restriction fragment lengthpolymorphism (RFLP) analysis, this combinesprobe and restriction enzyme technologies. Theassay format is generally known as Southern blot-


ting. Restriction enzymes cut DNA at sites charac-terized by short sequences of certain bases, result-ing in a number of fragments of different lengths.The number and sizes of the fragments vary con-siderably from one organism to another, evenwithin a species. The fragments can be separatedaccording to size and transferred directly to amembrane by electrophoretic techniques. Theimmobilized fragments are then probed, resultingin a DNA fingerprint that is characteristic of thesource DNA.

PCR technology: amplification-basedtechnology

The best-established DNA amplification method isthe polymerase chain reaction. This is based on therepetitive cycling of three simple reactions: denatu-ration of double-stranded DNA, annealing of sin-gle-stranded complementary oligonucleotides andextension of oligonucleotides to form a DNA copy.The conditions for these reactions vary only inincubation temperatures, all occurring in the sametube in a cascade manner. The repetitive cycling istherefore self-contained and can be automated in aprogrammable thermocycler. The first step is theheat denaturation of native DNA which melts asthe hydrogen bonds of the double helix break. Thesingle strands are then available to re-anneal withcomplementary-sequence DNA. The second steptakes place at a reduced temperature. Two shortDNA primers are annealed to complementarysequences on opposite strands of a template,thereby flanking the region to be amplified. Theyact as a starting point for DNA polymerase andconsequently define the DNA region to be ampli-fied. In the third step synthesis of complementarynew DNA occurs through the extension of eachannealed primer by the action of Taq polymerase inthe presence of excess deoxyribonuclease triphos-phates. The new strand formed consists of theprimer at its 5′ end trailed by a string of linkednucleotides that are complementary to those of thecorresponding template. An essential feature of the

amplification process is that all previously synthe-sized products in the previous cycle act as templatesfor the subsequent cycle, resulting in geometricamplification. Twenty cycles, which take as little as2 h, result in a millionfold increase in the amountof target DNA, which can be visualized by elec-trophoresis and staining with ethidium bromide, orby using a DNA probe.

The success of molecular instrumentation will belargely dependent on the development of appropri-ate sample extraction and purification technolo-gies. Successful systems will need to integrateautomated, high-throughput, extraction platformswith downstream detection systems. A number ofsystems are showing much promise. The QIAmp96 DNA blood kit and BioRobot 9604 (Qiagen,Valencia, USA) are able to automate the isolationof DNA from blood, plasma, serum, bone marrowand body fluids. The process uses silica gel mem-brane technology for isolation of nucleic acids anda buffer system that allows for selective binding ofnucleic acids to the membrane, washing and elu-tion of the nucleic acids.

The BioRobot is reported to process up to 96samples in microwell plate format within 2 h, anduses an automated tip change system to avoidcross-contamination and a barcode reader for sam-ple identification.

The Biomek 2000 Laboratory AutomationWorkstation from Beckman Coulter (Fullerton,CA, USA) and the Dynal (Oslo, Norway) DNADIRECT Auto 96 method based on magnetic-beadcapture of DNA from whole blood is anotherpromising high-throughput system. The BeckmanCoulter system allows the simultaneous processingof up to 96 samples in 10 min.

Other manufacturers are marketing walk-awayDNA extraction systems. Organon Teknika(Durhan, NC, USA) uses solid-phase technology inthe form of silicon dioxide particles to bind nucleicacids. Autogen (Framingham, MA, USA) producesvarious models of automated DNA and RNAextraction systems, which are based on traditionalnucleic acid extraction chemistry.

There are, however, many challenges to nucleicacid extraction. Automated instruments will need to

Molecular microbiology 125

consider quality control requirements before rou-tine implementation of this technology can beachieved, and the ability to interface with high-throughput PCR systems will be an important fac-tor. Roche Molecular Biochemicals (Indianapolis,IN, USA) and Applied Biosystems, Inc. (ABI; FosterCity, CA, USA) have announced plans to launchautomated nucleic acid extraction systems that inter-face with their corresponding systems for PCR and PCR product detection. The ABI PRISM 6700 instrument promises integration with the ABIPRISM 5700, and the Roche MagNApure will inte-grate with their LightCycler PCR instrument. Systemssuch as these can be programmed to automate bothextraction and PCR set-up, providing a benchmarkfor high-throughput extraction technology.

Polymerase chain reaction technology is continu-ally improving and now uses much shorter cycletimes and improved formats for detecting PCRproducts with reduced incidence of cross-contami-nation.

These features are incorporated in some of the com-mercially available analytical systems. The semiau-tomated COBAS AMPLICOR system (RocheDiagnostics Corp., USA) uses microwell plates con-taining appropriate reagents and probes, anddepends on a biotin-labelled, enzyme-linked methodto detect amplified nucleic acids following multiplethermocycling steps. This system completes a run inabout 4 h and is being used for qualitative testing ofhepatitis C virus (HCV), Mycobacterium tubercu-losis, M. avium, M. intracellulare, Chlamydia tra-chomatis and Neisseria gonorrhoeae, andquantitative testing of cytomegalovirus (CMV),hepatitis B virus (HBV), HCV and human immun-odeficiency virus type 1 (HIV-1). Livengood andWrenn (2001) have shown the system to be an accu-rate, rapid, and cost- and labour-efficient methodfor the detection of Chlamydia trachomatis andNeisseria gonorrhoeae.

‘Real-time’ PCR assays

‘Rapid-cycle real-time PCR’ is now available inseveral commercially available instruments, includ-

ing the LightCycler from Roche Applied Science,the Smart Cycler from Cepheid in Sunnyvale (CA,USA) and the GeneAmp 5700 and its updated ver-sion, Prism 7700, from Applied Biosystems. Otherrapid-cycle PCR instruments are available, but atthis time there are few published clinical data withregard to their application for microbiology test-ing. The principal advantage of real-time PCR isthat the formation of amplification products can bequantified and monitored in real time. The highsensitivity and short turnaround time of these sys-tems could soon have a significant impact onpatient care. Accuracy is improved and results areavailable rapidly, providing opportunities to diag-nose infections sooner, improve patient care anddecrease the overall costs of providing that care.

Microarray technology

Microarray technology will eventually be applica-ble to clinical microbiology laboratories and islikely to allow testing that may include detection ofmultiple gene targets for organism identification ordrug-resistance patterns (Marshall and Hodgson,1998; Tomb, 1998; Kozian and Kirschbaum, 1999;Diehn et al., 2000). The basic technology revolvesaround DNA complementary to the genes of inter-est being generated and laid out in microscopicquantities on a solid surface at predetermined posi-tions. DNA from samples is eluted over the surface,and complementary DNA binds and is generallydetected by fluorescence after excitation.

Chips from several manufacturers are presentlyavailable. The GeneChip array (Affymetrix SantaClara, CA, USA) has oligonucleotide probesequences attached to a 1 cm2 glass chip substrate.Each characterized probe is located in a specificand identifiable area on the probe array. TheGeneChip instrument system can be purchasedwith a fluidics station, a hybridization oven, anAgilent GeneArray to measure emitted light, a PCworkstation and GeneChip Data Analysis Suitesoftware. Oligonucleotides can be placed on chipsby a variety of technologies.


Current limitations of microarray technologyinclude the high costs of instrumentation and dis-posables and our current inability for easy analysisof the enormous amount of information availablethrough this technology. Production costs willdecline over the coming years, although it is unlikelythat this technology will play a significant role indiagnostic clinical microbiology in the immediatefuture. For a thorough review of suppliers ofmicroarrays, see Cummings and Rehman (2000).

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DIRECT EXAMINATION

Microscopic examination is a crucial part of thediagnostic process for most fungal infections andobservation of fungal elements in clinical materialcan greatly enhance the significance of a subse-quent isolation of a yeast or mould. Moreover,such examination can lead to a rapid presumptivediagnosis, often days before a positive culture isobtained. In tissue specimens such as biopsies, andnail and skin scrapings, the presence of hyphae isdiagnostic, but it is important to culture the mate-rial to establish the identity of the pathogen. Insome cases the pathogen may fail to grow in cultureand the microscopy result will be the only evidenceof a fungal aetiology.

Arrange hair, skin or nail fragments on a slide ina drop of mounting fluid (see below). Apply a coverglass and then leave for a few minutes or, inthe case of nail specimens, up to 30 minutes, untilthe preparation softens and ‘clears’. Then applygentle pressure to the top of the cover glass to pro-duce a monolayer of cells for microscopic examina-tion.

Use the same procedure for small fragments of tis-sue from biopsy samples and the residue from cen-trifuged Sputasol-treated sputum, brochoalveolarlavage (BAL), peritoneal dialysis or other body flu-ids. Make wet films of pus or other exudates inequal volumes of potassium hydroxide (KOH) andglycerol if a more permanent preparation isrequired. Mix cerebrospinal fluid (CSF) depositwith an equal volume of 2.5% nigrosin in 50%glycerol or Indian ink. Glycerol prevents mouldgrowth in the solution, delineates capsules moresharply and prevents preparations, which should

be thin, from drying out. Smear swabs from casesof mucosal candidiasis on a glass slide and stain byGram’s method. As the small budding yeast cells ofHistoplasma capsulatum are difficult to detect inwet mounts, add Calcofluor white, 0.1% in dis-tilled water or examine Giemsa-stained prepara-tions.

Fluorescence microscopy

An optical brightener, such as Calcofluor white,may be used to enhance the natural fluorescence offungal cell walls. Mixed in equal volumes withKOH, this will facilitate the microscopic examina-tion of specimens because it will bind to the chitinin the fungal cell walls. Examine preparationsunder a fluorescence microscope fitted with anultraviolet (UV) light source, and appropriate exci-tation and barrier filters.

Mounting fluid and stains

Potassium hydroxide Use a 20% solution of KOH in water to ‘clear’specimens so that fungal mycelia and yeast cellsmay be more easily seen.

Lactophenol–cotton blue This is commercially available or may prepared asfollows.

9

Mycological methods

LactophenolPhenol crystals 20 gLactic acid 20 mlGlycerol 40 mlDistilled water 20 ml

Heat gently to dissolve.

Lactophenol–cotton blueCotton blue 0.075 gLactophenol 100 ml

Store away from direct sunlight.

LactofuchsinAcid fuchsin 0.1 gLactic acid 100 ml

Store away from direct sunlight.Mount small portions of moulds from foods in

water or, if the material is water repellent, in lac-tophenol with or without cotton blue.

Polyvinyl alcohol-mounting fluid Polyvinyl alcohol granules 17 gLactic acid 85% 80 mlDistilled water 20 ml

Heat the polyvinyl alcohol (PVA) in the distilledwater in a 55°C water bath with frequent stirring.Add the lactic acid and transfer to a boiling waterbath, stirring occasionally. The resulting clear vis-cous fluid may be kept for several years at roomtemperature.

Direct methods for Pneumocystiscarinii (Pneumocystis jiroveci)

Cysts of P. carinii in induced sputum or BAL sam-ples may be stained with Grocott’s methenaminesilver (see original paper for method: Grocott,1955), toluidine blue-O or cresyl violet. Thetrophozoites stain less readily with these agents andGiemsa, Diff-Quik or Gram–Weigart stains areused instead. Currently, there are several kits avail-able for indirect immunofluorescence stainingusing monoclonal antibodies raised against P.

carinii, which work well although there may besome false positives as a result of cross-reactions.

Molecular methods are under development forthe detection of this organism.

Histological examination

Histological examination of biopsy material canhelp to distinguish hyalohyphomycosis (infectionswith moulds with hyaline or colourless hyphae),phaeohyphomycosis (infections with moulds withpigmented hyphae), zygomycosis in which thehyphae are broad and aseptate, and yeast infec-tions. There are several stains that can be used toenhance the microscopic appearance. Grocott’smethenamine silver stain (Grocott, 1955), a specificstain for fungal cell walls, is the most useful. Thisstains fungal cell walls dark brown or blackwhereas the host tissue is counterstained green.Haematoxylin and eosin (H and E) produces lessdistinction between fungus and host cells, but maybe useful for Zygomycetes and in the staining ofmycetoma granules. Mayer’s mucicarmine specifi-cally stains the polysaccharide capsular material ofCryptococcus neoformans. Periodic acid–Schiff(PAS) is also used to stain cryptococci. Zygomycetehyphae, unlike the hyphae of other moulds, arespecifically stained by cresyl fast violet. Giemsastain is useful for Histoplasma capsulatum andmay also be used on fresh specimens. Fungalimmunostaining with antibodies raised to cell wallantigens can also be employed using immunofluo-rescence (bright green) or immunoperoxidase(dark). Such staining can be useful for diagnosticpurposes when the organism has not been isolatedor when only formalin-fixed tissue is available.

Direct examination of mould growthson foods and other perishablematerials

Examine with a lens or low-power microscope insitu to see the arrangement of spores, etc. before

130 Mycological methods

disturbing the growth. Remove a small piece ofmycelium with a wire or needle. It may help if theend of the needle has been bent at right angles togive a short, sharp hook. Use the point of the nee-dle to cut out a piece of the growth near the edgewhere sporulation is just beginning. Transfer it to adrop of lactophenol–cotton blue, lactofuchsin orPVA mountant on a slide, apply a cover slip andthen examine after 30 min when the stain has pen-etrated the hyphae. If bubbles are present warmgently to expel them.

ISOLATION OF MOULDS ANDYEASTS

Primary isolation

Most fungi are not particularly fastidious in theirnutritional requirements and will grow quite read-ily on many bacteriological agars. However, themedium most commonly employed for the isola-tion of fungi is glucose peptone agar (Sabouraud’sagar) containing 0.5 g/100 ml chloramphenicol. Ifthe medium is to be used to isolate dermatophytesadd cycloheximide to inhibit overgrowth by con-taminating moulds. Use glucose peptone agar, maltagar or one of the proprietary media. Note, how-ever, that the culture media has a marked influenceon the colonial growth of most fungi, particularlythe dermatophytes, and most of the identificationmanuals describe or illustrate the colonial growthon glucose peptone agar. Place about 20 pieces ofhair or skin into the surface of the medium. Do notpush them into the agar. Cut nail clippings intosmall pieces with a scalpel before culturing. Piecesof skin, hair or nail powder may be picked up moreeasily if the heated inoculating needle is first pushedinto the medium to moisten it.

Spread pus, CSF, sputum or BAL deposits, orbiopsy material from suspected cases of the inva-sive mycoses, on slopes of glucose peptone agarwith chloramphenicol. Medical flats containingthis agar may be used because they provide a largersurface area to increase the chance of isolation.

Plates may also be used but they are more suscepti-ble to contamination by environmental moulds,and offer less protection in the case of isolation ofan unexpected Hazard Group 3 pathogen. It is alsouseful to set up a purity culture at the same time asthe sample to alert one to the presence of contami-nating moulds in the culture environment. Cut tis-sues into small pieces. Do not homogenize thembecause this would break fungal mycelium intonon-viable fragments. This is particularly true forthe Zygomycetes, which can be difficult to culturefrom necrotic tissue even when the hyphae havebeen seen on microscopic examination.

Wash mycetoma grains several times with sterilesaline to remove most contaminants before culturing.

Incubate cultures at 28°C but if the material isfrom a deep body site incubate also at 37°C to has-ten growth of the pathogen. Most yeasts grow in2–3 days, although Cryptococcus spp. may requireincubation for up to 6 weeks. Common moulds,too, will grow in a few days. Cultures for dermato-phytes should be kept for 2 weeks although mostare identifiable after 1 week. It is good practice toexamine all mould cultures once or twice a weekand ensure adequate aeration.

Dimorphic Hazard Group 3 pathogensSpecimens suspected of containing Hazard Group3 pathogens should be handled in the appropriatecontainment Level 3 Laboratory. The above meth-ods may be used but slopes or medical flats ratherthan plates should be used for isolation purposes.If the suspected pathogen is one of the group ofdimorphic fungi, an additional culture on a slopeof brain–heart infusion agar incubated at 37°Cmay yield the yeast form of the fungus and help toconfirm its identity.

Isolation from food, soil, plantmaterial, etc.

Inoculate plates of chloramphenicol-containingglucose peptone agar or other selective media oruse dichloran glycerol (DG18) medium.

Isolation of moulds and yeasts 131

Dip slides, coated with appropriate media, are use-ful for liquid samples or homogenates and are avail-able commercially. Zygomycetes, if present, willoutgrow everything else but are markedly restrainedwhen grown on Czapek-Dox agar. To grow mouldsfrom physiologically dry materials such as jam,increase the sucrose content of the Czapek-Dox agarto 20%. Some yeasts can tolerate low pH levels andmay be isolated on malt agar to which 1% lactic acidhas been added after melting and cooling to 50°C.Incubate cultures at room temperature and at 30°C.

Enumeration of moulds in foods, etc.

Make a 10% suspension of the material in sterilewater, Ringer’s or peptone water diluent. Process ina Stomacher (other blenders may raise the temper-ature and injure the moulds). Make tenfold dilutionsand surface plate on selective media (e.g. AspergillusFlavus Parasiticus agar [AFPA], oxytetracyclineyeast extract agar [OGYE] or, for xerophilicmoulds, DG18). Incubate and count colonies onplates that have between 50 and 100. Calculate thenumbers of mould propagules per gram.

Methods for individual foods are given in Chapter14. Useful sources of information are Pitt andHocking (1985), King et al. (1986), Krogh (1987),and Sampson and van Reenan-Hoekstra (1988).

Sampling air for mould spores

Various air-sampling instruments are described inChapter 19. A cheap, simple device is the Portonimpinger in which particles from a measured vol-ume of air are trapped in a liquid medium on whichviable counts are done. More information is gainedby using the Andersen sampler, which captures theairborne particles using the principle that thehigher the air speed the smaller the particle thatwill escape the airstream and be impacted on anagar surface. Plastic Petri dishes give lower countsthan glass ones but they make the whole machinemuch easier to handle.

The medium and time of incubation will dependon the organism sought. For general use one runwith Czapek-Dox agar and one with chloram-phenicol glucose peptone or malt agar will serve;for thermophilic actinomycetes use glucose pep-tone agar without antibiotics; incubate one set at40°C, the other at 50°C.

As invasive aspergillosis is often a nosocomialinfection and high-risk patients may be subjected tothe inhalation of potentially lethal fungal sporeswithin the hospital environment, it is helpful tomonitor for the presence of fungal spores in somepatient areas. The air-sampling techniques outlinedabove may be employed but will provide only asnapshot of the spores that were airborne at the timeof sampling and lack sensitivity unless a large vol-ume of air is sampled. A longitudinal approach is tosample horizontal surfaces for the presence of fun-gal spores. This can be made more quantitative bysampling a given area in a room. Moisten a swab insterile distilled water, wipe it over an area of 0.5 m2

and on the surface of a Sabouraud agar plate con-taining chloramphenicol. Incubation of the plate at45°C for 48 h will select for Aspergillus fumigatus.Incubation at 28°C will also allow the growth ofother environmental moulds. A modification of thesettle plate method may also be employed, in whicha high-sided, screw-capped receptacle with at leasta 5-cm aperture (‘honey pot’) is left open at a par-ticular point in the area to be sampled for 7 days.After this time any spores that it contains are har-vested by adding a volume of 4 ml sterile saline with0.01% Tween 80, closing the lid, shaking and thenplating on to Sabouraud’s agar with chlorampheni-col and incubating as before.

IDENTIFICATION OF MOULDS

Most molds may be identified by examining micro-scopic and macroscopic morphology. Colony form,site, surface colour and pigmentation are usefulfeatures but microscopic examination is essential.Well-sporing mould colonies may usually be read-ily identified. If spores are not seen, re-incubateand/or subculture on to other media (potato


sucrose agar, malt agar, Borelli’s lactrimel agar andhalf cornmeal agar are all useful for stimulatingsporulation). Sporulation may sometimes be stimu-lated by incubation under near-ultraviolet (‘black’)light or by exposing the colonies to alternatingcycles of light and dark (diurnal cycles).

The usual methods of examining sporing struc-tures are needle mounts and/or tape mounts. Toprepare a needle mount take a small fragment ofmycelium from the colony with a sharp needle andtease it out in a drop of mounting fluid. Place acover glass over the specimen and tap it a few timesto help to spread the mycelial fragments within themounting fluid. The spores are usually most visibleat the edges of the mycelial mass.

Tape mounts have the advantage that spores canbe viewed without disruption of their normal con-figuration. Cut a strip about 1.5 cm long from aroll of sticky tape preferably using double-sidedtape and a plastic dispenser. Attach one end to amounted needle, forming a flag. Touch the surfaceof the colony with this near the growing edge. Toimprove the optics mount the tape, sticky side up,in a drop of mountant, add another drop of moun-tant and overlay this with a cover glass, so that thestructures are not being examined through a layerof tape. Examine immediately, because tapemounts are not suitable for long-term preservation:the mounting fluid renders the sticky tape opaque.

The centre of a well-sporing colony may shownothing but spores, in which case it may be desir-able to make a second preparation nearer to theedge of the colony or to try a different preparationtechnique such as a needle mount. Conversely, apoorly sporing colony may show spores only in itscentre and it may be necessary to dig down into theagar with the tip of a needle. It will frequently benecessary to make several mounts to be able to dis-cern the sporing structures and precise mechanismof spore production.

Slide culture

This enables the spores and sporing structures to beexamined in situ and permits a more critical exam-

ination. Cut small blocks of medium (0.5 × 0.5 cm)from an appropriate agar-poured plate and transferthem to the centres of sterile slides. Inoculate theedges of each block with material from a well-growing colony and apply a sterile cover glass ontop of the block. Incubate in a Petri dish containingmoist filter paper and glass rods to support the slide(wet chamber). Once visible growth has developed,monitor its progress with the low-power objectiveof the microscope. When typical structures are vis-ible prepare a slide and cover slip with a drop ofmountant. Lift the cover glass carefully off the slideculture and mount it on the prepared slide. Lift offand discard the agar block. Place a drop of mount-ing fluid on the original slide to produce a secondmount. Examine both preparations after 30 min.

Exoantigen identification methods andGene–Probe

The exoantigen test (Immuno-Mycologics, Inc.) isdesigned to permit the identification of the dimor-phic fungal pathogens Histoplasma capsulatum,Coccidioides immitis and Blastomyces dermati-tidis. The method consists of a double-diffusiontest in which an antiserum specific to each of thesethree fungi is run against an extract of the isolate tobe identified. The production of precipitin lineswith one of the control extracts of the threepathogens confirms the identification. Anothercommercial method, Gen-Probe, has also beendeveloped for these Hazard Group 3 pathogens.

Molecular identification methods

Molecular identification methods have the advan-tage of speed and may allow identification of non-sporing moulds and the differentiation ofmorphologically similar groups of yeasts andmoulds. They may also prove useful in the identifi-cation of yeasts and moulds in tissue sections whenculture of the organism has failed or when only for-malin-fixed tissue sections are available.Amplification of the fungal DNA is carried out by

Identification of moulds 133

polymerase chain reaction (PCR) and then sub-jected to a series of species-specific probes or DNAsequencing, e.g. various primers have been devel-oped for the PCR amplification of fungal genomicribosomal DNA sequences. Species-specific probescan then be used to detect matching sections or theprecise order of bases may be sequenced and com-pared with stored database sequences for differentfungal species. These methods of mould and yeastidentification are in various stages of developmentand a careful morphological examination, andin the case of yeasts analysis of biochemicalprofiles, is an essential adjunct to confirm theidentification.

Molecular strain typing

Examination of the degree of similarity of fungi ofthe same species may be a useful tool for tracing thesource of infection in an outbreak. It can also helpto track the development of drug-resistant strainsover time in a given population or help to establishwhether a current infection is re-infection orrelapse. Most methods based on phenotypic char-acteristics have proved disappointing and recentdevelopments have concentrated on an assessmentof genetic relatedness. Various methods have beenexamined, including restriction fragment lengthpolymorphism (RFLP), random amplified poly-morphic DNA (RAPD), multilocus enzyme elec-trophoresis (MEE), probes based on repetitiveelements, analysis of microsatellite regions andelectrophoretic karyotyping. Numerous publica-tions attest to the relative merits of each approach.

EXAMINATION OF YEASTCULTURES

Microscopic morphology

To study yeast morphology use a Dalmau platecomprising cornmeal agar with 1% Tween 80.Streak the organism on a plate of the medium,

cover with a sterile cover glass and incubate at30°C. Examine after 3–4 days through the coverglass with × 10 and × 40 objectives. Look forcolony colour, cell shape, and the presence orabsence of mycelia, pseudomycelia, arthrospores,chlamydospores and capsules.

To encourage ascospore formation inoculatesodium acetate agar made by adjusting the pH of0.5% sodium acetate to 6.5 with acetic acid, thensolidifying it with agar. Stain by the malachitegreen–safranin method. Prepare a smear in a dropof water and dry in air. Fix by heat and cover with1% malachite green for 2 min. Heat until steamrises, wash with tap water and counterstain withsafranin for 1 min. Wash, blot and dry. Ascosporesare stained green and vegetative cells red.

The germ tube test

A test for the production of a true mycelium candifferentiate Candida albicans and the recentlydescribed Candida dubliniensis from other yeasts.Add a small portion of a colony (over inoculationcan lead to autoinhibition of germ tube produc-tion) to a tube containing 0.5 ml horse serum andincubate at 37°C for 3 h. Then remove a drop toview under a microscope. The production of ashort germ tube initially without constriction at thepoint of emergence from the blastospore is diag-nostic for these two species.

Fermentation tests

These differ from those used in bacteriology in thatthe sugar concentration is 3%. Durham tubes arealways used to detect gas formation and a 10-mlvolume of medium is preferable to encourage fer-mentation rather than oxidation.

Inoculate a set of the following sugar media:glucose, maltose, lactose, raffinose and galactose.Incubate cultures at 25–30°C for at least 7 days.Gas production indicates fermentation.


Auxanograms

Fermentation tests alone are insufficient to identifyspecies of yeasts and assimilation tests should alsobe performed. Culture the test organism on a slopeof pre-assimilation medium. Prepare a heavy sus-pension by adding about 5 ml sterile water to theslope and gently suspend the growth; it is not nec-essary to wash the organisms. Melt tubes of aux-anogram carbon-free base (for carbon assimilation)and nitrogen-free base (for nitrogen assimilation)and cool to 45°C. Add about 0.25 ml of the yeastsuspension to each 20 ml auxanogram medium andimmediately prepare pour plates in 9-cm Petridishes. When set, place up to five discs of carbon ornitrogen sources well apart on the surface of theappropriate medium. Incubate at 28°C and exam-ine the plates daily for up to 7 days for a halo ofgrowth around the substrate.

Carbon sources commonly used are glucose (con-trol), maltose, sucrose, lactose, inositol, galactose,raffinose, mannitol and cellobiose. Nitrogensources include sodium nitrate, asparagine andethylamine hydrochloride. Alternatively prepareslopes (or tubes of liquid) of a basal medium con-taining only the test sugar and an indicator.Inoculate with a suspension of the test organismtaken from pre-assimilation medium and note thepresence or absence of growth for a 3-week period.Much more extensive series of test sugars may beemployed to characterize the physiological proper-ties of the test yeast and these may be analysed byreference to specialist texts, which, however, arebeyond the scope of most routine microbiologylaboratories.

Commercial ‘kit’ tests for yeastidentification

There are several commercial kits that are satisfac-tory for the identification of most strains of yeastencountered in clinical laboratories. Some usemethods based on traditional criteria, whereas oth-ers rely on novel approaches. In all of them, how-ever, the databases are from a restricted list of

species and occasional misidentifications mayoccur. When using the kits, therefore, it is crucial toexamine the yeast morphology on a cornmeal agarplate; for many of them this forms an importantpart of the numerical identification profile.Examination of the morphology can help to mini-mize the danger of misidentification.

Chromogenic agars

There are commercial agars incorporating chro-mogenic substrates which lead to the production ofdifferent colony colours for different yeast species.Such agars may be used as the basis for identi-fication of a limited range of yeast species andare particularly useful for detecting mixed cultures.

Mycotoxins and seed-borne moulds

Mycotoxins are metabolites of fungi, which may beproduced during mould growth on foods and ani-mal feeds. The most heavily contaminated com-modities are usually cereals and oilseeds. Themycotoxigenic moulds most frequently encoun-tered belong to the genera Penicillium, Aspergillusand Fusarium (Moss, 1989). Repeated ingestion ofaflatoxin, produced by Aspergillus flavus, probablyhas the most serious long-term consequences,because it is a powerful carcinogen.

Surface sterilize seeds by immersion for 2 min insodium hypochlorite (0.4% available chlorine). Placeup to 10 small or 5 large seeds on DG-18 agar in aPetri dish and incubate at room temperature.Subculture from colonies to oatmeal agar (Aspergillus),Czapek yeast autolysate agar (Penicillium) or syntheticnutrient-poor agar (Fusarium).

SEROLOGICAL METHODS

Antigen production

There is now little call for laboratories to producetheir own antigen preparations because commer-

Serological methods 135

cially produced, standardized and quality controlledantigens are now available to test for antibody pro-duction to a wide range of fungal pathogens.

Antibody detection

Antibody detection is useful in immunocompetentpatients but is of less benefit for patients who havean invasive disease and often have an underlyingimmunological deficiency. Detection of antibody toAspergillus spp. can help to confirm aspergilloma,allergic aspergillosis, acute bronchopulmonaryaspergillosis and aspergillus endocarditis.Detection of antibodies to Candida spp. is thesingle most consistent diagnostic finding in patientswith candida endocarditis and can also be useful inpatients with invasive candida infections after sur-gery where an antibody titre of ≥ 1 : 8 is usuallyindicative of active infection. Complement fixationand immunodiffusion antibody tests are useful inthe diagnosis of blastomycosis, histoplasmosisand coccidioidomycosis. Both yeast and mycelialphase antigens of Histoplasma are used. All thereagents required for these tests are commerciallyavailable.

Immunodiffusion

In the double diffusion (DD) test, serum and anti-gen are allowed to diffuse towards each other fromwells cut in an agar gel. Lines of precipitation formafter several days where individual antibodies andantigen meet at optimum concentration. Counter-immunoelectrophoresis (CIE) provides a morerapid and sensitive method for the detection of theprecipitins. It differs from DD in that an electriccurrent is passed through the agar gel for 90 min tohasten the migration process. In CIE tests, condi-tions are chosen in which antigens migrate towardsthe anode because their isoelectric points are lowerthan the pH of the buffer, and antibodies migratetowards the cathode as a consequence of electroen-dosmosis. Non-specific reactions and other arte-

facts resembling precipitin lines are more commonand more obvious with CIE than DD.

Agar base

Dissolve 2 g agar (Oxoid No. 1) in 100 ml water byautoclaving. Add 100 ml buffer heated to 50°C andmix with the agar. Keep in a water bath at 50°Cand discard if not used within 48 h of preparation.

BufferBoric acid (H2BO3) 10 g Powdered borax (Na2B4O7·10H2O) 20 g EDTA, disodium salt 10 g Water to 1000 ml

The pH should be 8.2. This buffer is doublestrength. Dilute it with agar for plates and slides orwith an equal volume of water for use in elec-trophoresis tanks.

PlatesPlace 30 ml agar into a plastic Petri dish and fit aPerspex jig with metal pegs in place of the lid toproduce the pattern of wells shown in Figure 9.1.The large wells are 6 mm in diameter and thesmaller wells 2 mm. The distance between the cen-tral and peripheral wells is also 6 mm.

The test serum occupies the central well, antigens


Figure 9.1 Double diffusion tests

the pairs of large and small wells (shown shaded),and appropriate control sera the top and bottomwells. Two antigens can thus be tested and a singlesolution suffices because the relative volumes (60 and6 μl in the two holes) give high- and low-concentra-tion gradients in the agar. This arrangement alsoallows reactions of identity to be obtained betweentest and control sera, thus eliminating some anom-alous reactions, particularly among Aspergillus spp.

Counterimmunoelectrophoresis

Buffered gelVeronal buffer 4 lPurified agar 20 gAgarose 20 gSodium azide 2 g

Add agarose and purified agar to buffer and steamat 100°C for 1 h. Add 2 g sodium azide, cool to 56°Cand adjust pH to 8.2. Dispense in 200-ml amounts.

Veronal buffer 0.05 mol/lBarbitone (caution) 13.76 gBarbitone sodium (caution) 30.28 gDistilled water 4.0 l

Adjust pH to 8.2 before use.

Saline–trisodium citrateSodium chloride 40 gTrisodium citrate 100 gSodium azide 1 gDistilled water 2.0 l

Buffalo black staining solutionBuffalo black 0.5 gDistilled water 500 mlEthanol 400 mlAcetic acid 100 ml

Gel destaining solutionMethanol 900 mlGlacial acetic acid 200 mlDistilled water 900 ml

MethodTwo gels are required for Aspergillus fumigatusprecipitins (●): one for the somatic antigen at lowand high concentrations, and the second for theculture filtrate antigen at low and high concentra-tions (Figure 9.2). These should be labelled 1 and 2,3 and 4, etc. depending on the number of tests. Thetop right-hand corner of each gel should be labelledwith alcian blue.

• All wells marked ● must be filled with 10 μlof the appropriate antigen: 2 mg/ml (lowconcentration) or 20 mg/ml (highconcentration).

• All wells marked must be filled with 10-μlvolumes of the patient’s serum.

• The first row of each gel must contain thepositive control serum.

Place the slide in an electrophoresis tank contain-ing 0.05 mol/l veronal buffer. Connect the slide tothe buffer with lint wicks, ensuring that good con-tact is made along the full width of the gel. Theantigen wells should be adjacent to the cathode andthe serum wells adjacent to the anode. Set theequipment to deliver a constant voltage of 30 V perslide and run for 90 min.

Once the gel has been run, immerse in a Petri dishcontaining saline–trisodium citrate for 18 h(overnight). Rinse the gel with tap water, cover itwith a moistened filter paper square, and dry witha hair dryer or inside a heated cabinet for about1 h. Once the gel has dried, moisten the filter paperwith tap water, remove it and rinse the gel. Dryonce more for about 10 min.

To stain the precipitin lines immerse the slide for

Serological methods 137

S Lo S HiS Lo S HiSomatic antigen Culture filtrate antigen

Figure 9.2 CIE template

10 min in Buffalo black solution followed by twochanges of destaining solution until the back-ground is colourless.

If rapid results are required, wash the wet gel for1 h and examine by dark-ground illumination. Thisgives a preliminary result only and must be con-firmed after washing and staining.

Care must be taken in interpretation of the tests.Clearly defined lines between the serum and anti-gen wells are an indication of a positive result andthe number of lines seen is an indication of thestrength of the reaction. If necessary, the serumsample can then be tested at increasing dilutions ina doubling dilution series to determine the titre,which is the highest dilution at which an anti-body–antigen precipitin line is still visible. Linesthat appear as blurred smudges are unlikely to besignificant, and also those that follow around theedges of the well.

Enzyme-linked immunosorbent assaytest for antibody detection

The enzyme-linked immunosorbent assay (ELISA)test is a sensitive method and can be used to detecteither antibody or antigen. To detect antibody thewells of a 96-well microtitre plate are coated withthe appropriate antigen. Antigens are availablefrom commercial sources and include both mycelial(somatic) and culture filtrate preparations. Aftercoating, plates are washed three times in phos-phate-buffered saline (PBS)–Tween, then 0.1-mlvolumes of test and reference sera diluted 1 : 100 inPBS–Tween are added to designated wells. Afterincubation at room temperature for 2 hours thewashing steps are repeated. Various detectionmethods can be used at this stage, including goat orrabbit anti-human IgG coupled to an enzyme, usu-ally alkaline phosphatase or peroxidase, added in0.1-ml volumes in PBS–Tween with 1% bovineserum albumin and incubated at room tempera-ture. After 2 h, the washing steps are repeated and0.1 ml of the appropriate enzyme substrate isadded and the reaction is allowed to continue for

30 min, then stopped by the addition of 50 ml of 1mol/l sulphuric acid. The absorbance value of eachwell is determined and results can be calculated byreference to the control sera.

There have been many modifications to this basicmethod including incubation at 37°C to speed upreaction times and the introduction of a furtherstep known as sandwich ELISA to enhance sensi-tivity.

Commercial antibody detectionmethods

In addition to the commercial availability of alarge array of fungal antigens and antibodies,some companies have developed methods forthe detection of antibodies to Candida andAspergillus spp. Commercial ELISA kits are avail-able as well as others using the principle ofhaemagglutination.

Antigen detection

Commercial antigen detection kits are available forthe early diagnosis of infections by Cryptococcus,Candida and Aspergillus spp. Latex agglutinationkits have been available for the detection of crypto-coccal antigen for more than 20 years and havesensitivity and specificity that are > 90%. The kitcan be used with serum or CSF samples. It is advis-able to test neat serum or CSF at a 1 : 10 dilution inan initial test to overcome potential problems withthe prozone effect caused by an excess of antigen.In cases of a positive reaction, doubling dilutionsshould be tested until an endpoint is reached. Highor rising titres are indicative of progressive infec-tion and in some patient groups monitoring of anti-gen titres is a useful prognostic indicator. Antibodydetection is less useful in the initial diagnosis ofcryptococcosis, but can be indicative of a goodprognosis because it is often detectable only oncean infection has almost resolved.


Latex agglutination tests have proved less success-ful for the detection of infections with Aspergillusand Candida spp. because they have suffered froma lack of sensitivity and specificity. However, thereare more sensitive commercial sandwich ELISAtests for the detection of aspergillus galactomannanand candida oligomannosides. These tests haveproved useful in certain patient groups and showpromise in the diagnosis or confirmation of inva-sive infection in some immunocompromisedpatients.

For further information on the serodiagnosis offungal diseases, see MacKenzie et al. (1980).

MOLECULAR METHODS INDIAGNOSIS

Currently molecular methods for the detection ofcirculating fungal genomic sequences are underevaluation for the diagnosis of invasive fungalinfection. Panfungal, as well as species-specific,primers are employed with various detection meth-ods including nested PCR, PCR–ELISA, PCR–blot,real-time PCR with light-cycler analysis and PCRsequencing. Test specimens include whole blood,serum, CSF, BAL fluid and tissue samples.Published DNA extraction methods vary fromphysical freeze–thaw techniques to enzymaticextraction. Development is still required to pro-duce standardized tests with acceptable levels ofsensitivity, specificity and reproducibility.

ANTIFUNGAL DRUGS

There are several different techniques for assessingthe susceptibility of an organism to an antifungalagent. The simplest method, which is suitable forthose agents that are water soluble, is based onagar diffusion. There are disc diffusion methods forflucytosine (5-fluorocytosine), fluconazole andvoriconazole. This approach has been furtherrefined in the commercially available E test.

Recent years have seen much effort expended on

the development of reproducible, standardizedmethods for the susceptibility testing of yeasts andmoulds. The National Committee of ClinicalLaboratory Standards (NCCLS, 2002a, 2002b) hasproduced two approved methods: M27-A2 foryeasts and M38-A for filamentous fungi. Bothmethods are based on a broth dilution format thatcan be miniaturized and performed in microtitreplates. The test conditions are as follows.

Test conditions for M27-A2 and M38-A

Medium RPMI 1640 with0.165 mol/l MOPS pH 7.0(Sigma)

Drug solvents Dimethylsulphoxide(DMSO) or water

Inoculum (cells/ml) M27-A2 for yeasts0.5–2.5 × 103

M38-A for moulds0.4–5 × 104

Incubation temperature 35°CIncubation duration 48 h (rarely 72 h)

Endpoint readings Complete inhibition, 80%inhibition or 50% inhibitiondepending on drug–organism combination

QC and reference Candida albicans ATCCorganisms 90028

C. albicans ATCC 24433C. krusei ATCC 6258C. parapsilosis ATCC

22019C. tropicalis ATCC 750Aspergillus flavus ATCC

204304A. fumigatus ATCC 204305

Drug dilutions are prepared and placed into eithertubes (1-ml final volume) or the wells of microtitreplates (0.2-ml final volume). The inoculum (0.1 ml)is added, tubes or plates are incubated at 35°C and

Antifungal drugs 139

read after 48 h, although it is sometimes possible toread after 24 hours. The endpoint is the lowest con-centration at which there is complete inhibition ofgrowth (amphotericin B and all drugs exceptflucytosine, fluconazole and ketoconazole in M38-A), 80% (flucytosine and azoles in M27-A) or 50%reduction in turbidity compared with a drug-freecontrol (flucytosine and azoles in M38-A). Thereare proposed breakpoints for flucytosine, flucona-zole and itraconazole for yeasts based on invitro–in vivo correlations (Table 9.1).

Table 9.1 Proposed breakpoints (milligrams per litre) forantifungal susceptibility tests

Disc test sensitivity methods

Disc diffusion methods indicate relative susceptibil-ity depending on zone size and do not provide aminimum inhibitory concentration (MIC). Theseare still useful for flucytosine, fluconazole andvoriconazole which are relatively water soluble,although care should be exercised in selecting thebasic medium because components of some com-plex media can obfuscate the antifungal activity.

Yeast morphology agar (YMA) can be used forflucytosine and high-resolution medium (HR) orMueller–Hinton agar for fluconazole and voricona-zole. The appropriate control organisms areCandida kefyr National Collection of PathogenicFungi (NCPF) 3234 for flucytosine and Candidaalbicans American Type Culture Collection(ATCC) 90028 for fluconazole and voriconazole.

FlucytosineInvert the plate and bisect it with a line drawn onthe base. Use a sterile swab to inoculate half of the

plate with the control organism and the other halfwith the test organism, both at an inoculum con-centration of 1 × 106 cells/ml in sterile water. Oncethe agar has dried, use sterile forceps to place adrug disc (1 μg) in the centre of each plate.Incubate the plate at 30°C for 48 h, but examineafter 24 h for obvious resistance. If the inhibitionzone for test strain is 80% or more of that for thecontrol strain, the organism may be considered sen-sitive to flucytosine. Resistant isolates should betested by another method to determine the preciseMIC of the drug. Truly resistant organisms willoften grow right up to the disc; intermediate zonesor discrete colonies forming within the zone sug-gest the possibility of emergent resistance duringtherapy.

FluconazoleInoculate Mueller–Hinton agar supplemented with2% glucose and 0.5% methylene blue with a yeastinoculum adjusted to a 0.5 McFarland standard.Once the agar has dried place a 25-μg fluconazoledisc in the centre. Incubate at 35°C for 18–24 hand measure the zone of inhibition. Zone sizes canbe correlated with the NCCLS breakpoints:≥ 19 mm is susceptible, 13–18 mm is susceptible-dose dependent and ≤ 12 mm is resistant (Meis etal., 2000). The QC strain should produce a zone of32–43 mm. A similar method can be employed forvoriconazole but there are as yet no establishedbreakpoints for this drug.

E test methods

There has been a further refinement of the princi-ple of agar diffusion in the commercially availableE test strips, in which a concentration gradient ofthe drug is produced in an agar plate by surfaceapplication of a test strip. The technology hasevolved to allow the use of this method with themajority of systemically active antifungal agents.Even amphotericin B, which is a large moleculethat demonstrates notoriously poor agar diffusion,has been complexed with a carrier to allow a con-centration gradient to be produced. In the E test

Susceptible SDD Resistant

Flucytosine < 4.0 8.0–16.0 < 32.0Fluconazole < 8.0 16.0–32.0 < 64.0Itraconazole < 0.125 0.25–0.5 < 1.0

SDD, susceptible dependent on dose (intermediate)


method, a strip containing a concentration gradi-ent of the test antifungal agent is placed on thesurface of an agar plate that has been pre-inocu-lated with the test organism. After incubation azone of inhibition is visible and the MIC of theorganism is read at the point at which the inhibi-tion zone intersects the E test strip. This methodhas proved to be useful and to correlate well withother standardized methods for both yeasts andmoulds.

Other commercial methods forantifungal susceptibility testing

There is a commercial test system that follows theNCCLS methodology (Sensititre or Yeast one,TREK) in a microtitre format but with the incor-poration of a chromogenic substrate to facilitateendpoint readings. There are other tests that use abreakpoint system of growth or no growth inwells containing one low and one high concentra-tion of each of a panel of drugs (Fungitest, ATBFungus).

Methods of assay

Assay of antifungal drugs is necessary either to con-firm the attainment of therapeutic levels or toensure that toxic levels are not reached. There arecurrently two antifungal agents for which routineassay is recommended. These are flucytosine, tohelp manage the toxicity associated with high levelsof this drug, and itraconazole to ensure adequateabsorption following oral administration.

The preferred method for assay is high-perform-ance liquid chromatography (HPLC), which candetect the absolute level of an antifungal agent evenin the presence of another agent. This is an impor-tant advantage because antifungal agents areincreasingly used in combination. Moreover,HPLC assay will detect pure drug and, unlike aplate assay, will not be subjected to the vagaries ofan active metabolite.

Extraction of itraconazoleItraconazole is extracted from 1-ml volumes ofserum with heptane–isoamyl alcohol.

HPLC conditions for itraconazoleColumn Chromsep Hypersil 5 ODS (100

× 3 mm) (Chrompak cat no. 28260)

Guard column Chromsep Guard ColumnReversed Phase

(Chrompak cat. no. 28141)Mobile phase 60% acetonitrile + 40% water

with 0.03% diethylamine (adjustpH to 7.8 withorthophosphoric acid)

Flow rate 0.5 ml/minInjection volume 20 μlRun time 10 minWavelength 263 nmSensitivity 0.04 absorbance units fixed

sensitivity (AUF)

Bioassay of flucytosine (5-fluorocytosine)

MediumYeast Nitrogen Base Glucose Citrate (YNBGC)solution

Bacto Yeast Nitrogen Base 67 gGlucose 100 gTrisodium citrate 59 gDeionized water 1000 ml

Adjust the pH to 7.0. Filter sterilize and store in20-ml amounts.

AgarPurified agar 40 gCold deionized water 1800 ml

Heat to boiling to dissolve the agar and then auto-clave at 121°C for 15 min. Dispense in 180-mlamounts.

Antifungal drugs 141

Stock solutionsDissolve 50 mg flucytosine in 5 ml sterile water.Filter sterilize and add 1 ml of this solution to 9 mlsterile water to obtain a 1000 mg/l stock solution.This stock solution may be stored at –20°C for atleast 12 months. Use the 1000 mg/l stock solutionto make up the following standards: 12.5, 25, 50,100 and 200 mg/l and internal control solutions of35 and 70 mg/l. Dispense in 0.5-ml amounts andstore at –20°C until required. These solutions arestable for at least 12 months.

Preparation of inoculumAdd 2 ml concentrated YNBGC solution to 18 mlsterile water in a 25-ml glass bottle. Inoculate withthe sensitive Candida kefyr strain NCPF 3234, har-vested from a Yeast Morphology Agar (YMA)plate (up to 1-week old), to obtain a concentrationof about 1 × 107 cells/ml (absorbance reading ofabout 1.6–1.7 on the spectrophotometer at540 nm).

Assay procedureMelt 180 ml purified agar, cool to 80°C and add20 ml concentrated YNBGC solution. Pour themolten medium into a large square plate placed ona level surface and leave to set. Flood the entire sur-face of the agar with 20 ml of cell suspension inYNBGC. Pour off the excess and place the plate ina 37°C incubator with the lid removed. Leave untilthe surface of the agar has dried (about 20 min). Laythe plate on a sheet of paper (template) with a pat-tern of 30 positions to which test or standard num-bers are assigned at random. Punch 30 wells of4 mm diameter in the agar with a cork borer (sixrows of five wells) and remove the agar plugs. Place20 μl of standard internal control, or patient speci-men, in each well. Each specimen should be testedin triplicate and must be placed on the plate in a ran-domized distribution. Incubate the plate at 37°Covernight (about 18 h). Measure the diameters ofthe zone of inhibition around each well using cal-lipers or a zone reader. If performing a manual cal-culation, plot the mean diameters of the fivestandards against drug concentrations on semi-

logarithmic paper, and construct a standard curvefrom which the drug concentrations in the patientspecimens and internal controls may be interpo-lated. The drug concentrations should be plotted onthe logarithmic ordinate.

Bioassay of itraconazole

The procedure should be followed as above withthe following modifications:

Preparation of stock solutionsDissolve 10 mg itraconazole in 1 ml dimethylfor-mamide. Add 9 ml methyl alcohol to obtain a1000 mg/l stock solution. Take 1ml of this solutionand add 9 ml methyl alcohol to obtain a 100 mg/lstock solution. Use the 100 mg/l stock solution tomake up the following standards: 0.5, 1, 2, 4 and8 mg/l and internal control solutions of 3.5 and7 mg/l in horse serum. Dispense in 0.5-ml amountsand store at –20°C until required. These standardsare stable for at least 1 month.

Inoculum preparationThe indicator organism is Candida albicans strainNCPF 3281.

REFERENCES

Grocott (1955) A stain for fungi in tissue and smearsusing Gomori’s methenamine–silver nitratetechnique. American Journal of Clinical Pathology

25: 957–959.King, A. D., Pitt, J. I., Beauchat, L. R. and Corry, J. E.

L. (1986) Methods for the Mycological

Examination of Food, Plenum, New York.Krogh, P. (1987) Mycotoxins in Food. London:

Academic Press.MacKenzie, D. W. R., Philpot, C. M. and Proctor, A.

G. J. (1980) Basic Serodiagnosis Methods for

Diseases caused by Fungi and Actinomyces, PublicHealth Laboratory Service Monograph No. 12.London: HMSO,


Meis, J., Petrou, M., Bille, J et al. (2000) A globalevaluation of the susceptibility of Candida speciesto fluconazole by disk diffusion. Diagnostic

Microbiology and Infectious Disease 36: 215–223.Moss, M. O. (1989) Mycotoxins of Aspergillus and

other filamentous fungi. Journal of Applied

Bacteriology 87(suppl): 69S–82S.National Committee for Clinical Laboratory Standards

(2002a) Reference Method for Broth Dilution

Antifungal Susceptibility Testing of Yeasts. ApprovedStandard M27-A2, 2nd edn. Wayne, PA: NCCLS.

National Committee for Clinical Laboratory Standards(2002b) Reference Method for Broth Dilution

Antifungal Susceptibility Testing of Filamentous

Fungi. Approved Standard M38-A. Wayne, PA:NCCLS.

Pitt, J. J. and Hocking, A. D. (1985) Fungi and Food

Spoilage. Sydney: Academic Press.Samson, R. A. and van Reenan-Hoekstra, E. S. (1988)

Introduction to Food Borne Fungi. Baarn:Centraalbureau voor Schimmelcultures.

References 143

It is often necessary to report on the size of the bac-terial population in a sample. Unfortunately, indus-tries and health authorities have been allowed toattach more importance to these ‘bacterial counts’than is permitted by their technical or statisticalaccuracy (see also p. 198).

If a total count is required, which is usually doneby physical methods, many of the organismscounted may be dead or indistinguishable fromother particulate matter. A viable count, which is acultural method, assumes that a visible colony willdevelop from each organism. Bacteria are, how-ever, rarely separated entirely from their fellowsand are often clumped together in large numbers,particularly if they are actively reproducing. A sin-gle colony may therefore develop from one organ-ism or from hundreds or even thousands oforganisms. Each colony develops from one viableunit. Because any agitation, as in the preparation ofdilutions, will either break up or induce the forma-tion of clumps, it is obviously difficult to obtainreproducible results. Bacteria are seldom distrib-uted evenly throughout a sample and as only smallsamples are usually examined very large errors canbe introduced. Viable counts are usually given asnumbers of colony-forming units (cfu).

In addition, many of the bacteria present in a sam-ple may not grow on the medium used, at the pH orincubation temperatures or gaseous atmosphereemployed, or in the time allowed.

Accuracy is often demanded where it is notneeded. If, for example, it is decided that a certainproduct should contain less than, say, 10 viablebacteria/gram this suggests that, on average, of tentubes each inoculated with 0.1 g seven would showgrowth and three would not, and of ten tubes each

inoculated with 0.01 g only one or two wouldshow growth. If all the 0.1-g tubes or five of the0.01-g tubes showed growth, there would be morethan 10 organisms/g. It does not matter whetherthere are 20 or 10 000: there are too many. There isno need to employ elaborate counting techniques.

In viable count methods, it is recognized that largeerrors are inevitable even if numbers of replicateplates are used. Some of these errors are, as indi-cated above, inherent in the material, others in thetechnique. Errors of ± 90% in counts of the orderof 10 000–100 000/ml are not unusual even withthe best possible technique. It is, therefore, neces-sary to combine the maximum of care in techniquewith a liberal interpretation of results. The figuresobtained from a single test are valueless. They canbe interpreted only if the product is regularly testedand the normal range is known.

Physical methods are used to estimate total popu-lations, i.e. dead and living organisms. Theyinclude direct counting, usually by instrumentation(see Chapter 8) and measurements of turbidity.Biological methods are used for estimating thenumbers of viable units. These include the platecount, roll-tube count, drop count, surface colonycount, dip-slide count, contact plate, membrane-filter count and most probable number estimations.

DIRECT COUNTS

Counting chamber method

The Helber counting chamber is a slide 2–3 mmthick with an area in the centre called the plat-

10

Estimating microbial numbers

form and surrounded by a ditch. The platformis 0.02 mm lower than the remainder of theslide. The top of the slide is ground so that,when an optically plane cover glass is placedover the centre depression, the depth is uniform.On the platform an area of 1 mm2 is ruled sothat there are 400 small squares each0.0025 mm2 in area. The volume over eachsmall square is 0.02 × 0.0025 mm3, i.e.0.00005 ml.

Add a few drops of formalin to the well-mixedsuspension to be counted. Dilute the suspension sothat when the counting chamber is filled there willbe about five or ten organisms per small square.This requires initial trial and error. The best diluentis 0.1% peptone water containing 0.1% lauryl sul-phate and (unless phase contrast or dark field isused for counting) 0.1% methylene blue. Alwaysfilter before use.

Place a loopful of suspension on the ruled areaand apply the cover glass, which must be clean andpolished. The amount of suspension must be suchthat the space between the platform and the coverglass is just filled, and no fluid runs into the ditch;this requires practice. If the cover glass is appliedproperly, Newton’s rings will be seen. Allow 5 minfor the bacteria to settle.

Examine with a 4-mm lens with reduced light,dark-field or phase contrast if available. Countthe bacteria in 50–100 squares selected atrandom so that the total count is about 500.Divide the count by number of squares counted.Multiply by 20 000 and by the original dilutionfactor to obtain the total number of bacteria permillilitre. Repeat twice more and take the aver-age of the three counts. Clumps of bacteria,streptococci, etc. can be counted as units or eachcell counted as one organism. It is difficult tocount micro-organisms with a normal mode ofgrowth that is in large aggregates (e.g. myco-bacteria).

With experience, reasonably reproducible countscan be obtained but the chamber and cover glassmust be scrupulously cleaned and examined micro-scopically to make sure that bacteria are not leftadhering to either.

Counts of viable cells

Environmental microbiologists employ a variety ofmethods for counting and estimating the number ofviable cells. These include direct epifluorescenceusing stains such as acridine orange (see pp. 123and 150). A Live/Dead stain is available fromMolecular Probes Inc. (Eugene, OR, USA).

Opacity tube method

International reference opacity tubes, containingglass powder or barium sulphate, are availableand are known as Brown’s or McFarland opacitytubes. These are numbered narrow glass tubes ofincreasing opacity and a table is provided equatingthe opacity of each tube with the number oforganisms per millilitre. The unknown suspensionis matched against the standards in a glass tube ofthe same bore. It may be necessary to dilutethe unknown suspension. These physical methodsshould not be used with Hazard Group 3organisms.

Rapid automated methods

A number of rapid, automated methods have arenow in general use. These use: electronic particlecounting; bioluminescence (as measured by bacter-ial ATP); changes in pH and Eh by bacterialgrowth; changes in optical properties; detection of14C in CO2 evolved from a substrate; micro-calorimetry; changes in impedance or conductivity;and flow cytometry (see Chapter 8).

VIABLE COUNTS

In these techniques the material containing thebacteria is serially diluted and some of each dilu-tion is placed in or on suitable culture media. Eachcolony developing is assumed to have grown from

Viable counts 145

one viable unit, which, as indicated earlier, may beone organism or a group of many.

Diluents

Some diluents, e.g. saline or distilled water, may belethal for some organisms. Diluents must not be useddirect from a refrigerator because cold shock mayprevent organisms from reproducing. Peptone–water diluent (see p. 76), generally known as ‘max-imum recovery diluent’ (MRD), is most commonlyused. If residues of disinfectants are present add suit-able quenching agents, e.g. Tween 80 or lecithin forquaternary ammonium compounds and sodiumthiosulphate for chlorine and iodine (see Chapter 4,Bloomfield, 1991 and Clesceri et al., 1998).

Pipettes

The 10-ml and 1-ml straight-sided, blow-outpipettes are commonly used and are specified forsome statutory tests. Disposable pipettes are laboursaving. Mouth pipetting must be expressly forbid-den, regardless of the nature of the material undertest. A method of controlling teats is given on p. 38.Automatic pipettors or pipette pumps, describedon p. 39, should be used. Some of these can be pre-calibrated and used with disposable polypropylenepipette tips.

Pipettes used in making dilutions must be veryclean, otherwise bacteria will adhere to their innersurfaces and may be washed out into another dilu-tion. Hydrophobic micro-organisms (e.g. mycobac-teria and corynebacteria) adhere to glass and plasticquite readily. Siliconed pipettes may be useful. Fast-running pipettes and vigorous blowing out shouldbe avoided: they generate aerosols. As much as 0.1mlmay remain in pipettes if they are improperly used.

Preparation of dilutions

Dispense 9-ml volumes of diluent in screw-cappedbottles. These are the dilution blanks. If tubes with

loose caps are used, sterilize them first and dispensethe diluent aseptically, otherwise a significantamount of diluent may be lost during autoclaving.

When diluting liquids, e.g. milk for bacterialcounts, proceed as follows. Mix the sample byshaking. With a straight-sided pipette dipped in halfan inch (1–1.5 cm) only, remove 1 ml. Deliver intothe first dilution blank, about half an inch above thelevel of the liquid. Wait 3 s, then blow out carefullyto avoid aerosol formation. Discard the pipette.With a fresh pipette, dip half an inch (1–1.5 cm)into the liquid, suck up and down 10 times to mix,but do not blow bubbles. Raise the pipette andblow out. Remove 1 ml and transfer to the nextdilution blank. Discard the pipette. Continue forthe required number of dilutions, and remember todiscard the pipette after delivering its contents, oth-erwise the liquid on the outside will contribute to acumulative error. The dilutions will be:

Tube no. 1 2 3 4 5Dilution 10−1 10−2 10−3 10−4 10−5

The dilution represents the volume of the original fluid in 1 ml

When counting bacteria in solid or semisolidmaterial, weigh 10 g and place in a Stomacher orblender. Add 90 ml of diluent and homogenize.

Alternatively, cut into small pieces with a sterilescalpel, mix 10 g with 90 ml of the diluent andshake well. Allow to settle. Assume that the bacte-ria are now evenly distributed between the solidand liquid.

Both of these represent dilutions of 1 : 10, i.e.1 ml contains or represents 0.1 g, and further dilu-tions are prepared as above. The dilutions will be:

Tube no. 1 2 3 4Dilution 10−2 10−3 10−4 10−5

The dilution represents the weight of the original material in 1 g

Mechanical aids for preparing the initial 1 : 10dilutions are available commercially.

Plate count

Melt nutrient agar or other suitable media inbottles or tubes of appropriate volumes (usually 15

146 Estimating microbial numbers

or 20 ml) amounts. Cool to 45°C in a waterbath.

Set out Petri dishes, two or more per dilution tobe tested, and label with the dilution number.Pipette 1 ml of each dilution into the centre of theappropriate dishes, using a fresh pipette for eachdilution. Do not leave the dish uncovered forlonger than is absolutely necessary. Add the con-tents of one agar tube to each dish in turn and mixas follows: move the dish gently six times in aclockwise circle of diameter about 150 mm; repeatcounterclockwise; move the dish back and forth sixtimes with an excursion of about 150 mm; repeatwith to-and-fro movements. Allow the medium toset, invert and incubate for 24–48 h.

Economy in pipettesIn practice, the pipette used to transfer 1 ml of adilution to the next tube may be used to pipette1 ml of that dilution into the Petri dish.Alternatively, after the dilutions have been pre-pared a single pipette may be used to plate themout, starting at the highest dilution to avoid signifi-cant carry-over.

Surface count method

This is often used instead of the pour-plate methodin the examination of foods, particularly becausethe growth of some organisms (e.g. pseudomonads)may be impaired if agar is used at too high a tem-perature. Also, it is easier to subculture coloniesfrom surface plates than from pour plates.

Place 0.1 ml or another suitable amount of thesample measured with a standard (commercial)loop or a micropipette in the centre of a well-driedplate of suitable medium and spread it with a loopor spreader all over the surface (see p. 81).Incubate and count colonies. Before incubatingmedia spread with liquid, it is important to ensurethat the plate is completely dry. If liquid stillremains on the surface, micro-organisms can growin the liquid before it is absorbed into the medium.This results in the appearance of many clustered

colonies, especially for rapidly growing micro-organisms. If freshly poured plates are used, incu-bation brings liquid to the surface and confluentgrowth can occur, even with small numbers ofcolony-forming units in the original volumespread. Plates should be dried at least overnightbefore spreading.

Whether pour plates or surface spreading isemployed, aggregates of cells cause problems inobtaining accurate and reproducible colony counts,because an aggregate, if undisturbed by pipettingor spreading, will yield a single colony. If an aggre-gate is composed of phenotypically different cells(lactose fermenting and non-lactose fermenting),the resulting colony on a fermentation indicatormedium will be mixed (e.g. sectored). If suspen-sions of aggregates are spread, the extent of spread-ing (e.g. to dryness) and the age of the medium (e.g.fresh or old and dry) will influence the number ofcolonies. Lowest counts will be obtained by spread-ing the liquid to dryness on old and dry plates tothe point where the liquid disappears and itbecomes difficult to move the glass spreader overthe surface.

When counting colonies, difficulties arise withlarge ‘smears’ of small colonies caused when a largeviable unit is broken up but not dispersed duringmanipulation. Treat these as single units. Usually,other colonies can be seen and counted through‘smears’. There may be problems with swarmingorganisms, such as some Proteus spp. In this casepour melted agar over the inoculated plates.

Counting colonies on pour or surface platesTo count on a simple colony counter select plateswith between 30 and 300 colonies for micro-organ-isms forming colonies of 2–5 mm diameter. Highernumbers can be counted for organisms that formsmall colonies (e.g. < 1 mm). Place the open dish,glass side up, over the illuminated screen. Countthe colonies using a 75-mm magnifier and a hand-held counter. Mark the glass above each colonywith a felt-tip pen. Calculate the colony or viablecount per millilitre by multiplying the averagenumber of colonies per countable plate by thereciprocal of the dilution. Preferably count the

Viable counts 147

colonies from at least two dilutions and calculatethe colony forming units in the original sample bythe method of weighted means (BSI, 1991). Reportas ‘colony forming units/millilitre’ (cfu/ml) or as‘viable count/millilitre’ not as ‘bacteria/gram’ or‘bacteria/millilitre’.

If all plates contain more than 300 colonies rulesectors, e.g. of one-quarter or one-eighth of theplate, count the colonies in these and include thesector value in the calculations.

For large workloads semi-automatic or fully auto-matic counters are essential. In the former the penused to mark the glass above the colonies is con-nected to an electronic counter, which displays thenumbers counted on a small screen. In the fullyautomatic models a TV camera or laser beam scansthe plate and the results are displayed or recordedon a screen or read-out device.

Roll-tube count

Instead of using plates, tubes or bottles containingmedia are inoculated with diluted material androtated horizontally until the medium sets. Afterincubation, colonies are counted.

Dispense the medium in 2- to 4-ml amounts in25-ml screw-capped bottles. The medium shouldcontain 0.5–1.0% more agar than is usual. Meltand cool to 45°C in a water bath. Add 0.1 ml ofeach dilution and rotate horizontally in cold wateruntil the agar is set in a uniform film around thewalls of the bottle. This requires some practice. Analternative method employs a slab of ice takenfrom the ice tray of a refrigerator. Turn it upsidedown on a cloth and make a groove in it by rotat-ing horizontally on it a bottle similar to that usedfor the counts but containing warm water. Thecount tubes are then rolled in this groove. If theroll-tube method is to be used often a commercialroll-tube apparatus, which includes a water bath,saves much time and labour.

Incubate roll-tube cultures inverted so that con-densation water collects in the neck and does notsmear colonies growing on the agar surface. Tocount, draw a line parallel to the long axis of the

bottle and rotate the bottle, counting coloniesunder a low-power magnifier.

The roll-tube method is popular in the dairyand food industries. Machines for speeding upand taking the tedium out of this method areavailable.

A roll-tube method for counting organisms in bot-tles (container sanitation) is given on p. 249.

Drop count method

In this method, introduced by Miles and Misra(1938) and usually referred to by their names,small drops of the material or dilutions are placedon agar plates. Colonies are counted in the inocu-lated areas after incubation.

Pipettes or standard loops that deliver knownvolumes are required. Disposable cottonwool-plugged Pasteur pipettes are available commer-cially and will deliver drops of 0.02 ml. These aresufficiently accurate for the purpose, provided thatthe tips are checked immediately before use toensure that they are undamaged. It is advisable,however, to check several pipettes in each batchby counting the number of drops of distilled waterthat yield 1 g.

Dry plates of suitable medium very well beforeuse. Drop at least five drops from each dilution ofsample from a height of not more than 2 cm (toavoid splashing) on each plate. Replace the lid butdo not invert until the drops have dried. After incu-bation, select plates showing discrete colonies indrop areas, preferably one that gives less than 40colonies per drop (10–20 is ideal). Count thecolonies in each drop, using a hand magnifier.Divide the total count by the number of dropscounted, multiply by 50 to convert to 1 ml and bythe dilution used.

This method lends itself to arbitrary standards,e.g. if there are fewer than 10 colonies among fivedrops of sample, this represents fewer than 100colonies of that material. If these drops areuncountable (> 40 colonies), then the colony countper millilitre is > 2000.


Spread-drop plate count

This is a modification of the Miles and Misramethod, often used in the examination of foods.

Spread standard volume drops (e.g. 0.02 ml) onquadrants marked out on the bottom of the dish.Inoculate two dishes, each with drops from differ-ent decimal dilutions. Do counts on plates (usuallytwo) from dilutions that yield ≤ 50 colonies.

Spiral plate method

The apparatus is described on p. 82. The results aresaid to compare favourably with those obtained bythe surface spread plate method. A commercialimaged-based colony counting system is availablefor the rapid and accurate counting of colonies onspiral plates.

Membrane filter counts

These offer a more accurate alternative to the mostprobable number (MPN) estimation (see p. 150and Clesceri et al., 1998). Liquid containing bacte-ria is passed through a filter that will retain theorganisms. The filter is then laid on a sterileabsorbent pad containing liquid culture medium oron the surface of an agar medium and incubated,when the colonies that develop may be counted.

The filter-carrying apparatus is made of metal,glass or plastic, and consists of a lower funnel,which carries a fritted glass platform surroundedby a silicone rubber ring. The filter disc rests on theplatform and is clamped by its periphery betweenthe rubber ring and the flange of the upper funnel.The upper and lower funnels are held together by aclamp.

The filters are thin, porous cellulose ester discs,varying in diameter and about 120 μm thick. Thepores in the upper layers are 0.5–1.0 μm diameterenlarging to 3–5 μm diameter at the bottom.Bacteria are thus held back on top, but culturemedium can easily rise to them by capillary action.

Filters are stored interleaved between absorbentpads in metal containers. A grid to facilitate count-ing is ruled on the upper surface of each filter.

Membrane filter apparatus may be reusable orsold as kits by the companies that make mem-branes. Shallow metal incubating boxes with close-fitting lids, containing absorbent pads to hold theculture media, are also required.

The most convenient size of apparatus for count-ing colonies takes filters 47 mm in diameter, butvarious sizes larger or smaller are available.

SterilizationAssemble the filter carrier with a membrane filterresting on the fritted glass platform, which in turnshould be flush on top of the rubber gasket. Screwup the clamping ring, but not to its full extent.Wrap in oven-resistant plastic film or foil and auto-clave for 15 min at 121°C. Alternatively, looselyassemble the filter carrier, wrap and autoclave;sterilize the filters separately interleaved betweenabsorbent pads in their metal container also byautoclaving. This is the most convenient methodwhen a number of consecutive samples are to betested; in this case, sterilize the appropriate numberof spare upper funnels in the same way.

Sterilize the incubation boxes, each containing itsabsorbent pad, by autoclaving.

Culture mediaOrdinary or standard culture media do not giveoptimum results with membrane filters. It has beenfound necessary to vary the proportions of some ofthe ingredients. Membrane filter versions of stan-dard media may be obtained from most culturemedia suppliers. A ‘resuscitation’ medium to reviveinjured bacteria (e.g. damaged by chlorine) is alsodesirable for some purposes.

MethodErect the filter carrier over a filter flask connectedby a non-return valve to a pump giving a suction of25–50 mmHg. Check that the filter is in place andtighten the clamping ring. Pour a known volume,neat or diluted, of the fluid to be examined into theupper funnel and apply suction.

Viable counts 149

Pipette about 2.5–3 ml of medium on a Whatman5 cm, No. 17, absorbent pad in an incubation con-tainer or Petri dish. This should wet the pad to theedges but not overflow into the container.

When filtration is complete, carefully restore thepressure. Unscrew the clamping ring and removethe filter with sterile forceps. Apply it to the surfaceof the wet pad in the incubation box so that no airbubbles are trapped. Put the lid on the containerand incubate. A fresh filter and upper funnel can beplaced on the filter apparatus for the next sample.

For total aerobic counts, use tryptone soya mem-brane medium; for counting coliform bacilli, either‘presumptive’ or E. coli, see ‘Water examination’(Chapter 20); for counting clostridia in water, seep. 255.

For anaerobic counts, use the same medium incu-bated anaerobically, or roll the filter and submergein a 25-mm diameter tube of melted thioglycollateagar at 45°C and allow to set. To count anaerobescausing sulphide spoilage, roll the filter and sub-merge in a tube of melted iron sulphite medium at42°C and allow to set. For yeast and mould counts,use the appropriate Sabouraud-type or Czapek-type media.

CountingCount in oblique light under low-power magnifica-tion. For some methods (e.g. counts of E. coli indrinking water), indicator media are available andonly colonies with a specific colour are counted. Ifit is necessary to stain colonies to see them, removethe filter from the pad and dip in a 0.01% aqueoussolution of methylene blue for half a minute andthen apply to a pad saturated with water. Coloniesare stained deeper than the filter. The grid facili-tates counting. Report as membrane colony countper standard volume (100 ml, 1 ml, etc.).

Count anaerobic colonies in thioglycollate tubesby rotating the tube under strong illumination.Count black colonies in iron sulphite medium.

Micro-organisms in airMembrane filters may be used with an impinger orcascade sampler. Methods are given on p. 247.

Dip slidesThis useful method, commonly used in clinical lab-oratories for the examination of urine, now has amore general application, especially in food anddrinks industries for viable coliform and yeastcounts. The slides are made of plastic and areattached to the caps of screw-capped bottles. Thereare two kinds: one is a single- or double-sided traycontaining agar culture media; the other consists ofa membrane filter bonded to an absorbent pad con-taining dehydrated culture media. Both have ruledgrids, on either the plastic or the filter, which facil-itate counting.

The slides are dipped into the samples, drained,replaced in their containers and incubated.Colonies are then counted and the bacterial loadestimated.

Direct epifluorescence filtrationtechnique

This is a rapid, sensitive and economical methodfor estimating the bacterial content of milk andbeverages. It may also be used for foods if they arefirst treated with proteolytic enzymes and/or sur-factants.

A small volume (e.g. 2 ml) of the product is passedthrough a 24-mm polycarbonate membrane, whichis then stained by acridine orange and examinedwith an epifluorescence microscope. Kits are avail-able from several companies. For more informa-tion, see Chapter 8.

Most probable number estimates

Most probable number (MPN) estimates aredescribed in Standard Methods for theExamination of Water and Wastewater (Clesceri etal., 1998). These are based on the assumption thatbacteria are normally distributed in liquid media,i.e. repeated samples of the same size from onesource are expected to contain the same number oforganisms on average; some samples will obviously


contain a few more, some a few less. The averagenumber is the most probable number. If the num-ber of organisms is large, the differences betweensamples will be small; all the individual results willbe nearer to the average. If the number is small thedifference will be relatively larger.

If a liquid contains 100 organisms/100 ml, then10-ml samples will contain, on average, 10 organ-isms each. Some will contain more – perhaps one ortwo samples will contain as many as 20; some willcontain less, but a sample containing none is mostunlikely. If a number of such samples are inocu-lated into suitable medium, every sample would beexpected to show growth.

Similarly, 1-ml samples will contain, on average,one organism each. Some may contain two or three,and others will contain none. A number of tubes ofculture media inoculated with 1-ml samples wouldtherefore yield a proportion showing no growth.

Samples of 0.1 ml, however, could be expected tocontain only one organism per 10 samples andmost tubes inoculated would be negative.

It is possible to calculate the most probable num-ber of organisms/100 ml for any combination ofresults from such sample series. Tables have beenprepared (Tables 10.1–10.41) for samples of 10 ml,1 ml and 0.1 ml using five tubes or three tubes of

each sample size and, for water testing, using one50-ml, five 10-ml and five 1-ml samples.

This technique is used mainly for estimating col-iform bacilli, but it can be used for almost anyorganisms in liquid samples if growth can be easilyobserved, e.g. by turbidity, acid production.Examples are yeasts and moulds in fruit juices andbeverages, clostridia in food emulsions and rope-spores in flour suspensions. ‘Black tube’ MPNcounts for anaerobes are described on p. 377.

Mix the sample by shaking and inverting vigor-ously. Pipette 10-ml amounts into each of five tubes(or three) of 10 ml double-strength medium, 1-mlamounts into each of five (or three) tubes of 5 mlsingle-strength medium and 0.1-ml amounts (or1 ml of a 1 : 10 dilution) into each of five (or three)tubes of 5 ml single-strength medium. For testingwater, also add 50 ml water to 50 ml double-strength broth.

Use double-strength broth for the larger volumesbecause the medium would otherwise be too dilute.Incubate for 24–48 h and observe growth, or acidand gas, etc. Tabulate the numbers of positivetubes in each set of five (or three) and consult theappropriate table.

Viable counts 151

Table 10.1 MPN/100 ml, using one tube of 50 ml, and five tubesof 10 ml

50-ml tubes 10-ml tubes MPN/100 mlpositive positive

0 0 00 1 10 2 20 3 40 4 50 5 71 0 21 1 31 2 61 3 91 4 161 5 18 +

1Tables 10.1–10.3 indicate the estimated number of bacteria ofthe coliform group present in 100 ml of water, correspondingto various combinations of positive and negative results in theamounts used for the tests. The tables are basically those origi-nally computed by McGrady (1918) with certain amendmentsdue to more precise calculations by Swaroop (1951). A fewvalues have also been added to the tables from other sources,corresponding to further combinations of positive and negativeresults which are likely to occur in practice. Swaroop has tabu-lated limits within which the real density of coliform organismsis likely to fall, and his paper should be consulted by thosewho need to know the precision of these estimates.

These tables are reproduced by permission of theEnvironmental Agency from The Microbiology of DrinkingWater Supplies. Part 4. Methods for the isolation andenumeration of coliform bacteria and Escherichia coli(including E. coli O 157: H7). London, 2002.


Table 10.2 MPN/100 ml, using one tube of 50 ml, five tubes of 10 ml and five tubes of 1 ml

50-ml tubes 10-ml tubes 1-ml tubes MPN/100 mlpositive positive positive

0 0 0 00 0 1 10 0 2 20 1 0 10 1 1 20 1 2 30 2 0 20 2 1 30 2 2 40 3 0 30 3 1 50 4 0 51 0 0 11 0 1 31 0 2 41 0 3 61 1 0 31 1 1 51 1 2 71 1 3 91 2 0 51 2 1 71 2 2 101 2 3 121 3 0 81 3 1 111 3 2 141 3 3 181 3 4 201 4 0 131 4 1 171 4 2 201 4 3 301 4 4 351 4 5 401 5 0 251 5 1 351 5 2 501 5 3 901 5 4 1601 5 5 180 +

Viable counts 153

Table 10.3 MPN/100 ml, using five tubes of 10 ml, five tubes of 1 ml and five tubes of 0.1 ml

10-ml 1-ml 0.1-ml MPN/tubes tubes tubes 100 mlpositive positive positive

4 0 2 204 0 3 254 1 0 174 1 1 204 1 2 254 2 0 204 2 1 254 2 2 304 3 0 254 3 1 354 3 2 404 4 0 354 4 1 404 4 2 454 5 0 404 5 1 504 5 2 555 0 0 255 0 1 305 0 2 455 0 3 605 0 4 755 1 0 355 1 1 455 1 2 655 1 3 855 1 4 1155 2 0 505 2 1 705 2 2 955 2 3 1205 2 4 1505 2 5 1755 3 0 805 3 1 1105 3 2 1405 3 3 1755 3 4 2005 3 5 2505 4 0 1305 4 1 1705 4 2 2255 4 3 2755 4 4 3505 4 5 4255 5 0 2505 5 1 3505 5 2 5505 5 3 9005 5 4 16005 5 5 1800+

10-ml 1-ml 0.1-ml MPN/tubes tubes tubes 100 mlpositive positive positive

0 0 0 00 0 1 20 0 2 40 1 0 20 1 1 40 1 2 60 2 0 40 2 1 60 3 0 61 0 0 21 0 1 41 0 2 61 0 3 81 1 0 41 1 1 61 1 2 81 2 0 61 2 1 81 2 2 101 3 0 81 3 1 101 4 0 112 0 0 52 0 1 72 0 2 92 0 3 122 1 0 72 1 1 92 1 2 122 2 0 92 2 1 122 2 2 142 3 0 122 3 1 142 4 0 153 0 0 83 0 1 113 0 2 133 1 0 113 1 1 143 1 2 173 1 3 203 2 0 143 2 1 173 2 2 203 3 0 173 3 1 203 4 0 203 4 1 253 5 0 254 0 0 134 0 1 17

REFERENCES

Bloomfield, S. F. (1991) Method for assessingantimicrobial activity. In: Denyer, S. P. and Hugo,W. B. (eds) Mechanism of Action of Chemical

Biocides. Society for Applied BacteriologyTechnical Series No. 20. Oxford: Blackwells, pp.1–22.

BSI (1991) BS 5763: Part I: Enumeration ofmicroorganisms: Colony count technique at 30°C.London: British Standards Institution.

Clesceri, L. S., Greenberg, A. E. and Eaton A. D.(1998) Standard Methods for the Examination of

Water and Wastewater, 20th edn. Washington DC:American Public Health Association.

Jacobs, M. B. and Gerstein, M. J. (1960) Handbook of

Microbiology. Princeton, NJ: D Van MostrandCo., Inc.

McCrady, M. H. (1918) Tables for rapid interpretationof fermentation test results. Public Health Journal,

Toronto 9: 201–210.Miles, A. A. and Misra, S. S. (1938) The estimation of

Table 10.4 MPN/100 ml, using three tubes each inoculated with 10, 1.0 and 0.1 ml of sample

Tubes positive Tubes positive Tubes positive

10 ml 1.0 ml 0.1 mlMPN



0 0 1 3 1 2 0 11 2 3 3 530 0 2 6 1 2 1 15 3 0 0 230 0 3 9 1 2 2 20 3 0 1 390 1 0 3 1 2 3 24 3 0 2 640 1 1 6 1 3 0 16 3 0 3 950 1 2 9 1 3 1 20 3 1 0 430 1 3 12 1 3 2 24 3 1 1 750 2 0 6 1 3 3 29 3 1 2 1200 2 1 9 2 0 0 9 3 1 3 1600 2 2 12 2 0 1 14 3 2 0 930 2 3 16 2 0 2 20 3 2 1 1500 3 0 9 2 0 3 26 3 2 2 2100 3 1 13 2 1 0 15 3 2 3 2900 3 2 16 2 1 1 20 3 3 0 2400 3 3 19 2 1 2 27 3 3 1 4601 0 0 4 2 1 3 34 3 3 2 11001 0 1 7 2 2 0 21 3 3 3 1100 +1 0 2 11 2 2 1 281 0 3 15 2 2 2 351 1 0 7 2 2 3 421 1 1 11 2 3 0 291 1 2 15 2 3 1 361 1 3 19 2 3 2 44

From Jacobs and Gerstein’s Handbook of Microbiology, D. Van Nostrand Company, Inc., Princeton, NJ (1960). Reproducedby permission of the authors and publisher


the bactericidal power of the blood. Journal of

Hygiene (Cambridge) 38: 732–749.Swaroop, S. (1951) Range of variation of Most

Probable Numbers of organisms estimated bydilution methods. Indian Journal of Medical

Research 39: 107–131.

References 155

All clinical material should be regarded as poten-tially infectious. Blood specimens may containhepatitis B and/or human immunodeficiencyviruses. For details of special precautions, seeNational Institutes of Health (NIH, 1988), WorldHealth Organization (WHO, 1991), AdvisoryCommittee on Dangerous Pathogens (ACDP,1995, 2001), and Collins and Kennedy (1997,1999). Material from the central nervous systemshould also be treated with care because it maycontain prions. For details of special precautionssee ACDP (1998).

The laboratory investigation of clinical materialfor microbial pathogens is subject to a variety ofexternal hazards. Fundamental errors and omis-sions may occur in any laboratory. Vigorous per-formance control testing programmes and theroutine use of controls will reduce the incidence oferror but are unlikely to eliminate it entirely (seeChapter 2).

Most samples for microbiological examinationare not collected by laboratory staff and it must bestressed that, however carefully a specimen isprocessed in the laboratory, the result can only beas reliable as the sample will allow. The laboratoryshould provide specimen containers that are suit-able, i.e. leak proof, stable, easily opened, readilyidentifiable, aesthetically satisfactory, suitable foreasy processing and preferably capable of beingincinerated. It will be apparent that some of thesequalities are mutually exclusive and the best com-promise to suit particular circumstances must bemade. Containers are discussed on p. 41. The spec-imen itself may be unsatisfactory because of faultycollection procedures, and the laboratory must beready to state its requirements and give a reasoned

explanation if cooperation is to be obtained fromward staff and doctors.

Specimens should be:

• Collected without extraneous contamination.• Collected, if possible, before starting

antibiotic therapy.• Representative: pus rather than a swab with a

minute blob on the end; faeces rather than arectal swab. If this is not possible, then aclear note to this effect should be made on therequest form, which must accompany eachsample.

• Ideally collected and sent to the laboratory ina clear, sealed, plastic bag to avoid leakageduring transit, or placed in a transportmedium. The request form accompanying eachspecimen should not be in the same bag (bagswith separate compartments for specimens andrequest forms are available) and should beclearly displayed to minimize sorting errors.

• Delivered to the laboratory without delay. Ifthis is not possible, appropriate specimensmust be delivered in a commercial transportmedium. For other specimens, storage at 4°Cwill hinder bacterial overgrowth. If urinesamples cannot be delivered promptly andrefrigeration is not possible, boric acidpreservative may be considered as a lastresort. Dip-slide culture is to be preferred.Sputum and urine specimens probablyaccount for the greater part of the laboratoryworkload and the problems associated withtheir collection are particularly difficult toovercome because the trachea, mouth andurethra have a normal flora.

11

Clinical material

It must be stressed that, in any microbiologicalexamination, only that which is sought will befound and it is essential to examine the specimen inthe light of the available clinical information(which, regrettably, is often minimal). This is evenmore important with modern techniques that relyon the direct detection of bacterial antigens ornucleic acids in clinical specimens. The examina-tion of pathological material for viruses and para-sites is not within the scope of this book, but thepossibility of infection by these agents should beconsidered and material referred to the appropriatelaboratory.

The laboratory has an obligation to ensure that itsrules are well publicized so that the periods ofacceptance of specimens are known to all. Any teststhat are processed in batches should be arranged inadvance with the laboratory. This may be particu-larly important for certain tests, e.g. microbiologi-cal assays of antibiotics for which the laboratorymay need to subculture indicator organisms inadvance. In the UK, it is now a requirement forclinical laboratory accreditation that the labora-tory produces a user manual to enable the clini-cians to use its services in an appropriate way.

BLOOD CULTURES

Blood must be collected with scrupulous care toavoid extravenous contamination. Blood culture isan important diagnostic procedure in cases ofrecurrent fever, and the highest success rate is asso-ciated with the collection of cultures just as thepatient’s temperature begins to rise, rather than atthe peak of the rigor. In cases of septicaemia, thetiming is less important, and a rapid answer and asusceptibility test result are essential. Culturing alarger volume of blood may increase the yield fromblood cultures.

Most busy clinical laboratories now use auto-mated systems for blood culture (see Chapter 8) ora kit such the Oxoid Signal or the Difco Sentinel,but there is still a place for the Castenada system.In this system, the culture bottles are biphasic, i.e.they have a slope of solid medium and a broth that

may also contain Liquoid (sodium polyethanolsulphonate), which neutralizes antibacterial factorsincluding complement. Large amounts of blood (upto 50% of the total volume of the medium) may beexamined in Liquoid cultures; otherwise, it is nec-essary to dilute the patient’s blood at least 1 : 20with broth. The bottles are tipped every 72 h sothat the blood–broth mixture washes over the agarslope. Colonies will appear first at the interphaseand then all over the slope. The bottles should beretained for at least 4 and preferably 6 weeks. TheCastenada system avoids repeated subculture andlessens the risk of laboratory contamination. It isimportant to avoid contamination because patientsreceiving immunosuppressive drugs are likely to beinfected with organisms not usually regarded aspathogens. No organism can therefore be automat-ically excluded as a contaminant.

Vacuum blood collection systems also help toreduce contamination. Some of these ensure a 10%carbon dioxide atmosphere in the container. This isimportant when organisms such as Brucella spp.are sought. If other media are used they should beincubated in an atmosphere containing at least10% carbon dioxide.

Evacuated containers or those containing addi-tional gas may need ‘venting’ with a cottonwool-plugged hypodermic needle (caution) or acommercial device. This is important, especially forthe recovery of Candida spp. and Pseudomonasspp. from neonatal samples, where only a smallvolume of blood is available for culture.

Blood cultures should be checked for growth atleast daily and subcultured to appropriate mediawhen growth is evident, either by visual inspectionor by one of the current automated detection sys-tems (see Chapter 8).

Likely pathogens

These are Staphylococcus aureus, Escherichia coliand other enterobacteria, including salmonellas,streptococci and enterococci, Neisseria meningi-tidis, Haemophilus influenzae, Brucella spp.,

Blood cultures 157

clostridia and pseudomonads. In ill or immuno-compromised patients, any organisms, especially ifrecovered more than once, may be significant (e.g.coagulase-negative staphylococci or coryneforms).In some circumstances special media may have tobe used to look for specific organisms (e.g.mycobacteria in AIDS patients). Blood frompatients who have had surgery may yield a mixedgrowth of other organisms.

Commensals

Note that common contaminants (from skin)include coagulase-negative staphylococci andcoryneforms. Transient bacteraemia with otherorganisms may occur occasionally.

All manipulations with open blood culture bottlesshould be done in a microbiological safety cabinet.

CEREBROSPINAL FLUID

It is important that these samples should be exam-ined with the minimum of delay. As with other flu-ids, a description is important. It is essential to notethe colour of the fluid; if it is turbid, then the colourof the supernatant fluid after centrifugation may beof diagnostic significance. A distinctive yellow tinge(xanthochromia) usually confirms an earlier bleedinto the cerebrospinal fluid (CSF), e.g. in a sub-arachnoid haemorrhage as opposed to traumaticbleeding during the lumbar puncture. (It is neces-sary, of course, to compare the colour of the super-natant fluid with a water blank in a similar tube.)

Before any manipulation examine the specimenfor clots, either gross or in the form of a delicatespider’s web, which is usually associated with a sig-nificantly raised protein level and traditionally istaken as an indication that one should look partic-ularly for Mycobacterium tuberculosis. The pres-ence of a clot will invalidate any attempt to make areliable cell count.

If the cells and/or protein concentrations areraised (> 3 × 106/l and/or > 40 mg/100 ml), a CSF

sugar determination should be compared with theblood sugar level collected at the same time. Thisand the type of cell may indicate the type of infec-tion.

Raised polymorphs with very low sugar usuallyindicate bacterial infection or a cerebral abscess.Raised lymphocytes with normal sugar usuallyindicate viral infection. Raised lymphocytes withlowered sugar usually indicate tuberculosis. Raisedpolymorphs or mixed cells with lowered sugar usu-ally indicate early tuberculosis. It is important,however, to note that there are many exceptions tothese patterns.

Make three films from the centrifuged deposit,spreading as little as possible. Stain one by theGram method and one with a cytological stain, andretain the third for examination if indicated foracid-fast bacilli. A prolonged search may be neces-sary if tuberculosis is suspected.

Plate on blood agar and incubate aerobically andanaerobically and on a chocolate agar plate forincubation in a 5–10% CO2 atmosphere. Examineafter 18–24 h. If organisms are found, set up directsensitivity tests according to local protocol. Culturefor M. tuberculosis as indicated.

Commercial systems are now available for thedetection (e.g. by latex agglutination) of the com-mon bacterial causes of meningitis (Streptococcuspneumoniae, N. meningitidis, H. influenzae). Theyshould be used in all cases of suspected bacterialmeningitis, but they are particularly useful whenthe Gram stain is unhelpful or when the patient hasalready received antibiotics.

Likely pathogens

These include H. influenzae, N. meningitidis, S.pneumoniae, Listeria monocytogenes, M. tubercu-losis and, in very young babies, coliform bacilli,group B streptococci and Pseudomonas aerugi-nosa. Staphylococcal epidermidis and some micro-cocci are often associated with infections inpatients with devices (shunts) inserted to relieveexcess intracranial pressure.

158 Clinical material

Opportunists

Any organism introduced during surgical manipu-lation involving the spinal canal may be involvedin meningitis. Infections with other agents mayoccur in the immunocompromised patient (e.g.Cryptococcus neoformans in AIDS).

Commensals

There are none.

DENTAL SPECIMENS

These may be whole teeth, when there may havebeen an apical abscess, or scrapings of plaque orcarious material. Plate on selective media forstaphylococci, streptococci and lactobacilli.

Likely organisms

These include S. aureus, streptococci, especially S.mutans and S. milleri, and lactobacilli.

EAR DISCHARGES

Swabs should be small enough to pass easilythrough the external meatus. Many commerciallyavailable swabs are too plump and may cause painto patients with inflamed auditory canals.

A direct film stained by the Gram method is help-ful, because florid overgrowths of faecal organismsare not uncommon and it may be relatively difficultto recover more delicate pathogens. Plate on bloodagar and incubate aerobically and anaerobicallyovernight at 37°C. Tellurite medium may be advis-able if the patient is of school age. A medium thatinhibits spreading organisms is frequently of value;CLED or MacConkey, chloral hydrate orphenethyl alcohol agars are the most useful.

Likely pathogens

These include S. pyogenes, S. pneumoniae,S. aureus, Haemophilus spp., Corynebacteriumdiphtheriae, P. aeruginosa, coliform bacilli,anaerobic Gram-negative rods, and fungi.

Commensals

These include micrococci, diphtheroids, S. epider-midis, moulds and yeasts.

EYE DISCHARGES

It is preferable to plate material from the eyedirectly on culture media rather than to collect it ona swab. If this is not practicable, it is essential thatthe swab be placed in transport medium.

Examine a direct Gram-stained film before thepatient is allowed to leave. Look particularly forintracellular Gram-negative diplococci and issue atentative report if they are seen, so that treatmentcan be started without delay; this is of particularimportance in neonates. Infections occurring a fewdays after birth may be caused by Chlamydia tra-chomatis, which should be suspected if films haveexcess numbers of monocytes or the condition doesnot resolve rapidly. Detection of chlamydial infec-tions may be attempted by microscopy (withGiemsa stain), cell culture or immunological meth-ods, e.g. immunofluorescence (see p. 102).

Plate on blood agar plates and incubate aerobi-cally and anaerobically and on a chocolate agar or10% horse blood Columbia agar plate for incuba-tion in a 5% carbon dioxide atmosphere.

Likely pathogens

These include S. aureus, S. pneumoniae, viridansstreptococci, N. gonorrhoeae, and Haemophilus,Chlamydia and Moraxella spp., but rarely coliform

Eye discharges 159

organisms, C. diphtheriae, P. aeruginosa, andCandida, Aspergillus and Fusarium spp.

Commensals

These include S. epidermidis, micrococci anddiphtheroids.

FAECES AND RECTAL SWABS

It is always better to examine faeces than rectalswabs. Swabs that are not even stained with faecesare useless. If swabs must be used, they should bemoistened with sterile saline or water before useand care must be taken to avoid contaminationwith perianal flora. It may be useful to collectmaterial from babies by dipping the swabs into arecently soiled napkin (diaper). Swabs should besent to the laboratory in transport medium unlessthey can be delivered within 1 h.

As a result of the random distribution ofpathogens in faeces, it is customary to examinethree sequential specimens. Culture as belowaccording to clinical information:

• Plate on desoxycholate citrate agar (DCA),xylose–lactose desoxycholate citrate agar(XLD) and MacConkey agar for salmonellas,shigellas and enteropathogenic Escherichiacoli. Incubate at 37°C overnight.

• Inoculate selenite and/or tetrathionatebroths, incubate overnight and subcultureto DCA, etc.

• Plate stools from children, young adults anddiarrhoea cases on campylobacter mediumplus antibiotic supplements. Incubate for 48 hat 42°C and at 37°C in 5% oxygen and 10%carbon dioxide.

• In cases of haemorrhagic colitis orhaemolytic–uraemic syndrome, culture forE. coli O157 on sorbitol MacConkey agar.Culture also for Aeromonas andPlesiomonas spp.

• Plate on TCBS and inoculate alkaline peptone

water if cholera is suspected, if the patient hasrecently returned by air from an area wherecholera is endemic or if food poisoning causedby Vibrio parahaemolyticus is suspected.

• Plate on Yersinia Selective Agar and incubateat 32°C for 18–24 h (see also p. 318).

• Plate on cycloserine cefoxitin egg yolkfructose agar (CCFA) for Clostridium difficileand test the stool for C. difficile toxins eitherin cell culture or with a commercial kit.

• In cases of food poisoning caused by S. aureus,plate faeces on blood agar and on one of theselective staphylococcal media (see p. 329),and inoculate salt meat broth. Incubate allcultures overnight at 37°C and subculture thesalt meat broth to blood agar and selectivestaphylococcal medium. It should be notedthat here the symptoms relate to a toxin so theorganisms may not be recovered.

• In food poisoning where C. perfringens maybe involved, make a 10−1 suspension of faecesin nutrient broth and add 1 ml to each of twotubes of Robertson’s cooked meat medium.Heat one tube at 80°C for 10 min and thencool. Incubate both tubes at 37°C overnightand plate on blood agar with and withoutneomycin and on egg yolk agar. Place ametronidazole disc on the streaked-outinoculum. Incubate at 37°C anaerobicallyovernight.

Likely pathogens

These include salmonellas, shigellas, Vibrio sp.,enteropathogenic E. coli, C. perfringens (both heatresistant and non-heat resistant), S. aureus, Bacilluscereus, Campylobacter, Yersinia and Aeromonasspp., and C. botulinum.

Commensals

These include coliform bacilli, and Proteus,Clostridium, Bacteroides and Pseudomonas spp.


NASAL SWABS (SEE ALSO‘THROAT SWABS’)

The area sampled will influence the recovery ofparticular organisms, i.e. the anterior part of thenose must be sampled if the highest carriage rate ofstaphylococci is to be found. For N. meningitidis, itmay be preferable to sample further in and for therecovery of Bordetella pertussis in cases of whoop-ing cough a pernasal swab is essential.

Inoculate as soon as possible on charcoal agar con-taining 10% horse blood and 40 mg/l cephalexinand incubate at 35°C in a humid atmosphere.

Direct films are of no value. If M. leprae is sought,smears from a scraping of the nasal septum may beexamined after staining with Ziehl–Neelsen stainor a fluorescence stain.

Plate on blood agar and tellurite media and incu-bate as for throat swabs.

Likely pathogens

These include S. aureus, S. pyogenes, N. meningi-tidis, B. pertussis, C. diphtheriae and otherpathogens of uncertain significance.

Commensals

These include diphtheroids, S. epidermidis, S.aureus, Branhamella spp., aerobic spore bearersand small numbers of Gram-negative rods (Proteusspp. and coliform bacilli).

PUS

As a general rule, pus rather than swabs of pusshould be sent to the laboratory. Swabs are vari-ably lethal to bacteria within a few hours becauseof a combination of drying and toxic componentsof the cotton wool released by various sterilizationmethods. Consequently, speed in processing isimportant; a delay of several hours may allow

robust organisms to be recovered while the moredelicate pathogens may not survive. Swabs shouldbe moistened with broth before use on dry areas.

Examine Gram-stained films and films either director concentrated material (see p. 381) for acid-fastrods. If actinomycosis is suspected wash the pus withsterile water and look for ‘sulphur granules’ (seeChapter 47). Plate granules on blood agar and incu-bate aerobically and anaerobically. Include a chlo-ral hydrate plate in case Proteus sp. is present.Prolonged anaerobic incubation of blood agar cul-tures, in a 90% hydrogen–10% carbon dioxideatmosphere, possibly with neomycin or nalidixicacid, may allow the recovery of strictly anaerobicBacteroides spp. These organisms may be recoveredfrequently if care is taken and the number of ‘ster-ile’ collections of pus consequently reduced. Culturein thioglycollate broth may be helpful. The dilutioneffect may overcome specific and non-specificinhibitory agents. Culture pus from post-injectionabscesses or infected surgical wounds for mycobac-teria (see p. 382).

The recovery of anaerobes will be enhanced by theaddition of menadione and vitamin K to the medium.Wilkins and Chalgren’s medium is also of value.

If the equipment is available, gas–liquid chro-matography is useful for the detection of volatilefatty acids, which may indicate anaerobes (but thebacteriologist’s nose is cheaper, even if less sensi-tive).

Likely pathogens

These include S. aureus, S. pyogenes, anaerobiccocci, Mycobacterium, Actinomyces, Pasteurella,Yersinia, Clostridium, Neisseria and Bacteroidesspp., B. anthracis, Listeria, Proteus, Pseudomonasand Nocardia, spp., fungi, and other organisms inpure culture.

Commensals

There are none.

Pus 161

SEROUS FLUIDS

These fluids include pleural, peritoneal synovial,pericardial, hydrocele, ascitic and bursa fluids.

Record the colour, volume and viscosity of thefluid. Centrifuge at 3000 rev/min for at least15 min, preferably in a sealed bucket. Transfer thesupernatant fluid to another bottle and record itscolour and appearance. Make Gram-stained filmsof the deposit and also stain films for cytologicalevaluation. Plate on blood agar and incubate aero-bically and anaerobically at 37°C for 18–24 h.Culture on blood agar anaerobically plus 10% CO2

atmosphere and incubate for 7–10 days forBacteroides spp. Use additional material as out-lined for pus. Prolonged incubation may be neces-sary for these strictly anaerobic organisms.

Gonococcal and meningococcal arthritis must notbe overlooked; inoculate one of the GC media andincubate in a 10% CO2 atmosphere for 48 h at 22and 37°C. Culture, if appropriate, for mycobacteria.

Likely pathogens

These include S. pyogenes, S. pneumoniae, anaero-bic cocci, S. aureus, N. gonorrhoeae, M. tuberculo-sis and Bacteroides spp.

Commensals

There are none.

SPUTUM

Sputum is a difficult specimen. Ideally, it shouldrepresent the discharge of the bronchial tree expec-torated quickly with the minimum of contamina-tion from the pharynx and the mouth, anddelivered without delay to the laboratory.

As a result of the irregular distribution of bacteriain sputum, it may be advisable to wash portions ofpurulent material to free them from contaminatingmouth organisms before examining films and

making cultures. Gram-stained films may be useful,suggesting a predominant organism.

Homogenization of the specimen with Sputasol,Sputolysin, N-acetylcysteine or pancreatin, forexample, followed by dilution enables the signifi-cant flora to be assessed. In sputum cultures not sotreated initially, small numbers of contaminatingorganisms may overgrow pathogens.

The following method is recommended. Addabout 2 ml sputum to an equal volume ofSputolysin or Sputasol in a screw-capped bottleand allow to digest for 20–30 min with occasionalshaking, e.g. on a Vortex Mixer. Inoculate a bloodagar plate with 0.01 ml of the homogenate, using astandard loop (e.g. a 0.01-ml plastic loop) andmake a Gram-stained film. Add 0.01 ml with a sim-ilar loop to 10 ml peptone broth. Mix this thor-oughly, preferably using a Vortex Mixer. Inoculateblood agar and MacConkey agar with 0.01 ml ofthis dilution. Incubate the plates overnight at 35°C;incubate the blood agar plates in a 10% CO2

atmosphere. Chocolate agar may be used butshould not be necessary for growing H. influenzaeif a good blood agar base is used.

Five to 50 colonies of a particular organism onthe dilution plate are equivalent to 106–107 of theseorganisms/millilitre of sputum and this level is sig-nificant, except for viridans streptococci when oneto five colonies are equivalent to 105–106 organ-isms/millilitre and this level is equivocal.

Set up direct sensitivity tests if the Gram filmshows large numbers of any particular organisms.

Methods of examining sputum for mycobacteriaare described in Chapter 45.

For the isolation of Legionella spp. and similarorganisms from lung biopsies or bronchial secre-tions use blood agar or one of the commerciallegionella media plus supplement.

Likely pathogens

These include S. aureus, S. pneumoniae, H. influen-zae, coliform bacilli, Klebsiella pneumoniae,Pasteurella, Mycobacterium and Candida spp., M.catarrhalis, Mycoplasma, Pasteurella, Yersinia


spp., Y. pestis, L. pneumophila, Aspergillus,Histoplasma, Cryptococcus, Blastomyces andPseudomonas spp., particularly from patients withcystic fibrosis (also Chlamydia pneumoniae,C. psittaci and Coxiella burnettii).

Commensals

These include S. epidermidis, micrococci, col-iforms, Candida spp. in small numbers, and viri-dans streptococci.

THROAT SWABS

Collect throat swabs carefully with the patient in aclear light. It is customary to attempt to avoid con-tamination with mouth organisms. Evidence hasbeen advanced that the recovery of S. pyogenesfrom the saliva may be higher than from a ‘throat’or pharyngeal swab. Make a direct film, stain withdilute carbol–fuchsin and examine for Vincent’sorganisms, yeasts and mycelia.

Plate on blood agar and incubate aerobically andanaerobically for a minimum of 18 but preferably48 h in 7–10% CO2 at 35°C. Plate on tellurite mediumand incubate for 48 h in 7–10% CO2 at 35°C.

For details of the logistics of mass swabbing, e.g.in a diphtheria outbreak, see Collins and Dulake(1983).

Likely pathogens

These include S. pyogenes, C. diphtheriae, C. ulcer-ans, S. aureus, C. albicans, N. meningitides andB. vincenti (H. influenzae Pittman’s type b in theepiglottis is certainly a pathogen).

Commensals

These include viridans streptococci, S. epidermidis,diphtheroids, S. pneumoniae, and probablyH. influenzae.

TISSUES, BIOPSY SPECIMENS,POSTMORTEM MATERIAL

As a general rule, retain all materials submittedfrom postmortem examination until the forensicpathologists agree to their disposal. If it is essentialto grind up tissue, obtain approval.

Grind tissue specimens in a blender or, if the spec-imen is very small, in a Griffith’s tube or with glassbeads on a Vortex Mixer. The Stomacher Lab-Blender is best for larger specimens because it savestime and eliminates the hazard of aerosol forma-tion. Examine the homogenate as described under‘Pus’.

URETHRAL DISCHARGES

Collect swabs of discharges into a transportmedium or plate directly at the bedside or in theclinic. Examine direct Gram-stained films. Lookfor intracellular Gram-negative diplococci. Inmales a Gram-stained film of the urethral dis-charge is a sensitive and specific means of diag-nosing gonorrhoea. In the female, the recovery oforganisms is more difficult and the interpretationof films may occasionally present problemsbecause over-decolourized or dying Gram-positive cocci may resemble Gram-negative diplo-cocci.

Plate on chocolate agar and a selective mediumfor gonococci (e.g. Modified Thayer-Martin,Modified New York City medium) and incubatein a humid atmosphere enriched with 5%CO2.

Likely pathogens

These include N. gonorrhoeae, and Mycoplasmaand Ureaplasma spp. Occasionally other pathogensmay be isolated, particularly coliforms when aurinary catheter is present.

Urethral discharges 163

Commensals

These include S. epidermidis, micrococci, diph-theroids and small numbers of coliform organisms.

URINE

The healthy urinary tract is free from organismsover the greater part of its length; there may, how-ever, be a few transient organisms present, espe-cially at the lower end of the female urethra.

Collection

Catheters may contribute to urinary infections andare no longer considered necessary for the collec-tion of satisfactory samples from females.

A ‘clean catch’ mid-stream urine (MSU) is themost satisfactory specimen for most purposes if it isdelivered promptly to the laboratory. Failingprompt delivery, the specimen may be kept in arefrigerator at 4°C for a few hours or, exception-ally, a few crystals of boric acid may be added.

Dip slides are now widely used for assessing uri-nary tract infections. They do, however, precludeexamination for cells and other elements unless afresh specimen is also submitted.

Examination

This includes counting leucocytes, erythrocytes andcasts, estimating the number of bacteria/millilitreand identifying them. Normally, there will be noneor fewer than 20 × 106/l of leucocytes or other cellsand a colony count of less than 104 organisms/ml.

Mix the urine by rotation, never by inversion.Count the cells with a Fuchs Rosenthal or similarcounting chamber or use an inverted microscope.Inoculate a blood agar plate and a MacConkey,CLED or EMB plate with 0.001 ml using a stan-dard loop (disposable loops or micro-pipettes with

disposable tips are satisfactory alternatives). Also0.1 ml of a 1 : 100 dilution of urine may be used.Spread with a glass spreader, incubate overnight at37°C and count the colonies:

< 10 colonies = 104 organisms/ml not significant

10–100 colonies = 104–105 organisms/ml doubtfulsignificance

> 100 colonies = > 105 organisms/ml significantbacteriuria

If dip slides are submitted, incubate themovernight and follow the manufacturer’s directionsfor counting.

Note that the accuracy and usefulness of bacter-ial counts on urines are limited by the age of thespecimen. Only fresh specimens are worth exam-ining. Other limitations are the volume of fluidrecently drunk and the amount of urine in thebladder.

Various other techniques, e.g. a filter paper ‘foot’,multipoint inoculator, dip sticks for leucocyteenzymes or simple visual inspection, have beenadvocated for screening urines. These save mediaand may be appropriate in laboratories where mostsamples tested are ‘negative’.

For methods of examining urine for tuberclebacilli, see p. 382.

Automated urine examination

The Rapid Automatic Microbiological Screeningsystem (RAMUS, Orbec), is a particle volumeanalyser, capable of counting leucocytes and bacte-ria. Counts of bacteria > 105/ml are regarded assignificant.

Multipoint inoculation methods are very useful inthe examination of large batches of urines (seeFaiers et al., 1991, and Chapter 8). The techniquecan allow for tests for the presence of antibacterialsubstances, direct inoculation of identificationmedia and media for those fastidious organismsthat are now considered to be of importance in uri-nary tract infections.


Likely pathogens

These include E. coli, other enterobacteria, Proteusspp., staphylococci including S. epidermidis,S. saprophyticus, enterococci, salmonellas (rarely),Leptospira spp. and mycobacteria.

Commensals

There can be small numbers (< 104/ml) of almostany organisms. It is unwise to be too dogmaticabout small numbers of organisms isolated from asingle specimen; it is better to repeat the sample.

VAGINAL DISCHARGES

Bacterial vaginosis is a condition in which there is adisturbance in the vaginal flora. Lactobacillidecrease in numbers whereas those of a variety ofothers, e.g. Gardnerella vaginalis, and Mobiluncusand Prevotella spp. increase. This condition shouldbe differentiated from vaginitis caused by C. albi-cans. A Gram-stained film may reveal ‘clue cells’ –epithelial cells with margins obscured by largenumbers of adherent Gram-variable rods. Culturefor G. vaginalis is less helpful.

PUERPERAL INFECTIONS

A high vaginal swab is usually taken. Swabs thatcannot be processed very rapidly after collectionshould be put into transport medium. Examinedirect Gram-stained films. If non-sporing, square-ended, Gram-positive rods, not in pairs, and whichappear to be capsulated, are seen they should bereported without delay, because they may beC. perfringens, which can be an extremely invasiveorganism.

Plate on blood agar and incubate aerobically andanaerobically overnight at 37°C and also plate onMacConkey or other suitable selective medium.Plate any swabs suspected of harbouring C. per-

fringens on a neomycin half-antitoxin Nagler platefor anaerobic incubation.

Likely pathogens

These include S. pyogenes, S. agalactiae, L. mono-cytogenes, C. perfringens, Bacteroides spp.,G. vaginalis, anaerobic cocci, Candida spp., andexcess numbers of enteric organisms.

Commensals

These include lactobacilli, diphtheroids, micro-cocci, S. epidermidis, small numbers of coliformbacilli, and yeasts.

NON-PUERPERAL INFECTIONS

Examine a Gram-stained smear and also a wetpreparation for the presence of Trichomonasvaginalis. If the examination is to be immediate,a saline suspension of vaginal discharge will besatisfactory; otherwise a swab in transportmedium is essential. A wet preparation may stillbe examined for actively motile flagellates butculture in trichomonas medium usually yieldsmore reliable results, especially in cases of lightinfection. An alternative to the wet preparation isthe examination under the fluorescence micro-scope of an air-dried smear stained by acridineorange.

Examine a Gram-stained smear for Mobiluncusspp. If N. gonorrhoeae infection is suspected in afemale, swabs from the urethra, cervix and poste-rior fornix should be collected, placed into trans-port medium or plated immediately on selectivemedium. Many workers do not make films fromthese swabs because the difficulties of interpreta-tion are compounded by the presence of charcoalfrom the buffered swabs; they rely on cultivationusing VCN (vancomycin, colistin, nystatin) orVCNT (VCN + trimethoprim) medium as a selec-

Non-puerperal infections 165

tive inhibitor of organisms other than N. gonor-rhoeae.

Plate on blood agar and incubate, aerobically andanaerobically, on Sabouraud’s agar (when lookingfor Candida spp.) and chocolate agar or anothersuitable gonococcal medium for incubation in a5% CO2 atmosphere to encourage isolation ofN. gonorrhoeae.

If L. monocytogenes infection is suspected, plateon serum agar with 0.4 g/l nalidixic acid added.

Likely pathogens

These include N. gonorrhoeae, C. albicans andrelated yeasts in moderate numbers, S. pyogenes,Gardnerella spp., L. monocytogenes, Haemophilusand Bacteroides spp., anaerobic cocci, Mobiluncusspp., possibly Mycoplasma spp. and T. vaginalis.

Commensals

These include lactobacilli, diphtheroids, entero-cocci and small numbers of coliform organisms.

WOUNDS (SUPERFICIAL) ANDULCERS

Examine Gram-stained films and culture on bloodagar and proceed as for pus, etc. Examine Albert-stained smears for corynebacteria. Culture also onLöwenstein–Jensen medium and incubate at 30°Cbecause some mycobacteria that cause superficialinfections do not grow on primary isolation at35–37°C.

Likely pathogens

These include S. aureus, streptococci, C. diphthe-riae, C. ulcerans (both in ulcers), M. marinum,M. ulcerans and M. chelonei.

Contaminants

These include staphylococci, Pseudomonas spp.,and a wide variety of bacteria and fungi.

WOUNDS (DEEP) AND BURNS

The collection of these specimens and their conse-quent rapid processing are two vital factors in therecovery of the significant organisms. Burn casesfrequently become infected with staphylococci andstreptococci as well as with various Gram-negativerods, especially Pseudomonas spp. It may be diffi-cult to recover Gram-positive cocci because of theovergrowing Gram-negative organisms.

Plate on blood agar and incubate aerobically andanaerobically. Plate also on MacConkey agar andCLED medium (to inhibit the spreading of Proteusspp.). Robertson’s cooked meat medium may beuseful.

Plate swabs, etc. from burns on media selectivefor S. pyogenes and Pseudomonas spp. Anaerobesare rarely a problem in burns.

Likely pathogens

These include Clostridium spp., S. pyogenes,S. aureus, anaerobic cocci, Bacteroides spp. andGram-negative rods.

Contaminants

These include small numbers of a wide variety oforganisms.

REFERENCES

Advisory Committee on Dangerous Pathogens (ACDP)(1995) Protection against Blood-borne Infections

in the Workplace: HIV and hepatitis. London:HMSO.


ACDP (1998) Transmissible Spongiform

Encephalopathies: Safe working and the prevention

of infection. London: HMSO.ACDP (2001) Supplement to ACDP Guidance on

Protection against Blood-borne Infections in the

Workplace: HIV and hepatitis. London: HMSO.Collins, C. H. and Dulake, C. (1983) Diphtheria: the

logistics of mass swabbing. Journal of Infection 6:277–280

Collins, C. H. and Kennedy, D. A. (eds) (1997)Occupational Blood-borne Infections. Wallingford:CAB International.

Collins, C. H. and Kennedy, D. A. (1999) Laboratory-

acquired Infections, 4th edn. Oxford:Butterworth–Heinemann, pp. 240–244.

Faiers, M., George, R., Jolly, J. and Wheat, P. (1991)Multipoint Methods in the Clinical Laboratory.London: Public Health Laboratory Service.

National Institutes of Health (1988) Working Safelywith HIV in the Research Laboratory. Bethesda,MD: NIH.

World Health Organization (1991) Biosafety

Guidelines for Diagnostic and Research

Laboratories Working with HIV, WHO AIDSSeries 9. Geneva: WHO.

References 167

The primary purpose of antimicrobial susceptibil-ity testing is to guide the clinician in the choice ofappropriate agents for therapy. Agents are oftenused empirically and routine testing provides up-to-date information on the most suitable agents forempirical use. Apart from routine clinical work,antimicrobial susceptibility tests are used to evalu-ate the in vitro activity of new agents.

Antimicrobial susceptibility may be reportedqualitatively as: sensitive (S), which indicates thatthe standard dose of the agent should be appropri-ate for treating the patient infected with the straintested; resistant (R), which indicates that an infec-tion caused by the isolate tested is unlikely torespond to treatment with that antimicrobial agent;and intermediate (I), where the strains are moder-ately resistant or moderately susceptible and indi-cate that the strain may be inhibited by larger dosesof the agent or may be inhibited in sites where theagent is concentrated (e.g. urine). It may also beregarded as a ‘buffer-zone’, which reduces the like-lihood of major errors, i.e. reporting a resistant iso-late as susceptible or vice versa. Caution isnecessary when the basis of resistance is the pro-duction of inactivating enzymes because zone sizesor minimum inhibitory concentrations (MICs) (seebelow) may indicate intermediate susceptibilityrather than resistance. The routine use of the inter-mediate category on patient reports should beavoided because clinicians rarely find it helpful

Quantitative testing determines the MIC, which isthe lowest concentration of the agent that will pre-vent a microbial population from growing. Theminimum bactericidal concentration (MBC) is thelowest concentration that kills 99.9% of the popu-lation.

The concentrations of agents defining the ‘break-points’ between sensitive, intermediate and resist-ant are based on clinical, pharmacological andmicrobiological considerations. These breakpointconcentrations may be used to interpret MICresults, as test concentrations in breakpoint meth-ods, or as the basis for interpreting zone sizes indisc diffusion tests (Andrews, 2001a, 2001b; King,2001).

TECHNICAL METHODS

The three principal methods used for susceptibilitytesting, Disc Diffusion, MIC/MBC determinationand Breakpoint, are outlined briefly below, as arethe technical factors that influence the results.

Disc diffusion tests

Diffusion tests with agents in paper discs are stillthe most widely used methods in routine clinicallaboratories. They are technically straightforward,reliable if correctly standardized and also relativelycheap.

Minimum inhibitory concentration

In minimum inhibitory concentration methods, thesusceptibility of organisms to serial twofold dilu-tions of agents in agar or broth is determined. TheMIC is defined as the lowest concentration of theagent that inhibits visible growth. The methods are

12

Antimicrobial susceptibility tests

not widely used for routine testing in clinical labo-ratories in most countries, although commercialsystems with restricted-range MICs in a variety offormats are available from a number of companies.In clinical laboratories MIC methods are used totest the susceptibility of organisms that give equiv-ocal results in diffusion tests, for tests on organismswhere disc tests may be unreliable, as with slow-growing organisms, or when a more accurate resultis required for clinical management, e.g. infectiveendocarditis. MIC methods are accepted as thestandard against which other methods are assessedand for testing new agents. MICs may also bederived from the size of the zone of inhibition indisc diffusion tests using a regression curve pro-duced by plotting the diameter of the zone of inhi-bition against the MIC of a range of strains of agiven species. In some cases it is necessary to knowthe MBC of an organism. This is determined bysubculturing the broth of tubes showing no turbid-ity in an MIC test on to antimicrobial agent-freemedia and observing for growth after further incu-bation.

Breakpoint methods

Breakpoint methods are essentially MIC methodsin which only one or two concentrations are tested.The concentrations chosen for testing are thoseequivalent to the breakpoint concentrations sepa-rating different categories of susceptibility. Inrecent years such methods have been used routinelyas an alternative to disc diffusion methods.Breakpoint tests are suitable for automated inocu-lation and reading of tests, and may be combinedwith identification tests.

Factors affecting susceptibility tests

Many of the effects of technical variation onsusceptibility testing are widely known (Barry,1976, 1991; Brown and Blowers, 1978; Wheat,2001).

MediumThe medium should support the growth of organ-isms normally tested and should not contain antag-onists of antimicrobial activity or substances (e.g.albumin) that can bind to and inactivate microbialagents. Thymidine antagonizes the activity ofsulphonamides and trimethoprim, leading to falsereports of resistance to these agents. If the mediumis not low in thymidine, lysed horse blood orthymidine phosphorylase should be added toremove free thymidine. Monovalent cations such asNa+ increase the activity of bacitracin, fusidic acidand novobiocin against staphylococci, and of peni-cillins against Proteus spp. Divalent cations such asMg2+ and Ca2+ reduce the activity of aminoglyco-sides and polymyxins against Pseudomonas spp.,and of tetracyclines against a range of organisms.

Media specially formulated for susceptibility test-ing should be used. Iso-Sensitest agar (Oxoid) andsimilar media are generally used in the UK;Mueller–Hinton agar (various suppliers) is used inseveral other countries. There have been problemswith consistency of batches of Mueller–Hintonagar which has led to the publication of a perform-ance standard (National Committee for ClinicalLaboratory Standards or NCCLS, 1996, 1997,1999). Additional supplements are necessary forgrowth of some organisms (see also Chapter 2).

pHThe activity of aminoglycosides, macrolides, lin-cosamides and nitrofurantoin increases with increas-ing pH. Conversely, the activity of tetracycline,fusidic acid and novobiocin increases as the pH islowered. The microbial growth rate is also influencedby pH, thus indirectly affecting susceptibility.

Depth of agar mediumWith diffusion tests the size of the zone varies withthe depth of the medium. The agar in the plateshould be level and of a consistent depth – approx-imately 4 ± 0.5 mm.

Inoculum size Increasing the inoculum size reduces the suscepti-bility to agents in both diffusion and dilution tests.

Technical methods 169

Tests of susceptibility to sulphonamides, trimetho-prim and β-lactam agents, where resistance is basedon β-lactamase production in staphylococci,Haemophilus spp. and Neisseria spp., and methi-cillin resistance in staphylococci, are particularlyaffected by variation in inoculum size. Among Gram-negative organisms, the effect of inoculum size onMICs of those producing β-lactamase depends onthe amount of any β-lactamase produced, and theactivity of the enzyme against the particular peni-cillin or cephalosporin under test. Standardizationof the inoculum size is therefore necessary.

In most diffusion methods an inoculum size thatgives a semi-confluent growth is used. This has theadvantage that incorrect inocula can be readilyidentified and the test rejected. There is no evi-dence, however, that semi-confluent growth givesmore reliable results than just confluent growth, asused in the NCCLS (2003) diffusion method.

In agar dilution methods (MIC or breakpoint) aninoculum of 104 colony-forming units (cfu)/inocu-lum spot is the accepted standard. This should beincreased to 106 cfu/inoculum spot for methicillinsusceptibility tests on staphylococci. With organ-isms producing extracellular β-lactamases, thestandard inoculum may result in MICs only slightlyhigher than those obtained with susceptible strains.This can be avoided by increasing the inoculum to106 cfu/inoculum spot on agar dilution plates. Forbroth dilution methods the standard inoculumshould be 105 cfu/ml of test broth at the beginningof the incubation period.

Use four or five colonies of a pure culture to avoidselecting an atypical variant. Prepare the inoculumby emulsifying overnight colonies from an agarmedium, by diluting an overnight broth culture orby diluting a late log-phase broth culture. Thebroth should not be antagonistic to the agent tested(e.g. media containing thymidine should not beused to prepare inocula for tests on sulphonamidesand trimethoprim). Organisms should be diluted indistilled water to a concentration of 107 cfu/ml. A0.5 McFarland standard gives a density equivalentto approximately 108 cfu/ml. Alternatively, inoculamay be adjusted photometrically or by standarddilution of broth cultures if the density of such cul-

tures is reasonably constant. If there is any doubtabout whether a particular inoculum preparationprocedure yields the requisite density of organisms,it should be checked by performing viable countson a small number of representative isolates beforeembarking on a series of MIC tests. Inocula shouldbe used promptly to avoid subsequent changes intheir density. Plates should be inoculated within15 min of standardizing the inoculum.

Preincubation and prediffusion In diffusion tests preincubation and prediffusion ofthe inoculum decrease and increase the sizes ofzones respectively. In practice these effects areavoidable by applying discs soon after plates areinoculated and incubating plates soon after discsare applied.

Antimicrobial agents/discs Modern commercial discs are generally producedto a high standard and problems caused by incor-rect content or incorrect agents in discs are rare.Humidity will cause deterioration of labile agentsand many of the problems with discs are related toincorrect handling in the laboratory. Store discs insealed containers in the dark (some agents, e.g.metronidazole, chloramphenicol and quinilones,are sensitive to light); the containers should includean indicating desiccant. Discs may be kept at < 8°Cwithin the expiry date given by the maker. To min-imize condensation of water on the discs allow con-tainers to warm to room temperature beforeopening them. For convenience, leave closed con-tainers at room temperature during the day andrefrigerate them overnight only.

Problems with humidity also affect pure antimi-crobial powders used in MIC testing and similarprecautions to those described for discs should betaken. If powders absorb water the amount ofactive substance per weighed amount will bereduced even if the agent is not labile because of theweight of the water absorbed.

IncubationIncubate plates at 35–37°C in air. Use alternativeconditions only if essential for growth of the organ-

170 Antimicrobial susceptibility tests

isms. Incubation in an atmosphere containing addi-tional carbon dioxide may reduce the pH, whichmay affect the activity of some agents. Incubationat 30°C is usually necessary for methicillin/oxacillin susceptibility tests against staphylococci,but should not be used for other agents becausezone sizes will be increased. Do not stack platesmore than four high in the incubator in order toavoid uneven heating during incubation.

Reading results Unless tests are read by an automated system thereading is somewhat subjective without clear end-points. Criteria for reading and interpreting suscep-tibility tests – whatever basic method is used –should be written down and available to staff whodo these tests.

Definitions of endpoints for agar dilution testsdiffer, although up to four colonies or a thin hazein the inoculated spot are commonly disregarded.Endpoints trailing over several dilutions may indi-cate contamination or a minority resistant popula-tion.

For broth dilution tests the MIC is the lowest con-centration of the agent that completely inhibitsgrowth.

In reading diffusion tests take the point of abruptdiminution of growth as the zone edge. If there isdoubt read the point of 80% inhibition of growth.Measure zones at points unaffected by interactionswith other agents or by proximity to the edge ofthe plate. Ignore tiny colonies at the zone edge,swarming of Proteus spp., and traces of growthwithin sulphonamide and trimethoprim zones.Large colonies within zones of inhibition may indi-cate contamination or a minority resistant popula-tion.

Diffusion methods

The basic technique for disc diffusion tests has notchanged for many years. Paper discs containingknown amounts of antibiotic are applied to plates

inoculated with the test and/or control organism.After incubation the diameter of the zone of inhibi-tion of growth is used to decide whether the strainis susceptible to the agent being tested. The meth-ods of control of variation and the basis of inter-pretation have fallen into two groups: thecomparative methods and the standardizedmethods.

In the comparative methods the zone size of thetest strain is compared with that of a control strainset up at the same time. It is assumed that variationin the test affects the test and control strainsequally, and thereby cancels them out. The com-parison may be made with a control strain on aseparate plate or both control and test may beinoculated on the same plate. However, the lack ofstandardization and the difficulty in selecting suit-able controls make these methods less reliable thanstandardized methods. This is particularly so formany of the newer antibiotics. The lack of stan-dardization also makes the analysis of trends inantibiotic resistance by inter- and intra-laboratorycomparison difficult, and the epidemiological valueof results is low.

In the standardized methods all details of thetests are recommended in the report of an interna-tional collaborative study of susceptibility testing(Ericsson and Sherris, 1971). Interpretation ofzone diameters is from a table of zone size break-points. The zone size breakpoints equivalent todefined MIC breakpoints have been established byregression analysis of the relationship of zone sizeto MIC, by error minimization, by study of thedistribution of susceptibility of different species,and by clinical experience relating the results oftests to the outcome of treatment. These differentapproaches have been combined in some methods.An important feature of the standardized methodsis that the zone size breakpoints are valid only ifall aspects of the defined method are followed pre-cisely. Hence there are several standardized meth-ods that differ in detail and in the breakpointsused. Both the British Society for AntimicrobialChemotherapy (BSAC) method (Andrews, 2001b)and, in the USA, the NCCLS (2003) method areexamples.

Technical methods 171

STANDARDIZED METHODS

British Society for AntimicrobialChemotherapy method

For full details of this method, the BSACGuidelines (Andrews, 2001b) should be consulted.These are also available on the BSAC website(www.bsac.org.uk) which is updated regularly. Asummary of the method is given below.

MediaIso-Sensitest agar (ISTA, Oxoid) is used for testingEnterobacteriaceae, Pseudomonas spp., entero-cocci and staphylococci other than for thedetection of methicillin/oxacillin resistance.Mueller–Hinton or Columbia agar with 2%sodium chloride is used for the detection of methi-cillin/oxacillin resistance in staphylococci. Iso-Sensitest agar (ISTA) with 5% defibrinated horseblood (ISTHBA) is used for Streptococcus pneumo-niae, haemolytic streptococci, Moraxellacatarrhalis, Neisseria meningitidis, and N. gonor-rhoeae. ISTHBA supplemented with 20 mg/lβ-nicotinamide adenine dinucleotide (NAD) is usedfor Haemophilus spp. and may also be used fororganisms tested on ISTHBA. The medium shouldbe approximately 4 mm deep (25 ml in a 90-mmPetri dish). Plates should be dried to remove excesssurface moisture but it is important not to over-drythem.

ControlsControl organisms should be used to monitor theperformance of the test by ensuring that the zonesizes obtained fall within the recommended limits.These are published by the BSAC. Examples aregiven in Table 12.1. They are also used to confirmthat the method will detect resistance. They are notused for the interpretation of susceptibility. Thefollowing are the control organisms: Escherichiacoli NCTC 12241 (ATCC 25922) or NCTC10418; E. coli β-lactamase positive strain NCTC11560; Staphylococcus aureus NCTC 12981(ATCC 25923) or NCTC 6571; S. aureus methi-

cillin/oxacillin-resistant strain NCTC 12493;Pseudomonas aeruginosa NCTC 12934 (ATCC27853) or NCTC 10662; Enterobacter faecalisNCTC 12697 (ATCC 29212); H. influenzaeNCTC 12699 (ATCC 49247 or NCTC 11931);S. pneumoniae NCTC 12977 (ATCC 49619);N. gonorrhoeae NCTC 12700 (ATCC 49226).Control strains should be stored so as to minimizemutation. Storage at –70°C on beads in glycerolbroth is ideal. Two vials should be used for eachcontrol, one ‘in use’ which is subcultured weeklyon to a non-selective medium. If this subculture ispure it is used to prepare one subculture for each ofthe next 7 days. A bead from the second vial is sub-cultured when it is necessary to replenish the ‘in-use’ vial.

InoculumThe aim is to produce a semi-confluent growth.This has the advantage that an incorrect inoculumcan easily be seen. A suspension of organisms in asuitable diluent (broth or peptone water) with adensity equivalent to a 0.5 McFarland standard isprepared and then diluted appropriately. To pre-pare a 0.5 McFarland standard add 0.5 ml0.048 mol/l BaCl2 (1.17% w/v BaCl2·2H2O) to99.5 ml 0.18 mol/l H2SO4 (1% w/v) with constantstirring. This should be distributed into screw-capped bottles of the same volume and size as thoseused for the preparation of the test suspensions.These standards should be stored, thoroughlymixed before use and replaced every 6 months.Alternatively, pre-prepared standards can beobtained from commercial suppliers. There are alsopre-calibrated photometers available from a num-ber of suppliers. Organisms may be suspendeddirectly in Iso-Sensitest broth, or non-fastidiousorganisms inoculated into Iso-Sensitest broth andincubated at 37°C until the appropriate density hasbeen achieved. The density of the suspension isadjusted to match the 0.5 McFarland standard andthen further diluted, within 10 min of preparation,in sterile distilled water. Enterobacteriaceae, ente-rococci, haemolytic streptococci, Pseudomonasspp. Acinetobacter spp. and Haemophilus spp. arediluted 1 : 100, staphylococci, Serratia spp.,


www.bsac.org.uk

Standardized methods 173

Table 12.1 The zone diameters, in millimetres, of control strains used in the BSAC disc diffusion method growing on Iso-Sensitest agarincubated at 35–37°C in air

a

Antibiotic Disc Escherichia coli Pseudomonas Staphylococcus Enterobacter content aeruginosa aureus faecalis(μg)

NCTC ATCC NCTC NCTC ATCC NCTC ATCC ATCC 10418 25922 11560 10662 27853 6571 25923 29212

Amikacin 30 24–27 23–27 21–30 26–32Ampicillin 10 21–26 16–22 26–35

25 24–30Cefotaxime 30 36–45 34–44 20–29 20–24

5 26–32b

Ceftazidime 30 32–40 31–39 29–37 27–35Cefuroxime 30 25–32 24–29Ciprofloxacin 1 31–40 31–37 21–28 24–30 25–32 17–22Co-amoxiclav 3 27–32

30 18–31 20–26 18–23Erythromycin 5 22–31 22–29

5 25–29b

5 22–29c 23–29c

Fusidic acid 10 32–40 30–37Gentamicin 10 21–27 21–27 20–26 22–28 24–30 20–26

200 22–27Imipenem 10 32–37 33–37 20–27 23–28Methicillind 5 18–30 18–28Oxacillind 1 19–30 19–29Penicillin 1 unit 32–40 29–36

1 unit 30–41b 27–35b

1 unit 39–43b

Piperacillin/ 85 28–35 29–37Tazobactam

Teicoplanin 30 17–23 16–20 19–25Trimethoprim 2.5 28–34 20–26 25–30 20–28

5 24–34Vancomycin 5 14–20 13–17 13–19

H. influenzae S. pneumoniaeb

(with NAD)b

NCTC ATCC ATCC 11931 49247 49619

Ampicillin 2 22–30 6–13Cefotaxime 5 33–45 27–38Co-amoxiclav 3 20–27 10–20Erythromycin 5 12–23 9–16 23–36Oxacillin 1 8–16

aAfter BSAC guidelines (Andrews, 2001b) with permissionbUsing Iso-Sensitest agar with 5% horse blood with or without NAD incubated at 35–37°C in 5% CO2cUsing Iso-Sensitest agar with 5% horse blood with or without NAD incubated at 35–37°C in airdUsing Mueller–Hinton with 2% NaCl incubated at 30°C in air

S. pneumoniae, N. meningitides and M. catarrhalisare diluted 1 : 10. N. gonorrhoeae is not dilutedany further. This suspension is used to inoculateplates within 15 min of preparation. A sterile cot-tonwool-tipped swab is dipped into the suspension,and the excess fluid removed and used to spread theinoculum over the entire surface of the plate.

DiscsDiscs should be stored as detailed above. Up to sixmay be placed on the surface of a 90-mm Petri dish.

IncubationWith most organisms plates should be incubatedwithin 15 min of disc application at 35–37°C in airfor 18–24 h. For Neisseria spp., Haemophilus spp.and S. pneumoniae, however, 5% CO2 at 35–37°Cis required. Also 24-h incubation is essential beforereporting enterococci as sensitive to vancomycin orteicoplanin. Plates for methicillin/oxacillin suscep-tibility testing are incubated at 30°C in air beforeinoculation.

ReadingThe diameter of the zone of inhibition is measuredto the nearest millimetre with a ruler, callipers oran automated zone reader. A template may also beused for interpreting susceptibility (a programmefor constructing templates may be downloadedfrom the BSAC website: www.bsac.org.uk). Thesusceptibility of a strain is determined by compar-ing the zone size obtained with breakpoint sizesgiven in the table published in conjunction with themethod. The zone size for a given antibiotic willvary according to the species being tested and theconcentration of the disc. Examples of disc concen-trations, MIC and zone size breakpoints andtheir interpretation are shown in Tables 12.2 and12.3.

Anaerobes and fastidious organisms Anaerobes and fastidious organisms require specialconditions (King, 2001). For those that will growwell in 24 h the BSAC recommend Wilkins–Chaldren agar (Oxoid) with 5% horse blood. Asuspension equivalent to a 0.5 McFarland Standard

is prepared and diluted 1 : 100 for Gram-negativeorganisms and 1 : 10 for Gram-positive organisms.Incubation is at 35–37°C under strict anaerobicconditions for 18–24 h.

For slow growing anaerobes and Helicobacterpylori the E test (see below) is recommended. Whentesting Campylobacter spp. an undiluted 0.5McFarland suspension is inoculated on to Iso-Sensitest agar with 5% horse blood, incubated at42°C or 37°C under microaerophilic conditions for18–24 h. Disc testing is not suitable for Legionellaspp. MICs should be determined on Buffered YeastExtract agar with 5% horse blood to which differ-ent concentrations of the antimicrobials have beenadded (at 45°C).

National Committee for ClinicalLaboratory Standards method

This is the standard method used in the USA and issummarized below. For full details including con-trol organisms, disc concentrations and the inter-pretation of zone sizes consult relevant NCCLS(2003) publications.

MediaMueller–Hinton agar is used for most organisms.For Streptococcus spp. 5% defibrinated sheepblood is added. For Haemophilus spp. useMueller–Hinton agar supplemented with 15 mg/lβ-NAD, 15 mg/l bovine haemin and 5 g/l yeastextract. GC agar with a defined growth supplementis used for N. gonorrhoeae.

InoculumA suspension in broth or other suitable diluent,with a turbidity equivalent to a 0.5 McFarlandStandard, is used as the inoculum.

IncubationPlates are normally incubated at 35°C for 16–18 hin air. For streptococci, Neisseria spp. andHaemophilus spp. they are incubated in 5% CO2,and for anaerobes in an anaerobic cabinet.


www.bsac.org.uk

Standardized methods 175

Table 12.2 Examples of MIC breakpoints, disc contents and zone diameters for the BSAC susceptibility methoda

Organisms and antibiotic MIC breakpoint (mg/l) Disc content Interpretation of zone (μg) diameters (mm)

R ≥ I S ≤ R ≥ I S ≤

Enterobacteriaceae and AcinetobacterAmpicillin 16 – 8 10 17 – 18Cefotaxime 2 – 1 30 29 – 30 Cefuroxime 16 – 8 30 19 – 20 Ciprofloxacin 2 – 1 2 17 – 18 Co-amoxiclav 16 – 8 20/10 17 – 18 Gentamicin 2 – 1 10 19 – 20 Trimethoprim 4 1–2 0.5 2.5 14 15–19 20

Pseudomonas spp.Amikacin 32 8–16 4 30 17 18–21 22 Ceftazidime 16 – 8 30 23 – 24 Ciprofloxacin 8 2–4 1 1 9 – 10 Gentamicin 8 2–4 1 10 14 15–21 22 Imipenem 8 – 4 10 21 – 22 Piperacillin/Tazobactam 32 – 16 75/10 23 – 24

StaphylococciCiprofloxacin 2 – 1 1 17 – 18 Erythromycin 1 – 0.5 5 19 – 20 Fusidic acid 2 – 1 10 29 – 30 Gentamicin 2 – 1 10 19 – 20 Linezolid 8 – 4 10 19 – 20 Methicillin 8 – 4 5 14 – 15 Oxacillin 4 – 2 1 14 – 15 Penicillin 0.25 – 0.12 1 unit 24 – 25 Teicoplanin 8 – 4 30 14 – 15 Vancomycin 8 – 4 5 11 – 12

S. pneumoniaeCefotaxime 2 – 1 5 29 – 30 Erythromycin 1 – 0.5 5 19 – 20 Penicillin 2 0.12–1 0.06 Oxacillin 1 19 – 20

EnterococciAmpicillin 16 – 8 10 19 – 20 Gentamicin 1024 – 512 200 9 – 10 Linezolid 8 – 4 10 19 – 20 Teicoplanin 8 – 4 30 19 – 20 Vancomycin 8 – 4 5 12 – 13

Haemolytic streptococciPenicillin 0.25 – 0.12 1 unit 19 – 20 Erythromycin 1 – 0.5 5 19 – 20 Cefotaxime 2 – 1 5 27 – 28

(continued)


Table 12.2 (continued)

Organisms and antibiotic MIC breakpoint (mg/l) Disc content Interpretation of zone (μg) diameters (mm)

R ≥ I S ≤ R ≥ I S ≤

M. catarrhalisAmpicillin 2 – 1 2 29 – 30 Co-amoxiclav 2 – 1 2/1 18 – 19 Erythromycin 1 – 0.5 5 27 – 28 Cefuroxime 2 – 1 5 19 – 20

N. gonorrhoeaePenicillin 2 0.12–1 0.06 1 unit 17 18–25 26 Cefuroxime 2 – 1 5 19 – 20 Nalidixic acidb 30

N. meningitidisPenicillin 0.12 – 0.06 1 unit 24 – 25 Cefotaxime 2 – 1 5 29 – 30 Rifampicin 2 – 1 2 29 – 30 Ciprofloxacin 2 – 1 1 31 – 32

H. influenzaeAmpicillin 2 – 1 2 19 – 20 Co-amoxiclav 2 – 1 2/1 19 – 20 Cefotaxime 2 – 1 5 24 – 25

aAfter BSAC guidelines (Andrews, 2001b) with permission bQuinolone resistance is most reliably detected with nalidixic acid. Strains with reduced susceptibility to fluoroquinilones, e.g.ciprofloxacin, have no zone of inhibition with nalidixic acidR, resistant; S, sensitive; I, intermediate

Table 12.3 Examples of MIC breakpoints, disc contents and zone diameters for the BSAC susceptibility method for urinary tractinfections (Gram-negative rods)a

Antibiotic Disc Interpretation of zone diameters (mm)

MIC BP (mg/l)content

Coliforms Escherichia coli Proteus mirabilis

R ≥ I S ≤(μg)

R ≥ I S ≤ R ≥ I S ≤ R ≥ I S ≤

Ampicillin 64 – 32 25 15 – 16 24 – 25Cephalexin 64 – 32 30 15 – 16 11 – 12Ciprofloxacin 8 – 4 1 19 – 20 19 – 20 19 – 20Co-amoxiclav 64 – 32 20/10 17 – 18 17 – 18Trimethoprim 4 – 2 2.5 16 – 17 16 – 17 16 – 17Nitrofurantoin 64 – 32 200 19 – 20Mecillinam 16 – 8 10 13 – 14 13 – 14Gentamicin 2 – 1 10 19 – 20 19 – 20 19 – 20aAfter BSAC guidelines (Andrews, 2001b) with permissionR, resistant; S, sensitive; I, intermediate

COMPARATIVE METHODS

Stokes’ method

This is a commonly used disc method in the UK.The diameter of the zone of inhibition of testorganism is compared with that of an appropriatecontrol growing on the same plate (Phillips et al.,1991).

MediaAny medium designed specifically for susceptibilitytesting may be used. It must be supplemented with5% defibrinated horse blood for tests on fastidiousorganisms. If the medium is not free from thymi-dine, add lysed horse blood for tests onsulphonamides and trimethoprim.

ControlsUse E. coli NCTC 10418 and NCTC 11560 to con-trol discs containing both a β-lactam agent and aβ-lactamase inhibitor, e.g. a mixture of amoxicillinand clavulanic acid (co-amoxiclav), P. aeruginosaNCTC 10662, H. influenzae NCTC 11931,S. aureus NCTC 6571, Cl. perfringens NCTC11229, Bacteroides fragilis NCTC 9343.

InoculumIn general, an acceptable inoculum can be preparedfrom a fully grown, nutrient-broth culture or froma suspension of several colonies emulsified in brothto give a density similar to a broth culture. Itshould give semi-confluent growth of colonies onthe plates after overnight incubation. Reject testswith a confluent growth or clearly separatedcolonies and repeat.

Inoculation of platesWith the band plating method the control culture isapplied in two bands on either side of the plateleaving a central area uninoculated. Use pre-impregnated control swabs or apply a loopful of aninoculum prepared as described above to both sidesof the plate and spread it evenly in the two bandswith a dry sterile swab. Seed the test organism

evenly in the band across the centre of the plate bytransferring a loopful of the broth culture or sus-pension to the centre of the plate, and spreading itwith a dry sterile swab. This method usually allowsup to four discs to be applied to the plate.

If a rotary plating method is used, apply the con-trol to the centre of the plate leaving an uninocu-lated 1.5-cm band around the edge of the plate.Apply the test organism to the 1.5-cm band. Thismethod usually allows up to six discs to be appliedto the plate.

Application of discsAfter the inoculum has dried apply discs to theinoculated medium with forceps, a sharp needle ora dispenser, and press down gently to ensure evencontact. Suitable concentrations of agents in discsare listed in Table 12.2.

Place discs on the line between the test and con-trol organisms Four discs can be accommodated ona 9-cm circular plate. If a rotary plating method isused apply six discs on a 9-cm circular plate.

IncubationIncubate tests and controls overnight at 35–37°C(30°C is recommended for methicillin/oxacillin sus-ceptibility tests of staphylococci).

Reading of zones of inhibitionMeasures zones of inhibition from the edge of thedisc to the edge of the zone. As the control and testorganism are adjacent on the same plate the differ-ence between the respective zone sizes may be eas-ily seen. If the test zones are obviously larger thanthe control or give no zone at all, it is not necessaryto make any measurement. If there is any doubt,measure zones with callipers (preferably), dividersor a millimetre rule.

InterpretationInterpret each zone size as follows:

• Sensitive: zone diameter equal to, wider than,or not more than 3 mm smaller than thecontrol.

• Intermediate: zone diameter > 2 mm butsmaller than the control by > 3 mm.

Comparative methods 177

• Resistant: zone diameter ≤ 2 mm.

Exceptions• Penicillinase-producing staphylococci show

heaped-up, clearly defined zone edges, andshould be reported as resistant, irrespective ofzone size.

• Polymyxins diffuse poorly in agar so thatzones are small and the above criteria cannotbe applied. Tests with polymyxins shouldtherefore be interpreted thus:– sensitive: zone diameter equal to, wider

than, or not more than 3 mm smaller thanthe control

– resistant: zone diameter > 3 mm smallerthan the control.

• Zones around ciprofloxacin discs are largewith some of the sensitive control strains sotests should be interpreted thus (1) if S.aureus or P. aeruginosa is used as control; or(2) if H. influenzae or E. coli is used ascontrol:– sensitive: zone diameter equal to, wider

than or not more than 7 mm, or 10 mmsmaller than the control

– intermediate: zone diameter > 2 mm butsmaller than the control by > 7 mm or10 mm

– resistant: zone diameter ≤ 2 mm.

Agar dilution methods

The agar dilution method for determining MICshas advantages over broth dilution methods in thatcontamination is more easily seen and re-isolationof the required organism is usually not a problem.

MediumAs for diffusion tests a medium optimized for sus-ceptibility testing should be used.

Antimicrobial agentsUse generic rather than proprietary names ofagents. Obtain antimicrobial powders directly

from the manufacturer or from commercialsources. The agent should be supplied with a cer-tificate of analysis stating the potency (μg or inter-national unit [IU]/mg powder, or as percentagepotency), an expiry date, details of recommendedstorage conditions and data on solubility. Ideally,hygroscopic agents should be dispensed in smallamounts and one used on each test occasion. Allowcontainers to warm to room temperature beforeopening them to avoid condensation of water onthe powder.

Range of concentrations testedThe range of concentrations will depend on theparticular organisms and antimicrobial agent beingtested. Recommended ranges for major groups oforganisms are given by the BSAC (Andrews,2001b).

Preparation of stock solutionsTo ensure accurate weighing of agents, use an ana-lytical balance and, whenever possible, weigh atleast 100 mg of the powder. With a balance weigh-ing to five decimal places the amount of powderweighed may be reduced to 10 mg. Use the follow-ing formula to make allowance for the potency ofthe powder:

Weight of powder (mg) =

Alternatively, given a weighed amount of antimi-crobial powder, calculate the volume of diluentneeded from the formula:

Volume of diluent (ml) =

Concentrations of stock solutions should be madeup to ≥ 1000 mg/l although the insolubility of someagents will prevent this. The actual concentrationsof stock solutions will depend on the method ofpreparing working solutions (see below). Agentsshould be dissolved and diluted in sterile waterwhere possible and some agents may require othersolvents or diluents (Andrews, 2001b). If required,sterilize by membrane filtration and compare sam-ples before and after sterilization to ensure that

Weight (mg) × Potency (μg/ml)��

Concentration (mg/l)

Volume (ml) × Concentration (mg/l)��

Potency of powder (μg/mg)


adsorption has not occurred. Unless otherwiseinstructed by the manufacturer, store stock solu-tions frozen in small amounts at –20°C or below.Most agents will keep at –60°C for at least 6months. Use stock solutions promptly on defrost-ing and discard unused solutions.

Preparation of platesVolumes of 20 ml agar are commonly used in 9-cmPetri dishes for agar dilution MICs. Prepare a seriestwofold dilutions of the agent at 20 times the finalrequired concentration. Add 1 ml of dilution to19 ml cooled molten agar, mix and pour into pre-labelled plates immediately. Allow the plates to setat room temperature and dry them so that no dropsof moisture remain on the surface of the agar. Donot over-dry the plates.

It is best to use plates immediately, particularly ifthey contain labile agents such as β-lactams. If theplates are not used immediately, store them in arefrigerator (4–8°C) in sealed plastic bags and usewithin 1 week.

Preparation of inoculumStandardization of the density of inoculum is essen-tial if variation in results is to be avoided. Followthe procedures described above.

Inoculation of platesMark the plates so that the orientation is obvious.Transfer diluted bacterial suspensions to the inocu-lum wells of an inoculum-replicating apparatus.Use the apparatus to transfer the inocula to theseries of agar plates, including control plates with-out antimicrobial agent at the beginning and end ofthe series. Inoculum pins 2.5 mm in diameter willtransfer close to 1 μl, i.e. an inoculum of104 cfu/spot if the bacterial suspension contains107 cfu/ml. If in doubt as to the volume delivered,check with the supplier and adjust the inoculumpreparation procedure to yield a final inoculum of104 cfu/spot. Allow the inoculum spots to dry atroom temperature before inverting the plates forincubation.

Incubation of platesIncubate plates at 35–37°C in air for 18 h. Toavoid uneven heating do not stack them more thanfour high. If the incubation period is extended forslow-growing organisms, assess the stability of theagent over the incubation period. Avoid incubationin an atmosphere containing 5% CO2 unlessabsolutely necessary for growth of the organisms(e.g. Neisseria spp.). Incubation at 30°C should beused for methicillin/oxacillin susceptibility tests onstaphylococci.

Reading the resultsThe MIC is the lowest concentration of the agentthat completely inhibits growth, disregarding up tofour colonies or a thin haze in the inoculated spot.A trailing endpoint, with a small number ofcolonies growing on concentrations that are severaldilutions above that which inhibits most organismsshould be investigated. Subculture and re-testingmay be necessary because this may be caused bycontamination. It may also be the result of resistantvariants, β-lactamase-producing organisms or, ifincubation is prolonged, regrowth of susceptibleorganisms after deterioration of the agent. Withsulphonamides and trimethoprim. endpoints maybe seen as only a reduction in growth if the inocu-lum is too heavy or antagonists are present.

Broth dilution methods

This macrodilution method involves the use of testtubes and culture volumes of 1–2 ml, and is mostsuitable for small numbers of tests because prepa-ration of many sets of tubes is tedious.Microdilution methods involve the use of plasticmicrodilution trays with culture volumes of0.1–0.2 ml. In the microdilution method, multipleplates may be conveniently produced by the use ofapparatus for automatically diluting and dispens-ing solutions.

MediumThe requirements for broth media are similar to

Comparative methods 179

those for agar media. There are fewer broth mediathan agar media designed specifically for antimi-crobial susceptibility testing. Oxoid Iso-Sensitestand Mueller–Hinton media are available in bothagar and broth formulations. Supplements, otherthan blood, may be added to broth for fastidiousorganisms as for agar media.

Preparation of dilutionsThe total volume of each dilution prepared willdepend on the number of tests to be done. Thevolumes should be adjusted according to thenumber of tests to be done, but should not be lessthan 10 ml. Dispense the solutions in 1-ml vol-umes in tubes for the macrodilution method and0.1-ml volumes into the wells of the microdilutionplate for the microdilution method. Include con-trol tubes or wells containing broth withoutantimicrobial agent to act as growth and sterilitycontrols.

Macrodilution tubes are commonly used immedi-ately after preparation (preferable for labile agentssuch as β-lactam agents), but may be stored at 4°Cfor up to 1 week. Microdilution trays may be keptin sealed plastic bags stored at –60°C or below forup to 1 month. Do not refreeze trays once theyhave been thawed.

Preparation of inoculumPrepare the inoculum as for the agar dilutionmethod but adjust the density so that, after inocu-lation, the final volume of test broth contains105 – 5 × 105 cfu/ml. The density of the inoculumrequired depends on the volume of broth in thetubes or wells and the inoculation system used.

Inoculation of tubes/platesInoculate macrodilution tubes containing 1 mlbroth with 0.05 ml inoculum containing5 × 106 cfu/ml or add 1 ml inoculum of5 × 105 cfu/ml in broth to 1-ml volumes of broth.The latter inoculum requires the preparation of adouble-strength dilution series of the agent to com-pensate for the 1 in 2 dilution on inoculation.

Inoculate microdilution wells containing 0.1-mlbroth with 0.005 ml inoculum containing

5 × 106 cfu/ml. Manual inoculation with a pipette iseasier if 0.05 ml inoculum is added to 0.05-mlvolumes of broth in the dilution series. As with themacrodilution method, inoculum density andantimicrobial concentrations are adjusted to com-pensate for the 1 in 2 dilution of agents on inocula-tion.

Incubation of tubes/platesSeal microdilution plates in a plastic bag, with plas-tic tape or some other method to prevent the platesfrom drying. To avoid uneven heating, do not stackmicrodilution plates more than four high. Incubatetubes or trays at 35–37°C as with agar dilutionplates.

Reading resultsThe MIC is the lowest concentration of the agentthat completely inhibits growth. The control con-taining no agent should be turbid.

MINIMAL BACTERICIDALCONCENTRATION

The MBC is the lowest concentration of an agentthat kills a defined proportion (usually 99.9%) ofthe population after incubation for a set time. Themethod is usually an extension of the broth dilu-tion MIC. After reading the MIC, organisms arequantitatively subcultured from tubes or wellsshowing no growth to antimicrobial-free agarmedium. After incubating the plates, the propor-tion of non-viable organisms, compared with theoriginal inoculum, is assessed.

The MBC is used to guide therapy in situationssuch as endocarditis, where the bactericidal activityof an agent is considered essential.

TIME–KILL CURVES

These are an extension of the MBC and give infor-mation on the rates at which organisms are killed.


The rate is measured by counting the number ofviable cells at different time intervals after exposureto various concentrations of an agent.

TOLERANCE

This is the ability of strains to survive but not growin the presence of an agent and relates particularlyto Gram-positive organisms tested against agentsacting on the terminal stages of peptidoglycan syn-thesis, i.e. β-lactam and glycopeptide agents.Strains that have an MIC : MBC ratio of ≥ 1 : 32after 24-h incubation are usually termed ‘tolerant’.However, MICs and MBCs are determined afterfixed periods of incubation and tolerance may be areflection of a lower rate of killing. Technical vari-ation has a marked effect on the detection of toler-ance (Sherris, 1986; Thrupp, 1986).

BREAKPOINT METHODS

Agar dilution breakpoint susceptibility testingmethods are essentially highly abbreviated agardilution MIC tests, in which only one or two criti-cal antimicrobial concentrations are tested. With asingle breakpoint concentration growth = resistantand no growth = susceptible.

Using a lower and a higher breakpoint concentra-tion no growth at either concentration = suscepti-ble, growth at the lower concentration only =intermediate, and growth at both concentrations =resistant.

The general considerations detailed in the sectionon dilution methods apply equally to agar incorpo-ration breakpoint susceptibility testing methods. Itis essential to inoculate growth control plates of thesame basal medium as the antimicrobial-containingplates at the beginning and end of the inoculationrun and to incubate them under the same conditionsas the test. Adequate growth of the test isolates onthese control plates allows a confident interpreta-tion of susceptibility based on the absence of growthon antimicrobial containing plates because theantimicrobial content is the only variable.

Purity plates

Purity plates (which may be half, third or quarterplates) are essential and should be of a suitable non-selective growth medium, e.g. blood agar. Preparethese by subculturing a standard 1-μl loopful fromthe inoculum ‘pots’ used for the inoculation ’run’,after all antimicrobial test and growth controlplates have been inoculated. In this way mixed cul-tures should be detected and the use of a standardloop allows an approximate check on the actualinoculum density used for each test organism.

Preparation of breakpoint plates

Preparing sets of plates for breakpoint testing frompure antimicrobial agent powders is a complextask. Accurate weighing of powders, dilution of thesolutions and quality control of the plates areessential. Pre-prepared antimicrobials at specificconcentrations for addition to molten agar areavailable from a number of manufacturers (e.g.Mast Adatabs).

Breakpoint concentrations tobe tested

Some examples of appropriate concentrations areshown in Tables 12.2 and 12.3; for a full list con-sult the BSAC or NCCLS publications (Andrews,2001b; NCCLS, 1997, 1999).

Reading and interpretation ofbreakpoint susceptibility tests

The ‘interpretation’ of breakpoint sensitivity test-ing has two elements: the reading of the laboratorytests and the assessment of their clinical relevance.In practice, the selected breakpoints are those con-sidered to provide clinically relevant results and areusually based on one or two concentrations of a

Breakpoint methods 181

given agent. The results can then be interpreted as‘susceptible’ or ‘resistant’ when one concentrationis used or as ‘susceptible’, ‘intermediate’ and ‘resist-ant’ when two are used.

The reading of breakpoint plates is the final stageof a susceptibility testing process, which shouldadhere to standard methods for preparation andstorage of media and antimicrobial agents, forpreparation of the inoculum and for conditions ofincubation.

The results are usually unambiguous and freefrom subjective interpretation. Automated comput-erized equipment is available for reading plates andrecording the results, e.g. Mastascan Elite.

Quality assessment breakpointsusceptibility tests

Check that the inoculum is pure, of the expectedspecies and of approximately the correct density –the use of a standard loop to inoculate the purityplate provides for both of these requirements.

Check that the control has grown adequately. It isuseful to have at least one antimicrobial agent inthe test set to which isolates are likely to be resist-ant, e.g. aztreonam for Gram-positive and van-comycin for Gram-negative isolates. This acts as anadditional control of adequate growth and can aidthe recognition of mixtures of Gram-positive andGram-negative organisms. Computer printouts ofdata or manual lists are useful for this purpose.These lists should also be checked for unexpectedsensitivity patterns that could be caused by mixedcultures, incorrect subculture and inversion of theplates, or simply reading the wrong plate. They arealso useful for alerting the control of infection andmedical teams to potential epidemiological ortreatment problems.

Organization of breakpointsusceptibility testing schemes

The range of antimicrobial agents selected for inclu-

sion within a breakpoint testing scheme must berelated to the species of organisms to be tested andthe clinical site of infection from which they are iso-lated. Some laboratories use breakpoint methodsonly for infections for which the usual causativeorganisms can be predicted with some accuracy, andthe range of agents to be tested is limited. The mostcommon example is urinary tract infections. Othersadopt a broader approach and test all aerobic Gram-negative bacilli whatever their clinical source againstthe same set of antimicrobial agents. Similarly a setof antimicrobial agents may be selected for break-point testing against staphylococci and enterococci.If the susceptibility testing medium used is supple-mented with growth additives, e.g. 5% lysed horseblood and nicotinamide adenine dinucleotide(NAD), the set of antimicrobial agents may beextended to cover susceptibility testing of more fas-tidious species such as pneumococci, other strepto-cocci, H. influenzae and Branhamella catarrhalis.

One system described by Franklin (1990) uses asusceptibility testing medium capable of supportingthe growth of virtually all bacteria of medicalimportance. All bacterial isolates are tested againstthe same wide range of agents, usually at more thanone breakpoint concentration. The results pro-duced by such a scheme will not be relevant to aparticular species/genus or site of infection.Selectivity in reporting is necessary.

THE E TEST

This system for testing MICs represents a differentapproach from quantitative testing of antimicro-bial activity and has some advantages over conven-tional methods. The commercial E test strips are50 mm × 5 mm plastic carriers with an exponentialantimicrobial gradient dried on one side and agraduated MIC scale on the other. To set up an Etest, inoculate agar medium by flooding or with aswab in the same way that plates are inoculated fordisc diffusion susceptibility tests. Up to six E teststrips can be placed in a radial pattern on a 15-cmplate, or a single strip may be placed on a 9-cmplate. On incubation, elliptical zones of inhibition


are produced and the MIC is read directly from thegraduated E test strip at the point of intersection ofthe zone of inhibition with the strip. Include a con-trol strain with each E test by inoculating one halfof a plate with the test organism and the other halfof the plate with the control organism and applythe E test strip to the line between the two organ-isms (ABBiodisk, Sweden).

β-LACTAMASE TESTING

Rapid biochemical tests may be used to detect β-lac-tamase production (Livermore and Brown, 2001).There are three main methods: those based onchromogenic cephalosporins are the most sensitivebut are not suitable for staphylococci.Staphylococcal β-lactamase may require inductionand can be detected with iodometric or acidometricmethods. The later methods are also suitable fordetecting production by N. gonorrhoeae andH. infuenzae. Extended spectrum β-lactamases(ESBLs) are plasmid-mediated enzymes producedby a number of enterobacteria and P. aeruginosa. InE. coli and Klebsiella spp., these can be detected bya combined disc method (Livermore and Brown,2001). An Iso-Sensitest plate is inoculated with thetest strain to give a semi-confluent growth. A cef-tazidime 30 μg disc and an amoxicillin/clavulanicacid 20 + 10 μg disc are placed 25–30 mm apart.After overnight incubation at 37°C, if the zone ofinhibition round ceftazidime disc is expanded ESBLproduction is inferred. ESBL production may alsobe detected by an E-test ESBL strip and by inferencefrom cephalosporin resistance. Methicillin (5 μg) oroxacillin (1 μg) discs may be used to detect intrin-sic resistance to penicillin of pneumococci.

DIRECT (PRIMARY) SUSCEPTIBILITYTESTS

Tests in which the specimen is the inoculum can beuseful because the results are available a day earlierthan tests on pure cultures; isolation from mixed

cultures may be easier if there are differences in sus-ceptibility and small numbers of resistant variantsmay be seen within zones of inhibition in diffusiontests. There are some potential disadvantages, inthat the inoculum cannot be controlled, care mustbe taken to avoid reporting results on commensalorganisms, and the specimens on which tests arelikely to be useful are limited to those from sitesthat are normally sterile and, in the case of urine, tospecimens shown microscopically to containorganisms. Direct tests should not be undertakenwith specimens from patients on antimicrobialtherapy (Waterworth and Del Piano, 1976)

For direct tests by the Stokes’ method, pus swabsmay be streaked in place of the test organisms orwell-mixed urines used in place of broth cultures orsuspensions of pure cultures of organisms. UseE. coli NCTC 10418 as a control for urines andS. aureus NCTC 6571 for other specimens. Withbreakpoint methods only urine specimens can betested. With the important and common exceptionof direct susceptibility testing on urines, repeat alltests on pure cultures and only report direct tests as’provisional’.

QUALITY CONTROL

Departure from the usual range of routine suscepti-bility may indicate gross errors in the method.Routine quality control is necessary, however, tomaintain high standards (Franklin, 1990). Externalquality control schemes provide independentassessment of the performance of a test, and allowsperformance to be compared with that of other lab-oratories, although internal quality control is nec-essary to detect day-to-day variation.

Standard strains must be used to control the per-formance of the method. Test colonies of controlcultures in the same manner as test cultures.Include control cultures in each batch of tests anduse them to evaluate new lots of agar or brothbefore they are put into routine use.

Quality control 183

Diffusion tests

A rapid examination of control zone sizes will indi-cate major problems. Recording of control zonesizes on a chart allows problems to be more easilydetected. Greater control may be exercised byestablishing zone-size limits. If control tests indi-cate problems the source of error should be investi-gated. Common problems are:

• Inactivation of labile agents in discs as aresult of inadequate storage or handling in thelaboratory may be indicated by a gradualdecrease in zone sizes. New batches should betested before being put into routine use.

• Too heavy or too light an inoculum may beindicated by a general decrease or increase inzone sizes.

• Errors in transcription or measuring zonesizes. In particular, different observers readingzone edges differently will result in fluctuatingzone sizes.

• Problems with medium, e.g. high pH, willproduce larger zones with aminoglycosideantimicrobials and erythromycin, and smallerzones with tetracycline, methicillin and fusidicacid. The reverse may occur if the pH is toolow. New batches of medium should be testedbefore being put into routine use. Fluctuatingzone sizes may indicate variation in the depthof medium.

• Contamination or mutational changes in thecontrol strain.

Minimum inhibitory concentrations

In general, the MICs of control organisms shouldbe within plus or minus one dilution step of thetarget values. In addition:

• For broth dilution tests, incubate one set oftubes or one microdilution plate uninoculatedto check for sterility (media contaminants onagar dilution plates are usually obvious).

• The control without antimicrobial agentsshould show adequate growth of test and

control strains.• Plate a sample of inoculum prepared for each

strain on a suitable agar medium to ensurethat the inoculum is pure.

• Occasionally check that the inoculum densityis correct by counting organisms in inocula(taken from growth controls with brothdilution methods).

• Check that endpoints are read consistently byall staff independently reading a selection oftests.

Breakpoint methods

In addition to the foregoing comments on qualitycontrol, the following points should be borne inmind by users of breakpoint methods. Monitor theconcentrations of antimicrobial agents in the platesby using control strains with known MICs for theagents. To achieve suitable control these strainsshould have MICs only one or two dilutions above(for the resistant control) or below (for the sensitivecontrol) the concentration in the breakpoint plates.

When breakpoint control strains of known MICare used, the breakpoint testing method must beidentical to that used for the initial selection of thecontrol strains in order to ensure reliability of per-formance. However, a batch of ’breakpoint’ plateswith, say, six different antimicrobial agents withtwo breakpoints each would require up to 24 con-trol organisms with appropriate MICs to check thelevels of agents adequately in all plates. If per-formed every day, this extensive control procedurewould leave little room for testing the clinical iso-lates! As plates can be stored without deteriorationfor up to 1 week, a sample of each batch of mediamay be subjected to this degree of control. In addi-tion, new batches of media may be checked by ini-tially running them in parallel with the previouslycontrolled plates. For certain agents, where there issome evidence that degradation of antimicrobialactivity may occur during storage of plates (e.g. co-amoxiclav and imipenem), daily microbiologicalquality control may be advisable. Similarly it is


advisable to include a ‘difficult’ control strain ofmethicillin-resistant S. aureus (MRSA) on a dailybasis to ensure reliable detection of methicillin-heteroresistant staphylococci. (Control strains forsome breakpoints are available from the AntibioticReference Laboratory, Public Health LaboratoryService, Colindale, London.)

Franklin (1990) described a method to overcomethe practical difficulties of using numerous controlstrains. By cutting plugs of agar from a batch of theprepared plates and placing them on the surface ofa seeded, non-inhibitory culture plate, the antimi-crobial content can be monitored by the size of theinhibition zone around the agar plug. As deviationfrom the expected zone size indicates error, anacceptable range has first to be determined.Establishing and validating this method of qualitycontrol may be at least as complex as searching fora small number of strains with appropriate MICs.

Inoculum size is critical; therefore, the carefulcleaning, maintenance and calibration of inoculat-ing pins are necessary.

Transcription errors are always possible andappropriate staff training can reduce their likeli-hood. Automated reading of the plates reduces thisrisk. If plates are read visually then plates contain-ing antimicrobial agents with similar soundingnames (e.g. the various cephalosporins) should beseparated in the order of plate reading to avoidconfusion. A legibly labelled template showing pinpositions should be available to the plate reader.Worksheets should be simple and easy to fill in. Allisolates with unusual susceptibility patterns shouldbe checked and, if necessary, investigated further.

REFERENCES

Andrews, J. M. (2001a) Determination of minimuminhibitory concentrations. Journal of Antimicrobial

Chemotherapy 48(suppl 1): 5–16.Andrews, J. M. (2001b) BSAC standardized disc

susceptibility testing method. Journal of

Antimicrobial Chemotherapy 48(suppl 1): 43–57.Barry, A. L. (1976) Antimicrobial Susceptibility Test:

Principles and practice. Philadelphia, PA: Lea &

Febiger, pp. 163–179.Barry, A. L. (1991) Procedures and theoretical

considerations for testing antimicrobial agents inagar medium. In: Lorian, V. (ed.), Antibiotics in

Laboratory Medicine 3rd edn. Baltimore, MA:Williams & Wilkins, pp. 1–16.

Brown, D. F. J. and Blowers, R. (1978) Disc methodsof sensitivity testing and other semiquantitativemethods. In: Reeves, D. S., Phillips, I., Williams, J.D. et al. (eds), Laboratory Methods in

Antimicrobial Chemotherapy. Edinburgh:Churchill Livingstone, pp. 8–30.

Ericsson, M. M. and Sherris, J. C. (1971) Antibioticsensitivity testing. Report of an internationalcollaborative study. Acta Pathologica et

Microbiologica Scandinavica (Section B)Supplement 217.

Franklin, J. C. (1990) Quality control in agar dilutionsensitivity testing by direct assay of the antibioticin the solid medium. Journal of Clinical Pathology

33: 93–95King, A. (2001) Recommendations for susceptibility

tests on fastidious organisms and those requiringspecial handling. Journal of Antimicrobial

Chemotherapy 48(suppl 1): 77–80.Livermore, D. M. and Brown, D. F. J. (2001) Detection

of β-lactamase mediated resistance. Journal of

Antimicrobial Chemotherapy 48(suppl 1): 59–64

National Committee for Clinical Laboratory Standards(NCCLS) (1996) Evaluating Production Lots of

Dehydrated Mueller–Hinton Agar. Approvedstandard M6-A. Wayne, PA: NCCLS.

NCCLS (2003) Performance Standards for

Antimicrobial Disc Susceptibility Tests. Approvedstandard M2-A8. Wayne, PA: NCCLS.

Phillips, I., Andrews, J., Bint, et al. (1991) A guide tosensitivity testing. Report of the Working Partyon Antibiotic Sensitivity Testing of the BritishSociety for Antimicrobial Chemotherapy. Journal

of Antimicrobial Chemotherapy 27(suppl D): 1–50.

Sherris, J. C. (1986) Problems of in vitro determinationof antibiotic tolerance in clinical isolates.Antimicrobial Agents and Chemotherapy 30:633–637.

References 185

Thrupp, L. D. (1986) Susceptibility testing ofantibiotics in liquid media. In: Lorian, V. (ed.),Antibiotics in Laboratory Medicine. Baltimore,MA: Williams & Wilkins, pp. 93–150.

Waterworth, P. M. and Del Piano, M. (1976)Dependability of sensitivity tests in primary

culture. Journal of Clinical Pathology 29:179–184.

Wheat, P. (2001) History and development ofantimicrobial susceptibility testing methodology.Journal of Antimicrobial Chemotherapy 48(suppl1): 1–4.


In 1992, the Advisory Committee on theMicrobiological Safety of Food defined food poi-soning as ‘any disease of an infectious or toxic naturecaused by or thought to be caused by the consump-tion of food or water’. The definition had been pre-viously adopted by the World Health Organization(WHO, 1998). Although the words ‘food poison-ing’ are used in public health legislation, ‘food-bornedisease’ is now the generally preferred term for theseconditions (Department of Health, 1994). Some pre-dominantly food-borne infections, e.g. salmonel-losis, can also be transferred by the faecal–oral route,and some infections predominantly spread by thefaecal–oral route, e.g. hepatitis A and Shigella spp.,can occasionally be food borne.

An outbreak of food-borne disease is an incidentin which two or more people, thought to have a com-mon exposure, experience a similar illness or proveninfection with at least one of them having been ill.A general outbreak is one that affects members ofmore than one household, or residents of an insti-tution. Food-borne disease encompasses illnesscaused by a variety of different agents, including:

• bacterial infections (e.g. with salmonellas,campylobacters)

• pre-formed bacterial toxins (e.g. botulism)• other biological toxins (e.g. paralytic shellfish,

scombrotoxin, poisoning)• viral infections (e.g. noroviruses)• parasitic infections (e.g. protozoa)• toxic chemicals (e.g. heavy metals).

As a result of the diversity of the agents involved,the clinical features of food-borne disease vary con-siderably. Typical symptoms and incubation timesprovide an indication of the likely cause, but micro-biological tests are required to confirm any suchdiagnosis. Most food-borne disease organismsexert their influence on the gastrointestinal tract,giving rise to ‘classic’ food-borne disease symptomssuch as nausea, vomiting, diarrhoea, abdominalpains, fever, etc.

Some toxins, however, act on other systems (e.g.nervous, immune), producing a wide variety ofsymptoms. Some characteristics of the moreimportant food-borne disease bacteria are summa-rized in Table 13.1, which gives details of mainsources, the types of food they are most com-monly associated with and their preferred growthconditions.

The true incidence of food-borne infection is diffi-cult to determine because asymptomatic infectionis common, only a minority of people with sympto-matic infection will seek medical treatment andonly a minority of patients will be investigatedmicrobiologically. There is a statutory requirementto notify suspected food-borne disease in mostcountries, and many countries also conduct surveil-lance by collating laboratory results. Food-borneinfection appears to be one of the most commoninfectious diseases, and the incidence is increasing.For a review of the factors that have contributed tothe changing patterns of food-borne disease, seeWHO (1998).

13

Food poisoning and food-borne disease

Table 13.1 Food-borne disease: agents, symptoms and sources

Bacteria Symptoms and possible Incubation Duration of Infective dose Microbiology/Growth Sources consequences period illness (cells/g) characteristics

Salmonella spp. Diarrhoea, vomiting, Up to Up to Risk varies with Non-sporing, temperature Foods not cooked at an abdominal pain, high 4 daysa 3 weeksb nature of food range 10–50°C but some adequate temperature temperature and susceptibility grow down to 6–8°C), or contaminated by raw Blood poisoning of individual acid resistant (grow at foods(septicaemia) and pH > 4.0) Mainly meat (especially inflammation of the poultry), eggs and dairy abdominal wall (peritonitis productsin severe cases) Also infected food

handlers and pets

Clostridium perfringens Diarrhoea, abdominal pain 8–22 hc ~24 hours ∼1 000 000 cells Heat-resistant spores Cooked meat dishes in total Temperature 15–50°C stored at the wrong

Optimum pH 6–7, temperature anaerobe

Escherichia coli O157 Abdominal pain, vomiting, 12–60 h Varies Very high Non-sporing Beef and beef products, diarrhoea that may contain infectivity (e.g. Temperature 10–45°C unpasteurized dairy blood, kidney failure tens of cells) Acid resistant (grows productsCan be fatal at pH 4.5) Unpasteurized fruit Young children and elderly juice, raw vegetablespeople particularly vulnerable Direct contact with

animals, infected peopleand bathing incontaminated water

Bacillus cereus Nausea, vomiting, diarrhoea, 10 min–16 h 24–36 h ∼100 000 or Heat resistant spores Cooked food stored at abdominal pain preformed toxin Temperature 10–50°C the wrong temperature

pH > 4.3 – mainly rice dishes andpastry productscontaining meat orvegetablesOccasionally dairyproducts and bakeryproducts


Bacteria Symptoms and possible Incubation Duration of Infective dose Microbiology/Growth Sources consequences period illness (cells/g) characteristics

Campylobacter spp. Fever, headache and dizziness Up to Varies, usually ∼100 Non-sporing, only grows Raw or undercooked followed by abdominal pain 10 daysd a week > 30°C, pH 6.5–7.5, meat, especially poultry, and diarrhoea prefers low oxygen, does unpasteurized milk, milk

not generally grow in from bird-pecked foods bottles, untreated water

Also pets

Staphylococcus aureus Vomiting, abdominal pain, 1–7 he Up to 2 days 100 000 to Non-sporing, some Human contamination diarrhoea 1 000 000 cells strains grow down to of food

will produce 7°C, pH range 4.5–9.3 Mainly fish, prawns and enough toxin to (optimum ~7) cream cakes handled cause illness and stored at the

wrong temperature

Listeria monocytogenes Mild flu to septicaemia and 3–70 days Varies Relatively low Non-sporing, temperature Occurs naturally in the meningitis infectivity optimum 30–37°C but environmentCan cause miscarriage, grows down to –1°C, Mainly raw vegetables stillbirth or premature acid resistant (grows at and meat, pâtés and labour in pregnant women pH > 4.3) soft cheeses

a Usually 12–48 hoursb But may be a carrier for 12 weeks or more after symptoms have gonec Usually 12–18 hoursd Usually 2–5 dayse Usually 2–4 hoursSource: Health Protection Agency, Communicable Disease Surveillance Centre

INFECTIVITY

The infectivity of the different organisms dependson the following:

• Type of bacteria: relatively large numbers(e.g. ≥ 100 000 cells) of most types of food-borne disease bacteria are generally requiredto cause illness, but verocytotoxin-producingEscherichia coli (VTEC) and Campylobacterspp., and some species of Shigella are notableexceptions, where illness can result from justa few tens or hundreds of cells.

• Type of food: some types of food (e.g. highfat content) protect bacteria from the harshacidic conditions in the human stomach, sothat food-borne disease may result from verymuch lower doses of bacteria than wouldnormally be the case (e.g. Salmonella inchocolate).

• Susceptibility of the individual: in general,very young, old or immunocompromisedindividuals are most at risk.

DEALING WITH OUTBREAKS OFFOOD-BORNE DISEASE

In the UK, responsibility and management of out-breaks of food-borne disease fall jointly to thelocal authority (LA) and the Primary Health CareTrust (PHA). All LAs and PHAs have contingencyplans drawn up in consultation with other expertssuch as the Health Protection Agency (HPA), localconsultants in communicable disease control(CCDCs), chief environmental health officers, etc.Outbreaks are identified via surveillance systems,public complaints, etc. Physicians have a statutoryduty to notify the appropriate local officer of allcases (and suspected cases) of food-borne disease.Identification may also arise by other routes, suchas through diagnostic laboratories alerting CCDCsor LAs if they encounter pathogenic bacteria inspecimens that they have tested. Initial investiga-tions will lead to a preliminary hypothesis as tothe likely cause of the outbreak, in terms of the

food and the food-borne disease agent involved,the number of people exposed to it, etc. This, inturn, should lead to an indication of the controlmeasures needed to protect public health.

In the US, physicians report outbreaks of food-borne disease to the local Health Departments,which inform the State Health Departments. They,in turn, report to the Centers for Disease Controland Prevention.

TYPES OF FOOD-BORNE AGENTS

Salmonellas

The two major groups are those causing entericfevers (typhoid and paratyphoid) and those causingfood-borne disease (non-typhoid salmonellas). Theenteric fever group consists of Salmonella typhi andSal. paratyphi A and B. With rare exceptions theseorganisms infect only humans. The non-typhoidgroup consists of 2200 serotypes, all of which havetheir primary reservoirs in animals. Salmonellascan grow in many foods at temperatures from 10°Cto 50°C. Pasteurization temperatures destroy them.Salmonellas survive well outside a person or an ani-mal in faeces, on vegetables, in animal feeds and inmany other foods for long periods. Fewer than 10serotypes are responsible for most of the cases ofhuman salmonellosis in the UK – predominantlyS. enteritidis and S. typhimurium (PHLS, 2000).

Campylobacters

The species causing food-borne disease areCampylobacter jejuni and C. coli. They are presentas part of the normal gut flora of many animalsincluding mammals, birds and reptiles. Theirgrowth temperatures are more exacting than forsalmonellas, and the organisms do not grow infood under normal circumstances. Growth occursup to 42°C and the organisms survive well in waterand raw milk. Poultry appear to be responsible forsome sporadic outbreaks. The organisms are heatsensitive and destroyed by pasteurization, and do

190 Food poisoning and food-borne disease

not survive in acid foods. In common with salmo-nellas, control measures such as adequate cookingand prevention of recontamination are successful.

Clostridium perfringens

This organism is found in animal faeces, soil, dust,vegetation and elsewhere in the environment. It willgrow only anaerobically, and produces a variety ofenterotoxins, some of which act on the gastrointesti-nal tract. Spores may be formed in adverse conditionsand enable the organism to survive circumstances thatwould normally kill vegetative bacteria. Five typesare recognized on the basis of toxins and enzymesproduced. Type A strains are responsible for mostfood-borne disease outbreaks. Young and long-staypatients and hospital staff tend to become colonizedwith C. perfringens. Counts of 105/gram in the fae-ces of sufferers are not uncommon.

As the heat resistance of C. perfringensspores varies,the organisms survive some cooking processes. Muchdepends on the amount of heat that reaches the cellsand the period of exposure. If the spores survive andare given suitable conditions, they will germinate andmultiply. It is common practice to cook a large jointof meat, allow it to cool, slice it when cold and re-heat it before serving. This is hazardous unless cool-ing is rapid and re-heating is thorough and above80°C. Growth of heat-sensitive strains in cooked foodmay be the result of too low a cooking temperatureor recontamination after cooking.

Staphylococcus aureus

Many people carry S. aureus in their noses and manycarry it on their skin. Enterotoxin production is com-mon and there are eight serologically distinct types(A, B, C1, C2, C3, D, E and F), all of which cause anintoxication type of disease. Growth occurs on manyfoods, particularly those with a high protein con-tent, and the toxin is readily produced at ambienttemperatures. The organisms are killed at 60°C in30 min, but the toxin will withstand heating at100°C for 30 min. Infection occurs usually in the

kitchen from human contamination (infected cuts,boils and other lesions or from nasal carriers).

Bacillus cereus

This organism is widespread in the environmentand is particularly, but not exclusively, associatedwith foods involving grains and cereals. Spores areformed that resist boiling and frying for short peri-ods, and will germinate in ideal conditions forgrowth. Subsequent heating does not destroy anytoxins that may have formed. B. cereus strains pro-duce two kinds of toxin: diarrhoeal and emetic.The emetic type is more common. Occasionallyother Bacillus spp. may be responsible for similarsymptoms (see Gilbert et al., 1981).

Clostridium botulinum

The pre-formed toxin of C. botulinum affecting thecentral nervous system (botulism) is rare in the UK,but more prevalent in Europe and the USA wherehome canning of meat and vegetables is commonpractice. This anaerobic, spore-forming organism isextensively distributed in the environment. Theorganism will grow particularly well on low-acidfoods (i.e. with a pH value higher than 4.5) such asfish, fruit and vegetables. There are seven antigenictypes, A–G, each with its own distinct toxin. Humanintoxications are usually with types A, B and E,although type G has caused one outbreak. The tox-ins, powerful as they are, are readily destroyed byheat. The spores of type A resist boiling for severalhours; those of other types are slightly less resistant.Conditions must be optimum for toxin formation:complete anaerobiosis, neutral pH and absence ofcompeting organisms. Between 12 and 36 h aftereating food containing the toxin, patients developsymptoms including thirst, vomiting, double visionand pharyngeal paralysis, lasting 2–6 days and usu-ally fatal. Vehicles are usually home-preserved meator vegetables, contaminated with soil or animal fae-ces and insufficiently heated. Correct commercialcanning processes destroy the organisms.

Types of food-borne agents 191

Listeria monocytogenes

This organism is widely distributed in the gastroin-testinal tract of animals and therefore the environ-ment generally. L. monocytogenes can grow over awide range of temperatures from as low as –1°C,are comparatively resistant to disinfectants andchanges in pH, and can survive extreme environ-mental conditions (it is hardly surprising that theyare recovered from some ice-creams). Strains maybe distinguished by serotyping, types 1/2a and 4bbeing most commonly found in strains isolatedfrom pathological sources. Listeriosis affectsmainly young children, pregnant women and eld-erly people. The organisms are present in smallnumbers in many foods, but are unlikely to causedisease unless they have the opportunity to multi-ply. This may happen at quite low temperatures,e.g. in improperly maintained cold storage cabinetsand refrigerators.

Yersinia enterocolitica

This is an uncommon cause of food poisoning butits ability to grow at 4°C makes it unusual and apotential hazard. The organism is widely distrib-uted in animals and the environment. Foods impli-cated include milk, ice-cream and seafoods. Thesymptoms are diarrhoea, fever, abdominal painand vomiting.

Vibrio parahaemolyticus

This marine organism is found in coastal andbrackish waters. It is halophilic. Infection is rare inthe UK but it is one of the most common causes offood-borne disease in Japan and has been incrimi-nated in outbreaks in the USA and Australia.

Infection is associated with eating raw andprocessed fish products. The organism has been fre-quently isolated from seafoods, particularly thoseharvested from warm coastal waters. Symptoms ofinfection may appear from 2 to 96 h after inges-

tion, depending on the size of the infecting dose,type of food and acidity of the stomach. They varyfrom acute gastroenteritis with severe abdominalpain to mild diarrhoea.

Escherichia coli

Large numbers of E. coli are present in the humanand animal intestines. They are often found in rawfoods where they may indicate poor quality. Atleast four different types (see p. 287) may beinvolved in food poisoning. E. coli may cause trav-ellers’ diarrhoea when the source may be contami-nated food or water. Verocytotoxin-producing E.coli O157 is by far the most frequently reportedstrain in human cases and appears to be largely ofbeef or milk origin.

Shigellas

The dysentery group of organisms are pathogens ofhumans and are usually transmitted by the fae-cal–oral route in circumstances of poor generalhygiene. Food contamination readily occurs,although Shigella spp. are rarely associated withfood-borne disease in developed countries.

Mycotoxins and aflatoxins

Certain fungi, mainly Aspergillus spp., form toxinsin nuts and grains during storage. These toxins cancause serious disease in humans and animals. Thetoxins are detected chemically and commercial kitsare available. For information about these toxins,see Moss et al. (1989).

Other organisms

Other agents of food-borne disease includeBrucella spp. and Mycobacterium bovis (both milk

192 Food poisoning and food-borne disease

borne), Aeromonas spp. and enterobacteria otherthan those mentioned above. Some viruses, e.g.hepatitis A, and parasites, not considered in thisbook are also known to be associated with food-borne disease.

EXAMINATION OFPATHOLOGICAL MATERIAL ANDFOOD

Do a total viable count (methods are described inChapter 10) on all foods suspected of causing foodpoisoning. In most cases this will be high. Relatethe findings to conditions of storage after serving. Ifpossible count the numbers of causative organisms.

Table 13.1 suggests the causative organism.Isolation and identification methods are given inthe relevant chapters:

• Salmonellas: Chapter 28• Campylobacters: Chapter 33• Staphylococcus: Chapter 36• Y. enterocolitica: Chapter 34• B. cereus: Chapter 42• V. parahaemolyticus: Chapter 24• Botulism: Chapter 44• E. coli: Chapter 26• C. perfringens: Chapter 44• Listeria: Chapter 40.

For further information on food poisoning, seeEley (1996), Hobbs and Roberts (1987),Parliamentary Office of Science and Technology(POST, 1997) and the US Centers for DiseaseControl.

REFERENCES

Centers for Disease Control and Infection.http://www.cdc.gov.health/foodill.htm.http://www.cdc.gov/ncidod/eid/vol5no5/pdf/mead/pdf.

Eley, A. R. (ed.) (1996) Microbial Food Poisoning.London: Chapman & Hall.

Gilbert, R. J., Turnbull, P. C. B., Parry, J. M. andKramer, J. M. (1981) Bacillus cereus and otherBacillus species: their part in food poisoning andother clinical infections. In: Berkeley, R.C.M andGoodfellow, M. (ed.), The Aerobic Endosporing

Bacteria: Classification and Identification. London:Academic Press, pp. 297–314.

Hobbs, B. C. and Roberts, D. (eds) (1987) Food

Poisoning and Food Hygiene, 5th edn. London:Edward Arnold.

Moss, M. O., Jarvis, B. and Skinner, F. A. (1989)Filamentous Fungi in Foods and Feeds. Journal of

Applied Bacteriology [Symposium Supplement] 67:1S–144S.

Health Protection Agency (2001) Cases in Humans,England and Wales, by Serotype, 1981–2001(www.hpa.org.uk/infections/topics_az/salmonella/data_human.htm).

Parliamentary Office of Science and Technology (1997)Safer Eating. Microbiological Food Poisoning and

its Prevention. London: HMSO.World Health Organization (1998) Surveillance

Programme for Control of Foodborne Infectionsand Intoxicants in Europe. Newsletter 57 (October1998). Copenhagen: WHO.

References 193

http://www.cdc.gov.health/foodill.htm

http://www.cdc.gov/ncidod/eid/vol5no5/pdf/mead/pdf

http://www.cdc.gov/ncidod/eid/vol5no5/pdf/mead/pdf

www.hpa.org.uk/infections/topics_az/salmonella/data_human.htm

www.hpa.org.uk/infections/topics_az/salmonella/data_human.htm

There are many textbooks on the microbiologicalsafety and the keeping quality of food, e.g. those ofAdams and Moss (2000), Jay (2000), and Lund etal. (2000), as well as the publications of theInternational Commission for MicrobiologicalStandards for Foods (ICMSF, 1980, 1986, 1988,2001) (website: www.dfst.csiro.au/icmsf.htm).

Food microbiologists often employ media andmethods first developed for clinical microbiology.The International Standards Organization (ISO),European Standards Committee (CEN) andInternational Dairy Federation (IDF) have pub-lished various procedures that include generalmethods for the preparation of dilutions, totalcolony counts, and various plate counts and MPN(most probable number) counting methods for coli-forms, members of the Enterobacteriaceae andEscherichia coli. There are also standard methodsfor the detection of pathogens, including salmonel-las, thermophilic campylobacters, Listeria mono-cytogenes, E. coli O157:H7, Staphylococcusaureus and others, as well as methods for enumer-ation of spoilage organisms, including lactic acidbacteria, pseudomonads, Brochothrix thermos-phacta, and yeasts and moulds. Almost all thesemethods are also published by the BritishStandards Institution (BSI). In North America theAOAC International also publishes standard meth-ods, as does the Food and Drug Administration(FDA, 1998) and the American Public HealthAssociation (APHA) (Dowens and Ito, 2001). TheUK Public Health Laboratory Service (PHLS) hasalso published a book of methods (Roberts andGreenwood, 2002). Many laboratories use thesemethods, which are mostly traditionally cultural,but others use simplified or more rapid versions

involving molecular (polymerase chain reaction orPCR) or immunological methods (e.g. enzyme-linked immunosorbent assay or ELISA).

Food microbiology laboratories doing officialtesting are now required to be accredited to a qual-ity assurance scheme. In the UK this is the UKAccreditation Service (UKAS) scheme. Micro-biology laboratories attached to food factories inthe UK are normally also accredited to UKAS or tosimilar organizations (e.g. ‘LabCred’ run by LawLaboratories or ‘CLAS’ [Campden LaboratoryAccreditation Scheme], run by Campden andChorleywood Food Research Association])approved by the major supermarkets, such asTesco, J. Sainsbury and Marks and Spencer.

Many UKAS-accredited contract laboratorieshave opened up in the UK in recent years, takingover the testing previously done in laboratoriesowned by supermarkets and food manufacturingcompanies. Quality assurance systems required inaccredited laboratories include: traceability ofsamples and results, recording of results, standardoperating procedures, training of analysts, regularcalibration and checking of equipment, docu-mented methods (and validated, if these differfrom standard methods) and monitoring of thequality of media. Laboratories may have to vali-date non-standard methods themselves, or theymay be able to use methods validated by organiza-tions such as AOAC International or MicroVal. Inaddition, laboratories have to take part in anexternal proficiency testing scheme, such as thosein the UK run by the UK Central Public HealthLaboratory (see Snell et al., 1991) or the UKCentral Science Laboratory (FEPAS – FoodExamination Proficiency Scheme) as well as

14

Food microbiology: general principles

www.dfst.csiro.au/icmsf.htm

several commercial schemes. Participating labora-tories receive shelf-stable, simulated food sampleswith instructions to examine them for particulargroups of microbes (presence/absence or enumera-tion). Alternatively, information about the type offood and the illness associated with it are given,and the laboratory has to decide which organismsto look for.

SCOPE OF MICROBIOLOGICALINVESTIGATIONS IN FOODMICROBIOLOGY

Four kinds of investigations are usually carried out:

1. Estimation of the viable (colony) count(colony-forming units [cfu]/g, cfu/cm2 orcfu/ml).

2. Estimation of the numbers of E. coli,coliforms (more often Enterobacteriaceae)and/or Enterococcus spp. (‘faecalstreptococci’). These may indicate thestandard of hygiene during the preparation ofthe foods, especially where processing (e.g.heating) would be expected to kill theseorganisms (see ‘Indicator organisms’,below).

3. Detection of specific organisms known to beassociated with spoilage. This may be moreimportant than the viable count indetermining the potential shelf-life.

4. Detection of pathogens.

No single method is completely satisfactory forthe examination of all foodstuffs. The methodsselected will depend on local conditions, e.g. avail-ability of staff, space and materials, numbers ofsamples to be examined and the time allowed toobtain a result. To these ends, rapid, automated(validated) methods have much to offer (seeChapter 8). It may be more desirable to examine alarge number of samples by a slightly suboptimalmethod than a smaller number by an exhaustivemethod.

Many of the micro-organisms in foods may besublethally damaged, e.g. by heat, cold or adverse

conditions such as low pH, low water activity, orhigh salt or sugar content. On the other hand, theseconditions may favour the growth of yeasts andmoulds, when mycotoxins may be formed.

An important problem in the microbiologicalexamination of foods is that micro-organisms areusually not evenly distributed throughout a prod-uct. This affects any sampling procedure; there isno easy answer.

INDICATOR ORGANISMS

These are organisms that may indicate evidence ofcontamination or pollution, especially of a faecalnature, e.g. E. coli and coliform bacilli. The familyname Enterobacteriaceae is sometimes usedbecause ‘coliforms’ are poorly defined taxonomi-cally. Enterobacteriaceae includes other bacteria,e.g. important pathogens such as salmonellas andvarious non-lactose fermenters that may be presentin human and animal faeces. It should be noted,however, that the presence of these latter organ-isms is not evidence, at first sight, of faecal contam-ination: many of them are normally present in soiland on plant material.

MICROBIOLOGICAL CRITERIA(STANDARDS)

Limits for numbers of organisms (or their toxins)are given various names. The terms are defined bythe ICMSF (2001) and summarized here.

Standard

This is a criterion set out in a law or regulation con-trolling foods produced, processed or stored in thearea of jurisdiction, or imported into that area.Foods not meeting the standard would be removedfrom the market.

Microbiological criteria (standards) 195

Guidelines

These are criteria used by manufacturers or regula-tory agencies in monitoring a food ingredient,process or system (can be applied at points duringmanufacture). Guidelines are advisory, and neednot lead to rejection of the food – but could be usedto help bring about changes to improve the safetyor quality of the food.

Microbiological (purchasing)specifications

These are criteria that determine the acceptance ofa specific food or food ingredient by a food manu-facturer or other private or public purchasingagency.

QUALITY ASSURANCE SYSTEMS

It is widely appreciated that satisfactory results ofmicrobiological testing of the endproducts of foodmanufacture cannot guarantee that the whole con-signment is safe, although it may detect an unsatis-factory batch. It is enough here to state thatendproduct testing alone is not sufficient to check,with a reasonable degree of certainty, that a givenbatch of food is satisfactory unless an impractic-able number of samples is examined. Micro-biological testing of the endproduct is only one ofmany methods of checking that the system is pro-viding safe food and is useful only as a check on thewhole process. The food manufacturing and cater-ing industry is obliged by law, in the EuropeanUnion and many other countries, to use a hazardanalysis critical control point (HACCP) system,which attempts to anticipate possible hazards inthe manufacturing process and devise methods ofcontrolling or minimizing them, as well as methodsof checking and correcting deviations. An exampleis monitoring the temperature and time of pasteur-ization. Details can be found in one of the many

publications on this subject (see, for example,ICMSF, 1988).

If a sample of a perishable food is taken at a pointafter manufacture and after an unknown period onthe shelf, the total colony count result is often diffi-cult to interpret and is of little use in assessing thequality at the point of manufacture. It is moreappropriate to test for pathogens.

SAMPLING PLANS

Limits are normally set in terms of numbers oforganisms (cfu/g or ml) or presence/absence of apathogen in a given amount of the food. The exactnumber of organisms can never be determined pre-cisely and it is therefore inappropriate to set crite-ria with exact limits beyond which a food isunacceptable. For this reason it is customary to usethree-class sampling plans for the numbers oforganisms. These classes are: ‘acceptable’ – under aspecified number (m); ‘marginally acceptable’ –under a higher specified number (M); and ‘unac-ceptable’ when numbers exceed M. M is usually atleast 10 times higher than m, e.g. the total countlimit for liquid, pasteurized, whole-egg productssuggested by ICMSF is:

n = 5, c = 2, m = 5 × 104, M = 106

where n is the total number of samples tested and cthe number that may exceed m. None shouldexceed M.

Two-class plans are usual for salmonella testing,e.g. samples of 5, 10, 20 or > 25 g might be testedfor the presence/absence of salmonellas and thefood would be unacceptable if any sample was pos-itive (n = 5, 10, 20 or more, c = 0, m = 0). The num-ber of samples examined would depend on theperceived risk if the food was contaminated: foodswith the highest risk would be those eaten withoutprior heating and by a very susceptible class of con-sumer, e.g. dried milk for babies.

Limits in microbiological standards that areapplied by enforcement authorities normallyinclude those for pathogens and indictor organ-isms, not total colony counts.

196 Food microbiology: general principles

SAMPLING

The usefulness of laboratory tests depends largelyon correct sampling procedures. The sampling plan(see above) must be applied to each situation sothat the samples submitted to the laboratory arefully representative of the batch. In factory sam-pling it is more useful to take small numbers ofsamples at different times during the day thanlarger numbers at any one time. Where resourcesare limited sampling may concentrate on the fin-ished product, but if there is an increase in counts itis necessary to go back through the process andtake ‘in-line’ samples, especially of raw (starting)materials because they may be the source of con-tamination. It is also important to sample the food-processing instruments.

Containers and samplers

Use screw-capped aluminium or polypropylene jarsor plastic bags; never use glass containers whereany breakages could contaminate the product orcause injury to personnel. Instruments for samplingfrom bulk material vary: for frozen samples use awrapped sterilized brace and bit, a cork borer or achopper that has been washed in 70% ethanol ormethylated spirit and flamed. Take care to avoidcuts from ice splinters. Use individually wrappedspoons, spatulas or wooden tongue depressors forsoft materials. With pre-packed food take severalpackages as offered for sale.

When sampling carcass meat and fish, where con-tamination occurs only on the surface, use a surfaceswab over a defined area or excise a surface layer2–3 mm thick (see p. 205).

Transport and storage of samples

Carry samples (other than canned and bottledfoods) in insulated containers, cooled with icepacks (‘coolboxes’). The ice should not have meltedby the time the samples arrive at the laboratory.

Small refrigerated containers that are run off a carbattery can also be used. The samples should betransferred to a laboratory refrigerator and exam-ined as soon as possible. There is a code of practicefor sampling by authorized officers under the FoodSafety Act 1990 (see also Harrigan and Park, 1991;Roberts and Greenwood, 2002).

PRE-TESTING CONSIDERATIONS

Before examining any particular food check thetype of spoilage and/or pathogens that might beexpected. In general, products with low pH and/orlow water activity (aW) tend to be spoiled by yeastsand moulds. At intermediate pH levels and low aW

lactic acid bacteria and other Gram-positive organ-isms such as micrococci and enterococci may pre-dominate. At neutral pH and/or high aW spoilage ismostly the result of Gram-negative bacteria such asPseudomonas spp. In some heat-processed foods,such as meat pies, spoilage is the result of out-growth of surviving spores of Bacillus and/orClostridium spp., especially in the absence of com-peting flora.

GENERAL METHODS

If the organisms are expected to be distributedthroughout the material, homogenize 10 g of thefood (weighed aseptically) in 90 ml maximumrecovery diluent (MRD, see p. 76) in a homoge-nizer (e.g. a Stomacher). Make serial dilutions, e.g.10–1–10–4, and do colony counts on appropriateagar media and inoculate enrichment media for rel-evant pathogens (see below).

If the organisms are likely to be on the surface only(e.g. of pieces of vegetable or dried fruit) weigh 50or 100 g into a sterile jar and add 100 ml diluent.Shake for 10 s, stand on the bench for 30 min andshake again. The organisms are assumed to havebeen washed into the diluent, i.e. 100 ml now con-tains the organisms from the original weight ofsample. Make dilutions and proceed as above.

General methods 197

Total and viable counts

Methods are given in Chapter 10. For total counts,counting-chamber and DEFT (direct epifluores-cence technique) counts are useful and suggestappropriate dilutions for viable counts. For count-ing mould hyphae and yeasts the Neubauerchamber, used in haematology, is better than theHelber chamber because it is deeper.

The methods and incubation temperatures forviable counts depend on the nature and storageconditions of the food. For most foods use pourplates and incubate at 30°C for 24–48 h.

For chilled and frozen meat and frozen fish usesurface plating because the psychrophiles and psy-chrotrophs that predominate in such foods may bedamaged if exposed to the temperature of meltedagar used in pour plating. Incubate at 20–25°C. Tocount psychrophiles incubate at 1°C for 14 days. Amaximum of 200 μl can normally be spread on a 9-cm agar plate. Two plates of each medium are nor-mally inoculated from each dilution. Surfaceplating can be more economic than pour plating ifmore than one dilution is plated on each Petri dish.Commonly four dilutions (10–50 μl of each dilu-tion) per dish can be used if spread, or six if notspread.

Use the ‘black tube’ method (see p. 377) forcounting anaerobes. Report counts as colony-form-ing units per gram or millilitre. The tables onp. 146 show the amount of food in grams con-tained in the various dilutions.

Presence/absence tests

It may not be necessary to set up full viable counts.If a standard is set on the basis of experience thetechnique can be modified to give a present orabsent (‘pass’ or ‘fail’) response. If, for example, anupper limit of 100 000 cfu/g is set, then a platecount method that uses 1 ml of a 1 : 1000 dilutionwill suffice. More than 100 colonies, easilyobserved without laborious counting, will fail thesample. Similarly, more than 100 colonies in roll

tubes containing 0.1 ml of a 1 : 100 dilution, ormore than 40 per drop in Miles and Misra countsfrom a 1 : 50 dilution, will suggest rejection.

Enterobacteriaceae, coliforms andE. coli

There is a wide choice of media for these tests. Most‘standard’ tests specify EE broth (which is brilliantgreen glucose broth), violet red bile glucose agar(VRBG) and violet red bile lactose agar (VRBL orVRB). VRBG and VRB should not be autoclaved –see manufacturers’ instructions. If these are notavailable MacConkey broth and agar may be used.Lauryl sulphate tryptose broth is also specified forsome tests. Some workers prefer minerals modifiedglutamate medium (MMGM) instead of EE broth.

If small numbers are anticipated use the MPN orpresence/absence (P/A) methods, but for heavilycontaminated material plate or surface counts arebetter.

EnterobacteriaceaePresence/absenceIf resuscitation of sublethally damaged organismsis indicated add dilutions of the original suspension(see above) to MRD, incubate at room temperaturefor 6 h.

Add 10 ml suspension to 10 ml double-strengthEE broth and 1 ml suspension to 10 ml single-strength EE broth. Incubate at 30°C for 18–24 hand then plate on VRBG. Incubate plates at 30°Cand examine for typical colonies.

MPN methodFor this method, see p. 150.

Pour platesIf resuscitation is necessary incubate the dilutionsin MRD (see p. 76) for 90 min. Use freshly pre-pared VRBG for the counts.

Surface counts (with resuscitation)Spread 0.1-ml volumes of the dilutions on non-selective medium (e.g. plate count agar). Incubate


for 6 h at room temperature to resuscitate damagedcells, overlay with 15 ml VRBG and incubate at30°C overnight.

ColiformsUse the methods outlined above but with laurylsulphate tryptose broth. Subculture positive tubesto brilliant green lactose broth to check gas produc-tion. For colony counts use VRB agar (with lactose).

Incubation temperaturesIncubate coliform tests on dairy products, etc. at30°C. For other products use 37°C.

Escherichia coli (‘faecal coli’)Subculture suspect colonies or broth tubes to bril-liant green broth and peptone water and incubateat 44 ± 0.2°C overnight. Only E. coli produces gasand indole at 44°C. E. coli can be confirmed in themembrane method (above) by immersing the mem-brane in indole reagent and exposing it to ultravio-let (UV) light for 30 min. E. coli colonies are pink.For other confirmation tests, see Chapter 26.

Membrane methodPlace the membranes on MMGM or other non-selec-tive medium. Spread 1-ml volumes of the diluted sus-pension on the membranes (do not invert plates).Incubate for 4 h at 37°C. Transfer membranes totryptose bile agar and incubate at 44°C for 18–24 h.Count colonies and subculture if necessary.

Counting enterococci

Do surface counts as described on p. 147 withkanamycin azide aesculin agar, azide blood agar,MacConkey or Slanetz and Bartley medium.Consult manufacturers’ manuals for colonialappearance.

Counting clostridia and bacilli

As counting these organisms usually requiresidentifying them as well, the techniques are

described under Clostridium, in Chapter 44.Direct plating for C. perfringens can be done ontryptone sulphite cycloserine (TSC) or oleando-mycin polymyxin sulphadiazine perfringens(OPSP) agar. Bacillus cereus is counted on poly-myxin egg yolk mannitol bromothymol blue agar(PEMBA) or mannitol egg yolk polymyxin agar(MEYP).

Spoilage organisms

Table 14.1 shows organisms associated with non-sterile food. Methods for enumerating spoilageorganisms are given under the headings of thefoods concerned. Table 14.2 shows the media usedfor examining foods.

Food poisoning bacteria

‘Routine’ examination of all foods for salmonellasand staphylococci, or other food-poisoning organ-isms, is hardly worth while on account of the rela-tive infrequency of isolation. Only those foods andingredients known to be vehicles need be tested.Methods are given in Chapter 13 and under theappropriate headings in later chapters.

Yeasts and moulds

SamplingCircumstances vary. Obtain as much ‘background’information as possible before deciding on themethod of examination. The number of samplesshould be as large as is practicable. Sample wholepackages where possible and examine the casingfor damage and water staining.

Visual inspectionOpen carefully and look for evidence of moulds.Microscopy is rarely helpful.

General methods 199

Table 14.1 Types of organisms associated with various non-sterile foods

pH Water Food Comments (chilled Microbial floraactivity unless otherwise stated)(aw)

7.4–5.4 1.00–0.97 Animal protein – milk, whole Aerobic Pseudomonas, Acinetobacter and Aeromonas spp.,liquid egg, raw red meat, fish, Enterobacteriaceae (Serratia, Hafnia, Enterobacter, Yersinia spp.),poultry lactic acid bacteria, Bacillus spp. (in milk and egg), Brochothrix

thermosphacta (in meat), Shewanella spp. (in meat and poultry,pH > 6.0, and fish)

Vacuum or gas packed (high Lactic acid bacteria, B. thermosphacta, Pseudomonas spp.,CO2) Enterobacteriaceae, psychrotolerant clostridia

Cooked, sliced, non-cured meats As raw meats

Bean sprouts and raw salads High counts normal Pseudomonas spp., Erwinia spp., Salmonella spp., otherEnterobacteriaceae, E. coli

Raw bacon and ham with salt Aerobic Micrococcus, Acinetobacter and Vibrio spp. (at pH > 5.9), yeasts, and nitrite moulds

Vacuum or gas packed Lactic acid bacteria, Micrococcus and Enterococcus spp.,(high CO2) Enterobacteriaceae (low salt, high temperature and high pH

favour Gram-negative bacteria)

Semi-preserved, cooked, cured Cans or flexible packs Enterococcus spp. and other lactic acid bacteria can survive, often meats, e.g. ham pasteurized at 65–70°C and occurring in large numbers, occasionally causing spoilage by

stored chilled gelatin liquefaction, gas production or souring

Clostridium and Bacillus spp. spoil when salt is low andtemperature > 10°C

< 4.5 0.95–0.8 Cheeses, including cottage High numbers of lactic acid Yeasts and mouldscheese and yogurt bacteria present as these Yoghurt with added fruit is prone to spoil as a result of yeasts –

products are fermented causes lids to domeSurface (e.g. Brie) or mould-ripened cheeses (e.g. Stilton), whichraise pH locally, can be colonized by L. monocytogenes and cancontain (in cheese made from unpasteurized milk) low numbersof Enterobacteriaceae, including E. coli and salmonellas


pH Water Food Comments (chilled Microbial floraactivity unless otherwise stated)(aw)

Fermented raw meats, e.g. High numbers of lactic acid Yeasts and moulds on surfacesalami bacteria present as these Can contain low numbers of L. monocytogenes and

products are fermented Enterobacteriaceae, including E. coli and salmonellasMay also include enterococci, High numbers of S. aureus and/or staphyloenterotoxin if micrococci and staphylococci fermentation was delayed

4.5–3.0 1.0–0.97 Pasteurized fruit juices Ambient temperature Yeasts, lactic acid bacteria Fruit, including tomatoes Ambient temperature Yeasts and moulds Mayonnaise Yeasts and lactobacilli – salmonellas if raw eggs used

0.85–0.70 Dried fruits, jams Higher aw products contain Moulds and yeasts preservatives – SO2, sorbateAmbient temperature

Pickles and fermented vegetables, High numbers of lactic acid Yeasts, moulds, lactic acid bacteria. Bacillus spp. if pH > 4.0e.g. sauerkraut bacteria present in fermented

productsAmbient temperature

7.4–5.4 0.8–0.6 Grains and flour Ambient temperature Moulds

Table 14.2 Plating media commonly used to examine foods

Medium Group selected Selective agents Indicator agents Inoculation Incubation: time, temperature,atmosphere

Spoilage organismsCFC Pseudomonas spp. Cephaloridine, fucidin, None Surface 48 h, 25°C, aerobic

cetrimide

MRS Lactic acid bacteria None None Surface 5 days, 25°C, anaerobic

STAA Brochothrix thermosphacta Streptomycin, thallium None Surface 25°C, 48 h, aerobicacetate, cycloheximide (actidione)

Iron agar Shewanella putrefaciens and None Sulphite, cysteine and Pour plate 20 or 25°C for 2–5 daysother H2S producers thiosulphate (with overlayer)

DG18 Yeasts and moulds Chloramphenicol, dichloran, None Surface 25°C, 7 days, in dark, lids upglycerol

RBC Yeasts and moulds Chloramphenicol, Rose None Surface 25°C, 5 days in dark, lids upBengal

DRBC Yeasts and moulds Chloramphenicol, Rose None Surface 25°C, 5 days in dark, lids upBengal dichloran

OGY Yeasts and moulds Oxytetracycline None Surface 25°C, 5 days in dark, lids up

Indicator organismsVRBG Enterobacteriaceae Bile salts, crystal violet Glucose, neutral red Pour or surface 37 or 30°C for 24 h

with or without overlayer

VRBL Coliforms (E. coli) Bile salts, crystal violet Lactose, neutral red Pour or surface 37 or 30°C for 24 h (44°C 24 h for with or without E. coli)overlayer

KAA Enterococcus spp. Sodium azide, kanamycin Aesculin, ferric Surface 37 or 42°C 18–24 h, aerobic ammonium citrate

M-E Enterococcus spp. Sodium azide Triphenyltetrazolium Surface 37°C for 4 h, then 44°C for 44 h, air chloride

TBA Escherichia coli Bile salts None On membrane 44°C 18–24 h, aerobic

TBX Escherichia coli Bile salts 5-bromo-4-chloro-3- Pour or surfaceindolyl β-D glucoronic or membraneacid (BCIG)


Medium Group selected Selective agents Indicator agents Inoculation Incubation: time, temperature,atmosphere

PathogensBP Staphylococcus aureus Lithium chloride, tellurite Tellurite, egg yolk Surface 37°C, 24–48 h aerobicmCCD Campylobacter jejuni and Deoxycholate, cefoperazone, None Surface 37 or 42°C, 48 h microaerobic (5% O2,

C. coli amphotericin 10% CO2, 85% N2 or H2)PEMBA Bacillus cereus and other Polymyxin, cycloheximide Mannitol, bromothymol Surface 30°C, 18–24 h, and, if needed,

Bacillus spp. blue, egg yolk overnight at room temperature,aerobic

MEYP Bacillus cereus and other Polymyxin B Mannitol/phenol red, Surface 30°C, 24–30 h, aerobic Bacillus spp. egg yolk

Oxford Listeria spp. Lithium chloride, acriflavine, Aesculin, ferric Surface 30 or 37°C, 48 h aerobic colistin, fosfomycin, ammonium citrate cefotetan, cycloheximide

PALCAM Listeria spp. Lithium chloride, acriflavine, Aesculin, ferric Surface 30°C, 24–48 h, microaerobic (5% O2,polymyxin, ceftazidime ammonium citrate 10% CO2, 85% N2 or H2)

Pre-enrichment Enrichment Plating Details

Salmonella spp. 25 g food in 225 ml buffered 0.1 ml in 10 ml Rappaport Streak on to brilliant green Confirm biochemically and/or withpeptone water (37°C, Vassiliadis broth (42.5°C agar, XLD agar, Rambach salmonella antisera18–24 h) 24 h) or agar, 37°C 24 h

1 ml in 10 ml selenite broth(37°C 24 and 48 h)

ThermophilicCampylobacter spp. 25 g food in 225 ml Bolton Streak on mCCD agar, 37 Presumptive confirmation of oxidase-

broth 37°C 48 h or or 42°C 48 h, microaerobic positive Gram-negative curved rods4 h 37°C and 42°C 44 h. (see Table 25.3) not growing aerobically at 37°C

E. coli O157:H7 25 g food in 225 ml of Plate on CT-SMAC agar; Send sorbitol-negative, indole positivetryptone soy broth with incubate at 37°C for 24 h colonies agglutinating anti-0157novobiocin. Examine after polystyrene beads to a reference6–7 h and 41.5°C using laboratoryimmuno-magnetic beads (with anti 0157 antibodies)

Different manufacturers use alternative names for some mediaBP, Baird-Parker medium; CFC, cephaloridine fucidin cetrimide medium; CT-SMAC, cefoxine tellurite sorbitol MacConkey agar; DG18, dichloran glycerol medium; DRBC,dichloran Rose Bengal chloramphenicol agar; KAA, kanamycin aesculin azide medium; mCCD, membrane campylobacter-selective medium; M-E, membrane enterococcusmedium; MEYP, mannitol egg yolk polymyxin agar; MRS, deMan, Rogosa and Sharp agar; Oxford, Oxford listeria medium; PALCAM, Listeria selective medium; PEMBA,polymyxin egg yolk mannitol bromothymol blue agar; RBC, Rose Bengal chloramphenicol agar; STAA, Gardner’s (1966) medium; VRBL, violet red bile lactose agar

Direct examinationPre-incubation may be useful (Jarvis et al., 1983).Place a filter paper soaked with glycerol in a largeglass Petri dish and autoclave it. Suspend a sampleabove the filter paper, e.g. on glass rods, replace thelid and incubate for up to 10 days. Examine dailyfor moulds.

To estimate shelf-life relative to mould growthunder adverse conditions, incubate unopened pack-ages in a controlled humidity cabinet.

CulturePrepare a 10–1 suspension in MRD containing0.1% Tween 80. Use a Stomacher if possible. Makeserial dilutions in the same diluent and surfaceplate (in duplicate) on Rose Bengal chlorampheni-col agar. Incubate at 22°C for 5 days. For low aw

foods use DG18 agar.

REFERENCES

Adams, M. R. and Moss, M. O. (2000) Food

Microbiology, 2nd edn. London: Royal Society ofChemistry.

Dowens, F. P. and Ito, K. (eds) (2001) Compendium of

methods for the Microbiological Examination of

Foods, 4th edn. Washington DC: American PublicHealth Association (APHA).

Food and Drug Administration (1998) Bacteriological

Analytical Manual, 8th edn. Arlington, VA: AOACInternational.

Gardner, G. A. (1966) A selective medium for theenumeration of Microbacterium thermosphactum

in meat and meat products. Journal of Applied

Bacteriology 29: 455–460.

Harrigan, W. F. and Park, R. W. A. (1991) Making

Food Safe. London: Academic Press. International Commission of Microbiological Standards

for Foods (ICMSF) (1980) Cereal and CerealProducts. In: Microorganisms in Foods. 3.Microbial Ecology of Foods, Vol. 2. New York:Academic Press.

ICMSF (1986) Microorganisms in Foods. 2. Sampling

for Microbiological Analysis. Principles and

Specific Applications. Toronto: Toronto UniversityPress.

ICMSF (1988) Microorganisms in Foods. 4.

Application of the HACCP system to ensure

Microbiological Safety and Quality. Oxford:Blackwell Scientific.

ICMSF (1998) Microorganisms in Foods. 6. Microbial

Ecology of Food Commodities. London: BlackieAcademic & Professional. Available from KluwerPublishers.


Microbiological Testing in Food Safety

Management. New York: Kluwer Academic/Plenum Publishers.

Jarvis, B., Seiler, D. A. L., Ould, S. J. L and Williams,A. P. (1983) Observations on the enumeration ofmoulds in food and feedingstuffs. Journal of

Applied Bacteriology 55: 325–326.Jay, M. (2000) Modern Food Microbiology, 6th edn.

MA: Aspen Publishers.Lund, B. M., Baird-Parker, T. C. and Gould, G. W.

(eds) (2000) The Microbiological Safety and

Quality of Food. New York: Aspen Publishers.Roberts, D. and Greenwood, M. (eds) (2002) Practical

Food Microbiology, 2nd edn. Oxford: Blackwell.Snell, J. J. S., Farrell, I. D. and Roberts, C. (eds) (1991)

Quality Control. Principles and practice in the

microbiology laboratory. London: Public HealthLaboratory Service.


FRESH AND FROZEN RAWRED MEAT

Table 14.1 (see p. 200) summarizes the types ofbacteria that cause spoilage. Many different speciesof bacteria are present on carcasses immediatelyafter slaughtering, but Gram-negative organismsmultiply and predominate during chill storage. Thedeep muscle tissue is usually sterile but occasionallymay be contaminated. ‘Bone taint’ is revealed by anunpleasant odour when the carcass is boned, and ismost common in the hip and shoulder joints, whichcool most slowly. Microbiological investigationmost often reveals clostridia or enterococci. Thesource of contamination is not certain, but could befrom contaminated slaughter instruments, viainfected lymph nodes or by spread from the intes-tine at the time of death. Meat from stressed ani-mals may be of poor keeping quality because thepH is higher than normal (> 6.0).

Carcass sampling

Microbial contamination is usually confined to theskin or surface of carcasses. The most reliablemethods are destructive (which can affect the valueof the carcass), involving excising the top 2–3 mmof a measured area (e.g. 25 or 50 cm2). The mostpractical non-destructive method is swabbing.Impression plates or slides may also be used. Ingeneral, excision methods recover more organismsthan swab methods, which in turn recover morethan impression methods. Microbiological testingof five to ten carcasses on one day per week, afterdressing and before chilling (aerobic colony count

at 30°C and Enterobacteriaceae at 37°C) from foursites on each carcass and from the abattoir envi-ronment is now required in the European Union(EC, 2001; SI, 2002). Excision sampling is recom-mended, with swabbing as an alternative.

Excision samplingUse aluminium templates with, for example, 25or 50 cm2 apertures. Remove the top 2–3 mm of themeat defined by the template area with a scalpel or,for frozen meat, a cork borer.

Homogenize an appropriate volume (e.g. 25 ml)in 50 ml of maximum recovery diluent (MRD – seep. 76) and do counts as described in Chapter 10and above.

Swab samplingMoisten a large plain cottonwool swab with awooden shaft with MRD and then rub it vigorouslyover the area to be sampled, e.g. as defined by atemplate. Repeat with a dry swab. Break off theends of both swabs into 10 ml MRD in the samecontainer and do total counts at 30°C as describedin Chapter 10 and above. This method cannot beused for frozen meat.

Whole carcass samplingTo sample the whole carcass by a non-destructivetechnique, use the following method (Kitchell et al.,1973). Sterilize large cottonwool pads, wrapped incotton gauze in bulk, and transfer them to individ-ual plastic bags. Moisten a pad with diluent, e.g.MRD, and wipe the carcass with it, using the bagas a glove. Take a dry pad and wipe the carcassagain. Place both pads in the same bag, then sealand label it. Add 250 ml diluent to the pads and

15

Meat and poultry

knead, e.g. in a Stomacher, to extract the organ-isms. Prepare suitable dilutions from the extractand do counts as above. Inoculate other media asdesired. This method is not usually practicable forbeef carcasses.

The areas on beef carcasses that are most likely tobe contaminated after dressing are the neck, rump,brisket and flank – and similar parts of other redmeat carcasses. For excised samples, the daily logmean limits of aerobic colony counts at 30°C andof enterobacteria at 37°C for cattle, sheep, goatsand horses are shown in Table 15.1. Values for pigcarcasses are in most cases 0.5 log cycles higher.Values for swab samples are about 20% of theexcision sample values.

In dry (about 80% relative humidity) chill condi-tions, mould spoilage may occur on chilled (−1°C)carcasses stored for several weeks. Carcasses arenow, however, seldom stored chilled for long peri-ods. Mould and yeast colonies can also be seen onprimals (see below) in frozen storage when the cor-rect temperature (≤ −12°C) has not been main-tained. ‘Green spots’ are usually caused byPenicillium spp., ‘white spots’ by Sporotrichumspp., ‘black spots’ by Cladosporium spp. and‘whiskers’ by Mucor and Thamnidium spp.

Boned-out meat

Most carcasses are split into primals a few daysafter slaughter. These are large pieces of meat that

are sometimes stored at 0 ± 1°C in vacuum-packsfor several weeks before being divided up intosmaller cuts for retail sale. Much meat is nowretailed in modified atmosphere packs (about 80%O2, 20% CO2) in supermarkets. The oxygen pre-serves the red colour of oxymyoglobin for as longas possible, whereas the carbon dioxide inhibits thepseudomonads, which predominate in meat storedin aerobic packs.

As with carcass sampling, primals or smaller cutsof red meat are usually sampled either by excisionor by swabbing. It may also be possible to samplethe liquid exudate (‘purge’) that accumulates invacuum packs. Two areas of 5 or 10 cm2 are usu-ally sampled per piece of meat. These could includea sample from a cut edge and another from theoriginal exterior surface. The samples may bepooled before microbiological examination.Results of excision or swabbing are expressed ascolony-forming units per square centimetre(cfu/cm2). In a meat production facility, where thehygiene is good, more than 70% of samples may beexpected to have counts of less than 1000 cfu/cm2

(or log10 3 cfu/ml).

Microbial flora of raw meat duringstorage

In moist, aerobic chill conditions the predominantspoilage organisms are pseudomonads, with lower

206 Meat and poultry

Table 15.1 Acceptable and unacceptable daily mean values of colony and enterobacteria counts for cattle, sheep, pig, goat and horsecarcasses

Daily mean values (log cfu/cm2) as

Acceptable range for Marginal range (> m but Unacceptable range

Cattle/sheep/ Pig< M) for cattle/pig/sheep/ (> M) for cattle/pig/

goat/horsegoat/horse sheep/goat/horse

Total viable count < 3.5 < 4.0 3.5 (pig: 4.0) to 5.0 > 5.0 Enterobacteriaceae < 1.5 < 2.0 1.5 (pig: 2.0) to 2.5 (pig: 3.0) > 2.5 (pig: > 3.0)

See Chapter 14 for definitions of sampling plansFrom EC (2001) and SI (2002)

numbers of Acinetobacter spp., psychrotrophicEnterobacteriaceae, including Enterobacter andHafnia spp., and Brochothrix thermosphacta.Spoilage is imminent when numbers ofpseudomonads reach about 107/cm2.

In meat stored in a modified atmosphere or vac-uum packs, lactic acid bacteria (LAB) normally out-grow the pseudomonads; Lactobacillus sake andL. curvatus are the most common LAB in vacuum-packed raw meat. Carnobacterium, Leuconostocand Weissella spp. occur in lower numbers.Psychrotrophic enterobacteria and B. thermo-sphacta are sometimes present. B. thermosphacta ismore often found in lamb and pork. Numbers ofLAB on vacuum-packed meat frequently exceed 108

(log 8)/cm2 for several weeks before the meat isjudged spoiled (an initial rather cheesy smell oftendissipates if the pack is left open for a few minutes).This is because the LAB produce much less obnox-ious products than pseudomonads in aerobicallystored meat. There are a number of possible reasonswhy vacuum-packed meat has apparently spoiledbefore the end of its normal shelf-life. Meat with pH> 6 will spoil rapidly as a result of the multiplica-tion of Enterobacteriaceae and/or Shewanella putre-faciens. Meat with apparently normal pH sometimeshas elevated numbers of enterobacteria or B. ther-mosphacta (> 105/cm2), which may be the result oftemperature abuse during storage, or possibly poorhygiene. In addition, psychrotolerant clostridia,most commonly the psychrophilic species C. ester-theticum, have emerged during the last 15 years asa very important cause of spoilage in vacuum-packed beef and, to a lesser extent, lamb, stored atproper chill temperature and with normal pH. Packssometimes ‘blow’ (become grossly distended withgas, see Chapter 16) within 2 weeks of packing andeconomic losses can be extremely high. The gasconsists largely of a mixture of CO2 and H2. Themeat usually appears normal in colour, but has avery unpleasant vomit-like odour. Gram stains ofthe drip fluid sometimes reveal large Gram-positiverods among the normal small rod-shaped LAB and

the occasional yeast. Isolation of C. estertheticum isdifficult because there is no selective medium, butspores may be selected by use of mild heat (65°C)or ethanol for a few minutes before plating on toblood agar and incubation at 10°C anaerobically for2–3 weeks. Colonies of C. estertheticum are nor-mally β-haemolytic. A more reliable and rapidmethod of detection is to use a polymerase chainreaction (PCR)-type method (e.g. Helps et al., 1999).Fortunately, there is no evidence that C. ester-theticum is pathogenic or produces toxins.

Many of the well-known pathogens may be foundon red meat in low numbers, as a result of cross-contamination from hides and intestinal contents.Salmonellas are not often present, however, exceptin pork and minced products. Campylobacters aremost often found on offal, such as liver, and sur-vive poorly during their shelf-life. C. perfringens,and (less often) C. botulinum may be isolated fromraw meat, but pose no threat unless the meat is mis-handled during processing and/or storage.Enterohaemorrhagic E. coli O157:H7, whichcauses severe disease and has a low infectious dose,colonizes cattle and sheep, and so can be found onraw meat. Careful attention to hygiene duringslaughter will minimize its presence on meat, butcannot guarantee its absence.

The microflora detected on meat sampled duringchilled shelf-life will depend on the method of stor-age (aerobic, gas or vacuum pack) and the point inshelf-life. Thus, aerobic plate counts alone areunlikely to be meaningful. Enumeration ofpseudomonads on aerobically stored meat might beuseful, because > log 7 cfu/cm2 would indicateimminent spoilage. The finding of high numbers ofLAB (> log 8 cfu/cm2) on vacuum- or gas-packedmeat would only confirm the presence of normalflora. Detection of numbers of Enterobacteriaceaeor B. thermosphacta at 30°C (> log 5 cfu/cm2)would be cause for concern that the meat mighthave been stored at elevated temperature and/orthat hygiene had been lax during slaughter andcutting.

Fresh and frozen raw red meat 207

COMMINUTED MEAT

This is fresh meat, minced or chopped and with noadded preservative, sometimes prepared from trim-mings. Instead of being confined to the surface,organisms are therefore distributed throughout andfurther contamination may occur during mincing.In addition, the normal structure of the meat is dis-rupted, making the nutrients more readily accessi-ble to the microflora. Raw minced meat tends to bespoiled by the same micro-organisms as largerpieces of raw meat, with the exception that it oftencontains significant numbers of yeasts.

Pathogens

As with whole red meat, salmonellas, clostridia andstaphylococci may be present. Tests for E. coliO157:H7 in 25 g minced beef are indicated becauseminced beef is often used to make beefburgers(hamburgers in the USA), which have been impli-cated in outbreaks of disease. This organism ismore of a hazard in beefburgers than on wholepieces of meat because the microflora occurthroughout the meat, and are more likely to surviveunder-cooking.

The EU (EC, 1994) has set limits for freshly pre-pared minced meat (Table 15.2).

This standard is likely to be revised soon toinclude a requirement for the absence of E. coli

O157:H7. This standard is most appropriate forminced meat intended for consumption withoutfurther cooking, e.g. in steak tartare or filet ameri-cain.

Numbers of microbes in stored minced meat canreach considerably higher figures than those indi-cated above. As the product may not be homoge-neous, it is useful to take several samples. In a studyof retail minced beef in the UK (Nychas et al.,1991) the microflora were similar whether theproduct had been stored in air or with CO2. Countsof B. thermosphacta, pseudomonads, LAB,Enterobacteriaceae and yeasts were all in the regionof 104–107 cfu/g and total counts were around108 cfu/g. Aerobic colony counts at 30°C andEnterobacteriaceae at 30 or 37°C should bechecked. Numbers of E. coli may be useful as anindicator of hygiene during preparation, and E. coliO157:H7 could also be sought. This organism isalmost certain to be present occasionally, however,because it can be found in the faeces of many cattle.The action to be taken if it is detected should there-fore be decided in advance

BRITISH FRESH SAUSAGES

Fresh sausages contain comminuted raw meat (usu-ally pork) and fat, and water mixed with otheringredients such as phosphates, rind, rusk, season-ing, spices and herbs. This is filled into a natural or


Table 15.2 European Union limits of bacterial counts for freshly prepared minced meat

Microbes Limits Sampling plan

n c m (/g) M (/g)

Aerobic mesophilic bacteria 5 2 5 × 105 5 × 106

Escherichia coli 5 2 50 500Salmonella spp. Absence in 10 g 5 0Staphylococcus aureus 5 2 100 5000

See Chapter 14 for definitions of sampling plans; m, specified number; M, higher specified number; n, total number of samples tested;c, the number that may exceed mFrom EC (1994)

artificial casing, with sodium metabisulphite or sul-phite as preservative (not more than 450 ppmmeasured as SO2 at the point of sale). This productshould not be confused with smoked sausages,which are popular in Europe and America

Microbial content

Without the preservative and spices, the microflorathat would develop in this product would be simi-lar to that in raw minced meat. The preservativeselectively inhibits the Gram-negative organisms,allowing yeasts and B. thermosphacta to predomi-nate, with lower numbers of LAB. Spoilage occursas a result of slow souring. Shelf-life is longer thanfor minced meat, and is extended by gas or vacuumpacking, which slows yeast growth. STAA medium(Gardner, 1982) is useful for the isolation and enu-meration of B. thermosphacta. Freshly preparedsausages often contain about log 4 cfu/g yeasts andlog 5 cfu/g B. thermosphacta. After 7 days of chillstorage the numbers of yeasts can reach log 7 cfu/gand numbers of B. thermosphacta log 8 cfu/g,whereas numbers of pseudomonads, Entero-bacteriaceae and LAB remain static at around log4 cfu/g (Banks et al., 1985).

Pathogens

Sulphite may have played a role in the relativelygood safety record of sausages as a cause of salmo-nella food poisoning. Although salmonellas can beisolated from sausages, sulphite will prevent themmultiplying if the product is stored at elevated tem-perature. E. coli O157:H7 should be a hazard onlyin sausages containing beef or lamb.

Viable counts

The Institute of Food Science and Technology(IFST, 1999) suggests limits for aerobic platecounts (cfu/g) on sausages immediately after pro-

duction of < log 5 (and an upper limit of log 7, andof log 4 and log 6 respectively for yeasts).

Remove the casing, if present, and do total viablecounts using dilutions from 10−4 to 10−8. Incubate at30°C for 48 h and at 22°C for 3 days. The use ofsurfactants, e.g. Tween 80, does not seem particu-larly advantageous. Sometimes they are inhibitory.

Counts of several million colony-forming unitsper gram may be expected. Skinless sausages mayhave lower counts than those with casings becausethe process involves blanching with hot water toremove the casings in which they are moulded.

Sausage casings, whether natural (stripped smallintestine of pig or sheep) or artificial, usually do notpresent any microbiological problems.

MEAT PIES

Meat pies may be ‘hot eating’, e.g. steak andchicken pies, or ‘cold eating’, e.g. pork, veal andham pies, and sausage rolls. Consumers, however,cannot, be relied on to reheat ‘hot-eating’ pies thor-oughly before consumption. In both cases, the pas-try acts as a physical barrier against post-cookingcontamination of the filling. The filling in mostcold-eating pies includes preservatives, e.g. nitritein pork pies and sulphite in sausage rolls.Temperature fluctuations during storage of theseproducts in films can result in condensation, soggypastry and subsequent spoilage.

‘Hot eating’ pies

The meat filling is pre-cooked, added to the pastrycasing and the whole is cooked again.

Viable countsExamine the meat content only. Open the pie witha sterile knife to remove the meat. Prepare a 1 : 10suspension in MRD. Do counts on dilutions of10−1 and 10−2 and incubate aerobically and anaero-bically on blood agar at 37°C for 48 h. A reason-able level is not more than 100 cfu/g. Numbers in

Meat pies 209

excess of 1000 cfu/g may indicate temperatureabuse after cooking (Bacillus or Clostridium spp.),or under-cooking.

Little other than heat-resistant spores should sur-vive in these products.

‘Cold eating’ pies

These contain cured meat, cereal and spices whichare placed in uncooked pastry cases. The pies arethen baked and jelly, made from gelatin, spices,seasoning, flavouring and water, is added. Providedthat the jelly is heated to, and maintained at, a suf-ficiently high temperature until it reaches the pie,there should be no problem.

Bad handling, insufficient heating and re-contam-ination of cool jelly can cause gross contaminationby both spoilage organisms and pathogens.

The pies are cooled in a pie tunnel through whichcold air is blown. At this stage mould contamina-tion is likely if the air filters are not properlycleaned. Air in pie tunnels may be monitored byexposing plates of malt agar or similar medium. Aswith hot-eating pies, bad storage may result in out-growth of spores of Bacillus and Clostridium spp.

Viable countsExamine only the filling. Do counts on homoge-nized material using dilutions of 10−1 and 10–2.Incubate at 30°C for 48 h. A reasonable level is lessthan 1000 cfu/g.

CURED MEAT

Cured raw meat

This is pork or beef that has been treated with saltand nitrite, but could be applied to any meat. Thehaemoglobin is altered so that the characteristicpink colour is produced when the meat is cooked.This is sold as bacon, gammon, cured shoulder, saltbeef or brisket, and has a lower water activity (aw)and a higher salt content than raw meat.

Bacon has been studied most, but other cured rawmeats are likely to have similar characteristics.Numbers and species of microbes on sides of baconcan be very variable, as a result of the variety ofecological niches present – rind, fat, lean, varia-tions in pH, aw, curing salts, etc. Surface spoilage ofrefrigerated bacon is mainly caused byMicrococcus, non-pathogenic Vibrio spp. andAcinetobacter. Yeasts occur more commonly onsmoked and dried rather than ‘green’ (unsmoked)bacon. Their growth may sometimes be visible tothe naked eye, and they can be physically removedwithout any problem. The generally lower aw andhigher salt result in much lower total numbers ofmicrobes on the surface than on green bacon.Gram-negative bacteria are rarely found. As smok-ing and drying are sometimes carried out at ele-vated temperatures, and curing salts are ofteninjected, ‘bone taint’ can sometimes be a problem(see p. 205).

Vacuum-packed sliced bacon

Most bacon is now sold sliced and packed invacuum or CO2, which prevents the oxidation of thered-coloured nitrosohaemoglobin to brown prod-ucts such as metmyoglobin. This type of packagingalso extends the microbiological shelf-life to severalweeks at chill temperatures. The predominant floraconsist of LAB, with lower numbers of micrococci.Numbers of organisms on fat tend to be higher thanon lean. As with vacuum-packed raw red meats,numbers of these bacteria can reach high levels (upto 108 cfu/g) during chill storage, producing gradualsouring before spoilage is evident. Table 14.1 (seep. 200) indicates the types of spoilage organisms.Storage at temperatures above ambient can result inputrefactive spoilage as a result of multiplication ofVibrio spp., and enterobacteria such as Proteus,Serratia and Hafnia spp., Staphylococcus aureus,Clostridium perfringens and C. botulinum couldtheoretically multiply and produce toxins if baconwere stored at elevated temperatures, but in practicethis product has not been associated with food-


borne illness. This is most probably because theproduct is usually cooked before consumption andthe toxins produced are heat labile. In addition,these organisms do not compete well with themajority microflora. Spoilage also intervenes andthe product becomes inedible. Salmonellas mightalso be found on bacon, but this food has not beenassociated with disease caused by this organism.

Microbial content

The main groups of organisms found immediatelyafter packaging are micrococci and coagulase-negative staphylococci. During storage at 20°C,lactobacilli, enterococci and pediococci becomedominant. Yeasts may also be found. If largenumbers of Gram-negative rods are present it isusually an indication that the initial salt contentwas low.

Vacuum-packed whole and slicedcooked meats and cooked curedmeats

These meats include cured products, such as ham,as well as uncured ones, such as cooked beef,boned chicken or turkey, and pork. The meat isusually cooked inside a plastic film, which is left onduring distribution and storage, or removed aftercooking. In the former case the product normallyhas very low counts; in the latter case some con-tamination is inevitable. The meat may be used incatering, or sliced and sold in vacuum or gas packs.Spore formers will survive cooking, and sopathogens such as C. perfringens or C. botulinumcould germinate and multiply if the products weretemperature abused. Spoilage caused by psychro-tolerant clostridia has occasionally been reportedin cooked uncured meat after storage at slightly ele-vated temperatures (Lawson et al., 1994; Broda etal., 1997; Kalinowski and Tompkin, 1999).

Other organisms that sometimes survive cookinginclude enterococci and some lactobacilli. As with

vacuum- or gas-packed raw meats, LAB normallypredominate in packed sliced cooked meats, reach-ing high numbers before gradual spoilage as aresult of souring. Spoilage organisms in aerobicallypacked cooked sliced meat are similar to those inaerobically stored raw meats (predominantlypseudomonads). Multiplication of small numbersof S. aureus, introduced during slicing, may occur,especially in cured meats, such as ham, if the tem-perature is raised about 10°C. Outbreaks of poi-soning caused by staphylococcal toxins in ham arenot uncommon, but more often occur as a result ofpoor practices in the catering industry (e.g. leavingmeat for extended periods at ambient temperature)than in industrially produced sliced packed ham.Multiplication of L. monocytogenes, which cangrow at refrigeration temperature, is possibly amore important hazard, especially with the currenttrend towards longer shelf-life. Levels in excess of100 cfu/g at the end of the shelf-life are consideredpotentially hazardous (EC, 1999).

Laboratory examinations

Whole joints in cooking bagsDo surface counts on blood agar and onMacConkey agar for enterococci. These are usefulorganisms for assessing the efficiency of cooking.Total counts should be less than 1000 cfu/g.

Sliced meatsDo total (surface) counts immediately after slicingand packing. These should not exceed 104 cfu/g,but much higher numbers can be found duringshelf-life. Numbers of L. monocytogenes shouldnot exceed 100 cfu/g by the end of the shelf-life(EC, 1999), and numbers of S. aureus should be< 1000 cfu/g.

Cooked sausages and patéAs with joints cooked in sealed film, whole cookedsausages are protected by their casing and, pro-vided cooking is sufficient, only low numbers ofspore formers will survive. Sometimes, however, afew enterococci or lactobacilli may survive and

Cured meat 211

grow. The spore formers cause no problem if theproduct is stored at low temperature. Hydrogenperoxide produced by lactobacilli can sometimescause grey–green, yellow or white discolorationcaused by oxidation of the nitrosyl myoglobin.

Belgian-type paté has similar characteristics tocooked sausages, except that it is more prone topost-cooking contamination. It is sometimes deco-rated with gelatin and pieces of vegetable. LABmay found at levels > 105 cfu/g. L. monocytogenesshould not be present at levels > 100 cfu/g at end ofthe shelf-life.

POULTRY

Intensive rearing produces birds with very tenderflesh, but the close confinement necessary in thismethod of production often results in cross-infec-tion. Poultry meat is thought to be the source of alarge proportion of cases of salmonellosis, andmost cases of campylobacter diarrhoea in the UKand the USA. Salmonella infections occur either asa result of undercooking or of raw-to-cooked foodcross-contamination. Campylobacter infectionsprobably occur by a similar mechanism, as well asby the hand-to-mouth route after handling the rawproduct. Table 14.1 (see p. 200) indicates the typeof spoilage organisms.

Sampling

The neck skin is a good sampling site because (1) itis the lowest point during processing and all fluidspass over it, and (2) it is easy to sample and may beremoved without affecting the value of the remain-der of the carcass. A good alternative is to rinse thewhole carcass.

Place the carcass in a large sterile plastic bag. Add500 ml diluent (MRD) and (holding the bag tightlyclosed) shake thoroughly for approximately 1 min.Carefully decant the fluid back into the bottle thatheld the sterile diluent. Do viable plate countson tenfold dilutions of this fluid. Normal counts

on carcass rinsed immediately after chill are listedbelow.

Viable counts

Discard the subcutaneous fat from the neck skin,homogenize a known weight of the remainingmaterial, prepare dilutions as described in Chapter10 and use plate count agar. Incubate at 30°C or37°C for 24–48 h. Express the counts as colony-forming units per gram of neck skin. These countsmay be about 10 times higher than those obtainedfrom the skin on other parts of the carcass. Normalranges at the abattoir immediately after chill(log10 cfu/g neck skin) are: viable count, 4.5–5.3;coliforms, 2.7–3.8; pseudomonads, 2.9–3.9 (Meadet al., 1993). Counts on carcass rinse are expressedas log10 cfu or per millilitre rinse fluid: viable count3.8–6.0; Enterobacteriaceae 2.6–4.6; pseudo-monads 3.2–5.9 (Corry, unpublished, 2002). Aschlorine in wash water at levels above about 5 ppmis no longer permitted for washing poultry in theEU, numbers of coliforms/Enterobacteriaceae tendto be a little higher than pre-2001.

Pathogens

Examine the carcass rinse or neck skin. Culture forsalmonellas and campylobacters as described inChapters 28, 33 and 36 (see also Chapter 14). HighS. aureus counts are sometimes found; these resultfrom colonization of plucking machines. They arerarely toxin-producing, food-poisoning strains.

REFERENCES

Banks, J. G., Dalton, H. K., Nychas, G. J. et al. (1985)Review: sulphite, an elective agent in themicrobiological and chemical changes occurring inuncooked comminuted meat products. Journal of

Applied Biochemistry 7: 161–179.


Broda, D. M., DeLacey, K. M., Bell, R. G. and Penney,N. (1997) Prevalence of cold tolerant clostridiaassociated with vacuum-packed beef and lambstored at abusive and chill temperatures. New

Zealand Journal of Agricultural Research 40:93–98.

European Commission (EC) (1994) Minced MeatDirective. Council Directive 94/65/EEC ofDecember 1994 laying down the requirements forthe production and placing on the market ofminced meat and meat preparations. Brussels: EU.

EC (1999) Opinion of the Scientific Committee onVeterinary Measures relating to Public Health onListeria monocytogenes.

(europa.eu.int/comm/food/fs/sc/scv/out25_en.html.Brussels, EU).

EC (2001) Commission Decision 2001/471/EC: of 8June 2001 laying down rules for the regular checkson the general hygiene carried out by the operatorsin establishments according to Directive1964/433/EEC on health conditions for theproduction and marketing of fresh meat andDirective 1971/118/EEC on health problemsaffecting the production and placing on the marketof fresh poultry meat. Official Journal of the

European Communities L 165, 21/06/2001 P.0048–0053

Gardner, G. A. (1982) Microbiology of processing:bacon and ham. In: Brown, M.H. (ed.), Meat

Microbiology. Oxford: Elsevier Applied Science,pp. 129–178.

Helps, C. R., Harbour, D. A. and Corry, J. E. L. (1999)PCR-based 16S ribosomal DNA detectiontechnique for Clostridium estertheticum causing

spoilage in vacuum-packed chill-stored beef.International Journal of Food Microbiology 52:57–65.

Institute of Food Science and Technology (IFST) (1999)Development and Use of Microbiological Criteria

for Foods. London: IFST. Kalinowski, R. M. and Tompkin, R. B. (1999)

Psychrotrophic clostridia causing spoilage incooked meat and poultry products. Journal of

Food Protection 62: 766–772.Kitchell, A. G., Ingram, G. C. and Hudson, W. R.

(1973) Microbiological sampling in abattoirs. In:Board, R.G and Lovelock, D.W. (eds), Sampling –Microbiological Monitoring of the Environment.Society for Applied Bacteriology Technical SeriesNo. 7. London: Academic Press, pp. 43–54.

Lawson, P., Dainty, R. H., Kristiansen, N., et al.

(1994) Characterization of a psychrotrophicClostridium causing spoilage in vacuum-packedcooked pork: description of Clostridium

algidicarnis sp. nov. Letters in Applied


Mead, G. C., Hudson, W. R. and Hinton, M. H.(1993) Microbiological survey of five poultryprocessing plants in the UK. British Poultry Science

17: 71–82.Nychas, G. J., Robinson, A. and Board, R. G. (1991)

Microbiological and physico-chemical evaluationof ground beef from retail shops. Fleischwirtschaft

71: 1057–1059.Statutory Instrument (SI) (2002) Statutory Instrument

No. 889. The Meat (Hazard Analysis and Critical

Control Point) (England) Regulations 2002.London: HMSO.

References 213

FRESH FRUIT AND VEGETABLES

Spoilage of fresh fruit is mostly caused by moulds.Mould and yeast counts may be indicated in fruitintended for jam making or preserving. Orangeserum agar is useful for culturing fungi from citrusfruit (Lund and Snowdon, 2000).

Washed, peeled and chopped vegetables, in packswhere the atmosphere has been modified eitherdeliberately, or as a result of the metabolic activityof the vegetables, to give a raised level of CO2 andreduced O2 content, are sold in most supermarkets.They can include mixtures of vegetables for ‘stir-fry’ dishes or stews, as well as various types ofsalad and lettuce. High viable counts – up to107–108 – are normal; products such as beansprouts, cress, watercress and spring onions havethe highest counts (Roberts et al., 1981; Lund,2000; Nyugen-the and Carlin, 2000). Spoilage iscaused by pseudomonads, psychrotrophic non-pathogenic Enterobacteriaceae (mostly Entero-bacter, Erwinia and Rahnella spp.), variouspectinolytic bacteria and lactic acid bacteria, espe-cially leuconostocs, as well as lower numbers ofyeasts and moulds. With the exception of the lacticacid bacteria, which seem to be encouraged by theraised level of CO2, similar numbers and types ofmicroflora can be found on loose vegetables.

Although the incidence of contamination withpathogenic micro-organisms in plant foods is lowerthan that on foods of animal origin, they can befound from time to time. Contamination some-times occurs as a result of the use of sewage-polluted water for irrigation, or contaminationwith animal or human faeces during harvest. Seedsused to produce sprouts (e.g. mung beans for bean

sprouts) can sometimes contain low numbers ofsalmonellas that multiply during germination, andcan be extremely difficult to eliminate from theseeds. For this reason consumers are often advisedto blanch bean sprouts before eating them insalads. For products that will be cooked beforeconsumption, the risk posed by pathogens is clearlyless. There have been outbreaks of various types offood-borne disease from raw salad vegetables andfrom fruit (mostly soft fruit, pre-sliced fruit orunpasteurized juice), including Shigella sonnei,salmonellas, pathogenic E. coli (including E. coliO157:H7), hepatitis A virus, and the protozoaCryptosporidium, Giardia and Cyclospora spp. Avery large outbreak of E. coli O157:H7 infection,caused by contaminated radish sprouts, occurred inJapan (Michino et al., 1999). Listeria monocyto-genes can sometimes be isolated from packed veg-etable products, but rarely in numbers sufficient tocause concern (i.e. > 100 colony-forming units(cfu)/g).

Routine examination of these products for all thepathogens listed above is not practicable (or evenpossible). E. coli type 1 can be sought as an indica-tor of faecal contamination, but coliform, ‘faecalcoliform’ or Enterobacteriaceae counts are notappropriate because normal spoilage flora will beincluded. Limits at use-by date for vegetables inFrance are: salmonellas: absent in 25 g; E. coli pergram: m = 100, M = 1000, n = 5, c = 2; L. monocy-togenes: M = 100/g (Nyugen-the and Carlin, 2000).(For this m is the threshold value for bacterialcount, result satisfactory if not exceeded; M is themaximum value for bacterial count, result unsatis-factory if exceeded in any sample; n is the numberof sample units comprising the sample; and c is the

16

Fresh, preserved and extendedshelf-life foods

number of sample units that may have bacterialcounts between m and M if the bacterial count ofthe other samples is m or less.)

DEHYDRATED FRUIT ANDVEGETABLES

These rarely cause outbreaks of disease, althoughthey too may be contaminated in the same way asfresh fruit and vegetables. Total counts on driedvegetables are very variable, and can be as high as108 cfu/g. Yeasts and moulds are also often presentat levels up to 104 cfu/g. Numbers of bacterialspores can sometimes be raised, especially if thevegetables were blanched before drying. In prod-ucts that may be consumed with minimal heating(e.g. ‘instant’ soups and dried herbs and spices)salmonellas should be sought in 25 g, and counts ofE. coli type 1 (not more than 5/g) and Bacilluscereus and Clostridium perfringens (m = 102, M =104) should be done.

Total counts on dried fruit are dominated bymoulds and yeasts. Hazards are most likely tocome from mycotoxins – particularly aflatoxin, indried figs or nuts (e.g. peanuts, pistachios, cashews,Brazil nuts). Level should be < 4 ppb. Aflatoxin-producing moulds can be enumerated onAspergillus Flavus Parasiticus Agar (AFPA),although direct detection of aflatoxin is preferable,because the moulds may no longer be viable.Desiccated coconut has sometimes been found tobe contaminated with salmonellas.

FROZEN VEGETABLES AND FRUIT

Freezing affects the microbial flora little, and themicrobiological profile should be similar to that ofthe fresh product – similar tests should be carriedout (see above). Some vegetable products areblanched before freezing, lowering the total countby about 2 log cycles, but contamination frequentlyoccurs from the processing environment beforefreezing. Frozen peas sometimes carry large

numbers of Leuconostoc spp. and other lactic acidbacteria (Lund, 2000). A particular hazard tofrozen vegetables from the processing environmentcould be L. monocytogenes, although outbreaksfrom this source have not been reported. Freezingcannot be relied on to eliminate pathogens such assalmonellas or hepatitis A virus.

PICKLES

Vegetables are first pickled in brine. The salt is thenleached out with water and they are immersed invinegar (for sour pickles) or vinegar and sugar (forsweet pickles). Some products are pasteurized.Spoilage is the result of low salt content of brine,poor quality vinegar, underprocessing and poorclosures.

Counts

Use glucose tryptone agar at pH 6.8 for total countsand at pH 4.5 for counts of acid-producing colonies(these have a yellow halo caused by a colour changeof indicator). Count lactic acid bacteria on Rogosa,MRS (de Man, Rogosa and Sharp) or other suitablemedium. Estimate yeasts either by the countingchamber method (stain with 1 : 5000 erythrosin) oron Rose Bengal chloramphenicol agar.

Culture

Use glucose tryptone and Rogosa or MRS agar forlactic acid bacteria. Grow suspected film yeasts in aliquid mycological medium, containing 5 and 10%sodium chloride for 3 days at 30°C. For obligatehalophiles, use a broth medium containing 15%sodium chloride.

Microbial content

Pickles are high-acid foods. Counts are usually low,e.g. 1000 cfu/g. Yeasts are a frequent source of

Pickles 215

spoilage and may be either gas producing or filmproducing. In the former, enough gas may be gen-erated to burst the container. Bacterial spoilagemay be caused by acid-producing or acid-tolerantbacteria such as acetic acid bacteria, lactic acid bac-teria and aerobic spore bearers. Infected pickles areoften soft and slimy.

Pickles of the sauerkraut type are fermented andtherefore contain large numbers of lactic acid bac-teria (Lactobacillus and Leuconostoc spp.) whichare responsible for their texture and flavour.

Acid-forming bacteria are active at salt concen-trations below 15%. Above this concentration,obligate halophiles are found.

KETCHUPS AND SAUCES

The most common cause of spoilage isZygosaccharomyces (Saccharomyces) baillii. Thisyeast grows at pH 2, and from < 5°C up to 37°C, in50–60% glucose; it is relatively heat resistant.

SUGAR AND CONFECTIONERY

Sugars, molasses and syrups

The importance of micro-organisms is related to theintended use of the products, e.g. flat-sour bacteria(see pp. 221 and 365) are more important in bakingthan in canning. Do total counts, examine for spore-forming thermophiles (B. stearothermophilus,Desulfotomaculum (formerly Clostridium) nigrifi-cans, C. thermosaccharolyticum), and for yeasts andmoulds. Spoilage is often caused by osmotolerantyeasts; culture for these on a medium of low wateractivity (DG18 agar).

Chocolate and cocoa powder

Chocolate has been shown to be the vehicle of sal-monella infection in a number of cases (see thereview by D’Aoust, 1977).

The standard method of examination for salmo-nellas needs to be modified in order to neutralize thenatural inhibitory effect of cocoa products. Add 25 gcocoa powder, or shave 25 g chocolate into 225 mlbuffered peptone water with 10% non-fat dried milkand then proceed according to the standard method(enrichment in selenite or tetrathionate andRappaport Vassiliadis broth, etc. – see Chapter 14).Do not place large lumps of chocolate into liquidmedium. Automated methods, especially thoseemploying impedimetric principles, are useful forsalmonella screening in industry. Examine spoiledsoft-centred chocolates for yeasts and moulds bydirect microscopy and culture on DG18 medium.

CAKE MIXES AND INSTANTDESSERTS

Spores are likely to be present, e.g. of C. perfrin-gens and B. cereus. Salmonellas and staphylococcimay survive processing or be post-processing con-taminants. Any of these organisms may multiply ifthe product is reconstituted and then kept underunsuitable conditions. The powdered product mayalso cross-contaminate other products that couldprovide suitable conditions for growth.

Do total viable counts; test for C. perfringens andB. cereus (m = 102, M = 104), E. coli (m = 10, M =100), Staphylococcus aureus (M = 103) and salmo-nellas (absent in 25 g).

Salmonellas and E. coli are potentially much morehazardous in instant desserts than in cake mixes,which will be cooked before consumption.

CEREALS

Contamination occurs from many sources – soil,manure, insects, animals, birds and fungal infectionbefore or after harvest (see Legan, 2000).

Flour

White flour, freshly milled, contains relatively highnumbers of micro-organisms – total count 30°C:

216 Fresh, preserved and extended shelf-life foods

5 × 103 – 5 × 105; yeasts and moulds a factor of 10lower, with higher numbers in whole-grain flour.Numbers of vegetative bacteria, yeasts and mouldsdecline during storage. Low numbers of coliformscan be found in most samples of freshly milledwheat flour, with E. coli in about 50% and salmo-nellas in 0.3–2.3% of 1-g samples. Low numbers ofthe food poisoning bacteria S. aureus and B. cereusare commonly found (< 103/g) and could be asource of illness if allowed to multiply to highlevels. Mycotoxins are probably the most seriouspotential hazards in flour. Trichothecenes(deoxynivalenol and to a lesser extent nivalenol)and ochratoxin, produced by moulds of the genusFusarium, are relatively common in wheat, espe-cially wheat imported into the UK. Aflatoxin anddeoxynivalenol can sometimes be found in NorthAmerican maize. These toxins are formed in thefield, not during storage. If flour gets damp it willspoil as a result of the growth of moulds.

If the flour will be used to make bread withoutpreservatives (e.g. propionic or acetic acid), theconcentration of spore formers implicated in ‘ropeybread’ (B. subtilis and B. lichenformis) will be ofinterest. If it is to be used to make raw chilled pas-try, the numbers of lactic acid bacteria present inthe flour will affect its shelf-life – this should be< 1000/g. Numbers of spores of thermophilic sporeformers, such as B. stearothermophilus andB. coagulans, are relevant if the flour is to be usedas an ingredient of a canned food.

Pastry

Uncooked pastry, prepared for factory use or forsale to the public, may suffer spoilage caused by lac-tobacilli. Do counts of lactic acid bacteria on Rogosaor MRS agar, and incubate at 30°C for 4 days.Counts of up to 10 000/g are not unreasonable.

Pasta products

These are made without a heating step from wheatflour, semolina, farina and water. Egg (powdered

or frozen), spinach, vitamins and minerals may beadded. The egg in particular may contain salmonel-las and, although these may be destroyed in thesubsequent cooking, there is the possibility ofcross-contamination from uncooked to cookedproducts.

High levels of S. aureus and/or preformed staphy-lococcal enterotoxin have been reported in driedpasta with or without filling (InternationalCommission on the Microbiological Specificationsfor Foods, ICMSF, 1998).

ExaminationAs contamination is likely to occur during manu-facture, it is necessary to liberate the organismsfrom within the pasta.

Add 25 g pasta to 225 ml MRD and allow it tosoften at room temperature for about 1 h.Macerate, e.g. in a Stomacher, and do total, E. coliand S. aureus, B. cereus, and yeast and mouldcounts.

Inoculate salt meat broth. Incubate at 37°C for24 h and plate on Baird-Parker medium for staphy-lococci. If staphylococcal enterotoxin is suspected,in the absence of viable S. aureus (e.g. ifmicroscopy reveals large numbers of non-viableGram-positive cocci), use one of the commercialkits see (p. 332). Examine 25 g for salmonellas.

Extrusion-cooked products

These include breakfast cereals and crispbreads,and may be textured with vegetable protein andbread crumbs. The liquid and solid ingredients areblended, shaped and cooked within 1–2 min.Examine as for pasta.

GELATIN (DRY PRODUCT)

Gelatin is often used to top up pastry cases of cold-eating pies, in canned ham production and in ice-cream manufacture. It should be free from sporesand coliforms.

Gelatin (dry product) 217

If the process involves low-temperature reconsti-tution examine for S. aureus, salmonellas andclostridia.

Examination

Weigh 5 g gelatin into a bottle containing 100 mlsterile water and allow to stand at 0–4°C for 2 h.Place the bottle in a water-bath at 50°C for 15 minand then shake well. Mix 20 ml of this solutionwith 80 ml sterile water. This gives a 1 : 100 dilu-tion. Use 1.0 and 0.1 ml for total counts by thepour-plate method. Incubate at 37°C for 48 h.Examine 25 g of dry product for salmonellas.

Gelatin for ice-cream manufacture

Do a semi-quantitative coliform estimation usingMacConkey or similar broth. Add 10 ml 1 : 100gelatin to 10 ml double-strength broth; add 1 ml1 : 100 gelatin to 5 ml single-strength broth and0.1 ml 1 : 100 gelatin to 5 ml single-strength broth.Incubate at 37°C for up to 48 h and do confirma-tory tests where indicated (see p. 253). Thus, thepresence or absence of coliforms and E. coli in 0.1,0.01 or 0.001 g of the original material can bedetermined. It is desirable that coliforms should beabsent from 0.01 g and E. coli absent from 0.1 g.The total count in gelatin to be used for ice-creammanufacture should not exceed 10 000 cfu/g.

Gelatin for canned ham production

This should have a low spore count. After doingthe total counts, heat the remaining 1 : 100 solu-tion of gelatin at 80°C for 10 min. Plate 4 × 1 ml ofthis solution on standard plate count medium.Incubate two plates at 37°C and two plates at 55°Cfor 48 h. There should be no more than one colonyper plate. The total count should not exceed10 000 cfu/g.

SPICES AND ONION POWDER

A plastic bag inverted over the hand is a satisfac-tory way of sampling spices. Some of them aretoxic to bacteria and the initial dilution should be1 : 100 in broth (except for cloves which shouldbe 1 : 1000). Do total viable counts and, ifindicated by intended usage, consider inoculatingB. cereus-selective agar (e.g. PEMBA, see Table14.1) and one of the media for staphylococci andsalmonellas.

The total counts on these materials vary widely.Total viable counts of 108/g are generally accept-able, but low spore counts are important if thesematerials are to be used for canned foods: anacceptable level at 30 and 55°C is < 100/g.

COCONUT (DESICCATED)

In the 1950s and 1960s this product was often con-taminated with salmonellas. In spite of improvedprocessing it is still a potential hazard in the con-fectionery trade. Examine samples for salmonellasby the method described on p. 295.

OILY MATERIAL

Mix 1.5 g tragacanth with 3 ml ethanol and add10 g glucose, 1 ml 10% sodium tauroglycocholateand 96 ml distilled water. Autoclave at 115°C for10 min. Add 25 g of the oily material under testand shake well. Make dilutions for examination inwarm diluent.

Salad creams

These contain edible oils and spoilage may becaused by the lipolytic bacteria. Test for these withtributyrin agar. Examine for coliform bacilli andthermophiles. All these should be absent. Thereshould not be more than five yeasts or moulds pergram.


Mayonnaise-based salads

Examine for lactobacilli. Yeasts will also grow socolonies should be examined by Gram staining.Alternatively, do parallel counts on both MRSmedium (lactobacilli plus yeasts) and MRS con-taining 100 mg/ml chloramphenicol (yeasts only)(Rose, 1985).

CANNED, PREPACKED ANDFROZEN FOODS

The practice of prolonging the shelf-life of foods bysealing them in metal or glass containers and heatprocessing them has long been established, butmodern packaging materials have given the con-sumer a wider choice. Some products are sterilizedand then aseptically packed into sterile containers –usually flexible. Excellent quality assurance duringthese processes is of paramount importance, partic-ularly for low acid products (Codex AlimentariusCommission, CAC, 1993a), but the principles formicrobiological examination are similar to thosefor products sterilized in pack.

Eating habits have changed. Single portions thatcan be taken from the freezer or cupboard andheated in a microwave oven are popular. Withthese, packaging is important. It should:

• prevent contamination by micro-organisms• preserve quality and nutritional value• be inert and offer no hazard in use• be economic to manufacture and distribute• be easily labelled.

Routine control of the product in the factory isthe responsibility of the quality assurance and lab-oratory departments of the manufacturer, butother laboratories may be asked to help whendefects or spoilage has developed after the productshave left the factory or where the product is sus-pected of having caused food poisoning.Occasionally, arbitration between suppliers ofingredients and manufacturers of finished goods isnecessary.

Sealed containers intended to be stored at ambienttemperature for long periods pose the highest risk iferrors occur during or after processing. Cannedfoods are divided into two categories, dependingon whether they can support germination, multipli-cation and toxin production by C. botulinumspores. This organism cannot grow in a productwith a pH < 4.5. Hence only products with a pH> 4.5 (‘low acid’) need to be heated sufficiently tokill C. botulinum. This type of product is usuallygiven at least a ‘12D’ heat treatment, i.e. the time ata particular temperature sufficient to kill 1012

spores of C. botulinum (3 min at 121°C, or equiv-alent treatment at another temperature). Low acidfoods include meat and fish, as well as many veg-etables. Acid canned foods are mostly fruits, andcan be preserved by a more gentle heat treatment(< 100°C), although they are still occasionallyspoiled by spore formers if the raw material isheavily contaminated. These can be Bacillus orClostridium spp. or ascospores of Byssochlamysfulva and B. nivea or spores of Alicyclobacillus.

Microbiological examination of canned foodsshould be carried out by specially trained person-nel. Detailed instructions can be found in variouspublications, e.g. CAC (1979b, 1993b), Food andDrug Administration (FDA, 1998). Figures 16.1and 16.2 are flow charts of procedures adaptedfrom CAC publications.

Investigations normally include detailed exami-nation of the can seams. The following sectionsprovide information about the defects that canoccur during or after canning. Faults are usuallyuncommon (< 1 per 1000 cans) and cannot bedetected by post-production quality control sam-pling – the canning process must be run using aquality assurance system such as the HazardAnalysis Critical Control Point system (HACCP)(ICMSF, 1988a; Shapton and Shapton, 1991). It iscustomary, however, to hold all cans at 37°C forabout 14 days after production, while routinequality control checks are carried out (e.g. exami-nation of can seams) and to examine the cansvisually for signs of spoilage (i.e. bulging –Shapton and Shapton, 1991).

Canned, prepacked and frozen foods 219

Types of container

CansTin plate and aluminium are widely used.Lacquering gives resistance to corrosion. Weldedthree-piece cans have largely superseded thesoldered type. Base metal thickness has beenreduced, giving lighter and cheaper cans, but thesehave to be ‘beaded’ to withstand processing.Two-piece cans with ‘easy-open ends’ arepopular.

Jars and bottlesThese are made of glass and closed with metal capand resilient seals, sometimes under vacuum. Jarsand bottles should be tamper proof.

TraysThese are made of aluminium or aluminium/polypropylene laminated, with lids.

Semi-rigid flexible plastic and laminatecontainersThese are easy to stack, light and allow a shelf-lifeof up to 2 years. Plastic-sided, metal-ended con-tainers are used for fluids. For a review of contain-ers, see Dennis (1987).

Types of blown can

Gas-producing organisms cause the can to swell.The first stage is the ‘flipper’ when the end of the


Sample

Aerobic broth culture

NAMn, 35°C upto 4 days 55°C up to 4 days

55°C up to 4 days

NAMn, 55°C up to 4 days

NAMn, 35°C up to 4 days, 55°C up to 4 daysHeat for 10 min

at 100°C

Heat for 10 min at 100°CNo spores, indicates post-processing contamination

Mixed flora: cocci, yeasts,moulds indicate post-process contamination

Growth suggestsinsufficient heating

Growth suggestsinsufficient heating

Suggests insufficientheating process

Normal, unlessproduct stored at high

temperature

GrowthMicroscopy

Growth (rods)

Microscopy shows spores

Mesophilic aerobicBacillus spp.

Thermophile aerobicBacillus spp.

Microscopy showsspores

Rods only present

30–37°Ca up to 4 days

NAMnb 35°C up to 10 days

Figure 16.1 Flow diagram for the aerobic culture of low-acid canned foods.a Incubation temperature may be varied according to environmental conditions. b Nutrient agar plus 0.4 mg/l manganese chloride toencourage sporulation of Bacillus subtilis (adapted from Codex Alimentarius Commission, 1979b)

can flips outward if the can is struck sharply. A‘springer’ is caused by more gas formation.Pressing the end of the can causes the other endto spring out. The next stage is the ‘swell’ or‘blower’ when both ends bulge. A ‘soft swell’ canbe pressed back but bulges again when the pres-sure is released. ‘Hard swells’ cannot be com-pressed.

Spoilage may not result in gas formation andmay be apparent only when the can is opened.This kind of spoilage includes the ‘flat-sour’defect. In addition, hydrogen gas is sometimesproduced in cans where microbial spoilage hasnot occurred. This is usually the result of a reac-tion between the contents of the can and themetal.

Spoilage caused by micro-organisms Microbiological examination of cans can be donewith or without prior incubation. If a can alreadyshows signs of spoilage, e.g. swelling, it may besufficient to open it with suitable precautions andexamine the contents. Cans that appear normalmay be incubated for up to 14 days at 37°C,examining at intervals for progressive swelling.Swollen cans are removed, and examined. Cansthat do not swell are examined after 14 days.Initial microscopic examination is followed by cul-tural tests. Cans must be opened in a way that theseams are not damaged and so that the possibilityof contamination from outside the can is mini-mized. This involves the use of a laminar flow orsafety cabinet, decontamination of the outside of


Rods with sporespresent: indicatesinsufficient heating.If clostridia checkculture for botulinumtoxin

Rods withoutspores: culture onother anaerobicmedia; absence ofspores indicatespost-processcontamination

Shorter rods: possiblyfacultative Bacillusssp. If product darkpossibly sulphidespoilage sporebearerEither indicatesinsufficient cooling orhigh temperaturespoilage. Does notindicate insufficientheating

Long, thin, poorlystained rods: probably thermophilicanaerobes; indicatesinsufficient cooling,high temperaturestorage or thatheating to >118°Cbe used. Does notindicate insufficientheating

Heat for 10 min at100°C in anaerobicbroth medium;incubate at 35°C for14 days

Mixed flora: cocci,yeasts, moulds,indicate post-process contamination

GrowthMicroscopy

GrowthMicroscopy

Sample

Anaerobic broth culture

30–37°Ca up to 10 days 55°C up to 4 days

Figure 16.2 Flow diagram for the anaerobic culture of low-acid canned foods.Note that spores of thermophilic anaerobes are rarely seen or found in microscopy or first subculture, hence no heating is suggested. Itmay be used, however, followed by anaerobic subculture at 55°C. a Incubation temperature may be varied according to environmentalconditions (adapted from Codex Alimentarius Commission, CAC, 1979b)

the can, e.g. with 4% iodine in 70% ethanol, andsterile instruments to open the can and sample thecontents. Samples of the contents should be takenfrom below the surface. Blown cans (grossly dis-tended) should be refrigerated before opening, andprecautions taken to prevent spillage of contentsduring opening (e.g. put a cloth or funnel over thecan).

Leaker spoilageContrary to the usual understanding of this word,leakage in canning usually refers to entry inwardsrather than exit outwards. After autoclaving andduring cooling there is negative pressure in the can,and bacteria may be drawn in through minuteholes. These holes can occur because of faults incan seams, particularly where the end-seams crossthe body seams, but leakage can occur in cans withnormal seams if hygienic conditions have not beenmaintained during cooling and drying of the cans.It is important therefore that water used to coolcans is treated, e.g. with chlorine, to minimizenumbers of micro-organisms in the water. Even drysurfaces in contact with wet cans need to becleaned and disinfected frequently in order to min-imize contamination. Leakage can also be causedby rough treatment of cans during processing orcooling. When the seams are dry, the chances ofcontamination are slight. Wetting cans duringshelf-life can also result in leakage. Cans contami-nated by leakage usually contain a mixture ofbacteria, which can include pathogens such asS. aureus or even C. botulinum, as well as (morecommonly) spoilage organisms. The cans may ormay not appear ‘blown’. Presence of holes can bechecked on the empty and thoroughly cleaned canby use of vacuum or pressure.

Underprocessing or no processing

Gross underprocessing or no processing will usu-ally have been found by the manufacturer’s tests.Mixtures of micro-organisms (rods, cocci, yeastsand moulds) will be found. When the processingtreatment is only slightly suboptimal, it is common

to find only one type of (spore-forming) organismconsistently from can to can, often in a similarposition in each can.

Inadequate cooling

If cooling is too slow or inadequate or the cans arestored at very high temperatures, e.g. in tropicalcountries, there may be sufficient time for thehighly resistant spores of B. stearothermophilus togrow and multiply. The optimum growth tempera-ture for this organism is between 59 and 65°C. Itwill not grow at 28°C or at a pH of less than 5.Together with B. coagulans, it is the chief cause of‘flat-sour’ spoilage in canned foods. Most strains ofB. coagulans will grow at 50–55°C. They will alsogrow in more acid conditions.

Preprocessing spoilage

This can occur when the material to be canned ismishandled before processing, e.g. if precookedmeats with a large number of surviving spores arekept for too long at a high ambient temperature,this may result in the outgrowth of spores and theproduction of gas. When canned, the organismswill be killed, leaving the gas to give the appearanceof a blown can.

Types of food and organisms causingspoilage

Low-acid foodsIf the pH is 4.5 or above, as in canned soups, meat,vegetables and fish (usually about pH 5.0), the cansare given a ‘12D’ process. One of the followingthermophiles is usually found in spoilage caused byunderprocessing.

• B. stearothermophilus, causing ‘flat-sour’spoilage


• C. thermosaccharolyticum, causing ‘hardswell’

• Desulfotomaculum (previously Clostridium)nigrificans, causing ‘sulphur stinkers’

• Mesophilic spore bearers, obligate orfacultative anaerobes, causing putrefaction.

Leaker spoilageThis may be the result of a variety of organisms:aerobic and anaerobic spore bearers, Gram-nega-tive non-sporing rods and various cocci, includingLeuconostoc and Micrococcus spp., may be found.S. aureus (food poisoning type) has been isolated.

Acid foodsCanned foods with pH 3.7–4.5 include mostcanned fruits. As C. botulinum cannot grow at orbelow pH 4.5, these foods are usually given amuch milder heat treatment than low acid foods.Aerobic and anaerobic sporebearers may causespoilage but this is not common. Lactobacilli andLeuconostoc spp. have been reported. Osmophilicyeasts and the mould Byssochlamys spp., whichproduces heat resistant spores, are sometimesfound. Acid canned foods are too acidic for thegrowth of most bacteria and leaker spoilage isuncommon.

High-acid foodsThese foods are given a mild heat treatment, aswith acid foods. Spoilage is rare in processed foodswith a pH of 3.7 or less, e.g. pickles and citrusfruits. Spores of Alicyclobacillus spp. sometimesspoil these products. Yeasts may be found whenthere has been serious underprocessing.

Canned cured meats, e.g. ham,corned beef

As these are preserved with salt and nitrite, theycan be given a less severe heat treatment than otherlow acid foods because, although C. botulinumspores can survive this treatment, they are unableto germinate and multiply.

Some cured meats are deliberately given a non-sterilizing process because they are rendered lesspalatable by autoclaving. Catering-size cans of hamand mixtures of ham and other meat are preservedwith salt and nitrite, and given the minimum heattreatment. The manufacturers do not claim sterilityand the label on the can invariably recommendscold or cool storage. The pH, salt, and nitrite con-tent and storage temperature should be such thatthe bacteria in the can are prevented from multi-plying. There is evidence that some species of ente-rococci produce antibacterial substances in cannedhams, which act antagonistically on some speciesof clostridia, lactobacilli and members of the genusBacillus. This is most likely a factor in the success-ful preservation of commercial products of thisnature (Spencer, 1966; Kafel and Ayres, 1969;Gardner 1983).

Frozen foods

Some frozen convenience foods are mentionedhere, others – frozen meat, fish and ice-cream – areincluded under their appropriate headings inChapters 15, 17 and 18. For frozen vegetables, seep. 215 (see also CAC, 1999; Lund, 2000).

The minimum temperature for multiplication ofmicrobes in foods is generally accepted to be about–10°C. Frozen foods are normally stored at –18 to–20°C, and so should be microbiologically stable.If the temperature is allowed to rise to around–10°C, spoilage is likely to occur as a result ofgrowth of yeasts and moulds. Freezing cannot berelied on to destroy micro-organisms, however,although it can be used in inactivate parasites suchas Trichinella spp. Frozen foods will therefore havea microflora similar to the fresh foods from whichthey were prepared. Their shelf-life is normallylimited by biochemical deterioration.

ExaminationThis can be carried out in a similar way to examin-ing fresh foods, except that it should be understoodthat a proportion of the population is likely to be


sublethally damaged by freezing (Mackey, 2000).This means, for instance, that colony counts ofEnterobacteriaceae or coliforms may be affected bybile salts in VRB or VRBG agar. To compensate forthis, a resuscitation step can be inserted, such asinoculating plates of non-selective agar for a fewhours before over-layering with selective medium,or by putting the inoculum on a membrane filter thatcan be transferred from a non-selective to a selec-tive medium. Enrichment protocols for recoveringpathogens such as salmonellas or listerias fromfoods usually include a resuscitation step, so specialprocedures are not usually needed for frozen foods.

Precooked chilled (‘cook–chill’)products

The safety and shelf-life of these foods depend onsublethal treatment and aseptic packaging. Majoradvances in transportation and handling of chilledand frozen products have increased the availabilityof these foods. Chilled foods account for over 40%of expenditure on foods in the UK. Temperaturesshould be controlled between +1°C and +4°Cthroughout handling and storage. As many of thesefoods are capable of supporting the growth offood-poisoning organisms, it is important that theyare not mishandled.

The packaging and the food are sterilized sepa-rately and united under ‘commercially sterile’ con-ditions. Flexible pouches and semi-rigid pots andcartons are used for low acid foods, e.g. ice-cream,custards, soups and sauces.

Cook–chill cateringThis system is used by hospitals, works canteens,prisons, schools, and banqueting and travel organ-izations. It demands a high level of technical con-trol to ensure safety and quality. If handled andstored according to the guidelines (Department ofHealth, 1989) they should be safe.

Bacteriological examinationThese products may be examined by the samemethod as other foods, and the same standards

should apply. These foods should conform to thefollowing standards:

• Viable count after 48 h at 37°C – < 100 000/g• E. coli – < 10/g• S. aureus – < 100/g• Salmonella spp. not present in 25 g• Listeria monocytogenes not present in 25 g• C. perfringens not present in 100 g.

‘Sous vide’ productsThese are cook–chilled foods that are vacuumpacked and given a mild heat treatment in therange 65–75°C. They are used primarily in thecatering industry, The packaging prevents post-cook recontamination, but the mild heat treatmentallows spores to survive, so these products must bestored below 3°C in order to prevent growth ofpsychrotrophic spore formers – especially non-pro-teolytic C. botulinum. Shelf-life can be severalweeks. Clearly, it is important that the storage tem-perature of these products is carefully controlled. Ithas been suggested (Gould, 1999) that productsintended to have a long shelf-life should either betreated in order to give a 6-log cycle reduction ofpsychrotrophic C. botulinum spores, which is 90°Cfor 10 min, or equivalent, or have other preserva-tive factors (pH < 5 or water activity (aw) < 0.97 orNaCl > 3.5% [salt on water]). The AdvisoryCommittee on the Microbiological Safety of Food(ACMSF, 1992) recommended that ‘sous vide’products given a mild heat treatment should have ashelf-life not exceeding 10 days.

Nissen et al. (2002) found that all studied ‘sousvide’ meals heat treated at 85–100°C for 10–60min had an initial total viable count (TVC) < 103/g,and 75% < 10/g. Similar numbers were found after3–5 weeks’ shelf-life, storing at 4°C for the firstthird and 7°C for the rest of the time. In anotherstudy (Nyati, 2000), meat and other dishes wereheated to internal temperatures varying from 68 to85°C. Reductions in total count varied from 4 to 6log cycles and satisfactory results were obtainedafter storage at 3°C for 4 weeks, but after 2 weeksat 8°C 8 of the 18 products were spoiled – withhigh counts of lactic acid bacteria and sometimes


pseudomonads. B. cereus (> 3 × 104 cfu/g) wasfound in one chicken product.

Apart from B. cereus and non-proteolytic C. bot-ulinum, other psychrotrophic pathogens that mightmultiply in sous vide foods and hence could betested for, are Listeria spp., Yersinia spp. andAeromonas spp.

BABY FOODS

Direct breast-feeding allows little chance of infec-tion of the infant with enteric pathogens. Testing ofhuman milk is described on p. 243. With otherfeeds contamination may occur during the time lagbetween production and consumption.

There are many milk preparations and weaningformulae, as well as dried, bottled and cannedfoods designed for small children. Dehydrated milkmay contain organisms that have survived process-ing and these may multiply if the product is notstored correctly after it is reconstituted. Dirtyequipment may contribute bacteria to an otherwisesterile material. Unsuitable water may be used forreconstitution. Since an outbreak of salmonellosisin the UK, from a contaminated dried milk-based

formula, attention has been paid to sampling plansfor salmonellas in dried milks. Sampling plans inthe Codex Code of Hygienic Practice for Foods forinfants and children (CAC, 1979a) are shown inTable 16.1. Enterobacter sakazaki has recentlybeen found in infant food formulae and implicatedas the cause of severe disease in babies. It may besought using a sampling plan similar to that forsalmonellas (Iverson et al., 2004).

Central milk kitchens in maternity units shouldhave very high standards of hygiene (see p. 245 forsampling surfaces, etc.). In-bottle terminal heating,with teat in place, is good practice.

Viable counts should be low and pathogensshould be absent (see Robertson; 1974; Collins-Thompson et al., 1980). Nasogastric feeds shouldconform to the same standards.

Heat-treated jars and cans should be examinedas described for extended shelf-life foods onp. 219.

SOFT DRINKS

Total counts should be low and coliform bacilliabsent, as in drinking water. Membrane filters can

Soft drinks 225

Table 16.1 Advisory microbiological specifications for foods for infants and children

Product Test n c Limit per g

m M

Dried biscuit type products:Plain None – – – –Coated Coliforms 5 2 < 3 (by MPN) 20

Salmonellasa (chocolate coatings only) 10 0 0 –Dried and instant products (e.g. milk) Mesophilic aerobic bacteria 5 2 103 104

Coliforms 5 1 < 3 (by MPN) 20Salmonellasa 60 0 0 –

Dried products requiring heating before Mesophilic aerobic bacteria 5 3 104 105

consumption Coliforms 5 2 10 100Salmonellasa 5 0 0 –

a 25 g of sample examinedm is the threshold value for bacterial count, result satisfactory if not exceeded; M is the maximum value for bacterial count, resultunsatisfactory if exceeded in any sample; n is the number of sample units comprising the sample; and c is the number of sample unitsthat may have bacterial counts between m and M if the bacterial count of the other samples is m or less (from Codex AlimentariusCommission, (1979a)

be used to test water intended for soft drinkproduction as well as the end product. Milliporepublish a very useful booklet on the microbio-logical examination of soft drinks. Yeast andmould spoilages are not uncommon and they mayraise the pH and allow other less acid-tolerantorganisms to grow.

In non-carbonated fruit drinks, yeasts are notinhibited by the amounts of preservatives that arepermitted by law. Spoilage is generally controlledby acidity (except Zygosaccharomyces baillii) (see‘Ketchups’, p. 216). In both these cases and car-bonated drinks, the microbial count diminisheswith time.

FRUIT JUICES

Lactic acid and acetic acid bacteria may grow atpH 4.0 or less in some fruit juices.

Heat-resistant fungi can cause problems in con-centrated juices. Screen by heating at 77°C for30 min. Cool and pour plates with 2% agar.Incubate for up to 30 days. Dip slides, includingthose with medium for yeasts and fungi, are useful.

Automated counting methods (e.g. ATP assay) areuseful. Fruit juices are incubated at 25°C for24–48 h (72 h for tomato juice), the reagent isadded and incubated for 45 min when the instru-ment gives the result.

BOTTLED WATERS

For information on bottled waters, see p. 257.

MILK-BASED DRINKS

For information on milk-based drinks, see p. 239.

VENDING MACHINES

Water and flavoured drinks in vending machinesmay be of poor quality, with high counts

(> 1000 cfu/ml) and coliforms. This may be theresult of inadequate cleaning (see Hunter andBurge, 1986).

USEFUL REFERENCES

Food microbiology is a very large subject and inaddition to the references cited in this chapter andthose preceding it the following are recommended:Hersom and Hulland (1980); Jowitt (1980);Harrigan and Park (1991); Roberts and Skinner(1983); ICMSF (1986, 1988, 1998a, 1998b,2002); Shapton and Shapton (1991); Hayes (1992);Jay (2000); Lund et al. (2000); Dowens and Ito(2001).

REFERENCES

Advisory Committee on the Microbiological Safety ofFood (ACMSF) (1992) Report on Vacuum

Packaging and Associated Processes. London:HMSO.

Codex Alimentarius Commission (CAC) (1979a)Recommended International Code of Hygienic

Practice for Foods for Infants and Children.CAC/RCP 21-1979.

CAC (1979b) Recommended International Code of

Hygienic Practice for Low and Acidified Low Acid

Canned Foods. CAC/RCP 23-1979, rev. 2 (1993) –see Appendix V.

CAC (1993a) Code of Hygienic Practice for Aseptically

Processed and Packaged Low Acid Canned Foods.CAC/RCP 40-1993.

CAC (1993b) Guideline Procedures for the Visual

Inspection of Lots of Canned Foods. CAC/GL 17.CAC (1999) Code of Hygienic Practice for Refrigerated

Packaged Foods with Extended Shelf Life.CAC/RCP 46-(1999).

Collins-Thompson, D. L., Weiss, K. F., Riedel, G. W.and Charbonneau, S. (1980) Microbiologicalguidelines and sampling plans for dried infantcereals and dried infant formulae. Journal of Food

Protection 43: 613–616.


D’Aoust, J. Y. (1977) Salmonella and the chocolateindustry: a review. Journal of Food Protection 40:718–726.

Dennis, C. (ed.) (1987) Symposium on the

Microbiological and Environmental Health

Problems in Relation to the Food and Catering

Industries. Chipping, Campden: Campden FoodPreservation Association.

Department of Health (1989) Chilled and Frozen Food.

Guidelines on Cook-Chill and Cook-Freeze

Catering Systems. London: HMSO. Dowens, F. P. and Ito, K. (eds) (2001) Compendium of

Methods for the Microbiological Examination of

Foods, 4th edn. Washington DC: American PublicHealth Association.

Food and Drug Administration (1998) Bacteriological

Analytical Manual, 8th edn. Arlington, VA: AOACInternational.

Gardner, G. A. (1983) Microbiological examination ofcuring brines. In: Board, R. G. and Lovelock, D.W. (eds), Sampling – Microbiological Monitoring

of the Environment. Society for AppliedBacteriology Technical Series No. 7. London:Academic Press.

Gould, G. W. (1999) Sous vide foods: conclusions ofan ECFF Botulinum Working Party. Food Control

10: 47–51.Harrigan W. F. and Park, R. W. A. (1991) Making

Food Safe. London: Academic Press. Hayes, R. (1992) Food Microbiology and Hygiene, 2nd

edn. London: Elsevier. Hersom, A. C. and Hulland, E. D. (1980) Canned

Foods. Thermal processing and microbiology.London: Churchill-Livingstone.

Hunter, P. R. and Burge, S. H. (1986) Bacteriologicalquality of drinks from vending machines. Journal

of Hygiene (Cambridge) 97: 497–504.International Commission on the Microbiological

Specification for Food (ICMSF) (1986)Microorganisms in Foods. 2. Sampling for

Microbiological Analysis. Principles and specific

applications. 2nd edn. Toronto: University ofToronto Press (updated as ICMSF 2002).

ICMSF (1988) Microorganisms in Foods. 4. Application

of the HACCP system to ensure Microbiological

Safety and Quality, Oxford: Blackwell Scientific.

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Ecology of Food Commodities. London: BlackieAcademic & Professional. Available from KluwerPublishers.

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Iverson, C., Druggan, P. and Forsythe, S. (2004) Aselective medium for Enterobacter sakazaki.International Journal of Food Microbiology, inpress.

Jay, M. (2000) Modern Food Microbiology, 6th edn.New York: Aspen Publishers.

Jowitt, R. (ed.) (1980) Hygienic Design and Operation

of Food Plants. Chichester: Ellis Horwood.Kafel, S. and Ayres, J. C. (1969) The antagonism of

enterococci on other bacteria in canned hams.Journal of Applied Bacteriology 32: 217–232.

Legan, J. D. (2000) Cereals and cereal products. In:Lund, B., Baird-Parker, A. C. and Gould, G. W.(eds), The Microbiological Safety and Quality of

Food, Vol. 1. London: Chapman & Hall, pp.759–783.

Lund, B. M. (2000) Freezing. In: Lund, B., Baird-Parker, A. C. and Gould, G. W. (eds), The

Microbiological Safety and Quality of Food, Vol.1. London: Chapman & Hall, pp. 122-145.

Lund, B. M. and Snowdon, A. L. (2000) Fresh andprocessed fruits. In: Lund, B., Baird-Parker, A. C.and Gould, G. W. (eds), The Microbiological

Safety and Quality of Food, Vol. 1. London:Chapman & Hall, pp. 738-758.

Mackey, B. M. (2000) Injured bacteria. In: Lund, B.,Baird-Parker, A. C. and Gould, G. W. (eds),The Microbiological Safety and Quality of

Food, Vol. 1. London: Chapman & Hall, pp.315–341.

Michino, H., Araki, K., Minami, S. et al. (1999)Massive outbreak of Escherichia coli O157:H7infection in schoolchildren in Sakai City, Japan,associated with consumption of white radish

References 227

sprouts. American Journal of Epidemiology 150:787–796.

Nissen, H., Rosnes, J. T., Brendehaug, J. and Kleiberg,G. H. (2002) Safety evaluation of sous vide-processed foods. Letters in Applied Microbiology

35: 433–438.Nyati, H. (2000) An evaluation of the effect of storage

and processing temperatures on the microbiologicalstatus of sous vide extended shelf-life products.Food Control 11: 471–476.

Nyugen-the, C. and Carlin, F. (2000) Fresh andprocessed vegetables. In: Lund, B., Baird-Parker, A.C. and Gould, G. W. (eds), The Microbiological

Safety and Quality of Food, Vol. 1. London:Chapman & Hall, pp. 620–684.

Roberts, T. A. and Skinner, F. A. (eds) (1983) Food

Microbiology. Society for Applied Bacteriology

Symposium Series No. 11. London: AcademicPress.

Roberts, T. A., Hobbs, G., Christian, J. H. B. andSkovgaard, N. (eds) (1981) Psychrotrophic

Microorganisms in Spoilage and Pathogenicity.London: Academic Press.

Robertson, M. H. (1974) The provision ofbacteriologically safe infant feeds in hospitals.Journal of Hygiene (Cambridge) 73: 297–303.

Rose, S. A. (1985) A note on yeast growth in mediaused for the culture of lactobacilli. Journal of

Applied Bacteriology 59: 53–56.Shapton, D. A. and Shapton, N. F. (eds) (1991)

Principles and Practices for the Safe Processing of

Foods. London: Butterworths.Spencer, R. (1966) Non-sterile canned cured meats.

Food Manufacture 41: 39–41, 43.


Examples of these three broad groups are shown inTable 17.1. Quality is assessed best by appearanceand odour. As with other foods, bacteriologicalexamination involves assessing the presence andnumbers of micro-organisms that affect the qualityof the food (i.e. the spoilage flora that influence thefreshness or shelf-life of the product) and those thatmay affect human health.

FRESH FISH

Microbial content

Total bacterial counts are still used today, althoughusually in combination with other microbiologicalindices such as numbers of spoilage bacteria andthe presence of potentially pathogenic bacteria.Fish muscle taken aseptically from a newly killedfish is sterile, but micro-organisms increase in num-bers during storage. Numbers on the surface of fishdiffer depending on where the sample was taken.Liston et al. (1976) found that numbers on freshlycaught fish varied between 102 and 106 colony-forming units (cfu)/cm2 on the skin, 1 log less in the

gills and up to 107 per g in the intestines of activelyfeeding fish. Large numbers of pseudomonads,flavobacteria, coryneforms, and Acinetobacter,Aeromonas and Cytophaga spp. are often associ-ated with the slime that occurs naturally on theskin. Micrococci and photobacteria (luminescent inthe dark on some media) are often present.Mycobacteria are also found in fish and causeproblems, especially in farmed fish raised in largeimpoundments of water or in tanks in the southernUSA.

Numbers of bacteria increase with time of storagebut only a critical proportion of these affects theorganoleptic quality of the food. The total countmethod should vary depending on the conditionsunder which or where the fish was caught. In theUK, local sea temperatures are generally low andthe temperature of agar plate incubation shouldreflect this to estimate bacterial numbers as accu-rately as possible. The former fisheries researchinstitute at Torry in Aberdeen, UK recommendedincubation at 20°C, which is the optimum growthtemperature for the majority of bacteria found inlocal waters. Some fish-associated micro-organismsgrow rather slowly, however, and an incubationperiod of 5 days was also recommended. This incu-bation time produced maximum counts, althoughnumbers at 3 days were sometimes not significantlylower.

Viable count

The method may vary depending on the type of fishunder investigation. For whole fish, select severalsites and aseptically peel back the skin and remove

17

Fresh fish, shellfish and crustaceans

Table 17.1 Broad definition of fish species

Fish type Examples of species

Finfish Cod, plaice, mackerel, salmon, trout

Molluscan shellfish Mussels, oysters, scallops, clams Crustaceans Shrimp, prawns, crab, lobster

25 g muscle for testing. In the case of small fishsuch as whitebait, the whole fish is often consumedand so the whole should be tested. For fillets, selectfrom the cut surface. Counts will be influencedgreatly by any inclusion of skin or intestines in theexamination of filleted fish portions

Add 225 ml chilled phosphate-buffered saline to25 g fish and homogenize for 2 min. Plate 0.1-mlvolumes (in duplicate) of serial dilutions on nutri-ent agar containing 0.5% NaCl (some nutrientmedia do not normally contain NaCl), and incu-bate at 20°C for 3–5 days. Count the colonies onplates containing between 30 and 300, and calcu-late the numbers of colony-forming units per gram.Counts of > 106/g in most fish indicate little or noremaining shelf-life but if intestinal or skin tissuewere assayed then this value could be one- to ten-fold greater.

Selective counts

A significant proportion of the viable count maynot contribute to the spoilage of the fish duringstorage. Some, e.g. Moraxella spp. (nowPsychrobacter spp.) and Acinetobacter spp. arebiochemically inactive (relative to others found infish) and large numbers of them do not significantlyreduce shelf-life or cause food poisoning if con-sumed. Others, however, including Shewanellaspp. and pseudomonads, are metabolically activeand are mainly responsible for spoilage. An inde-pendent count of these is of more use than the total

count because it can be used to estimate remainingshelf-life. Spoilage bacteria may be selectivelyassessed (Wood and Baird, 1943) by their produc-tion of hydrogen sulphide (black colonies) on cys-teine-iron agar. Gibson and Ogden (1987) usednumbers of H2S-producing bacteria to predict theshelf-life of packaged fish, as did Gram and Huss(1999), who observed good correlation betweennumbers of H2S-producing bacteria and remainingshelf-life of iced cod (Table 17.2).

Plate homogenates directly on Iron Agar (Oxoid)or add 1 : 10 dilutions to the melted medium (at45°C), mix and allow to solidify. Then pour on anoverlayer (1–2 ml) of melted medium. This mini-mizes oxidation of ferrous sulphide, which wouldcause instability of the black precipitate. Incubatethe plates at 20–25°C for 3–4 days and count thenumbers of black colonies.

Rapid methods

Conductance techniques (Richards et al., 1978)may be particularly useful to estimate the microbi-ological status of fish because the metabolism ofthe spoilage flora gives rise to significant changes inconductance (see Chapter 8). Ogden (1986)showed acceptable correlation between conduc-tance measurements and total viable counts in dif-ferent fish species and improved correlation whenplotted against numbers of spoilage bacteria(counted selectively using the Wood and Bairdmethod).

230 Fresh fish, shellfish and crustaceans

Table 17.2 Relationship between numbers of H2S-producing bacteria and remaining shelf-life for iced cod

Shelf-life remaining (days) Log10 cfu/g H2S-producing bacteria

14 2 11 3 9.5 4 7 5 5 6 2 7

Adapted from Gram and Huss (1999)

SHELLFISH AND CRUSTACEANS

These are usually harvested from inshore watersor river estuaries that are prone to sewage pollu-tion. The bivalve molluscs, in particular, are filterfeeders and hence tend to concentrate bacteriaand viruses from their environment. Escherichiacoli is used as an index of pollution. The mostprobable number (MPN) technique is generallyused but alternatives have been investigated(Ogden et al., 1998) because the MPN method istime-consuming, labour intensive and of limitedaccuracy. Although the methods given below areprimarily for shellfish, they may also be used forcrustaceans.

MPN method for E. coli determinationin shellfish

Use protective gloves to select 15–30 shellfish.Reject any that show signs of gaping or damage.Scrub them clean under running water of potablestandard and open with a sterile shucking knife.Collect both meat and liquor in a weighed sterilecontainer (beaker or bag) and transfer to aStomacher for homogenization for 2-3 min in a fil-ter bag.

Prepare 10−1 and 10−2 dilutions in sterile 0.1%peptone water. Use minerals modified glutamatemedium (MMG) for the MPN test described onp. 150, adding 10 ml of the 10−1 dilution to five10-ml tubes of double-strength MMG, 1 ml of thesame dilution to five 9-ml tubes of single-strengthMMG and 1 ml of the 10−2 dilution to five 9-mltubes of single-strength MMG media. Thus, thereare 1 g, 0.1 g and 0.01 g tissue per tube in eachset.

Incubate all tubes at 37 ± 1°C for 18–24 h in awater bath and note gas production. Re-incubatethe tubes that show no gas for a further 18–24 h.Absence of gas confirms a negative result forE. coli.

Subculture tubes with gas into (1) 5 ml brilliantgreen bile broth (BGBB) and (2) 5 ml 1% tryptone

water, and incubate both at 44 ± 0.5°C for 24 h.Positive tubes for gas production in BGBB andindole production from tryptone (Kovacs reagent)confirm E. coli. Alternatively, the presence ofβ-glucuronidase confirms E. coli and is recognizedby fluorescent colonies grown on MUG agar orblue colonies on BCIG agar, the latter being prefer-able because the colour remains within thebacterial colony (unlike fluorescence), which aidsaccurate counting. Calculate numbers from con-ventional MPN tables (see p. 152).

Crustaceans

Wash crabs, lobsters, etc. in running water asabove. Break open, remove some meat and proceedas above. Wash prawns, shrimps, etc. in runningwater and proceed as above.

Standards

The European Union (EC, 1991) has set categoriesof shellfish for human consumption. These areshown in Table 17.3.

Those in category A are fit for human consump-tion whereas those in category B require furthertreatment. This can include repositioning in cleanseawater and waiting for E. coli levels to fall natu-rally (relaying), or transferring the animals to atank where the water is circulated through a ultra-violet (UV) source to kill bacteria (depuration).Category C shellfish are deemed unfit for humanconsumption.

Shellfish and crustaceans 231

Table 17.3 Microbiological definition of shellfish categories

Category Numbers of E. coli/100 g shellfish

A < 230 B 230–4600 C > 4600

Pathogens

The pathogens that have been particularly associ-ated with sea food poisoning include vibrios,notably Vibrio parahaemolyticus (see Chapter 24),Staphylococcus aureus (see Chapter 36), Listeriamonocytogenes (see Chapter 40) and Clostridiumbotulinum type E (see Chapter 44). Aeromonads(see Chapter 24) and salmonellas (see Chapter 28)have also been incriminated. Methods for the isola-tion of these micro-organisms from foods are givenin the appropriate chapters.

The possibility that fish might be contaminated bypathogenic viruses should be considered. Molluscsare filter feeders and depuration, which removesbacteria, may not remove viruses; they are notalways killed by heat treatment either. Bivalves aretherefore often associated with outbreaks of viraldiseases such as hepatitis A and winter vomitingdisease.

Molluscs are sometimes vehicles for algal toxins.Consumption may result in diarrhoeic and amnesicshellfish poisoning, the onset of which may be asshort as 30 min. The origins of these toxins are thevaried marine phytoplankton, which are the natu-ral food of the shellfish. Some 40 of the 5000species (mainly dinoflagellates and diatoms) areable to produce powerful toxins, which give arange of symptoms after consumption (Whittle andGallacher, 2000).

In the last 25 years there has been a markedincrease in the incidence of scombroid fish poison-ing. The symptoms resemble those of food poison-ing (i.e. diarrhoea and vomiting, headache,giddiness, rash on head and neck) 10 min to a fewhours after ingestion of scombroid fish, e.g. mack-erel, tuna, etc. Raised levels of histamine resultingfrom the decarboxylation of histidine in fish tissueby bacteria during poor storage of the fish is alwaysinvolved. Levels of ≥ 5 mg/100 g fish are consideredindicative of mishandling, and ≥ 50 mg/100 ghazardous (International Committee for theMicrobiological Standards of Foods or ICMSF,1998).

REFERENCES

EC (1991) Council Directive laying down the healthstandards for the production and placing on themarket of bivalve molluscs. 91/492/EEC. Official

Journal of the European Communities 24September, 1991. No. L268/1.

Gibson, D. M. and Ogden, I. D. (1987) Estimating theshelf life of packaged fish. In: Kramer, D. E. andListon, J. (eds), Developments in Food Science:

Seafood quality determination. Amsterdam:Elsevier, pp. 437–451.

Gram, L. and Huss, H. H. (1999) Microbiology of fishand fish products. In: Lund B, Baird-Parker, A. C.and Gould, G. W. (eds), The Microbiological

Safety and Quality of Foods. London: Chapman &Hall, pp. 472–506.

International Committee for the MicrobiologicalStandards of Foods (ICMSF) (1998) Micro-

organisms in Foods 6. Microbial Ecology of Food

Commodities. Amsterdam: Kluwer, pp. 130–189.Liston, J., Stansby, M. E. and Olcott, H. S (1976)

Bacteriological and chemical basis for deteriorativechanges. In: Stansby, M. E. (ed.), Industrial Fishery

Technology. New York: Kreiger, pp. 345–358.Ogden, I. D. (1986) The use of conductance methods

to predict bacterial counts in fish. Journal of

Applied Bacteriology 61: 263–268.Ogden, I. D., Brown, G. C., Gallacher, S. et al. (1998)

An interlaboratory study to find an alternative tothe MPN technique for enumerating Escherichia

coli in shellfish. International Journal of Food

Microbiology 40: 57–64.Richards, J. C. S., Jason, A. C., Hobbs, G. et al. (1978)

Electronic measurement of bacterial growth.Journal of Physics E. Scientific Instruments 11:560–568.

Whittle, K. J. and Gallacher, S. (2000) Marine toxins.British Medical Journal 56: 236–253.

Wood, A. J. and Baird, E. A. (1943) Reduction oftrimethylamine oxide by bacteria, 1. TheEnterobacteriaceae. Journal of Fisheries Research

Board of Canada 6: 194–201.

232 Fresh fish, shellfish and crustaceans

These foods have a long history of spoilage and asvehicles for microbial diseases, including tubercu-losis, scarlet fever, diphtheria, and various kinds offood poisoning and food-borne illnesses, all ofwhich result from low standards of hygiene inproduction and distribution. This has resultedin legislation that impinges on bacteriologicalexaminations.

MILK

In Europe, the European Commission (EC, 1992)lays down microbiological tests and standards formilk, etc. These are given under each test descrip-tion. Similar tests and criteria are used in othercountries such as the USA (Downs and Ito, 2001).

Raw milk

Raw milk, which includes that from cows, goats,ewes and buffaloes, is milk that is intended for:

1. The production of heat-treated drinking milk,fermented milk, junket, jellies and flavouredmilk drinks

2. The manufacture of milk-based productsother than those specified in (1), e.g. cows’milk, after packing for drinking in that state;and cows’, ewes’ or goats’ milk sold directlyto the ultimate consumer.

The bacteriological tests required to satisfy legalstandards for these milks are:

• the plate count (total viable count, TVC) at30°C

• enumeration of coliforms and Staphylococcusaureus

• detection of salmonellas.

Pasteurized milk

This is raw milk that meets the above requirementsand has been heated at 71.7°C for 15 s or an equiv-alent time/temperature combination, and thenimmediately cooled to below 10°C. The testsrequired by legislation are:

• the plate count at 21°C• enumeration of coliforms• detection of pathogens (Listeria

monocytogenes and salmonellas)• the phosphatase test.

Ultra-heat-treated (UHT) milk

This is raw milk, as above, that has been heated at135°C for 1 s. The statutory bacteriological test isthe plate count at 30°C.

Sterilized milk

This is raw milk, as above, that has been heated at100°C for such a time that it will pass the turbiditytest. The statutory tests are:

• the plate count at 30°C• the turbidity test.

18

Milk, dairy produce, eggs and ice-cream

Collection and transport of samples

Raw milk intended for the manufacture of milkdrinks or milk-based products should be sampledat the point of production using sampling pro-grammes drawn up on risk assessment principles.At least two samples a month should, however, besampled for the plate count. Cows’ milk packed fordrinking should be sampled at the point of sale.Samples should be transported to the laboratory at0–4°C.

Statutory testing

For legal purposes the reference methods specifiedin the EC (1992) Directive or other internationallyaccepted methods of analysis must be used, but forroutine practice the methods described below areadequate. The targets, i.e. acceptable highest levels,usually corresponding to legal standards, are givenbelow (see, however, the comments on the accu-racy of viable counts in Chapter 10).

Total viable count

The method is that given on p. 145. Test raw milkson arrival, use standard milk plate count agar andfor raw milks incubate at 30°C for 72 h. Pre-incubate pasteurized milks at 6°C for 5 days beforetesting and then incubate plate counts at 21°C. Thetargets levels (as total viable counts per millilitre orTVC/ml) are:

• Raw milk not intended for drinking in thatstate: < 100 000

• Raw milk packed for drinking: < 50 000• Raw milk sold directly to the ultimate

consumer: < 20 000• Pasteurized milk: m = 50 000; M = 500 000;

n = 5; c = 1; where m is the threshold valuefor bacterial count, result satisfactory if notexceeded; M is the maximum value forbacterial count, result unsatisfactory ifexceeded in any sample; n is the number ofsample units comprising the sample; and c is

the number of sample units that may havebacterial counts between m and M if thebacterial count of the other samples is m orless. The test fails if any sample has a countexceeding M.

Total viable count, UHT and sterilizedmilk

Pre-incubate samples at 30°C for 15 days or 55°Cfor 7 days after sampling at point of production.Perform plate counts in duplicate using 0.1-ml vol-umes of milk and plate count agar. Incubate at30°C for 72 h. The target level is:

• UHT and sterilized milk: < 100/ml.

Coliform count

Make serial tenfold dilutions and perform platecounts with violet red bile lactose agar. Incubate at30°C for 24 h. Count only red colonies that are0.5 mm in diameter or larger. Confirm if necessaryby subculture in brilliant green bile lactose broth at30°C for 24 h. It is useful to test confirmed col-iforms by the 44°C and indole tests (see p. 254) todetermine whether they are Escherichia coli. Thetarget coliform level per millilitre is:

• Raw milk: < 100/ml• Pasteurized milk: m, 0; M, 5; n, 5; c, 1.

Pathogens

Methods for these are given elsewhere: salmonel-las, p. 295; S. aureus, p. 339; Listeria, p. 356;E. coli O157, p. 288; and campylobacters, p. 314.Pathogens should be absent in 25 ml in milk tested.

Phosphatase test

The enzyme phosphatase in milk is destroyed by thetime/temperature conditions used for pasteurization.

234 Milk, dairy produce, eggs and ice-cream

Detection of the enzyme indicates inadequatepasteurization.

The fluorimetric method is an internationally rec-ognized method and is more sensitive and repro-ducible than the non-fluorimetric technique(Greenwood and Rampling, 1997; BSI, 2000). Thealkaline phosphatase activity of the sample is meas-ured by a continuous fluorimetric direct kineticassay. A non-fluorescent aromatic monophos-phoric ester substrate, in the presence of any alka-line phosphatase (ALP) derived from the sample,undergoes hydrolysis of the phosphatase radical,producing a highly fluorescent product.Fluorimetric measurement of ALP activity is meas-ured at 38°C for a 3-min period (BSI, 1997). Thefluorimeter will perform the necessary calculationsand display the sample number, the averageincrease in fluorescence and the ALP activity inmilliunits per litre.

The legal level of phosphatase activity permittedin pasteurized milk is 4 μg phenol/ml. This is equiv-alent to 500 mU/l by the fluorimetric test and 10 μgp-nitrophenol/ml by the Aschaffenberg–Mullen(A–M) test.

The A–M test (SI, 1989) may be used but shouldbe validated against the European referencemethod (EC, 1992a).

Turbidity test, sterilized milk

Weigh 4.0 g ammonium sulphate (AnalaR) into a50-ml conical flask. Add 20 ml milk and shake for1 min to ensure that the ammonium sulphate dis-solves. Stand for 5 min and filter through a 12.5-cmWhatman No. 12 folded filter paper into a testtube. When 5 ml clear filtrate has collected, placethe tube in a water-bath at 100°C for 5 min.Remove the tube and allow to cool. Examine thefiltrate for turbidity. Properly sterilized milk givesno turbidity.

Sterilization alters the protein constituents andthe ammonium sulphate precipitates all non-heat-coagulable protein. If heating is insufficient, someprotein remains unaltered and is not precipitated

by the ammonium sulphate. It coagulates, givingturbidity when the filtrate is boiled.

Microbial content of milk

Milk is at risk of faecal contamination from thecow or other producer species, and is subject topotential contamination from equipment and theenvironment during milking and handling.Refrigerated storage, which is normal practice aftermilking, retards the multiplication of bacteria pres-ent. Pasteurization, intended to kill pathogenic bac-teria, does not necessarily reduce the count of otherorganisms. It may increase the numbers of ther-mophiles.

The ‘normal microflora’ depends on temperature:at 15–30°C S. lactis predominates and many strep-tococci and coryneform bacteria are present, but at30–40°C they are replaced with lactobacilli andcoliform bacilli. All of these organisms ferment lac-tose and increase the lactic acid contents, whichcauses souring. The increased acid content preventsthe multiplication of putrefactive organisms.Spoilage during cold storage is caused by psy-chrophilic pseudomonads and Alcaligenes spp.,psychrotrophic coliforms, e.g. Klebsiella aerogenesand Enterobacter liquefaciens, which are anaero-genic at 37°C; in pasteurized milk, thermoduriccoryneform organisms (Micrococcus lactis) may besignificant. These coryneforms probably comefrom the animal skin or intestine and from utensils.At temperatures above 45°C, thermophilic lacto-bacilli (Lactobacillus thermophilus) rapidlyincrease in numbers.

Gram-negative bacilli are rarely found in quartersamples collected aseptically. These enter from theanimal skin and dairy equipment during milkingand handling. Alcaligenes spp. are very common inmilk. Pseudomonas fluorescens gels UHT milk.

Undesirable micro-organisms responsible for ‘offflavours’ and spoilage include psychrophilicPseudomonas, Achromobacter, Alcaligenes andFlavobacterium spp., which degrade fats and pro-teins and give peculiar flavours. Coliform bacilli

Milk 235

produce gas from lactose and cause ‘gassy milk’.S. cremoris, Alcaligenes viscosus and certainAerobacter species, all capsulated, cause ‘ropymilk’. Oospora lactis and yeasts are present in stalemilk. P. aeruginosa is responsible for ‘blue milk’and Serratia marcescens for ‘red milk’ (differentiatefrom bloody milk).

Pathogenic organisms that may be present includeE. coli O157, S. aureus, campylobacters, salmonel-las, Yersinia enterocolitica, listerias, Streptococcuspyogenes and other streptococci from infectedudders in mastitis. Tubercle bacilli may be foundby culture of centrifuged milk deposits and gravitycream. B. abortus is excreted in milk and can beisolated by culture, or antibodies can be demon-strated by the ring test or whey agglutination test.Rickettsia burnetti, the agent of Q fever, may befound by animal inoculation of milk from sus-pected animals.

Mastitis

Looking for evidence of mastitis in bulk milk isobviously unrewarding in any but farm or othersmall samples. Heat the test sample in the waterbath at 35°C. Mix the test sample carefully andcool to 20°C. Place 0.01 ml of the prepared testsample on a clean slide and make a film. Dry thefilm on a level hot plate. Dip the dried film in dyesolution containing methylene blue for 10 min.Dip the film in tap water to wash away surplusdye, and dry (BSI, 1997). Examine for somaticcells and count the number. For bacteriologicalexamination and identification of causative organ-isms, see p. 335.

EXAMINATION OF DAIRYEQUIPMENT

See Chapter 19 for methods, etc.

Microbial content

The types of organisms found will be similar to thosein milk. Soil organisms, e.g. Bacillus spp., may bepresent. B. cereus reduces methylene blue very rap-idly and may also cause food poisoning. Organismsoriginating from faeces, e.g. various coliforms andPseudomonas spp. are found. Coliform bacilli alsorapidly reduce methylene blue. Pseudomonads mayproduce oily droplets in the milk.

CREAM

Natural cream

The Dairy Products (Hygiene) Regulations (SI,1995) specify the temperature requirements forproduction of all types of heat-treated cream exceptclotted cream in the UK. These are national ratherthan EC requirements which have been retained onpublic health grounds. They do not apply to creamfrom sheep and goats. There is a requirement to sat-isfy the phosphatase test. The bacteriological testsand targets required for cream are as follows.

Pasteurized cream

• Salmonellas: absent in 25 ml• L. monocytogenes: absent in 1 ml• Coliforms (guideline level): m, 0; M, 5; n, 5;

c, 2 (see above).

Sterilized and UHT cream

Incubate in a closed container at 30°C for 15 daysor 55°C for 7 days.

• Plate count at 30°C: < 100/ml.

Coliform test for pasteurized cream

Place 10 g of the test sample into a containercontaining glass beads. Add 90 ml buffered


peptone diluent (see p. 76) and shake to disperse. AStomacher may be used, but omit the glass beads.The temperature of the diluent should be approxi-mately that of the test sample. Prepare further dec-imal dilutions if required.

The following method combines detection of col-iforms and presumptive E. coli in a liquid mediumcontaining 4-methylumbelliferyl-β-D-glucuronide(MUG) (from which fluorescent umbelliferone isreleased by glucuronidase activity) and calculationof the number of coliforms and/or presumptiveE. coli by the most probable number (MPN) tech-nique after incubation at 30°C. It is particularlysuitable for use when levels are likely to be low(< 10/ml). The enumeration of coliforms andE. coli by the MPN technique involves six stages:

1. Inoculation of three tubes of lauryl tryptosebroth with Durham tubes and containing0.01% MUG per dilution of the test sample,using those dilutions appropriate to obtainingthe required detection requirements for thatproduct.

2. Incubation of those tubes at 30°C for 48 h.3. Identification of tubes showing gas production

as positive for presumptive coliforms.4. Confirmation of coliform presence by

subculture to brilliant green bile broth anddetection of gas formation after incubation at30°C for 24 h.

5. Identification of tubes showing fluorescenceand formation of indole as positive forpresumptive E. coli.

6. Determination of the MPN index from thenumber of positive tubes of selected dilutionsusing an MPN table and calculating thecoliform count and/or E. coli count per gramor millilitre of sample.

Plate (total viable) count

Do plate counts or Miles and Misra counts (seep. 148) on serial dilutions ranging from 10−2 to 10−6

in a 0.1% peptone water solution, using milk platecount agar.

If the cream is solid or difficult to pipette, makethe initial 10−1 dilution by weight as described onp. 197. The 10−1 dilution may be used, with appro-priate media, for spiral plating for total viable, col-iform and S. aureus counts.

Culture for pathogens

• Plate serial dilutions on Baird-Parker mediumfor S. aureus.

• For salmonellas pre-enrich a 1 in 10 mixtureof cream (preferably 25 g) in buffered peptonewater (BWP) containing glass beads andshake to disperse. Proceed as on p. 295.

• For campylobacters, use a 10-g sample andthe method on p. 314.

• For L. monocytogenes, use a 25-g sample andthe method on p. 356.

Phosphatase test

This is as for milk (see p. 234).

Microbial content

Fresh cream may contain large numbers of organ-isms, including coliforms, B. cereus, E. coli, S.aureus, other staphylococci, micrococci and strep-tococci. Most fresh cream has been pasteurized andtherefore contamination by many of these organ-isms is the result of pasteurization failure or faultydairy hygiene.

Cream should not be used in catering in circum-stances where these organisms can multiply rapidly.

Imitation cream

Do plate counts as for milk, at 30°C, coliform counts,and also examine for S. aureus (see p. 330). Examinefor staphylococci only, as for natural cream.

Cream 237

PROCESSED MILKS

Dried milk

This often has a high count and, if stored underdamp conditions, is liable to mould spoilage.Spray-dried milk may contain S. aureus. The bacte-riological tests and targets required for dried milkare:

• Salmonellas: absent in 25 g; n, 10; c, 0• L. monocytogenes: absent in 1 g• S. aureus (per g): m, 10; M, 100; n, 5; c, 2• Coliforms 30°C (per g, guideline level): m, 0;

M, 5; n, 5; c, 2.

TVC, coliform and S. aureus tests fordried milk

Warm a solution of dipotassium hydrogen phos-phate pH 7.5 to 45°C in a water-bath.Thoroughly mix the contents of the closed samplecontainer by repeatedly shaking and inverting. Ifthe volume of the contents is too great to allowthorough mixing, transfer the contents to a largercontainer and mix. Remove 10 g sample and addto a bottle containing 90 ml dipotassium hydrogenphosphate. To aid dissolution, swirl slowly to wetthe powder, then shake the bottle (e.g. 25 times in7 s). The addition of sterile glass beads may helpreconstitution, particularly with roller-dried milk.A Stomacher may be used, but omit the glassbeads. Replace the bottle in the water bath for5 min, shaking occasionally. Prepare further deci-mal dilutions if required:

• Do total counts as for fresh milk but prepareextra plates for incubation at 55°C for 48 hfor thermophiles.

• Enumerate coliforms using the MPN methodas described for coliform testing of cream (seep. 237).

• The 1 in 10 and further serial dilutions maybe used to plate on Baird-Parker medium forS. aureus (see p. 330).

Salmonellas in dried milk

Thoroughly mix the contents of the sample con-tainer as described above. Place 225 ml sterile dis-tilled water in an appropriate container and add1 ml brilliant green solution. Weigh 25 g of the testsample and pour it over the surface of the liquid.Close the container but do not shake. Allow tostand undisturbed for 60 ± 10 min before incuba-tion at 37°C. If after 3 h incubation the dried milkhas not dissolved, shake the container to mix thecontents. Proceed as on p. 295.

Yeasts, moulds and spores

Examine for yeasts and moulds as described forbutter examination (see p. 239).

The number of spore bearers is important in milkand milk powder intended for cheese making.Inoculate 10-ml volumes of bromocresol purplemilk in triplicate with 10-, 1.0- and 0.1-g amountsof milk. Heat at 80°C for 10 min, overlay with 3 ml2% agar and incubate for 7 days. Read by notinggas formation and use Jacobs and Gerstein’s MPNtables to estimate the counts (see p. 154).

Condensed milk

This contains about 40% of sugar and is sometimesattacked by (osmophilic) yeasts and moulds thatform ‘buttons’ in the product. Examine as forbutter (see p. 239).

Evaporated milk

This is liable to underprocessing and leakerspoilage. Clostridium spp. may cause hard swelland coagulation. Yeasts may cause swell. Openaseptically and examine as appropriate (seep. 219).


Fermented milk products

Yoghurt, leben, kefir, koumiss, etc., are made by fer-mentation (controlled in factory-made products) ofmilk with various lactic acid bacteria, and strepto-cocci and/or yeasts. The pH is usually about 3.0–3.5and only the intended bacteria are usually present,although other lactobacilli, moulds and yeasts maycause spoilage. The presence of large numbers ofyeasts and moulds can be indicative of poor hygiene.Plate on potato glucose agar (pH adjusted to 3.5)and incubate at 23 ± 2°C for 5 days.

Yoghurt is made from milk with Lactobacillusbulgaricus and Streptococcus thermophilus. In thefinished product there should be more than 108/mlof each and they should be present in equal num-bers. Lower counts and unequal numbers predis-pose to off-flavours and spoilage.

Make doubling dilutions of yoghurt in 0.1% pep-tone water and mix 5 ml of each with 5 ml melted LSDifferential Medium. Pour into plates, allow to set andincubate at 43°C for 48 h. Both organisms producered colonies, because the medium contains triphenyltetrazolium chloride. Those of L. bulgaricus are irreg-ular or rhizoid, surrounded by a white opaque zone,whereas those of S. thermophilus are small, round andsurrounded by a clear zone. Count the colonies andcalculate the relative numbers of each organism.

Post-pasteurization contamination with coliformsmay occur. One indicator of contamination orspoilage is an increased pH, which is usually very acid.If this is pH > 5.6, examine for coliforms, E. coli, sal-monellas and other enteric pathogens. The bacterio-logical tests and targets required for yoghurt are:

• Salmonellas: absent in 25 ml; n, 5; c, 0• L. monocytogenes: absent in 1 ml• Coliforms (per ml, guideline level): m, 0;

M, 5; n, 5; c, 2.

The methods are as described for cream above.

MILK-BASED DRINKS

The bacteriological tests and targets required forliquid milk-based drinks are:

• Salmonellas: absent in 25 ml; n, 5; c, 0• L. monocytogenes: absent in 1 ml• Coliforms (per ml, guideline level): m, 0; M,

5; n, 5; c, 2.


Phosphatase test

This is as for milk (see p. 233).

BUTTERMILK

Test for coliforms only, using the MPN method asdescribed for coliform testing of cream (see p. 237).

BUTTER

Soured cream is pasteurized and inoculated withthe starter, usually S. cremoris or a Leuconostoc sp.Salted butter contains up to 2% of sodium chlorideby weight.

Examination and culture

Emulsify 10 g butter in 90 ml warm (45°C) pep-tone/saline diluent and do plate counts on tryptoseagar. Culture on a sugar-free nutrient medium andtributyrin agar.

The bacteriological tests and targets required forbutter made from pasteurized milk or cream are:

• Salmonellas: absent in 25 g; n, 5; c, 0• L. monocytogenes: absent in 1 g• Coliforms (per gram, guideline level): m, 0;

M, 10; n, 5; c, 2.


Microbial content

Correct flavours are caused by the starters, whichproduce volatile acids from the acids in the souredraw material.

Butter 239

Rancidity may be caused by Pseudomonas andAlcaligenes spp., which degrade butyric acids.Anaerobic organisms (Clostridium spp.) may pro-duce butyric acid and cause gas pockets. Some lac-tic acid bacteria (Leuconostoc spp.) cause slimesand others give cheese-like flavours. Coliforms,lipolytic psychrophiles and casein-digesting prote-olytic organisms, which give a bitter flavour, maybe found. To detect these, plate on Standard PlateCount agar containing 10% sterile milk and incu-bate at 23 ± 2°C for 48 h. Flood the plates with 1%hydrochloric acid or 10% acetic acid. Clear zonesappear round the proteolytic colonies.

Yeasts and moulds may be used as an index ofcleanliness in butter. Adjust the pH of potato glu-cose agar to 3.5 with 10% tartaric acid and inocu-late with the sample. Incubate at 23 ± 2°C for 5days. Report the yeast and mould count per millil-itre or gram. Various common moulds grow on thesurface but often these grow only in water dropletsor in pockets in the wrapper.

CHEESE

Many different cheeses are now available, includ-ing varieties that contain herbs which may con-tribute to the bacterial load. Some are traditionallymade from raw milk and may contain pathogenssuch as S. aureus, brucellas, campylobacters and/orlisterias.

Milk is inoculated with a culture of a starter, e.g.S. lactis, S. cremoris, S. thermophilus, and rennetadded at pH 6.2–6.4. To make hard cheeses, thecurd is cut and squeezed free from whey, incubatedfor a short period, salted and pressed. The strepto-cocci are replaced by lactobacilli (e.g. L. casei) nat-urally at this stage and ripening begins. Some cheesesare inoculated with Penicillium spp. (e.g. P. roque-fortii), which give blue veining and characteristicflavours as a result of the formation, under semi-anaerobic conditions, of caproic and other alcohols.

Soft cheeses are not compressed, have a highermoisture content and are inoculated with fungi,usually Penicillium spp., which are proteolytic andflavour the cheese.

Types and flavours of cheeses are caused by theuse of different starters and ripeners and varyingstorage conditions.

Examination and culture

Take representative core samples aseptically, gratewith a sterile food grater, thoroughly mix and sub-sample. Fill the holes, left as a result of boring, withHansen’s paraffin cheese wax to prevent aeration,contamination and texture deterioration.

Make films, and de-fat with xylol and stain.Bacteria may be present as colonies: examine thinslices under a lower-power microscope. Weigh 10 gand homogenize it in a blender with 90 ml 1 mol/lsodium citrate solution or dipotassium hydrogenphosphate pH 7.5. The methods for coliforms,E. coli, S. aureus, L. monocytogenes and salmonel-las (pre-warm BPW to 45°C) are as described forcream above.

The bacteriological tests and targets required forcheese are as follows.

Hard cheese made from raw milk or thermisedmilk

• Salmonellas: absent in 25 g; n, 5; c, 0• L. monocytogenes: absent in 1 g• S. aureus (per gram): m, 1000; M, 10 000;

n, 5; c, 2• E. coli (per gram): m, 10 000; M, 100 000;

n, 5; c, 2.

Hard cheese made from heat-treated milk

• Salmonellas: absent in 25 g; n, 5; c, 0• L. monocytogenes: absent in 1 g.

Soft cheese made from raw or thermised milk

• Salmonellas: absent in 25 g; n, 5; c, 0• L. monocytogenes: absent in 25 g; n, 5; c, 0• S. aureus (per gram): m, 1000; M, 10 000;

n, 5; c, 2• E. coli (per gram): m, 10 000; M, 100 000;

n, 5; c, 2.


Soft cheese made from heat-treated milk

• Salmonellas: absent in 25 g; n, 5; c, 0• L. monocytogenes: absent in 25 g; n, 5; c, 0• S. aureus (per gram): m, 100; M, 1000; n, 5;

c, 2• E. coli (per gram): m, 100; M, 1000; n, 5; c, 2• Coliforms 30°C (per gram): m, 10 000; M,

100 000; n, 5; c, 2 (guideline level).

Fresh cheese

• Salmonellas: absent in 25 g; n, 5; c, 0• L. monocytogenes: absent in 25 g; n, 5; c, 0• S. aureus (per gram): m, 10; M, 100; n, 5; c, 2.

Microbial content

Apart from streptococci, lactobacilli and fungi thatare deliberately inoculated or encouraged, othermicro-organisms may be found. Contaminantmoulds, and Penicillium, Scopulariopsis, Oospora,Mucor and Geotrichum spp., give colours and off-flavours. Putrefying anaerobes (Clostridium spp.)give undesirable flavours. Rhodotorula sp. givespink slime and Torulopsis sp. yellow slime.Gassiness (unless deliberately encouraged by propi-onibacteria in Swiss cheeses) is usually caused byEnterobacter spp., but these are not found if themilk is properly pasteurized.

Gram-negative rods, some of which may hydrol-yse tributyrin, may also be present. Psychrophilicspoilage is common and is caused by Alcaligenesand Flavobacterium spp. Counts may be very high.Bacteriophages that attack the starters and ripenerscan lead to spoilage.

Butyric blowing

Clostridium butyricum and C. tyrobutyricum maycause gas pocket spoilage during the ripening ofhard cheeses.

Homogenize cheese samples in 1 mol/l sodium cit-rate solution or dipotassium hydrogen phosphatesolution pH 7.5 and add dilutions to meltedsodium polyethanol sulphate (SPS) agar in tubes.

Layer paraffin wax on the agar and incubate at30°C. Butyric clostridia will produce gas bubbles inthe medium. The organisms may be enumerated bythe three- or five-tube MPN method with inoculaof 10, 1 and 0.1 ml homogenate.

Propionibacteria

Although deliberately introduced into somecheeses, propionibacteria can cause spoilage inothers. Accurate counts require microaerophilicconditions and media.

Prepare doubling dilutions of cheese homogenatein 1 mol/l sodium citrate solution or dipotassiumhydrogen phosphate solution pH 7.5, and add 1 mlof each to tubes of yeast extract agar containing2% sodium lactate or acetate. Mix and seal thesurface with 2% agar. Incubate anaerobically orunder carbon dioxide at 30°C for 7–10 days.Propionibacteria produce fissures and bubbles ofgas in the medium. Choose a tube with a countablenumber of gas bubbles and calculate the numbersof presumptive propionibacteria (some otherorganisms may produce gas bubbles).

EGGS

Bacterial contamination of eggs can occur at differ-ent stages ranging from production to storage,processing, distribution and preparation.Contaminants on the shell are potential contami-nants of the liquid egg. Washing eggs in water is aneffective method of removing shell contaminants.Spoilage bacteria of eggs are mostly Gram-negativerods (P. fluorescens, Alicaligenes bookeri,Paracolobacterium intermedium, P. melanovo-genes, S. putrefaciens, P. vulgaris, Flavobacteriumspp.). Salmonella serotypes (enteritidis, gallinarum,pullorum) are associated with eggs and egg prod-ucts. The EC Directive on egg products (EC,1992b) contains microbiological standards for sal-monellas, Enterobacteriaceae, S. aureus, and TVCsin the product from the treatment establishment.

Eggs 241

Shell eggs

Shell eggs may be contaminated with various sal-monellas during their passage down the oviduct orfrom the environment after laying. On the shell,Salmonella spp. may be present with other bacte-ria including coliforms, Pseudomonas spp.,Bacillus spp., staphylococci and faecal strepto-cocci. Shell eggs are normally examined for thepresence of Salmonella spp. Batch examination ofeggs can be carried out by testing pools of sixeggs.

Disinfect the shells by swabbing or immersing withindustrial alcohol. Break the eggs in batches of six,keeping the shells and contents in separate, sterilizedcontainers, e.g. screw-capped jars. Add to each anequal volume of buffered peptone water (BPW) (pre-enrichment). Incubate at 37°C for 24 h and sub-culture 0.1 ml to 10 ml Rappaport–Vassiliadis SoyaBroth (RVSB) and 1 ml to 10 ml selenite broth (SB).Incubate the RVSB at 42°C and SB at 37°C for up to48 h. Subculture to xylose lysine deoxycholate(XLD) and brilliant green agar (BGA) or mannitollysine crystal violet agar (MLCB), and proceed as onp. 295.

Pasteurized liquid egg

The American regulations require that liquid wholeegg be heated to at least 60°C and held for 3.5 min,whereas the EC Directive specifies a heat treatmentat 64°C for 2.5 min. The EC Directive bacteriolog-ical tests required are:

• TVC at 30°C• enumeration of staphylococci (see p. 329) and

coliforms (see p. 284), and detection ofsalmonellas (as above and see p. 295).

The targets levels are:

• Plate count: M, 100 000/g or ml• Enterobacteriaceae: M, 100/g or ml• Staphylococci: absent in 1 g or ml• Salmonellas: absent in 25 g or ml.

Liquid egg albumen and crystalline eggalbumen

Bacteriological tests and target levels required areas above for pasteurized liquid egg.

Powdered egg

Powdered egg products including whole egg, eggyolk and albumen are recognized vehicles of salmo-nellas and should be examined for them.Bacteriological tests and target levels required areas above for pasteurized liquid egg.

Frozen egg

Frozen egg must be pasteurized or otherwisetreated to destroy salmonellas present. The α-amy-lase, bacteriological tests and target levels requiredare as above for pasteurized liquid egg.

ICE-CREAM AND EDIBLE ICES (ICE-LOLLIES)

These products vary in their composition and maybe ice-cream, as defined in the various food regula-tions, mixtures of ice-cream and fruit or fruitjuices, or contain no ice-cream. In general, viable(plate) counts, coliforms and tests for pathogensshould be applied to ice-cream and commoditiesthat are said to contain it whether as a core or as acovering. Otherwise the pH should be ascertained.If this is ≤ 4.5 there is no point in proceedingfurther. The EC (1992a) Directive specifies thetemperature requirements for production ofpasteurized and sterilized ice-cream. The bacterio-logical tests and targets required for ice-cream andother frozen milk-based products are:

• Salmonellas: absent in 25 g; n, 5; c, 0• L. monocytogenes: absent in 1 g• S. aureus (per gram): m, 10; M, 100; n, 5; c, 2


• Coliforms 30°C (per gram): m, 10; M, 100;n, 5; c, 2 (guideline level)

• Plate count 30°C (per gram): m, 100 000;M, 500 000; n, 5; c, 2 (guideline level).

Sampling

Send samples to the laboratory in a frozen condi-tion. Remove the wrappers of small samples asepti-cally and transfer to a screw-capped jar. Sampleloose ice-cream with the vendor’s utensils (whichcan be examined separately if required, see Chapter19). Keep larger samples in their original cartons orcontainers.

Hold edible ices by their sticks, remove the wrap-per and place the lolly in a sterile screw-capped500-ml jar. Break off the stick against the rim ofthe jar. Reject samples received in a melting condi-tion in their original wrappers. Allow the samplesto melt in the laboratory but do not allow theirtemperature to rise above 20°C.

Examination

Weigh 10 g of the test sample into a container.Place the container in a water-bath at 37°C untilthe whole test portion has just melted. Add 90 mlBPW diluent pre-warmed to 37°C. Mix in thestomacher until thoroughly dispersed (1 min).

The methods for coliforms, S. aureus, L. monocy-togenes and salmonellas are as described for creamabove.

Do plate counts using plate count agar and incu-bate at 30°C for 72 h. The spiral plating method(see p. 82) may be used for total counts.

Miles and Misra counts (see p. 148) are very con-venient. Dilute the ice-cream 1 : 5 and drop three0.02-ml amounts with a 50-dropper on blood agarplates. Allow the drops to dry, incubate at 37°Covernight and count the colonies with a hand lens.

Multiply the average number of colonies per dropby 250 (i.e. dilution factor of 5 and volume factorof 50) and report as count per millilitre. If there areno colonies on any drops, the count can be

reported as < 100/ml. It is difficult to count morethan 40 colonies per drop; therefore, report suchcounts as > 10 000 cfu/ml.

In counts, note the presence of B. cereus. Thissporebearer may be present in the raw materials. Itsspores resist pasteurization.

Counts at 0–5°C for psychrophiles may be usefulin soft ice-cream.

Ingredients and other products

Do plate counts at 5, 20 and 37°C. Although ice-cream is pasteurized and then stored at a low tem-perature, only high-quality ingredients with lowcounts will give a satisfactory product.

In cases of poor grading or high counts on the fin-ished ice-cream, test the ingredients and also themix at various stages of production. Swab rinses ofthe plant are also useful (see p. 248).

HUMAN MILK

There are effective bactericidal and bacteriostaticsystems within raw breast milk that prohibit thefurther proliferation of micro-organisms in themilk. There is therefore no need for routine micro-biological screening. Microbiological testing ofmother’s own milk should be carried out onlywhen there is a clinical indication.

Bacteriological criteria for feeding raw humanmilk to babies on neonatal units have been recom-mended by Carroll et al. (1979). Samples contain-ing 100 000 cfu/ml or less are classified as:

• class I – milk considered sterile (no growthafter overnight incubation)

• class II – milk containing saprophytes• class III – milk containing potential pathogens

(S. aureus, enterococci, group B streptococci,Enterobacteriaceae and P. aeruginosa) – thisis rejected.

Samples containing more than 100 000 cfu/ml arerejected. Pasteurization is recommended when thebacterial contamination is too high and when

Human milk 243


potential pathogens are present in the milk. Thephosphatase test should be used to establish theefficiency of pasteurisation (as for cows’ milk,p. 234). For more information see Carroll et al.(1979) and DHSS (1982).

For further information on the microbiology ofmilk and milk products see Greenwood (1995),Rampling (1998) and Robinson (1990a, 1990b).

REFERENCES

British Standards Institution (BSI) (1997) BS EN ISO13366-1: Milk – Enumeration of somatic cells.

Part 1. Microscopic method. London: BritishStandards Institution.

BSI (2000) BS EN ISO 11816-1: Milk and milk-based

drinks – Determination of alkaline phosphatase

activity – Fluorimetric method. London: BritishStandards Institution.

Carroll, L., Davies, D. P., Osman, M. et al. (1979).Bacteriological criteria for feeding raw breast-milkto babies on neonatal units. Lancet ii: 732–733.

Department of Health and Social Security (DHSS)(1982) The Collection and Storage of Human

Milk, Reports on Health and Social Subjects No.29. London: HMSO.

Downs, F. P. and Ito, K. (eds) (2001). Compendium of


Foods, 4th edn. Washington: American PublicHealth Association.

EC (1989) European Council Directive 89/437/EEC of20 June 1989 on hygiene and health problems

affecting the production and placing on the marketof egg products. Official Journal of the European

Communities L212: 87–100.EC (1992) European Council Directive 92/46/EEC of

16 June 1992 laying down the health rules for theproduction and placing on the market of raw milk,heat-treated milk and milk-based products. Official

Journal of the European Communities L268: 1–32.Greenwood, M. (1995) Microbiological methods for

the examination of milk and dairy products inaccordance with the Dairy Products (Hygiene)Regulations 1995. PHLS Microbiological Digest

12: 74–82.Greenwood, M. H. and Rampling, A. M. (1997)

Evaluation of a fluorimetric method for detectionof phosphatase in milk. PHLS Microbiology Digest

14: 216–217.Rampling, A. (1998) The microbiology of milk and

milk products. In: Topley & Wilson’s

Microbiology and Microbial Infections, 9th edn,Vol. 2, Systematic Bacteriology. London: Arnold,pp. 367–393.

Robinson, R. K. (1990a) Dairy Microbiology. The

Microbiology of Milk, Vol 1, 2nd edn. London:Elsevier Applied Science.

Robinson, R. K. (1990b) Dairy Microbiology. The

Microbiology of Milk Products. Vol. 2, 2nd edn.London: Elsevier Applied Science.

Statutory Instrument (SI) (1989) SI 2383 The Milk

(Special Designation) Regulations. London:HMSO.

SI (1995) SI 1986 The Dairy Products (Hygiene)

Regulations. London: HMSO.

It may be necessary to estimate the numbers ofviable organisms on surfaces and in the air of build-ings, in containers and on process equipment:

• to verify the standard of hygiene andefficiency of cleaning procedures

• to trace contamination from dirty to cleanareas

• to test for the escape of ‘process’ organisms inindustrial microbiology and biotechnologyplants

• to assess the level of bacterial contaminationin ’sterile’ environments

• to investigate premises where humidifier fever,sick building syndrome, etc. are suspected.

SURFACE SAMPLING

Few standards or guidelines have been publishedon what is an acceptable level of micro-organismcontamination on surfaces. The EC (2001)Decision calls for regular checks to be carried outon the general hygiene by meat establishmentoperators. It provides that cleaned and disinfectedsurfaces sampled using the agar contact plate orswab technique should have an acceptable range of0–10 colony-forming units (cfu)/cm2 for total viablecounts (TVCs) and 0–1 cfu/cm2 for Entero-bacteriaceae. As a guideline, the US Public HealthService (Downs and Ito, 2001) recommends thatadequately cleaned and disinfected food serviceequipment should have a total number of not morethan 100 viable micro-organisms per utensil or sur-face area of equipment sampled.

Agar contact plates

Contact plates consist of an agar surface that isapplied to the surface to be sampled. They are com-mercially available as Rodac (Replica OrganismDirect Contact) plates. Non-selective or selectivemedia may be used. If the surface to be sampled hasbeen cleaned with a phenolic or quaternary ammo-nium agent, 0.5% Tween 80 and 0.7% soyalecithin should be added to the medium to neutral-ize them.

To sample a surface remove the lid of the Rodacplate and press the agar firmly on to the surface,applying a rolling uniform pressure on the back ofthe plate to effect contact. Replace the lid and incu-bate the plate at an appropriate temperature. Whenused on some surfaces, agar debris left on the sur-face during sampling will have to be removed, e.g.with an alcohol-impregnated wipe, to preventmicrobiological growth associated with such nutri-ent debris.

Contact plates can be used only on flat non-porous surfaces that are relatively easy to cleanand disinfect. They are suitable only to assess sur-face populations in a range that can be accuratelycounted on their surface, i.e. 10–100 colonies.The technique itself is less accurate in predictingsurface populations than the surface swab tech-nique (Holah, 1999). This is not a problem, how-ever, when used for historical trend analysisbecause it is the deviation from normal internalstandards in results over a period of time that isimportant, not the accuracy of the results them-selves.

19

Environmental microbiology

Surface swab counts

The swab method may be used to sample any sur-face up to 1 m2. Wipes and sponges are preferredfor large surface areas (floors, walls), and fabric-tipped swabs or sponges for equipment and smallfood contact surfaces (100 cm2). They are com-mercially available in a range of sizes as individu-ally packaged units in a purpose-designedtransport case, or individually wrapped in a dis-posable sleeve and packed in bulk. Sterile tem-plates are required to define the swabbing area forflat surfaces, and are also commercially availablein a range of sizes.

Place the sterile template on the surface that isto be swabbed. There should be one sterile pre-moistened swab, cloth or sponge per container.Open the package containing the sterile swab,and aseptically remove it. Reseal the package.Depending on the entire surface to be sampled,either sterile forceps may be used or a sterileglove worn to hold the swab. Rub swab firmlyand thoroughly over the surface to be sampled.Rub five times from the bottom to the top andfive times from the left to the right. Return theswab to its container and reseal. If fabric-tippedswabs have been used, ensure that the part han-dled during swabbing is broken off. For those sur-faces that are difficult to swab because of theircontour, it may not be possible to swab a definedarea with this procedure.

Soak and squeeze swabs in nutrient broth or anappropriate selective medium to recover the organ-isms. If the surface has been washed or treated witha phenolic or quaternary ammonium (QAC) disin-fectant, add 0.5% Tween 80 and 0.7% soyalecithin to the medium. Swabs are available com-mercially with neutralizing buffer. The count per25 cm2 (or the area swabbed) is given by the num-ber of colony-forming units per millilitre of rinse orsolvent multiplied by 10. With Miles and Misracounts (see p. 148), it is given by the number ofcolony-forming units per five drops multiplied by100.

Rapid methods

Rapid monitoring methods generate results in asufficiently short timeframe (usually within 20 min)to allow process control, e.g. by measuring ATPlevels after cleaning to determine whether re-clean-ing is necessary. Current methods allow the quan-tification of micro-organisms (DEFT, DEM, ATP),food soils (ATP, protein) or both (ATP). There areno commercially available methods that allow thedetection of specific microbial types within thistimeframe. For more details on rapid samplingmethods, see Holah (1999).

AIR SAMPLING

Contamination of air in hospitals and other work-places may be responsible for the spread of infec-tion. In addition, in offices and factories, especiallywhere there is ‘air conditioning’, it may be relatedto allergic illnesses such as humidifier fever and the‘sick building syndrome’. It may also lead to con-tamination of products (e.g. foods, pharmaceuti-cals).

Airborne contaminants may be released by peo-ple, industrial processes or poorly maintained air-conditioning plants. Bacteria and fungal sporesmay be suspended in the air singly, in large clumps,or on the surfaces of skin scales and dust fromthe materials being processed. Small particles (e.g.5 μm) may remain suspended in the air for longperiods and be moved around by air currents.Filtration, as in some air-conditioning plants, maynot remove them. Larger particles settle rapidlyand contaminate surfaces.

Settle plates

Settle plates are typically 90-mm Petri dishes con-taining appropriate agar. Non-selective or selectivemedia may be used. Several plates are exposed fora given time, incubated at the appropriate temper-ature, and colonies counted. Settle plates do not

246 Environmental microbiology

give an assessment of the number of micro-organ-isms in the air, only those that settle on the surface.The accuracy of the technique is limited to thenumber of colonies that can be accurately countedon the surface of the plate, which in turn is relatedto exposure time. Therefore, prior trials arerequired to establish such exposure times. Thosewho need to monitor the air over long periods, e.g.in hospital cross-infection work, favour settleplates.

Mechanical air samplers

In principle, known volumes of air are drawnthrough the equipment and micro-organisms andother particles are deposited on the surface of agarmedia, on membrane filters or into broth mediawhere they may be enumerated. Some samplers(e.g. the Andersen) and other cascade samplers notonly allow colony counting, but also provide infor-

mation about the sizes of the particles from whichthe colonies develop. Particles deposited on mem-branes may be analysed chemically, immunologi-cally or by ATP detection kits.

Table 19.1 lists the characteristics of severaldevices. Choice depends on the volume of air to besampled and the convenience of handling, e.g.portability and power supply. The SAS and RCSmodels are hand held and battery operated. Each ofthe monitoring methods has limitations of whichthe user should be aware. None of these samplersgives the total number of viable organisms per unitvolume. Not all the bacteria or spores are trappedon to the medium; some adhere to other parts ofthe equipment. In addition, viability may beimpaired by physical conditions or inactivated byimpact. Nevertheless they are useful in the assess-ment of the microbiological quality of air.

For more detailed information on air sampling seeBennet et al. (1991), Ashton and Gill (1992),Hambleton et al. (1992), Holah (1999), andDowns and Ito (2001).

Air sampling 247

Table 19.1 Examples of commonly used air-sampling devices

Type of sampler Advantages Disadvantages

Cascade sieve impacter, e.g. Reliable results, information on Not practical in industry use Andersen 6-stage sampler particle size distribution

Single-stage sieve impacter, e.g. MAS-100 and SAS are practical in Inefficient at small particle sizes Andersen one-stage industrial use (< 5 μm)Surface Air System (SAS)MAS-100 and MicroBio MB2

Slit impacter, e.g. Casella Efficient, easy to use Static, bulky MK II

Centrifugal airstream, e.g. Practical in industrial use Selectivity for larger particles Reuter Centrifugal Sampler (RCS)

Impingement, e.g. All Glass Efficient for sampling of bacteria, Not practical in industry use Impinger (AGI-30) yeast, and spores Multistage Liquid ImpingerBiosampler three-nozzle impinger

Filtration, e.g. MD8 Practical in air sampling of isolaters; Desiccation of vegetative microbial efficient for sampling of spores cells

WASHED BOTTLES ANDCONTAINERS

Swab-rinse method for utensils,cartons and containers

Dip a cotton-tip swab in sterile 0.1% peptonewater and rub it over the surface to be tested, e.g.the whole of the inside of the carton. Use one swabfor five such articles. Use one swab for a predeter-mined area of a worktop surface, chopping board,etc. Swab both sides of knives, ladles, etc. Returnthis swab to the tube and swab the same surfacesagain with another, dry swab.

Add 10 ml 0.1% peptone water to the tube con-taining both swabs. Shake and stand for20–30 min. Do plate counts with 1.0- and 0.1-mlamounts on yeast extract agar (YEA). Divide thecount per millilitre by 5 to obtain the count perarticle. Inoculate three tubes of single-strengthMacConkey broth with 0.1-ml amounts to obtaincoliform counts.

Rinse method

For churns, bins and large utensilsAdd 500 ml quarter-strength Ringer’s solution con-taining 0.05% sodium thiosulphate to the vessel toneutralize chlorine, if present. Rotate the vessel towash the whole of the inner surface and then tip therinse into a sterile screw-capped jar.

Do counts with 1.0- and 0.1-ml amounts of therinse in duplicate. Incubate one pair at 37°C for48 h and one at 22°C for 72 h. Take the meancounts/ml at 37°C and 22°C and multiply by 500to give the count per container.

For churns, cans and other large containers,counts of not more than 10 000 cfu/container areregarded as satisfactory, between 10 000 and100 000 the cleaning procedure needs improve-ment, and over 100 000 as unsatisfactory(Harrigan, 1998).

For milk bottles, soft drink bottles and jars

If bottles are sampled after they have been througha washing plant in which a row of bottles travelsabreast through the machine, test all those in onesuch row. This shows if one set of jets or carriers isout of alignment. In any event, examine not lessthan six bottles. Cap them immediately. To eachbottle add 20 ml quarter-strength Ringer’s solutioncontaining 0.05% sodium thiosulphate and recap.Roll the bottles on their sides to rinse all of theinternal surface. Leave the bottles for 15–30 min ina horizontal position. Roll the bottles again toachieve thorough wetting.

Pipette 5 ml of the rinse from each bottle into eachof two Petri dishes. Add 15–20 ml molten YEA,mix and incubate one plate from each bottle at37°C for 48 h and one at 22°C for 72 h. Incubationof both plates at 30°C or 25–30°C is also accept-able. Pipette 5 ml from each bottle into 10 ml dou-ble-strength MacConkey broth and incubate at37°C for 48 h.

Take the mean of the count of the 37°C and 22°Cplates and multiply by 4 to give the count per bot-tle. Find the average of the counts, omitting any fig-ure that is 25 times greater than the others(indicating a possible fault in that particular line).

The figure obtained is the average colony countper container. Milk bottles giving average colonycounts ≤ 200 cfu are regarded as satisfactory, from200 to 1000 indicate that the cleaning procedureneeds improvement, and over 1000 as unsatisfac-tory (Harrigan,1998).

Coliforms should not be present in 5 ml of therinse.

Membrane filter method

Examine clean bottles and jars by passing the rinsethrough a membrane filter. Place the membrane ona pad saturated with an appropriate medium, e.g.double-strength tryptone soya broth for totalcount. Incubate at 35°C for 18–20 h. Stain themembrane with methylene blue to assist in count-ing colonies under a low-power lens. Examineanother membrane for coliforms using MacConkeymembrane broth or membrane-enriched lauryl


sulphate broth. Incubate at 35°C for 18–24 h.Staining to reveal colonies is unnecessary.Subculture suspect colonies into lactose peptonewater and incubate at 37°C for 48 h to confirm gasproduction.

Roll-tube method for bottles

Use nutrient or similar agar for most bacteria,deMan, Rogosa and Sharp (MRS) medium for lac-tobacilli, reinforced clostridial medium (RCM) forclostridia and buffered yeast agar for yeasts.Increase the agar concentration by 0.5%. Use theroll-tube MacConkey agar for coliforms.

Melt the medium and cool it to 55°C. Add 100 mlmedium to 1-litre bottles and proportionally less tosmaller bottles. Stopper the bottles with sterile rub-ber bungs and roll them under a cold water tap toform a film of agar over the entire inner surface (see‘Roll-tube counts’, p. 148). Incubate vertically andcount the colonies. For lactobacilli and clostridia,replace the bung with a cottonwool plug and incu-bate anaerobically.

Vats, hoppers and pipework

Large pieces of equipment are usually cleaned inplace (CIP). Test flat areas by agar contact or swab-bing. Take swab samples of dead ends of pipeworkand crevices. CIP pipelines are examined by therinse method where large quantities are pumpedaround the system. Samples of rinse fluid are col-lected from the discharge end of the system fromthe first, middle and final portions of rinse fluid.The first will contain any residual bacteria notkilled by CIP treatment. If this has not been effec-tive, the first sample will give a higher count thanthe middle and final samples. If all three samplesare satisfactory then the cleaning was efficient.Rinse fluid samples may also be taken at variouspoints throughout the system.

Examination of cloths, towels, etc.

To demonstrate unhygienic conditions and thenecessity for frequent changes of cloths, examine asfollows.

Spread a washcloth or drying cloth over the top ofa screw-capped jar of known diameter. Pipette10 ml 0.1% peptone water on to the cloth so thatthe area over the jar is rinsed into it. Do platecounts. If a destructive technique is possible, cutportions of cloths, sponges or brushes with sterilescissors and add to diluent. Where the cloth is of asmall size add directly to diluent.

Mincers, grinders, etc.

After cleaning, rinse with 500 ml 0.1% peptonewater. Treat removable parts separately by rinsingthem in diluent in a plastic bag. Do colony counton rinsings as for milk bottles.

Sampling dried material (scrapings,sweepings, etc.)

Take a sample at each site selected, using appropri-ate sterile equipment, and insert into sterile con-tainers. If sufficient sample is available, thequantity should approximate that used for determi-nations applied to the finished product.

Biotechnology plant

To detect the release of aerosols sample the air byone of the methods described on p. 246. To detectleakage around pipe joins and valves use the swabrinse method (see p. 248). Test surface contamina-tion with Rodac plates (see p. 245). For moredetails on sampling and monitoring in processbiotechnology, see Bennet et al. (1991) andTuijnenburg-Muijs (1992).

Washed bottles and containers 249

Pharmaceutical, etc. ‘clean rooms’

Use one of the mechanical air samplers (see p. 247),preferably placed outside the room and samplingthrough a probe. There are several different levelsof clean rooms and the standards, i.e. the numberof particles per unit volume, are set out in a BritishStandard (BSI, 1999). Settle plates are also usefulbut do not give comparable results.

Humidifier fever, etc.

Use one of the mechanical samplers (see p. 247) totest the air in premises where there are suspectedcases of humidifier fever, sick building syndrome,extrinsic allergic alveolitis, etc. Do counts for bothfungi and bacteria.

Sample the water and the biofilm in the humidifierreservoir for bacteria and fungi (see pp. 256 and257). See also Collins and Grange (1990),Flannigan et al. (1991), and Ashton and Gill(1992).

For further information on effective environmen-tal microbiological sampling, see Holah (1999) andDowns and Ito (2001).

REFERENCES

Ashton, I. and Gill, F. S. (eds) (1992) Monitoring

Health Hazards at Work, 2nd edn. Oxford:Blackwells.

Bennet, A. M., Hill, S. E., Benbough, J. E. andHambleton, P. (1991) Monitoring safety in processbiotechnology. In: Grange, J. M., Fox, A. andMorgan, N. L. (eds), Genetic Manipulation:

Techniques and Applications. Society for AppliedBacteriology Technical Series No. 28. London:Academic Press, pp. 361–376.

BSI (1999) BS EN ISO 14644-1: Cleanliness and

associated controlled environments. Part 1.

Classification of air cleanliness. London: BritishStandards Institution.

Collins, C. H. and Grange, J. M. (1990) The

Microbiological Hazards of Occupations,Occupational Hygiene Monograph No. 17. Leeds:Science Reviews.

Downs, F. P. and Ito K. (eds) (2001) Compendium of


Foods, 4th edn. Washington: American PublicHealth Association.

EC (2001) European Commission Decision2001/471/EC laying down rules for the regularchecks on the general hygiene carried out byoperators in establishments according to Directive64/433/EEC on health conditions for theproduction and marketing of fresh meat andDirective 71/118/EEC on health problems affectingthe production and placing on the market of freshpoultry meat. Official Journal of the European

Communities L165: 48–53.Flannigan, B., McCabe, M. E. and McGarry, F. (1991)

Allergenic and toxigenic micro- organisms inhouses. In: Austin, B. (ed.), Pathogens in the

Environment. Society for Applied MicrobiologySymposium Series No. 20. London: Blackwells, pp.S61–S74.

Hambleton, P., Bennett, A. M., Leaver, J. andBenbough, J. E. (1992) Biosafety monitoringdevices for biotechnology processes. Trends in

Biotechnology 10: 192–199.Harrigan, W. E. (ed.) (1998) Laboratory Methods in

Food Microbiology. London: Academic Press.Holah, J. (ed.) (1999) Effective Microbiological

Sampling of Food Processing Environment.

Guideline No. 20. Chipping Campden: Campden& Chorleywood Food Research Association.

Tuijnenburg-Muijs G. (1992) Monitoring andvalidation in biotechnological processes. In:Collins, C. H. and Beale, A. J. (eds), Safety in

Industrial Microbiology and Biotechnology.

Oxford: Butterworth–Heinemann, pp. 214–238.


The microbiological examination of water is usedworld wide to monitor and control the quality andsafety of various types of water. These includepotable waters, treated recreational waters anduntreated waters, such as the sea, used for recre-ational purposes. Microbiological examination ofwater samples is usually undertaken to ensure thatwater is safe to drink or to bathe in. The quality ofwater intended for human consumption in the EUis covered by a Directive (EC, 1998). This has beenenacted into UK law by the Water Industry Act1991, the Water Supply (Water Quality)Regulations 2000 (SI, 2000) and the Private WaterSupplies Regulations 1991 (SI, 1991a). In the UKbottled water is subject to the Natural MineralsWater, Spring Water, and Bottled Drinking WaterRegulations 1999 (SI, 1999). For a review ofmicrobiological standards of water and their rela-tionship to health risk, see Barrell et al. (2000).

Officially approved methods for the bacteriologi-cal examination of water are given by the UKEnvironment Agency (EA, 2002) and in the USA bythe American Public Health Association (APHA,1998). In relation to public health the principal testsapplied to water are the viable plate or colony countand those for coliforms, Escherichia coli, faecal col-iforms (includes species of Klebsiella, Enterobacterand Citrobacter spp.), enterococci and sulphite-reducing clostridia (Clostridium perfringens). Inaddition to the conventional microbiologicalparameters, UK legislation requires continuousmonitoring of ‘at-risk’ water treatment works forcryptosporidial oocysts (the Water Supply [WaterQuality] Regulations – SI, 2001). Table 20.1 pro-vides the UK microbiological guidelines and stan-dards for drinking water. In the UK, the Health

Protection Agency (HPA) provides a water micro-biology external quality assurance scheme forlaboratories to assess proficiency in testing.

SAMPLING

Water samples are usually collected by environ-mental health officers and water engineers, who usesterile 300-ml or 500-ml plastic or glass bottlessupplied by the laboratory.

For samples of chlorinated water the bottles mustcontain sodium thiosulphate (0.1 ml of a 1.8% solu-tion per 100 ml capacity) to neutralize chlorine.

COLONY COUNTS

These are required under UK regulations and ECDirectives and are standard procedures in the USA.The technique is described in Chapter 10. Yeastextract agar (YEA) is used and tests are done induplicate with undiluted and serially diluted sam-ples. One set is incubated at 22°C for 72 h and theother at 37°C for 24 or 48 h. Permitted values areprovided in Table 20.1.

TOTAL COLIFORMS ANDE. COLI TEST

Most probable number method withminerals modified glutamate medium

The principle of the most probable number (MPN)method for estimating the numbers of micro-

20

Water microbiology

252 Water microbiology

Table 20.1 Microbiological guidelines and standards for drinking water

Type of water Parameters Permitted Guide level Imperative concentration levelor value

Mains water(continuous sampling Coliforms/E. coli 0/100 ml < 10/100 l recommended) Cryptosporidium

oocystsa

Private suppliesClasses A–E and Coliforms/E. coli 0/100 ml 1–4 Colony counts at 22°C No significant increase

and 37°C over normal levels

Class F Coliforms/E. coli No organism detrimentalto public health

Natural mineral watersSampled any time Coliforms/E. coli 0/250 mlup to sale Enterococci 0/250 ml

Pseudomonas aeruginosa 0/250 mlSulphite-reducing clostridia 0/50 mlParasites/pathogens Absent

Within 12 h of bottling As above + colony count 22°C/72 h 100/mlColony count 37°C/48 h 20/ml

Drinking water in containersAt any time Coliforms/E. coli 0/100 ml

Enterococci 0/100 mlSulphite-reducing clostridia ≤ 1/20 mlColony counts at 22°C Should show noand 37°C appreciable increase

after bottlingWithin 12 h of bottling As above +:

Colony count 22°C/72 h 100/mlColony count 37°C/48 h 20/ml

Vending machinesColiforms/E. coli 0/100 mlColony count 22°C/72 h ≤ 10 000/mlColony count 37°C/24 h ≤ 1000/mlb

Meat premisesRoutine sampling Coliforms/E. coli 0/100 ml

Colony count 22°C/72 h ≤ 100/mlColony count 37°C/48 h ≤ 10/ml

If coliforms found Resample as for routine plus test for: Enterococci 0/100 mlSulphite-reducing clostridia 0/20 ml

a Applies to treatment works shown by risk assessment to be potentially contaminatedb Provided that colony counts are no more than 10 times greater than those in water entering the machineReproduced from Barrell et al. (2000) with permission of the publishers, HPA Communicable Disease Surveillance Centre

organisms is given in Chapter 10. For water exam-ination the test involves the addition of known vol-umes of the sample to a range of bottles or tubescontaining a fluid medium, in this case mineralsmodified glutamate medium (MMGM), which hasreplaced the original MacConkey broth.

Select the range according to the expected purityof the water:

• Mains chlorinated water: A and B• Piped water, not chlorinated: A, B and C• Deep well or borehole: A, B and C• Shallow well: B, C and D• No information: A, B, C and D.

A: Add 50 ml water to 50 ml double-strengthmedium.

B: Add 10 ml water to each of five tubes of 10 mldouble-strength medium.

C: Add 1 ml water to each of five tubes of 5 mlsingle-strength medium.

D: Add 0.1 ml water to each of five tubes of 5 mlsingle-strength medium.

Incubate the bottles or tubes at 37°C for 18–24 hand examine for acid production (as demonstratedby the presence of a yellow coloration) and againafter a further 24 h. Retain all positive culturesbecause they are required for confirmatory testingfor coliforms and E. coli.

The number of tubes or bottles for each series ofvolume of sample is recorded where a positivereaction is given, demonstrated by growth withinthe medium and the production of a yellowcoloration.

When dilutions of sample have been used, a con-secutive series of volumes is chosen whereby someof the tubes or bottles show a positive reaction andsome show a negative reaction.

From the results determine the MPN of bacteriain the sample from the probability tables inChapter 10 (Tables 10.1–10.3).

Confirmation of coliformsThe confirmation procedure outlined here is basedon the demonstration that lactose fermentation is

indicative of the possession of the β-galactosidaseenzyme. Alternative procedures based on the directdetection of this enzyme, e.g. using the substrate o-nitrophenyl-β-D-galactopyranoside, may be moreappropriate in some cases.

Subculture from each tube or bottle showinggrowth in the MMGM medium to MacConkeyagar (MA) and nutrient agar (NA). Incubate at37°C for 18–24 h. If a pure culture is obtained onNA, do the oxidase test. If the isolate is oxidasenegative, test for lactose fermentation or possessionof β-galactosidase (see p. 98). If there is any doubtabout the purity of the culture, subculture at leastone typical colony from MA to NA, incubate at37°C for 18–24 h and carry out the oxidase test.

For each isolate to be tested, subculture to lactosepeptone water (LPW), incubate at 37°C and exam-ine for acid production after 24 h. If the results arenegative, re-examine after a further 24 h.Confirmation of acid production is demonstratedby the change of colour from red to yellow. Furtheridentification may be carried out using characteris-tic colonies on MA by means of appropriate bio-chemical and other tests. Commercial kits may beused.

Read the most probable number of coliformsfrom Tables 10.1–10.3.

Confirmation of E. coliIn addition to confirmatory tests for coliforms,subculture to MA each tube or bottle showinggrowth in the medium and incubate at 44°C for18–24 h. Inoculate tryptone water (TW) with typi-cal coliform colonies and incubate at 44°C for24 h. Test for indole production by Kovac’smethod (see p. 97). E. coli gives a positive reaction.A test for β-glucuronidase (see p. 286) may assist inthe early confirmation of E. coli. Suitable commer-cial test kits may be used.

Read the most probable numbers of E. coli fromTables 10.1–10.3. Permitted values for coliformsand E. coli are provided in Table 20.1.

Rapid methodsRapid test procedures are now available commer-cially to detect coliforms and E. coli in water

Total coliforms and E. coli test 253

samples. The Colilert 18 medium (Indexx Inc.,Portland, Maine) is one of these (EA, 2002).

Membrane filter method

Advantages of using membrane filter techniquesfor waters

• Speed with which results can be obtained asdirect results

• Saving of labour, media, glassware and costof materials if the filter is washed, sterilizedand re-used

• Incubation conditions can be varied easily toencourage the growth of attenuated or slow-growing organisms

• False-positive reactions that occur with somemedia in the MPN method for coliforms as aresult of the growth of aerobic and anaerobicspore-bearing organisms, or to mixtures oforganisms, are unlikely to occur withmembranes.

Disadvantages of using membrane filtertechniques for waters

• There is no indication of gas production(some waters contain large numbers of non-gas-producing lactose fermenters capable ofgrowth in the medium).

• Membrane filtration is unsuitable for waterswith high turbidity because the filter willbecome blocked before sufficient water canpass through it, whereas the accumulateddeposit on the membrane may also inhibit thegrowth of indicator organisms.

• Membrane filtration is unsuitable for waterswith low numbers of coliforms in the presenceof large numbers of non-coliform organismscapable of growth on the media used. Thesewill overgrow or obscure colonies ofcoliforms.

If large numbers of water samples are to be exam-ined and much fieldwork is involved, the mem-brane method is undoubtedly the most convenient.

For each sample, pass two separate 100-ml

volumes of the water sample through 47-mm diam-eter sterile membrane filters of 0.45 μm pore size. Ifthe supply is known or is expected to contain morethan 100 coliforms/100 ml, either filter smaller vol-umes or dilute the sample before filtration, e.g.10 ml water diluted with 90 ml quarter-strengthRinger’s solution.

Place sterile absorbent pads of at least the samesize as the membranes and approximately 1 mmthick in sterile Petri dishes and pipette 2.5–3 mlmembrane lauryl sulphate broth (MLSB) over thesurface. Place a membrane face up on each pad.

Incubate both membranes at 30°C for 4 h thentransfer one membrane to 37°C for 14 h for totalcoliforms and the other to 44°C for 14 h for E. coli.

CountingCount all yellow colonies within a few minutes ofbeing removed from the incubator and report aspresumptive coliform and E. coli count/100 ml ofwater. Membrane counts may be higher than MPNcounts because they include all organisms produc-ing acid, not only those producing acid and gas.

Confirmatory tests for coliforms Subculture colonies to LPW and incubate at 37°C.After 6 h, subculture to MA and NA. (Largecolonies may be subcultured to MA and NAdirectly from the membrane.) Examine the LPWcultures at 37°C after 24 h for acid production and,if the results are negative, after a further 24 h.

Incubate the MA and NA plates at 37°C for 24 hand do oxidase tests on colonies only from the NA.Coliforms are oxidase negative, possess β-galac-tosidase and produce acid from lactose within 48 hat 37°C. Further identification may be carried outusing colonies on characteristic colonies on MA bymeans of appropriate biochemical and other tests(Barrow and Feltham, 1993). Commercial kits maybe used.

Confirmatory tests for E. coliSubculture yellow colonies from the membranesincubated at 37°C and 44°C to MA and NA, toLPW (containing a Durham tube if informationregarding gas production is required) and to TW.


Incubate all media at 44°C for 24 h. E. coli is oxi-dase negative, produces acid (and usually gas) inLPW, and indole in TW. Tests for β-glucuronidasemay assist in the early confirmation of E. coli.Commercial test kits may be used.

FAECAL STREPTOCOCCI INWATER

These organisms are useful indicators when doubt-ful results are obtained in the coliform test. Theyare more resistant than E. coli to chlorine and aretherefore useful when testing repaired mains. Onlygroup D streptococci are significant.

Membrane method

Prepare and filter the sample as described above forcoliforms and E. coli. Place the membrane on thesurface of a well-dried plate of membrane entero-coccus agar (MEA). For potable waters, incubate at37°C for 48 h and for untreated waters incubate at37°C for 4 h followed by 44°C for 44 h.

Count all red, maroon or pink colonies. These arepresumptive faecal streptococci. Subculture a suit-able number of red, maroon, pink or colourlesscolonies from the membrane to bile aesculin agarand incubate at 44°C for 18 h. Development ofdiscrete colonies, surrounded by a brown or blackhalo from aesculin hydrolysis, within a few hoursconfirms the presence of faecal streptococci.

SULPHITE-REDUCING CLOSTRIDIA

Membrane method

Pass 100 ml of the sample (heated at 75°C for 10min) through a 47-mm diameter sterile membranefilter of 0.45 μm pore size. Place the membrane faceupwards on the tryptose suphite cycloserine agarwith egg yolk (TSCA) and pour 15–20 ml TSCA,

pre-cooled to 50°C, over the membrane and allowto set. Incubate anaerobically at 37°C. Examine theplates after 24 and 48 h and count all blackcolonies. The specificity of TSCA is such that con-firmation of isolates is not required (EA, 2002). Ifthere is any doubt about the organisms isolated bythis method, examine Gram-stained films from thecolonies for spore-forming rods.

Permitted values for sulphite-reducing clostridiaare provided in Table 20.1.

PATHOGENS IN WATER

Methods for the detection of certain bacterialpathogens are given below, but other pathogens,outside the scope of this book, such as viruses (e.g.norovirus, rotaviruses, hepatitis A) and parasites(e.g. Cryptosporidium and Giardia spp., andamoebae) should not be overlooked.

As a general rule the direct search for pathogenicbacteria has no place in routine microbiologicalexamination of a water supplier. There may beoccasions, for example, when the water is pollutedby E. coli, when there is, however, a need to searchfor faecal pathogens.

Filter the sample using 47-mm diameter sterilemembrane filter of 0.45 μm pore size (0.2 μm poresize for campylobacters).

Salmonellas

Transfer the membrane to 90 ml enrichmentmedium (e.g. BPW) in a wide-mouthed screw-capped container. Incubate at 37°C for 24 h andsubculture 0.1–10 ml Rappaport–Vassiliadis SoyaBroth (RVSB). Incubate the RVSB at 42°C for 24 h.Subculture to xylose lysine deoxycholate (XLD)and brilliant green agar (BGA) and incubate at37°C for 24 h. Return the RVSB to the incubator at42°C for a further 24 h. Again, subculture to XLDand BGA and incubate at 37°C for 24 h. Proceed ason p. 295.

Pathogens in water 255

Vibrio cholerae and other vibrios

Transfer the membrane to 100 ml alkaline peptonewater. Incubate at 25°C for 2 h followed by12–16 h at 37°C. Subculture to thiosulphate–citrate–bile salt–sucrose agar (TCBSA) and incu-bate at 37°C for 16–24 h. Proceed as on p. 275.

Legionellas

Legionellas may be present in the water in coolingtowers, humidifier tanks, etc. and also in thebiofilm that is usually attached to the walls of thecontainers. Examination of water alone may give afalse impression.

Membrane filtration is the usual method of testingthe water for legionellas (BSI, 1998). Examine sev-eral samples. If the water is cloudy, pre-filter itthrough glass fibre pads.

Transfer the membrane to 5–25 ml peptone salinediluent in a screw-capped bottle. Vortex mix fornot less than 2 min to detach the micro-organisms.Decant the diluent and centrifuge it for 30 min at3000 × g or 10 min at 6000 × g.

Inoculate buffered charcoal yeast extract agarmedium (BCYE) or other legionella-selective mediawith 100-μl amounts of resuspended deposit.Incubate under high humidity at 37°C. Includestock control cultures.

If the samples are highly contaminated use one ofthese decontamination methods:

• Heat the deposit at 50°C for 30 min and thenculture 100-μl amounts.

• Suspend 0.1 ml of the deposit in 0.9 ml acidbuffer– KCl (0.2 mol/l, 14.91 g/l) 25 ml – HCl (0.2 mol/l, 17.2 ml conc. acid/l) 3.9 ml – sterilize by autoclaving.

Plate out 100-μl amounts every min for 5 min.Identify legionellas as described on p. 322.Centrifuge biofilm suspensions and treat thedeposit as described for contaminated samples(above).

Campylobacters

Transfer the membrane in a suitable screw-cappedcontainer filled almost completely with 90 mlcampylobacter-enrichment broth. Seal tightly andincubate at 37°C for 24 h and then at 42°C for afurther 24 h. Subculture to campylobacter-selectiveagar or the blood-free modification of this agar andincubate in a gas jar containing a microaerobic gas-generating kit at 37°C for 48 h. Proceed as onp. 314.

E. coli O157

Transfer the membrane into containers with 90 mlmodified tryptone soya broth (MTSB) or BPW.MTSB is suitable for polluted water, whereas BPWis more appropriate for the recovery of stressedE. coli O157 from drinking water and relativelyunpolluted waters (EA, 2002). Incubate the MTSBat 42°C for 24 h, and the BPW at 37°C for 24 h.Subculture on to cefoxime tellurite sorbitolMacConkey agar (CT-SMAC) and incubate at37°C for 24 h, and proceed as on p. 287.

Recovery of E. coli O157 from enrichment brothis improved by using immunomagnetic separationwith antibody-coated magnetic beads (Wright et al.1994; EA, 2002).

Pseudomonas aeruginosa

Place the membranes on pseudomonas agar andincubate at 37°C for 48 h. Count all colonies thatproduce a green, blue or reddish–brown pigmentand those that fluoresce under ultraviolet (UV)light. Regard those colonies that producepyocyanin (green/blue pigmented) as P. aeruginosa.

Confirm fluorescent colonies by subculture on milkagar with cetyl trimethylammonium bromide incu-bated at 37°C for 24 h. Record as confirmedP. aeruginosa colonies that are 2–4 mm in diameter,show typical pigment production and possess a ‘haloof clearing’ around the colony where the casein has


been hydrolysed. Where pigment is not produced,and P. aeruginosa is suspected, subculture coloniesto a fresh plate of milk agar with cetyl trimethylam-monium bromide; incubate at 37°C for 24 h. Thisprovides a pure culture that can then be confirmedusing commercially available test kits. For rapididentification of P. aeruginosa, which grows at42°C, incubate subcultures at that temperature.

Microfungi and actinomycetes

These organisms cause odours and taints and oftengrow in scarcely used water pipes, particularly inwarm situations, e.g. basements of large buildingswhere the drinking water pipes run near to theheating pipes.

Sample the water when it has stood in the pipesfor several days. Regular running or occasionallong running may clear the growth. Centrifuge 50ml of the sample and plate on Rose Bengal agar andmalt agar medium. Add 100 μg/ml kanamycin tothe malt medium to suppress most eubacteria.

Alternatively, pass 100 ml or more of the samplethrough a membrane filter and apply this to a padsoaked in liquid media of similar composition.

BOTTLED NATURAL WATERS

These may be ‘still’ or carbonated. The tests used,with guidance levels at the point of sale and within12 h of bottling, are given in Table 20.1.Environmental mycobacteria have been found inmoderate numbers in some bottled waters.

SWIMMING POOLS

As with drinking water, microbiological examina-tion of recreational water is aimed largely at detect-ing markers of faecal pollution. However, colonycounts play a rather greater role as a marker of gen-eral water quality, and tests for P. aeruginosa arealso often carried out. P. aeruginosa is associated

with disease, usually folliculitis or otitis externa, inpeople who have used swimming pools or spapools (Hunter, 1996).

Use a commercial kit to determine the chlorinecontent. This may be more useful than bacterio-logical examination but, if the latter is required,take 300-ml samples in thiosulphate bottles (seep. 251) from both the deep and the shallow ends.Do plate counts and test for coliforms, E. coli andpseudomonads. In satisfactory pools target levelsare as follows:

Guide level Target levelColony count, 37°C/24 h < 100/ml < 10/mlColiforms < 10/100 ml 0/100 mlE. coli 0/100 ml P. aeruginosa (optional) 0/100 ml

Staphylococci are more resistant than coliforms tochlorination. They tend to accumulate on the sur-face of the water in the ‘grease film’. To findstaphylococci take ‘skin samples’. Open the samplebottle so that the surface water flows into it. Filterthe test volume and place each test membrane onthe agar surface in a Petri dish containing modifiedlipovitellin–salt–mannitol agar (M-5LSMA). Incu-bate at 30°C for 48 h. Count and record presump-tive staphylococci, and proceed as on p. 330.

SPA, JACUZZI ANDHYDROTHERAPY POOLS

These have been associated with human infectionswith pseudomonads and legionellas. As they aremaintained at 37°C the chlorine (or bromine) levelsshould be closely monitored.

Do plate counts on tryptone agar and test for coli-forms, E. coli and pseudomonads. Target levels are:

Guide Target level level

Total viable count, 37°C/24 h < 100/ml < 50/mlColiforms and E. coli 0/100 ml P. aeruginosa 0/100 ml

Pools with colony counts > 100 cfu/ml need furtherinvestigation (PHLS, 1994, 1999; HPA, 2003).

Spa, jacuzzi and hydrotherapy pools 257

BATHING BEACHES AND OTHERRECREATIONAL WATERS

Test undiluted and serially diluted samples for col-iforms, faecal coliforms and enterococci. Test forsalmonellas and enteroviruses when there aregrounds for suspecting deterioration in water qual-ity or that the organisms are present. The EU guidelevels (SI, 1991b) are as follows:

Guide level Imperative levelTotal coliforms 500/100 ml 10 000/100 mlFaecal coliforms 100/100 ml 2000/100 mlEnterococci 100/100 mlSalmonella 0.l(Enteroviruses 0 plaque-forming units (PFU)/10 l)

OTHER ORGANISMS OF PUBLICHEALTH IMPORTANCE

These are not considered in this book althoughsome references are cited.

Viruses

Viruses (especially rotaviruses) have been impli-cated in outbreaks associated with recreational andpotable waters (see Sellwood, 1998).

Cyanobacteria

Blue-green algae produce a variety of dangeroustoxins. Serious illnesses have been associated withtheir presence in recreational and potable watersand fish (see Codd, 1994).

Cryptosporidium spp.

Oocysts of this organism may be present inrecreational and potable waters and offer a serioushazard to health (see Badenoch, 1995).

WATER TESTING IN REMOTEAREAS

Problems arise in the bacteriological examinationof water in remote parts of tropical and subtropicalcountries. Some of the culture media describedabove are expensive or may not be available. It maybe necessary to use simpler media such asMacConkey and litmus lactose broths and agars.These will affect results and standards. In addition,the 44°C tests may be unreliable because someindigenous, non-faecal organisms may grow atthose temperatures.

Water samples collected in remote areas andtransported over long distances to laboratories areunlikely to give reliable results. A portable kit, theOxfam-Del Agua, devised for the WHO project fordrinking water in rural areas, will give reliable fae-cal coli counts, turbidity and free chlorine levels. Itis available from Del Agua (RCPEH, Guildford,UK; see Lloyd and Helmer, 1991) and Septi-Check,Indexx Inc. Portland, Maine, USA).

SEWAGE

Effluents and sludge are usually monitored micro-biologically to ensure that treatment has reducedthe loads of salmonellas, listerias and campylobac-ters, all of which can survive in water; if untreatedsludge is spread on land.

For further information on water microbiologyand technical directions on sampling see: APHA(1996); EA (2002); Barrell et al. (2000); HSE(2001); and HPA (2003).

REFERENCES

APHA (1986) Standard Methods for the Examination

of Water and Wastewater, 20th edn. Washington:American Public Health Association,

Badenoch, J. (1995) Cryptosporidium in Water

Supplies. Report of a group of Experts. London:HMSO.


Barrell, R. A. E., Hunter, P. R. and Nichols, G. (2000)Microbiological standards for water and theirrelationship to health risk. Communicable Disease

and Public Health 3: 8–13. Corrections publishedin Communicable Disease and Public Health 3:221.

Barrow, G. I. and Feltham, R. K. A. (eds) (1993)Cowan and Steel’s Manual for the Identification of

Medical Bacteria, 3rd edn. London: CambridgeUniversity Press.

BSI (1998) British Standard BS ISO 11731: Water

quality – Part 4: Microbiological methods – Section

4.12: Detection and enumeration of Legionella.London: British Standards Institution.

Codd, G. A. (1994) Blue-green algal toxins: water-borne hazards to health. In: Golding, A. M. B,Noah, N. and Stanwell-Smith, R. (eds), Water and

Public Health. London: Smith Gordon, pp.271–278.

EA (2002) The Microbiology of Drinking Water, Parts

1–10: Methods for the Examination of waters and

Associated Materials. London: EnvironmentAgency

EC (1998) European Council Directive 98/83/EC of 3November 1998 on the quality of water intendedfor human consumption. Official Journal of the

European Communities L330: 32–54.HPA (2003) Guidelines for water quality on board

merchant ships including passenger vessels.

London: Health Protection Agency.HSE (2001) Legionnaire’s Disease. The control of

Legionella bacteria in water systems. Approved

Code of Practice and Guidance. London: Healthand Safety Executive.

Hunter, P. R. (1996) Outbreaks of disease associatedwith treated recreational waters. Microbiology

Europe 4: 10–12.Lloyd, B. and Helmer, R. (eds) (1991). Surveillance of

Drinking Water in Rural Areas. London:Longman.

Public Health Laboratory Service (1994) Hygiene for

Spa pools. London: PHLS.PHLS (1999) Hygiene for Hydrotherapy pools, 2nd

edn. London: PHLS. Sellwood, J. (1998) Viruses implicated as potential

emerging pathogens. In: de Louvois, J. andDadswell, J. (eds), Emerging Pathogens and the

Drinking Water Supply. London: PHLS, pp. 55–1.SI (1991a) The Private Water Supply (Water Quality)

Regulations. SI 2790. London: HMSO.SI (1991b) The Bathing Waters (Classification)

Regulations SI 1597. London: HMSO.SI (1999) The Natural Minerals Water, Spring Water

and Bottled Drinking Water Regulations. SI 1540.London: HMSO.

SI (2000) The Water Supply (Water Quality)

Regulations. SI 3184. London: HMSO.SI (2001) The Water Supply (Water Quality)

(Amendment) Regulations. SI 2885. London:HMSO.

Wright, D. J., Chapman, P. A. and Siddons, C. A.(1994) Immunomagnetic separation as a methodfor isolating Escherichia coli O157:H7 from foodsamples. Epidemiology and Infection 113: 31–39.

References 259

21

Key to common aerobic, non-sporing, Gram-negative bacilli

Aerobic Gram-negative non-sporing bacilli thatgrow on nutrient and usually on MacConkey agarsare frequently isolated from human and animalmaterial, from food and from environmental sam-ples. Some of these bacilli are confirmed pathogens;others, formerly regarded as non-pathogenic, arenow known to be capable of causing human dis-ease under certain circumstances, e.g. in ‘hospitalinfections’, after chemotherapy or treatment withimmunosuppressive drugs; others are commensalsand many are of economic importance.

The organisms described in this and Chapters 22and 23 have received much attention from taxono-mists in recent years. Names of species and generahave been changed and new taxa created. Thenomenclature used here is that in common use atthe time of going to press, mostly as in the currentedition of Topley and Wilson’s Microbiology andMicrobial Infections (Collier et al., 1998). See alsoBergey’s manual (Holt et al., 1994). Table 21.1 liststhe general properties of aerobic Gram-negativerods that grow on nutrient agar.

Table 21.1 Properties of Gram-negative rods that grow on nutrient agar

Organism O/Fa Oxidase Arginine Gelatin Motilityhydrolysis liquefaction

Pseudomonas spp. OA + v v +Acinetobacter spp. ON - - - -Alcaligenes spp. N - - - +Flavobacteria O + - + -Chromobacterium spp. O + - + +Janthinobacterium spp. F + + + +Aeromonas spp. F + + + +Plesiomonas spp. F + + - +Vibrio spp. F + + - +Enterobacteria F - v v v Pasteurella spp. F + - - -Yersinia spp. F - - - vMoraxella spp. N + - - vBordetella spp. N v - - v

a Oxidation/fermentation test (Hugh and Liefson)

O, oxidative; A, alkaline; N, no reaction; F, fermentative; v, variable

References 261

REFERENCES

Collier, L., Balows, A. and Sussman, M. (eds) (1998)Topley and Wilson’s Microbiology and Microbial

Infections, 9th edn. Vol. 2, Systematic

Bacteriology. London: Arnold.

Holt, J. G., Kreig, N. R., Sneath, P. H. A. et al. (eds)(1994) Bergey’s of Determinative Bacteriology, 9thedn. Baltimore, MA: Williams & Wilkins.

Up to 1984, over 100 species were included in thegenus Pseudomonas. These were mainly plantpathogens and only a minority was pathogenic forhumans and animals. Today only members of theribosomal RNA (rRNA) group I (of five rRNAgroups) are included in the genus. Other rRNAgroups have been reclassified in the generaBurkholderia (group II), Comamonas (nowDelftia, group III), Brevundimonas (group IV) andStenotrophomonas (group V). They are all aero-bic, Gram-negative, non-sporing rods about3 μm × 0.5 μm, they are motile by polar flagellaand, with the exception of Stenotrophomonasmaltophilia, they are usually oxidase positive, useglucose oxidatively (or produce no acid) and donot produce gas. Some produce a fluorescentpigment. In this book ‘pseudomonad’ is used as ageneral term for Pseudomonas spp. and relatedspecies

A recent phylogenetic analysis of the genusPseudomonas which used combined gyrB andrpoD nucleotide sequences identified two intra-generic subclusters. Cluster I was further dividedinto two complexes and cluster II into three com-plexes (Yamamoto et al., 2000).

Pseudomonads commonly occur in soil andwater. Some species are recognized human and ani-mal pathogens but some others, formerly regardedas saprophytes and commensals, have been incrim-inated as opportunist pathogens in hospital-acquired infections and susceptible patient groupssuch as those with cystic fibrosis (CF). In hospitalsthey are commonly isolated as contaminants ofintravenous fluids, distilled water supplies, soaps,disinfectants and antiseptics, infusions and otherpharmaceuticals. Species colonizing swimming

pools, particularly P. aeruginosa, have been associ-ated with skin and ear infections.

ISOLATION AND IDENTIFICATION

Inoculate nutrient agar, MacConkey agar, nutrientagar containing 0.03% cetrimide, pseudomonasagar with supplements and King’s medium A.Incubate at 20–25°C (food, environment, etc.) or35–37°C (human and animal material).Temperature and length of incubation are crucialfor accurate identification.

Colonies on nutrient agar vary in appearanceaccording to species (see below). A greenish-yellowor bluish-yellow fluorescent pigment may diffuseinto the medium. Other less common pigments arepyomelanin (brown) and pyorubrin (port-wine).Mucoid strains are sometimes found in clinicalmaterial, particularly in sputum from patients withCF, and may be confused with klebsiellas.

Do oxidase test, reaction in Hugh and Leifsonmedium, and inoculate Thornley’s arginine broth(Moeller’s method is too anaerobic) andMacConkey agar. Inoculate nutrient broths andincubate at 4 and 42°C. Use a subculture from asmooth suspension for this test. Examine growthon King’s B medium with a Wood’s lamp for fluo-rescence. If this is seen, test for reduction of nitrateand acid production from glucose and mannitol inammonium salt sugars and gelatin liquefaction(Tables 22.1 and 22.2).

The API 20NE kit, designed for non-fermentingGram-negative rods, is useful for identifyingpseudomonads. Other kits include Minitek NF andOxi-ferm.

22

Pseudomonas and Burkholderia andother pseudomonads

Table 22.1 Reactions of medically important pseudomonads in biochemical tests

Species Pigment Oxidase O/F Growth Growth Arginine ONPG NO3 reduction NO2 reductiontesta at 42°C at 4°C

Pseudomonas aeruginosa Green +b O + − + − + +P. fluorescens − + O − + + − v −P. putida − + O − v + − v −P. alcaligenes − + A + − v − v −P. pseudoalcaligenes − + A + v v − + −P. stutzeri Yellow/− + O + v v − + +Burkholderia cepacia −/Yellow v v v − − v v −B. pseudomallei − + O + − + − + −B. mallei − v O v − + − + −Delftia acidovorans − + A/− − − − − + −Brevundimonas diminuta − + A − − − − − −Brevundimonas vesicularis Orange + O v − − v − −Sphingomonas paucimobilis Yellow + O − − − + − −Stenotrophomonas maltophilia − v O/A/− v − − v v −

a F, fermentative; O, oxidative; A, alkaline; −, no change; v, variableb +, ≥ 90% positive; −, ≤ 10% positive; v, 11–89% positive

Pseudomonads are motile (occasionally non-motile strains may be met), oxidative and non-reac-tive, or produce alkali in Hugh and Leifsonmedium. The oxidase test is usually positive exceptfor Stenotrophomonas (formerly Xanthomonas)maltophilia and Burkholderia cepacia. Thosepseudomonads that give a positive arginine test arecommon and give a positive arginine test morerapidly than other Gram-negative rods. This test isvery useful for recognizing non-pigmented strains.Alcaligenes and Vibrio spp. do not hydrolysearginine. Pseudomonads vary in their ability toliquefy gelatin and grow at 4 and 42°C (see Table22.1).

Some of the strains associated with food spoilageare psychrophiles and may be lipolytic. Brine-tolerant strains and phosphorescent strains are notuncommon.

THE FLUORESCENTPSEUDOMONADS

Pseudomonas aeruginosa

The colonies of P. aeruginosa on nutrient agar after24 h incubation in air are usually large (2–4 mm),flat and spreading Many strains produce a dif-fusible pigment. Some, however, form ‘coliform-like’ colonies with entire edges and a creamyconsistency. Other colonial forms are rarely iso-lated from clinical material and include rough anddry ‘pepper-corn’ colonies, gelatinous and mucoidforms. The last two are most commonly found in

cultures of sputum from CF patients and if not pig-mented can be confused with klebsiellas, particu-larly on MacConkey agar. Small colony variant‘dwarf forms’ may appear on extended incubation.P. aeruginosa characteristically forms a blue–greendiffusible pigment on King’s A agar which is causedby pyocyanin (blue) and fluorescein (yellow) pig-ments (see above).

Production of the blue–green pigment and a posi-tive oxidase test reaction is sufficient to indicatethat a strain is P. aeruginosa. Other confirmatorytests for the species are oxidation of glucose inHugh and Leifson medium, deamination of argi-nine in Thornley’s agar, and growth on three suc-cessive subcultures in nutrient broth at 42°C.Non-pigmented cultures should be examined onKing’s B medium for fluorescence and, if positive,should be tested further for reduction of nitrate tonitrogen gas and failure to grow at 4°C. Anaerobicgrowth is possible only in the presence of an alter-native terminal electron acceptor such as nitrate.The demonstration of motility in Craigie tubes istherefore inappropriate. Sugar use is best demon-strated in ammonium salts-based medium becausethe weak acid formed by oxidation of the sugar isneutralized by alkali from the breakdown of pep-tone.

Pseudomonas aeruginosa is ubiquitous in natureand about 10–15% of humans will carry the organ-ism transiently in the bowel with a rapid turnoverof strain types. Infections in healthy individuals arerare but skin infections after prolonged exposure tocontaminated recreational waters are not uncom-mon. Less frequent infections of the healthy includeendophthalmitis and otitis externa. In hospital

264 Pseudomonas and Burkholderia and other pseudomonads

Table 22.2 Fluorescent pseudomonads

Species Growth at NO3 Acid from

4°C 42°C Mannitol Maltose

P. aeruginosa − + + + −P. fluorescens + −a + + +P. putida +a − − − −

a 40% of strains

patients, P. aeruginosa is a common cause of infec-tion ranging from relatively benign colonization ofthe upper airways after intubation, surgicalwounds and the catheterized bladder to blood-stream infections with a mortality rate of 60%.

As many as 80% of CF patients may be colonizedin the lung with P. aeruginosa and once establishedit is particularly refractory to antibiotic treatment.Many isolates grow as mucoid colonies but mixturesof different colonial forms are frequently found onprimary plates. These variants invariably prove tobe genetically identical. P. aeruginosa from CFpatients is often atypical in growth requirements andmay be auxotrophic for specific amino acids, be nonmotile and a minority may exhibit extreme suscep-tibility to semi-synthetic penicillins.

For epidemiological studies, isolates of P. aerugi-nosa can be serotyped by slide agglutination of livecultures. There are 21 internationally accepted Oserotypes but four types account for approximately50% of clinical and environmental isolates. DNAfingerprinting using pulsed-field gel electrophoresisof XbaI restriction endonuclease digests canachieve further discrimination between serotypes.Other typing systems used include ribotyping andrandom polymerase chain reaction (PCR) typing(Grundmann et al., 1995).

Compared with the Enterobacteriaceae, P. aerugi-nosa is relatively resistant to antibiotics but the greatmajority of strains from acute infections aresusceptible to ticarcillin, piperacillin, ceftazidime,imipenem, gentamicin, tobramycin, ciprofloxacinand aztreonam. Resistance to antibiotics is conferredby mechanisms including low cell permeability,chromosomal and plasmid-mediated β-lactamases,aminoglycosidases and carbapenemases, alteredantibiotic binding proteins, and an efflux systemactive against a wide range of compounds(Livermore, 2002).

Pseudomonas fluorescens and P. putida

Two other species, P. fluorescens and P. putida, arefluorescent pseudomonads but these are rare in

clinical specimens because they are psychrophileswith optimal growth temperatures below 30°C.P. fluorescens has often been implicated as acontaminant of stored blood products, which whentransfused into a patient may cause endotoxic shock.The species may occasionally be recovered from thesputum of CF patients. It is also a recognized foodspoilage agent, particularly of refrigerated meat, andit may gel UHT milk if this is stored above 5°C. Theymay be recovered from fresh vegetables or plants,and sinks, taps and drains in the hospital environ-ment. P. fluorescens grows at 4°C, produces fluo-rescein pigment on King’s B agar and some isolateswill not grow at 37°C. It is resistant to a wide rangeof antibiotics including imipenem.

Pseudomonas alcaligenes and P.pseudoalcaligenes

Both of these species grow at 42°C but neithergrows at 4°C. They are ubiquitous and are oppor-tunist pathogens. They do not liquefy gelatin andmay or may not grow on cetrimide agar. Somestrains of P. alcaligenes do not hydrolyse arginine.

Pseudomonas stutzeri

Colonies are commonly rough, dry and wrinkledand can be removed entire from the medium. Oldercolonies turn brown. It may hydrolyse arginine andmay not grow at 4°C but does grow at 42°C. It issalt tolerant but not halophilic. A ubiquitousorganism, it has been found in clinical material butis not regarded as a pathogen.

BURKHOLDERIA SPP.

The genus Burkholderia was defined in 1992 byYabuuchi et al. for Pseudomonas species formerlybelonging to rRNA group II. There are 30 validlynamed species in the genus but the medically

Burkholderia spp. 265

important species are B. cepacia, B. pseudomalleiand B. mallei. The organism formerly known asPseudomonas pickettii was reassigned by Yabuuchiet al. (1992) to Burkholderia, but has subsequentlybeen reclassified as Ralstonia pickettii. Occasionalclinically significant isolates of R. pickettii arerecovered from hospital patients but they are mostoften isolated from the ward environment and ascontaminants of antiseptic and disinfectant solu-tions. It is oxidase and nitrate variable and does nothydrolyse arginine.

Burkholderia cepacia

Burkholderia cepacia is a complex of at least sevendifferent genomovars (phenotypically similar,genetically distinct species). These are B. cepacia(genomovars I, III and IV), B. multivorans (II),B. vietnamensis (V), B. stabilis (VI) and B. ambi-faria (VII). The genus, Pandoraea, comprising fivespecies, clusters closely to the complex in phyloge-netic trees based on 16S rRNA gene sequences(Coeyne et al., 2001). Genomovar I strains areoften isolated from the environment butgenomovars II and III, respectively, are principallyassociated with hospital-acquired infections andlung infections of a minority of CF patients inwhom it may prove fatal. The species can surviveand multiply in pharmaceuticals and cosmetics,and is able to grow in distilled water.

These organisms grow moderately well on nutri-ent agar and a variety of non-fluorescent pigmentsmay be produced by some strains. About half of theisolates are oxidase positive; none hydrolyse argi-nine. They grow slowly at 37°C and extended incu-bation for 48 h is recommended to optimize theirrecovery from sputum. Cultures often die rapidlyon storage on nutrient agar slopes, but surviveremarkably well suspended in sterile distilledwater. Selective media, based on their constitutiveresistance to colistin and bile salts, have beendescribed but other colistin-resistant, Gram-nega-tive rods may also be recovered on these media.Members of the complex are not differentiated well

by phenotypic tests, although PCR assays specificfor individual genomovars have been developed(see Coeyne et al., 2001).

Burkholderia cepacia has high intrinsic resistanceto antimicrobial agents and is generally resistant tothe antibiotics active against P. aeruginosa. It isresistant to aminoglycosides, colistin, ticarcillin,azlocillin and imipenem. Variable susceptibility isshown to temocillin, aztreonam, ciprofloxacin andtetracycline and about two-thirds of strains fromCF patients are susceptible to ceftazidime,piperacillin/tazobactam and meropenem.

Burkholderia pseudomallei

Cultures on blood agar and nutrient agar at 37°Cgive mucoid or corrugated, wrinkled, dry coloniesin 1–2 days, and an orange pigment may developon prolonged incubation. There is no growth oncetrimide agar. Variation between rough andsmooth colonies is frequent. Cells may also exhibitbipolar staining in Gram stains. B. pseudomallei isa strict aerobe; it is motile, oxidizes glucose andbreaks down arginine. Most isolates are reliablyidentified by API 20NE microgalleries but must bedistinguished from non-pigmented strains ofP. aeruginosa, P. stutzeri and B. mallei. It is resist-ant to colistin and gentamicin but isolates are gen-erally susceptible to imipenem, piperacillin,amoxicillin–clavulanic acid, doxycycline, cef-tazidime, aztreonam and chloramphenicol.

Caution: B. mallei and B. pseudomallei are inHazard (Risk) Group 3 and should be handledonly under full Biosafety Containment Level 3conditions. Although odour is said to be a usefulcharacteristic, cultures should never be sniffed.Laboratory-acquired infections are not un-common.

Burkholderia pseudomallei is an importantpathogen of humans (melioidosis) and farm animalsin tropical and subtropical areas, where it is endemicin rodents and is found in moist soil, and on vegetablesand fruit. A closely related but non-pathogenicspecies, B. thailandensis, has been described from


environmental samples in south-east Asia. Culturesshould be sent to a reference laboratory for speciesconfirmation. For further information on these organ-isms and melioidosis, see Dance (1999).

Burkholderia mallei

In exudates this organism may appear granular orbeaded and may show bipolar staining. It gives 1-mm, shining, smooth, convex, greenish–yellow,buttery or slimy colonies, which may be tenaciouson nutrient agar, no haemolysis on blood agar andit does not grow on MacConkey agar in primaryculture. Growth may be poor on primary isolation.It grows at room temperature, does not change theHugh and Leifson medium, produces no acid inpeptone carbohydrates, is nitrate positive, andgives variable urease and oxidase reactions.

Burkholderia mallei is the causative agent of glan-ders, a rare disease of horses. It has not been iso-lated from animals or humans in the UK sinceWorld War II.

DELFTIA ACIDOVORANS

This organism was reclassified from the genusComamonas. It is found on occasion in clinicalspecimens and the hospital environment. Isolatesgrow as non-pigmented colonies overnight at 37°Cbut incubation should be extended to 48 h for slowgrowing strains. Some strains are resistant to col-istin and gentamicin and may grow on B. cepacia-selective media. Antimicrobial susceptibility isvariable but most isolates are susceptible to urei-dopenicillins, tetracycline, the quinolones andtrimethoprim–sulphamethoxazole.

BREVUNDIMONAS DIMINUTA ANDBREVUNDIMONAS VESICULARIS

These are closely related species previously of rRNAhomology group IV of Pseudomonas spp. Theygrow slowly on nutrient agar and require 48 h incu-

bation at 37°C. B. vesicularis grows as orange pig-mented colonies on nutrient agar and gives a weakoxidase reaction. B. diminuta is not pigmented.Both species are negative in Hugh and Leifson’s testbut B. vesicularis will oxidize glucose in an ammo-nium salts medium. They are rare in clinical speci-mens and of doubtful clinical significance.

STENOTROPHOMONAS MALTOPHILIA

Colonies resemble those of P. aeruginosa but a yel-low or brown diffusible pigment may be produced;on blood agar they can appear as faint lavendercolour. It is usually oxidase negative, does nothydrolyse arginine and does not grow on cetrimideagar. It is the only pseudomonad, along withB. cepacia (63%), that gives a positive lysinedecarboxylase reaction. It has been isolated from avariety of hospital environmental sources and isusually of clinical significance in severely immuno-compromised patients. The extensive use ofimipenem, to which S. maltophilia is resistant,appears to be associated with nosocomial out-breaks. S. maltophilia is increasingly isolated fromthe sputum of CF patients and is often misidentifiedas B. cepacia because it grows reasonably well oncolistin-containing media. Most strains are suscept-ible to co-trimoxazole, doxycycline and mino-cycline, and third-generation cephalosporins but areresistant to aminoglycosides.

SPHINGOMONAS PAUCIMOBILIS

This species produces a non-diffusible yellow pig-ment and is most likely to be confused withflavobacteria. Motility is poor and best seen in cul-tures incubated at room temperature. It forms acidfrom a wide range of sugars in ammonium salts-based medium and is consistently ONPG positive.It has been found in clinical material and recoveredfrom hospital equipment. Most strains are suscep-tible to erythromycin, tetracycline, chlorampheni-col and aminoglycosides.

Sphingomonas paucimobilis 267

For further information about the classificationand identification of the pseudomonads seeKersters et al. (1996) and Cowan and Steel’sManual (Barrow and Feltham, 1993) and Pitt(1999).

REFERENCES

Coeyne, T., Vandamme, P., Govan J. R. W. et al.

(2001) Taxonomy and identification of theBurkholderia cepacia complex. Journal of Clinical

Microbiology 39: 3427–3436.Barrow, G. I. and Feltham R. K. A. (eds) (1993)

Cowan and Steel’s Manual for the Identification of


Dance, D. A. B. (1999) Melioidosis. In: Guerrant, R.L., Walker, D. H. and Weller, P. F. (eds), Tropical

Infectious Diseases: Principles, Pathogens and

Practice, Vol. 1. Philadelphia, PA: ChurchillLivingstone, pp. 430–407.

Grundmann, H., Schneider, C., Hartung, D. et al.

(1995) Discriminatory power of three DNA-basedtyping techniques for Pseudomonas aeruginosa.Journal of Clinical Microbiology 33: 528–534.

Kersters, K., Ludwig, W., Vancanneyt, M. et al. (1996)Recent changes in the classification of thepseudomonads; an overview. Systematic and

Applied Microbiology 19: 465–477.Livermore, D. M. (2002) Multiple mechanisms of

antimicrobial resistance in Pseudomonas

aeruginosa: our worst nightmare? Clinical

Infectious Diseases 34: 634–640.Pitt, T. L. (1999) Pseudomonas, Burkholderia and

related genera. In: Collier, L., Balows, A. andSussman, M. (eds), Topley and Wilson’s

Microbiology and Microbial Infections, 9th edn,Vol 2, Systematic Bacteriology, CD-ROM.London: Arnold, Chapter 47.

Yabuuchi, E., Kosaka, Y., Oyaizu, H. et al. (1992)Proposal of Burkholderia gen. nov; and transfer ofseven species of the Pseudomonas homology groupII to the new genus, with the type speciesBurkholderia cepacia (Palleroni and Holmes 1981)comb. nov. Microbiology and Immunology 36:1251–1275.

Yamamoto, S., Kasai, H., Arnold, D. L. et al. (2000)Phylogeny of the genus Pseudomonas: intragenericstructure reconstructed from the nucleotidesequences of gyrB and rpoD genes. Microbiology

146: 2385–2394.


ACINETOBACTER

Organisms have been moved into and out of thisgenus for several years. At present it seems to con-tain two species – Acinetobacter calcoaceticus (for-merly A. anitratum) and A. lwoffii – although someworkers use the name A. baumannii for both or forvarious ‘subspecies’. It is difficult to distinguishbetween the two species.

Acinetobacters are Gram-negative, non-motile,obligate aerobes, oxidase and nitrate reductionnegative, catalase positive, and are oxidative orgive no reaction on Hugh and Leifson medium (seebelow) and grow on MacConkey agar.

They are ubiquitous in the environment and havebeen found in clinical material.

Isolation and identification

Plate material on nutrient, blood and MacConkeyagar (and Bordetella or similar media if indicated).Incubate at 22°C and at 37°C for 24–48 h. Dooxidase and catalase tests. Inoculate nutrient brothfor motility, Hugh and Leifson medium, glucosepeptone water, nitrate broth and urea medium.

Species of Acinetobacter

Acinetobacter calcoaceticus ssp. anitratusThis gives large non-lactose fermenting colonies onMacConkey and DC agars and grows at room tem-perature. On Hugh and Leifson medium it is oxida-tive, producing acid but not gas from glucose. Itgives a variable urease reaction.

Infections in a wide variety of sites in humanshave been reported. Many strains are resistant toseveral antibiotics. Hospital cross-infections withresistant strains cause problems. Sometimes indirect films this organism may resemble Neisseriaspp.

Acinetobacter calcoaceticus ssp. lwoffiiColonies on blood agar are small and haemolytic,and may be sticky. There is no change in Hugh andLeifson medium; it fails to produce acid from glu-cose and is urease negative. It can cause conjunc-tivitis. This and the other two biotypes aredistinguished by the API 20NE.

For more information about infections withAcinetobacter spp., see Vivian et al. (1981) andBergogne-Bérézin et al. (1996).

ALCALIGENES

These organisms resemble acinetobacters in manyways and some workers include them in the genusAcinetobacter. They are widely distributed in soil,fresh and salt water, and are economically impor-tant in food spoilage (especially of fish and meat).Many strains are psychrophilic. Some species havebeen isolated from human material and suspectedof causing disease.


Plate on blood and MacConkey agars. Incubate at20–22°C (foodstuffs) or 35–37°C (pathologicalmaterial) for 24–48 h. Subculture white colonies of

23Acinetobacter, Alcaligenes, Flavobacterium,

Chromobacterium and Janthinobacterium, andacetic acid bacteria

Gram-negative rods on agar slopes for oxidase test,in peptone water for motility, in Hugh and Leifsonmedium, glucose peptone water, bromocresol pur-ple milk, arginine broth and gelatin.

Species of Alcaligenes

The genus Alcaligenes is restricted here to motile,oxidase-positive, catalase-positive Gram-negativerods which give an alkaline or no reaction in Hughand Leifson medium, an alkaline reaction inbromocresol purple milk and grow on MacConkeyagar. Arginine is not hydrolysed and gelatin is notusually liquefied. Most strains are resistant to peni-cillin (10-unit disc). There is one species, A. fae-calis, which now includes A. odorans and severalstrains of doubtful taxonomic status.

Alcaligenes faecalis

This culturally resembles Bordetella bronchisepticabut is urease negative. It is a widely distributedsaprophyte and commensal. Cultures of somestrains, especially those associated with foodspoilage, have a fruity odour, e.g. of apples or rot-ting melons. At least one strain of Alcaligenes isassociated with ropy milk and ropy bread.

ACHROMOBACTER

This is no longer officially recognized, but foodbacteriologists find it a convenient ‘dustbin’ fornon-pigmented Gram-negative rods which are non-motile, oxidative in Hugh and Leifson medium,oxidase positive, produce acid but no gas from glu-cose, acid or no change in purple milk, do nothydrolyse arginine and usually liquefy gelatin.

FLAVOBACTERIUM

The genus Flavobacterium contains many speciesthat are difficult to identify and some have been

transferred to other genera (Murray et al., 1999).Food bacteriologists, however, frequently isolateGram-negative, bacilli-forming, yellow coloniesfrom their samples and, although some undoubt-edly belong to other genera, it suits the convenienceof many food bacteriologists to call them ‘flavobac-teria’. Some of these organisms are proteolytic orpectinolytic and are associated with spoilage offish, fruit and vegetables.

There is one species of medical importance.

Flavobacterium meningosepticum

This has been isolated from cerebrospinal fluid(CSF) in meningitis in neonates, from other humanmaterial, and from hospital intravenous and irriga-tion fluids.

It grows on ordinary media; colonies at 18 h are1–2 mm, smooth, entire, grey or yellowish andbutyrous. There is no haemolysis on blood agar, nogrowth on DCA and none on primary MacConkeycultures, but growth on this medium may occurafter several subcultures on other media. A yellow-ish–green pigment may diffuse into nutrient agarmedia.

Flavobacterium meningosepticum is oxidative inHugh and Leifson medium, non-motile, oxidaseand catalase positive, liquefies gelatin in 5 days,does not reduce nitrates and does not grow onSimmons’ citrate medium. Rapid hydrolysis of aes-culin is a useful characteristic. The API ZYM sys-tem is useful. Acid is produced from 10% glucose,mannitol and lactose in ammonium salt mediumbut not from sucrose or salicin. Indole productionseems to be positive by Ehrlich’s but negative byKovac’s methods (Snell, 1973). See Table 23.1 fordifferentiation from pseudomonads and Acineto-bacter. For more information see Holmes (1987).

CHROMOBACTERIUM ANDJANTHINOBACTERIUM

An important characteristic of these organisms isthe production of a violet pigment (unpigmented

270 Acinetobacter, Alcaligenes, Flavobacterium, Chromobacterium and Janthinobacterium etc.

strains are not unknown), although this may not beapparent until the colonies are several days old.They grow well on ordinary media, giving cream oryellowish colonies that turn purple at the edges.The pigment is soluble in ethanol but not in chlo-roform or water, and can be enhanced by addingmannitol or meat extract to the medium. Citratebut not malonate is used in basal syntheticmedium; the catalase test is positive and the ureasetest negative. Ammonia is formed. Gelatin is lique-fied. The oxidase test is positive but difficult to doif the pigment is well formed.

Chromobacterium violaceum

This species is mesophilic, growing at 37°C but notat 5°C. It is fermentative in Hugh and Leifsonmedium, hydrolyses arginine and casein but notaesculin (Table 23.2). It is a facultative anaerobe.

The violet pigment is best seen in cultures on man-nitol yeast extract agar incubated at 25°C.Colonies of non-pigmented variants may be seen incultures.

Although usually a saprophyte, cases of humanand animal infection have been described inEurope, the USA and the Far East.

Janthinobacterium lividum

This organism is psychrophilic, growing at 5°C butnot at 37°C. It is oxidative in Hugh and Leifsonmedium, does not hydrolyse arginine or casein, andhydrolyses aesculin. It is an obligate aerobe.

The violet pigment is best seen in cultures on man-nitol yeast extract agar incubated at 20°C.

More information about the identification ofthese organisms is given by Logan and Moss(1992).

Chromobacterium and Janthinobacterium 271

Table 23.1 Flavobacterium meningosepticum, Pseudomonas and Acinetobacter spp.

Species Oxidase Nitrate Citrate reduction

F. meningosepticum + − −Pseudomonas spp. + + +Acinetobacter spp. − − v

v, varies with subspecies, see text

Table 23.2 Chromobacterium violaceum and Janthinum lividum

Species Growth at Hydrolysis

5°C 37°C HL testa Arginine Casein Aesculin

C. violaceum - + F + + -J. lividum + - O - - +a Hugh and Leifson (fermentation oxidation) testF, fermentative; O, oxidative

ACETIC ACID BACTERIA

These bacteria are widely distributed in vegetation.They are of economic importance in the fermenta-tion and pickling industries, e.g. in cider manufac-ture (Carr and Passmore, 1979), as a cause of ropybeer and sour wine, and of off-odours and spoilageof materials preserved in vinegar. Ethanol is oxi-dized to acetic acid by Gluconobacter spp. butAcetobacter spp. continue the oxidation to producecarbon dioxide and water. They also differ in thatAcetobacter produces carbonate from lactate andhas peritricate flagella whereas Gluconobacter doesnot produce carbonate and has polar flagella. Sevenspecies of Acetobacter and three of Gluconobacterare recognized but identification is difficult.

Isolation and identification to genus

Identification to species is rarely necessary. It isusually sufficient to recognize acetic acid bacteriaand to determine whether the organisms produceacetic acid and/or destroy it.

Inoculate wort agar or Universal Beer Agar oryeast extract agar containing 10% glucose that hasbeen filter sterilized and added at 50°C and 3% cal-cium carbonate at pH 4.5. Incubate at 25°C for24–48 h. Colonies are large and slimy. Subcultureinto yeast broth containing 2% ethanol and Congored indicator at pH 4.5. Acid is produced.

Test the ability to produce carbon dioxide fromacetic acid in nutrient broth containing 2% aceticacid. Use a Durham tube.

Plant bacterial masses (heavy inoculum, do notspread) on yeast extract agar at pH 4.5 containing

5% glucose and at least 3% finely divided calciumcarbonate in suspension. Clear zones occur aroundmasses in 3 weeks but if less chalk is used somepseudomonads can do this.

Culture on yeast extract agar containing 2% cal-cium lactate. Acetobacter spp. grow well and pre-cipitate calcium carbonate. Gluconobacter spp.grow poorly and give no precipitate.

Carr and Passmore (1979) describe a medium todifferentiate the two genera. It is yeast extract agarcontaining 2% ethanol and bromocresol green(1 ml 2.2% solution/l) made up in slopes. BothGluconobacter and Acetobacter spp. produce acidfrom ethanol and the indicator changes fromblue–green to yellow. Acetobacter spp. then use theacid and the colour changes back to green (Table23.3).

For more information, see Carr and Passmore(1979) and Swings et al. (1992).

REFERENCES

Bergogne-Bérézin, E., Joly-Guillou, N. L. and Towner,K.J. (1996) Acinetobacter: Microbiology,

Epidemiology, Infections and Management. BocaRaton, FL: CRC Press.

Carr, J. G. and Passmore, S. M. (1979) Methods foridentifying acetic acid bacteria. In: Skinner, F. A.and Lovelock, D. W. (eds), Identification Methods

for Microbiologists, 2nd edn. Society for AppliedBacteriology Technical Series No. 14. London:Academic Press, pp. 33–45.

Holmes, B. (1987) Identification and distributionFlavobacterium meningosepticum in clinicalmaterial. Journal of Applied Bacteriology 43:29–42.

272 Acinetobacter, Alcaligenes, Flavobacterium, Chromobacterium and Janthinobacterium etc.

Table 23.3 Acetic acid bacteria

Genus Acid from CO2 from acetic Calcium carbonateethanol acid precipitation

Gluconobacter + − −Acetobacter + + +

Logan, N. A. and Moss, M. O. (1992) Identification ofChromobacterium, Janthinobacterium andIodobacter species. In: Board, R. G., Jones, D. andSkinner, F. A. (eds), Identification Methods in

Applied and Environmental Microbiology. Societyfor Applied Bacteriology Technical Series No. 29.London: Blackwells, pp. 183–192.

Murray, P. R., Baron, J. O., Pfaller, M. A. et al. (eds)(1999) Manual of Clinical Microbiology, 7th edn.Washington: American Society of Microbiology.

Snell, J. J. S. (1973) The Distribution and Identification

of Non-fermenting Bacteria. Public Health

Laboratory Service Monograph No. 4. London:HMSO.

Swings, J., Gillis, M. and Kersters, K. (1992)Phenotypic identification of acetic acid bacteria. In:Board, R. G., Jones, D. and Skinner, F. A. (eds),Identification Methods in Applied and

Environmental Microbiology. Society for AppliedBacteriology Technical Series No. 29. London:Blackwells, pp. 103–110.

Vivian, A., Hinchcliffe, E. J. and Fewson, C. A. (1981)Acinetobacter calcoaceticus: some approaches to aproblem. Journal of Hospital Infection 2: 199–204.

References 273

The differential properties of the organisms in thisgroup and those of the enterobacteria are summa-rized in Table 24.1.

VIBRIOS

The genus Vibrio includes at least 40 species ofGram-negative, non-sporing rods, motile by polarflagella and enclosed within a sheath. Some havelateral flagella and may swarm on solid media.They are catalase positive, use carbohydrates fer-mentatively and rarely produce gas. All but onespecies (Vibrio metschnikovii) are oxidase andnitrate reductase positive. They are sensitive to150 μg discs of the vibriostatic agent 2,4-diamino-6,7-di-isopropyl pteridine (0/129). An exception isV. cholerae O:139 which is resistant to 0/129. Awide range of extracellular enzymes is produced.Vibrios occur naturally in fresh and salt water.Species include some human and fish pathogens.See Table 24.1 for differentiation of Vibrio,

Plesiomonas, Aeromonas and Pseudomonas spp.and enterobacteria.

Isolation

Vibrios grow readily on most ordinary media butenrichment and selective media are necessary forfaeces and other material containing mixed flora.

FaecesAdd 2 g of 20-ml amounts of modified alkalinepeptone water (MAPW – see p. 75). Inoculate thio-sulphate citrate bile salt sucrose agar (TCBS)medium. Incubate MAPW at 37°C for 5–8 h or at20–25°C for 18 h and subculture to TCBS.Incubate TCBS cultures at 37°C overnight.

Other human materialVibrios may occur in wounds and other material,particularly if there is a history of sea bathing.Examine primary blood agar plates (see below forcolony appearance).

Water and foodsAdd 20 ml water to 100 ml MAPW. Add 25 g foodto 225 ml MAPW and emulsify in a Stomacher.Incubate at 37°C for 18 h and subculture on TCBS.If V. parahaemolyticus is specifically sought, enrichthe food sample in salt polymyxin broth (SPB) inaddition to MAPW.

For further information on the choice of mediacombinations for specific vibrios from food andenvironmental samples, see Donovan and vanNetten (1995).

24

Vibrio, Plesiomonas and Aeromonas

Table 24.1 Characteristics that distinguish genera

Genus O/F Oxidase 0/129 NaCl test 150 μg enhancement

Vibrio F + S +Plesiomonas F + S −Aeromonas F + R −Enterobacteria F − R −Pseudomonas O/N + R −

F, fermentative; O/N, oxidative/no acid produced; R, resistant;

S, sensitive

Enumeration of vibrios

Use liquid samples neat and diluted 10−1. Make 10−1

homogenates of solid samples. Spread 50 or 100 μlon TCBS medium, incubate 20–24 h and countvibrio-like colonies. Report as colony-formingunits (cfu) per gram.

For the three-tube MPN (most probable number)method prepare dilutions of 10−1, 10−2 and 10−3.Add 1 ml of each dilution to each of three tubes ofAPW and/or SPB and incubate at 30°C overnight.Subculture to TCSB and incubate at 37°C for20–24 h. Examine for vibrio-like colonies, identifythem and use the three-tube table for food samples(Roberts et al., 1995).

Colonial morphology

Table 24.2 lists the colony appearances of vibriosand related organisms on TCBS medium. On non-selective media colonial morphology is variable.The colonies may be opaque or translucent, flat ordomed, haemolytic or non-haemolytic, smooth orrough. One variant is rugose and adheres closely tothe medium.

Identification

The following tests enable the common species tobe identified. For more detailed tests, use the API20E and 20NE systems (Austin and Lee, 1992) butinclude 0/129 sensitivity and growth on CLED(cystine lactose electrolyte-deficient) medium.

Oxidation/fermentation testUse Hugh and Leifson method (see p. 96).

Oxidase testUse the method on p. 99 to test colonies from non-selective medium. Do not use colonies from TCBSor other media containing fermentable carbohy-drate because changes in pH may interfere with thereaction.

Arginine dihydrolaseUse arginine broth or Moeller’s medium with 1%NaCl (see p. 95).

Decarboxylase testsUse Moeller’s medium containing 1% NaCl, butdo not read the results too early because there is aninitial acid reaction before the medium becomesalkaline. The blank should give an acid reaction;

Vibrios 275

Table 24.2 Growth of vibrios on thiosulphate citrate bile salt agar (TCBS), 37°C for 18 h

Species of Vibrio Growth Acid from Sucrose Colour Colony size (mm)

V. cholerae + + Y 2–3 V. parahaemolyticus + − G 2–5 V. vulnificus + − G 2–3 V. alginolyticus + + Y 3–5 V. fluvialis + + Y 2–3 V. furnissi + + Y 2–3 V. metschnikovii + + Y 2–4 V. anguillarum + + Y 1–3 V. hollisae − − −V. damsela + − G 2–3

G, colonies green; Y, colonies yellow

failure to do so may suggest poor growth andthe electrolyte supplement should be added (seep. 75).

Carbohydrate fermentation testsUse peptone water sugars (see p. 73) with 1%NaCl.

IndoleUse peptone water containing 1% NaCl andKovac’s method (see p. 97).

Voges–Proskauer test Use a semisolid medium under controlled condi-tions. If incubation is prolonged and a sensitivemethod is used almost any vibrio may give positiveresults (Furniss et al., 1978).

Gelatin liquefactionUse gelatin agar (see p. 96).

ONPG testTest for β-galactosidase activity in ONPG brothwith 1% NaCl or use discs (see p. 99).

Growth at 43°CInoculate nutrient broth with 1% NaCl and incu-bate in a water bath.

Sensitivity to 2,4-diamino-6,7-di-isopropylpteridine (0/129)This was originally used as a disc method to distin-guish between vibrios (sensitive) and aeromonads(resistant), but the use of two discs (150 μg and10 μg) enables two groups of vibrios to be distin-guished. Make a lawn of the organisms on nutrientagar, and place one disc of each concentration of0/129 on it and incubate overnight. Do not useany special antibiotic sensitivity-testing mediumbecause the growth of vibrios and the diffusioncharacteristics of 0/129 differ from those onnutrient agar (Furniss et al., 1978).

Growth on CLED mediumSome vibrios require the addition of sodium chlo-ride to the medium whereas others do not. Culture

on an electrolyte-deficient medium, e.g. CLED.This permits two groups, halophilic and non-halophilic vibrios, to be distinguished. InoculateCLED lightly with the culture and incubate at 37°Covernight.

Salt toleranceThis varies with species. Inoculate peptone watercontaining 0, 3, 6, 8 and 10% NaCl. This tech-nique must be standardized to obtain consistentresults (Furniss et al., 1978).

Toxin detectionThere are kit tests (e.g. Oxoid) for detectingcholera toxin in culture filtrates. However, cultureswill need to be referred to international referencelaboratories in the case of isolates of V. choleraeO:1 and O:139.

PROPERTIES OF VIBRIOS,PLESIOMONAS AND AEROMONAS

These are shown in Tables 24.2 and 24.3.

Species of vibrios

Vibrio choleraeThis is sensitive to 0/129 and oxidase positive,decarboxylates lysine and ornithine, but does nothydrolyse arginine produces acid from sucrose,although no acid from arabinose or lactose. It growson CLED medium and is therefore nonhalophilic.

All strains possess the same heat-labile H antigenbut may be separated into serovars by their O anti-gens. There are 198 ‘O’ serotypes recognized at thepresent time.

Serovar O:1: the cholera vibrioThis is the causative organism of epidemic orAsiatic cholera. It is agglutinated by specific O:1cholera antiserum. Phage typing (Lee and Furniss,1991) is of epidemiological value. Non-toxigenicstrains of V. cholerae O:1 have been isolated andhave shown distinctive phage patterns.

276 Vibrio, Plesiomonas and Aeromonas

Table 24.3 Vibrio species and allied genera

Species Oxidase 0/129 Growth on NaCl VP Lysine Arginine Ornithine ONPG Acid CLED decarboxylase dihydrolyase decarboxylase hydrolysis from

10 μg 150 μg 0% 6% sucrose

V. cholerae + S S + + − + + − + + +V. parahaemolyticus + R S − − + − + − + – –V. vulnificus + S S − − + + + − + + –V. alginolyticus + R S − − + + + − + – +V. fluvialis + R S − − + − − + – + +V. furnissi + R S + v + − − + – + +V. metschnikovii − S S + + v v v + – v +V. anguillarum + S S v v v + − + – + +V . mimicus + S S + + − − + − + + –Aeromonas + R R + + − v v + – + +Plesiomonas + S S + + − − + + + + –

S, sensitive; R, resistant; v, variable

It is possible, by using carefully absorbed sera, todistinguish two subtypes of V. cholerae O:1. Theseare known as Ogawa and Inaba but as they are notcompletely stable and variation may occur in vitroand in vivo subtyping is of little epidemiologicalvalue.

Although there are two biotypes of V. choleraeO:1 – the ‘classic’ (non-haemolytic) and the ‘eltor’(haemolytic) – the former now virtually non-exis-tent. Biotyping is not, therefore, a useful epidemio-logical tool (Table 24.4).

Serovars other than O:1, non-O:1 V. choleraeThese serovars have the same biochemical charac-teristics as V. cholerae O:1 and the same H antigenbut possess different O antigens and are not agglu-tinated by the O:1 serum. They have been called‘non-agglutinating’ (NAG) vibrios. This is an obvi-ous misnomer; they are agglutinated both bycholera H antiserum and by antisera preparedagainst the particular O antigen they possess.Another term that has been used is ‘non-choleravibrio’ (NCV); as both terms have been used in dif-ferent ways, this has resulted in considerable con-fusion. It is best if all these vibrios are referred to asnon-O:1 V. cholerae, or, if a strain has beenserotyped, by that designation.

Vibrio cholerae O:139 (Bengal)In 1992 a new outbreak of cholera occurred in theIndian subcontinent. The causative organism wasidentified as V. cholerae serotype O:139 (Bengal).

This strain produces cholera toxin (CT) and didnot type with the existing antisera set (1–138) atthat time. The strain is resistant to the vibriostaticagent 0/129 and has not spread from the Indiansubcontinent.

Some of the other serotypes are undoubtedlypotential pathogens and produce a toxin similar to,if not identical with, that of the cholera vibrio.Some outbreaks have occurred but most isolateshave been from sporadic cases. These vibrios arewidespread in fresh and brackish water in manyparts of the world, including the UK. They do not,however, cause true epidemic cholera.

Vibrio parahaemolyticusThis halophilic marine vibrio will not grow onCLED. It does not ferment sucrose and thereforegives a (large) green colony on TCBS agar. O and Kantigens may be used to serotype strains. It is asso-ciated with outbreaks of gastroenteritis followingthe consumption of seafood and was recognized asthe most common cause of food poisoning inJapan. It is usually present in coastal waters,although only in the warmer months in the UK.The Kanagawa haemolysis test is said to correlatewith pathogenicity (see Barrow and Feltham,1993). Controlled conditions for testing thehaemolysis of the human red blood cells are essen-tial. Only laboratories with sufficient experienceshould do this test. Most environmental strains arenegative.

Vibrio vulnificusThese vibrios were first called L+ vibrios after theirability to ferment lactose. They are ONPG positiveand sensitive to 10 μg 0/129 disc. Care needs to betaken to distinguish them from V. parahaemolyti-cus. V. vulnificus produces sulphatase (opaque halo)on sodium dodecylsulphate polymyxin B sucrose(SPS) agar; V. parahaemolyticus does not (Kitauraet al., 1983; Donovan and van Netten, 1995).

Vibrio alginolyticusA very common marine (halophilic) vibrio ofworldwide distribution. It grows well in MAPWand on TCBS and other plating media. It produces


Table 24.4 Vibrio cholerae O:I biovars (biotypes)

Classic Eltor

Haemolysis − VVP − +Chick cell agglutination − +Polymyxin B 50 IU S R Classic phage IV S R Eltor phage 5 R S

S, sensitive; R, resistant

large sucrose-fermenting colonies on TCBS andmost strains will show swarming growth on marineor seawater agars. It is biochemically similar toV. parahaemolyticus, but is distinguished by theability to ferment sucrose. It does not cause humangastroenteritis. As a result of its presence in mostseawater, this vibrio has value as an indicatororganism for cooked seafoods.

Vibrio fluvialisFirst reported as group F vibrios, this is common inrivers, particularly in the brackish water of estuar-ies, and may cause gastroenteritis in humans.V. fluvialis needs to be distinguished fromV. anguillarum (see Table 24.3).

Vibrio furnissiThis was previously classified as V. fluvialis biovarII. It has been associated with human gastroenteri-tis in Japan and other parts of the East, and hasbeen isolated from the environment and seafood.

Vibrio metschnikoviiThis is both oxidase and nitrate reduction negative,but its other characters are typical of vibrios. Itincludes the now illegitimate species V. proteus. Itis frequently isolated from water and shellfish, butthere is little evidence of pathogenicity for humans.

Vibrio anguillarumThis is phenotypically far from being a uniformspecies. Some strains are pathogenic for fish,although not especially for the eel, as its namemight suggest. Many strains will not grow at 37°Cbut do grow at 30°C and would be missed in labo-ratories that confine their incubation temperaturesto about 37°C.

Vibrio hollisaeA halophilic vibrio isolated from human cases ofgastroenteritis, this does not grow on TCBS. It isdistinguished from other halophilic vibrios fromhuman sources by its negative lysine, arginine andornithine tests, and the limited range of carbohy-drates fermented. It has not been recovered fromthe environment and a clear relationship withhuman disease has not been established.

Vibrio damselaA marine bacterium isolated from human woundsand from water and skin ulcers of damsel fish. It isarginine positive, produces gas from glucose,requires NaCl for growth, is not bioluminescent,and ferments glucose, mannose and maltose. It isdistinguished from other named vibrios byDNA–DNA hybridization.

Vibrio mimicusResembles non-O:I V. cholerae but is VP negativeand does not ferment sucrose. Colonies on TCBSmedia are therefore green. It has been isolated fromthe environment and is associated with seafoods. Ithas also been isolated from human faeces. Somestrains produce a cholera-like toxin.

Plesiomonas shigelloides

This bacterium has had a varied taxonomic historyhaving been labelled C27 Paracolon, Pseudomonas,Aeromonas and Vibrio shigelloides. It is close to theenterobacteria and some strains are agglutinated byShigella sonnei antisera, hence the shigella-like nameshigelloides. However, this bacterium is oxidasepositive and sensitive to 0/129. These characteristicsdistinguish it from the enterobacteria.

It is now thought to be a cause of gastroenteritisbecause most isolates are from patients with diar-rhoea following travel.

There are no common specific enrichment meth-ods or media for Plesiomonas spp. It grows poorlyon TCBS, but well on enteric media such as DCAand MacConkey. Colonies are shigella-like and areidentified using a range of tests, as shown in Table24.3, with key characteristics being: sensitive to0/129 lysine, arginine and ornithine decarboxylasepositive, oxidase positive and cross-reactions withshigella antisera.

Aeromonas species

These are Gram-negative rods that are motile bypolar flagella. They are oxidase and catalase

Properties of vibrios, Plesiomonas and Aeromonas 279

positive, and resistant to 0/129. They metabolizecarbohydrates fermentatively (gas may be producedfrom glucose), liquefy gelatin and reduce nitrates.The arginine dihydrolase test using arginine brothis positive for most strains. Indole and VP reactionsvary with species. Some strains are psychrophilicand most grow at 10°C. See Tables 24.2 and 24.3for differentiation from other groups.

The genus is divided into two groups,Salmonicida and Hydrophila (Table 24.5).

The Salmonicida group This contains psychrophilic, non-motile aeromon-ads. They may be divided at the subspecies level andare associated with furunculosis of fish, a diseaseof economic importance in fish farming (Frerichsand Hendrie, 1985). The aeromonads in theSalmonicida group are not associated with humandisease and are not known to be found in food.

The Hydrophila groupAeromonads in this group are motile, mesophilicand grow at standard laboratory temperatures of30 and 37°C. This group contains a number ofclusters or species. Most workers recognize thespecies A. hydrophila, A. sobria and A. caviae

(Table 24.6). Other species, e.g. A. media,A. veronii and A. schuberti, have been described inrecent years. Until satisfactory identification sys-tems are available and validated it is prudent todivide the Hydrophila group only into the speciesA. hydrophila, A. sobria and A. caviae.

Species within this group may be the cause ofhuman gastroenteritis. Several workers havedetected virulence associated factors, e.g. entero-toxin production with clinical isolates. Thesefactors are found mainly in A. hydrophila andA. sobria, but less frequently with A. caviae iso-lates. In countries with high ambient temperatures,where aeromonads are common in drinking water,they are frequently isolated in both normal anddiarrhoeal faecal specimens. Aeromonads in thisgroup have been found in wound infections and asrare isolates from blood cultures.

Aeromonads in the Hydrophila group grow rap-idly in food even at refrigeration temperatures(Mattrick and Donovan, 1998a,b) and were foundin 54% of salad samples in a European CommunityCo-ordinated Food Control Programme in Englandand Wales in 1995 (Little et al., 1997). They arewide spread in nature and are frequently found inwater, animals, sewage, dairy products and fish.They may be misidentified as ‘coliforms’ becauselactose-fermenting strains are common.

IsolationAn incubation temperature of 30°C is recom-mended for all procedures. Aeromonas species willgrow on non-selective media, such as blood agar,which is suitable for clinical specimens other thanfaeces.


Table 24.5 Aeromonas groups

Group Motility Growth Brown at 37°C pigment

Hydrophila + + vSalmonicida − − +

Table 24.6 Aeromonas Hydrophila group

Species Aesculin Gas from Acid from Lysine VP Haemolysis H2Shydrolysis glucose arabinose decarboxylase

A. hydrophila + v + + + ++ +A. caviae + − + − − ± −A. sobria − + − + + ++ +

v, variable

FaecesPlate on selective agar such as bile salt irgasan bril-liant green agar (BSB) or Ryan’s modification ofXLD (RXLD (Mattrick and Donovan, 1998a).Aeromonas spp. produce non-xylose-fermentingcolonies on BSB and RXLD. Enrichment is not usu-ally needed for faeces.

FoodPlate food homogenates on BSB or RXLD andstarch ampicillin agar (SAA). This will detect largenumbers and may be used for quantitative proce-dures. For enrichment inoculate alkaline peptonewater (APW) and tryptone soy broth (TSB) con-taining 30 mg/l ampicillin and 40 mg/l irgasan(Mattrick and Donovan, 1998a).

WaterUse a membrane filtration method with SAA forthe quantitative procedures for both treated andraw water.

IdentificationFor a presumptive test do oxidase and spot indoletests on suspected colonies. Aeromonas spp. give apositive reaction in both.

To distinguish between the psychrophilicSalmonicida group and the mesophilic Hydrophilagroup, test for oxidase, fermentation on glucoseand resistance to 0/129 discs. For separation intothe two groups, test for growth at 20 and 37°C, formotility and a brown pigment on media containing1% tyrosine incubated at 20°C (see Table 24.5).

For identification to species level within theHydrophila group, test for aesculin hydrolysis,lysine decarboxylase, VP, gas from glucose, H2Sproduction, acid from arabinose and salicin, andarginine dihydrolase test for confirmation ofgeneric identification (see Table 24.6).

REFERENCES

Austin, B. and Lee, J. V. (1992) Aeromonadaceae andVibrionaceae. In: Board, R. G., Jones, D., Skinner,F. A. (eds), Identification Methods in Applied and

Environmental Microbiology. Society for AppliedBacteriology Technical Series No. 29. Oxford:Blackwells, pp. 163–183.

Barrow, G. I. and Feltham, R. K. A. (1993) Cowan and

Steel’s Manual for the Identification of Medical

Bacteria. Cambridge: Cambridge University Press.Donovan, T. J. and van Netten, P. (1995) Culture

media for the isolation and enumeration ofpathogenic Vibrio species in foods andenvironmental samples. International Journal of

Food Microbiology 26: 77– 91.Frerichs, G. N. and Hendrie, M. S. (1985) Bacteria

associated with disease of fish. In: Collins C. H.and Grange , J. M. (eds), Isolation and

Identification of Micro-organisms of Medical and

Veterinary Importance. Society for AppliedBacteriology Technical Series No. 21. London:Academic Press, pp. 355–371.

Furniss, A. L., Lee, J. V. and Donovan, T. J. (1978)The Vibrios. Public Health Laboratory ServiceMonograph No. 11. London: HMSO.

Kitaura, T., Doke, S., Azuma, I. et al. (1983) Haloproduction by sulphatase activity in V. vulnificus

and V. cholerae 0:1 on a new selective sodiumdodecyl sulphate containing medium: a screeningmarker in environmental surveillance. FEMS

Microbiology Letters 17: 205–209.Lee, J. V. and Furniss, A. L. (1981) Discussion 1. The

phage-typing of Vibrio cholerae serovar O1. In:Holme, T., Holmgren, J., Mersom, M. H. et al.(eds), Acute Enteric Infections in Children. New

prospects for treatment and prevention.Amsterdam: Elsevier, pp. 191–222

Little, C. L., Monsey, H. A., Nichols, G. L. et al. (1997)The microbiological quality of refrigerated salads andcrudités. PHLS Microbiological Digest 14: 142–146.

Mattrick, K. L. and Donovan, T. J. (1998a) Optimisationof the protocol for the detection of Aeromonas

species in ready-to-eat salads, and its use to speciateisolates and establish their prevalence. Communicable

Disease and Public Health 1: 263–266.Mattrick, K. L. and Donovan, T. J. (1998b) The risk to

public health of aeromonas in ready-to-eat saladproducts. Communicable Disease and Public

Health 1: 267–270.

References 281

25

Key to the enterobacteria

This informal group of Gram-negative non-sporingrods contains a number of species ofEnterobacteriaceae as well as some other species.Only those that are relatively common are consid-ered in the following chapters. They are aerobic,facultatively anaerobic, oxidase negative and fer-mentative in Hugh and Leifson medium. Details ofothers may be found in the latest edition of Topleyand Wilson’s Microbiology and MicrobialInfections (Collier et al., 1998) and in Cowan andSteel’s Manual for the Identification of MedicalBacteria (Barrow and Feltham, 1993).

The genera considered here and in the followingchapters may be conveniently divided into fourgroups:

1. Escherichia, Citrobacter, Klebsiella andEnterobacter spp., which usually produce acidand gas from lactose. For historical reasonsthey are known collectively as the coliformbacilli (see Chapter 26).

2. Edwardsiella, Hafnia and Serratia spp., whichusually do not ferment lactose, although someworkers include them in the coliforms (seeChapter 27).

3. Salmonella and Shigella spp., which do notferment lactose and are important intestinalpathogens (see Chapter 28).

4. Proteus, Providencia and Morganella spp.,which do not ferment lactose and differ fromthe other groups in their ability to deaminatephenylalanine (see Chapter 29).

Table 25.1 shows the general biochemical proper-ties of these genera.

The genus Yersinia is included among the enter-obacteria by some workers but for convenience isconsidered in this book in Chapter 34.

REFERENCES

Barrow, G.I. and Feltham, R. K. A. (1993) Cowan and


Bacteria, 3rd edn. Cambridge: CambridgeUniversity Press.

Collier, L., Balows, A. and Sussman, M. (eds) (1998)Topley and Wilson’s Microbiology and Microbial


Bacteriology. London: Arnold.

Table 25.1 General properties of enterobacteria

Genus Indole Motility Acid from Citrate Urease H2S Gelatin PPA* ONPG

Lactose Mannitol Inositolutilization liquefaction hydrolysis

Escherichia + + + + − − − − − − +Citrobacter v + v + − + − + − − +Klebsiella v + v + + + (+) − − − +

Ch. 26

Enterobacter − + v + − + − − − − +�

Erwinia − + v + − v* − − v − +Edwardsiella + + − − − v* − + − − −Hafnia − + − + − + − − − − +

Ch. 27

Serratia − + − + − + − − + − +�

Salmonella − + − + + + − + − − −Shigella v − − v − − − − − − + � Ch. 28

Proteus v + − v v v + v v + −Providencia + + − v + + − − − + − Ch. 29Morganella + + − − − v + − − + − �v, variable; (+) slow; v*, varies with method; * phenylalanine deaminase

These genera are included in the group of lactose-fermenting, Gram-negative, non-sporing bacilliinformally known as ‘coliform bacilli’, and aretherefore considered together in this chapter. Theyare oxidase negative and fermentative (as distinctfrom Pseudomonas spp.) and nutritionally non-exacting, and grow well on ordinary culture media.They also grow in the presence of bile, whichinhibits many cocci and Gram-positive bacilli.

Table 25.1 shows their general properties andthose that distinguish them from the other Gram-negative bacilli considered in Chapters 27–29.

ISOLATION

Pathological material

Plate stools, intestinal contents of animals, urinedeposits, pus, etc., on MacConkey, eosin methyl-ene blue (EMB) or Endo agar and on blood agar.Cystine lactose electrolyte-deficient medium(CLED) is useful in urinary bacteriology. Proteusdoes not spread on this medium. Incubate at 37°Covernight.

Foodstuffs

The organisms may be damaged and may not growfrom direct plating. Make 10% suspensions of thefood in tryptone soy broth. Incubate at 25°C for2 h and then subculture into brilliant green bilebroth and lauryl tryptose broth. MacConkey brothshould not be used because it supports the growth

of Clostridium perfringens, which produces acidand gas at 37°C. Minerals modified glutamatebroth may be useful for the recovery of ‘damaged’coliforms. Incubate at 37°C overnight and plate onMacConkey, Endo, EMB, violet red bile agar orCLED media.

IDENTIFICATION

Coliform bacilli show pink or red colonies,2–3 mm in diameter, on MacConkey agar.Klebsiella colonies may be large and mucoid. OnEMB agar, colonies of Escherichia coli areblue–black by transmitted light and have a metallicsheen by incident light. Colonies of klebsiellas arelarger, brownish, convex and mucoid, and tend tocoalesce. On Endo medium, the colonies of bothare deep red and colour the surrounding medium.They may have a golden yellow sheen.

For full investigation test as follows: H2S produc-tion (Triple Sugar Iron agar or Kligler medium),growth on Simmon’s citrate agar, lysine andornithine decarboxylases, arginine dihydrolase,urea hydrolysis, growth in KCN broth, phenyl-alanine deaminase, gelatin liquefaction and β-glucuronidase (β-GUR – see below and Table 26.1).

The following short cuts are useful.

Strains from clinical material

Examine a Gram-stained film: some cocci andGram-positive bacilli grow on selective media suchas MacConkey and CLED agar.

26

Escherichia, Citrobacter, Klebsiella andEnterobacter

Table 26.1 Escherichia, Citrobacter, Klebsiella and Enterobacter spp.

Indole Lysine Ornithine Arginine Citrate H2S Acid from Urease ONPG decarboxylase decarboxylase dihydrolase utilization hydrolysis37°C 44°C Adonitol Inositol

Escherichia coli + + + v v - - - - - vCitrobacter freundii v - - - + + + + - v +C. koseri + - - + + + - + - v +Klebsiella v - + - - + - + + + +

pneumoniaeK. oxytoca + - + - - + - + + + +K. ozaenae - - v - v + - + v - +K. rhinoscleromatis - - - - - - - + + - -Enterobacter - - + + - + - + + - +

aerogenesE. cloacae - - - + + + - v - v -

v, variable

From Donovan (1966)

Inoculate peptone water to test for indole produc-tion (at 37 and 44°C), motility and β-glucuronidase(β-GUR) test. Inoculate ONPG broth to test for β-galactosidase.

Non-clinical strains

Examine a Gram-stained film. Inoculate lactosebroth containing an indicator and a Durham’stube, and tryptone peptone water for indole test:incubate in a water bath at 44 ± 0.2°C. Include thefollowing controls:

• Stock E. coli, which produces gas and isindole positive

• Stock K. aerogenes which produces no gasand is indole negative.

Confirm lactose-fermenting colonies on mem-brane filters and in primary broth cultures by sub-culturing to tryptone peptone water and lactosebroth, testing for indole and gas production at 37and 44°C. Plate on MacConkey agar for colonyappearance. Do identification tests below.Fermentation of adonitol and inositol may be use-ful (Table 26.1).

β-GUR and MUG tests

These tests, together with the indole test, are veryuseful in distinguishing between Escherichia coliand other coliforms, especially in the examinationof foods.

The enzyme β-GUR is produced by over 90% ofstrains of E. coli but not by the O157 strain (seebelow). It is also produced by some other enter-obacteria (Perez et al., 1986).

Inoculate 0.5 ml of a 0.1% solution of p-nitro-phenyl-β-D-glucopyranoside in 0.067M phosphatebuffer with a loopful of an 18 h peptone waterculture. Incubate at 37°C for 4 h. A yellow colourindicates the release of p-nitrophenyl. A solidmedium, allowing several spot tests on one plate, isavailable (MAST).

MUG is a fluorescent agent – 4-methyl umbellif-eryl-β-D-glucuronide – which is commerciallyavailable for addition to culture media. Colonies ofE. coli fluoresce under ultraviolet (UV) light(source not greater then 6 W).

‘Short set’ method

A modification of the ‘short set’ system of Donovan(1966) uses tubed media (Table 26.2). There aretwo API systems: RAPIDEC coli and RAPIDECGUR, both of which include a β-GUR test.

Multipoint inoculation tests are cost-effective fortesting large number of strains. A ‘short set’ fromthe MAST range may be used, e.g. β-GUR, inositol,aesculin, hydrogen sulphide or the full MAST 15system. There are two that are aimed at E. coli: theColi strip (LabM, UK), which uses a fluorogenicsubstrate and an indole test; and ChromocultColiform agar (Merck, Germany) which usesβ-GUR and indole.

286 Escherichia, Citrobacter, Klebsiella and Enterobacter

Table 26.2 ‘Short set’ for coliforms

Species β-GUR Motility Inositol Aesculin H2S Indole

E. coli + + − − − + Klebsiella spp. − − + + − vC. freundii − + − − + − C. koseri − + − − − + Enterobacter spp. − + − v − −

v, variable; β-GUR, β-glucuronidase

ANTIGENS OF ENTEROBACTERIA

There are four kinds of antigens. The O or somaticantigens of the cell body are polysaccharides andare heat stable, resisting 100°C. The H or flagellarantigens are protein and destroyed at 60°C. The Kand Vi antigens are envelope, sheath or capsularantigens and heat labile. There are three kinds of Kantigen: L, which is destroyed at 100°C and is anenvelope, occasionally capsular; A, which isdestroyed at 121°C and is capsular; and B, which isdestroyed at 100°C and is an envelope. These Viand K antigens mask the O, and agglutination withO sera will not occur unless the bacterial suspen-sions are heated to inactivate them.

SPECIES OF COLIFORM BACILLI

Escherichia coli

This species is motile, produces acid and gas fromlactose at 44°C and at lower temperatures, is indolepositive at 44°C and 37°C, and fails to grow in cit-rate. It is MR positive, VP negative and H2S nega-tive and usually decarboxylates lysine. It does nothydrolyse urea or grow in KCN broth and does notliquefy gelatin. Most strains give positive β-GURand MUG tests.

This is the so-called ‘faecal coli’ which occursnormally in the human and animal intestine, and itis natural to assume that its presence in food indi-cates recent contamination with faeces. It is wide-spread in nature, however, and, although moststrains probably had their origin in faeces, its pres-ence, particularly in small numbers, does not neces-sarily mean that the food contains faecal matter. Itdoes suggest a low standard of hygiene. It seemsadvisable to avoid calling them ‘faecal coli’ and toreport the organisms as E. coli.

Although normally present as a commensal in thehuman and animal intestine, E. coli is also associ-ated with human and animal infections. It is themost common cause of urinary tract infections inhumans and is also found in suppurative lesions,

neonatal septicaemias and meningitis. In animals itcauses mastitis, pyometria in bitches, coli granulo-mas in fowls and white scours in calves.

Pathotypes of E. coli causing intestinal diseaseThere are at least four of these (Gorbach, 1986,1987), described as: enteropathogenic (EPEC),enterotoxigenic (ETEC), enteroinvasive (EIEC) andverotoxigenic (VTEC). A number of different Oserotypes in each pathotype have been incrimi-nated. The lists of these change frequently.

The EPEC strains have been associated with out-breaks of infantile diarrhoea and also travellers’diarrhoea, and are identified serologically (seeabove), but this is necessary only in outbreaks. It isnot recommended that single cases of EPEC beinvestigated in this way.

ETEC strains cause gastroenteritis in both adults(travellers’ diarrhoea) and children (especially indeveloping countries). The vehicles are contami-nated water and food. They produce enterotoxins,one of which is heat labile (LT) and the other heatstable (ST).

EIEC strains cause diarrhoea similar to that inshigellosis. The strains associated with invasivedysenteric infections are less reactive than typicalE. coli. They may be lysine negative, lactose nega-tive and anaerogenic, and resemble shigellas. Thecorrelation between serotype and pathogenicity isimperfect. Reference laboratories use tissue cultureand other specialized tests.

VTEC strains derive their name from their cyto-toxic effect on Vero cells in tissue culture. Theiralternative name is enterohaemorrhagic E. coli(EHEC). They have been associated withhaemolytic–uraemic syndrome and haemorrhagiccolitis, and are known to be responsible for food-borne disease (see Chapter 13).

Several kits are available for detecting and identi-fying these pathogens: Phadebact UK, VET-RPLA(Oxoid), E. COLI ST EIA (Oxoid).

Serological testing of E. coliScreen first by slide agglutination with the (com-mercially available) antisera. Heat saline suspen-

Species of coliform bacilli 287

sions of positives in a boiling water bath for 1 h todestroy H and K antigens. Cool, and do tube Oagglutination tests. A single tube test at 1 : 50 (witha control) is adequate. Read at 3 h and then leaveon the bench overnight and read again.

Serotype O157:H7 has been responsible for seri-ous outbreaks in recent years. It does not fermentsorbitol and may be recognized on SorbitolMacConkey agar and identified with commercialO157 antisera (Oxoid latex).

Kits for the identification of O157 strains includeMicroscreen (Microgen), Prolex (Prolab) andCaptivate (LabM).

Citrobacter freundii

This organism is motile, indole variable, grows incitrate and KCN media, and is ONPG positive. It isH2S positive but the lysine decarboxylase test isnegative. Urea hydrolysis is variable. Non- or late-lactose-fermenting strains occur. This organismoccurs naturally in soil. It can cause urinary tractand other infections in humans and animals.

Citrobacter koseri

This differs from C. freundii in that it is H2S nega-tive and does not grown in KCN medium. It isfound in urinary tract infections and occasionallyin meningitis.

Klebsiella pneumoniae

Also known as Friedlander’s pneumobacillus, thisis indole negative and gives a positive lysine decar-boxylase reaction, but does not produce H2S. It isurease and β-galactosidase positive. It may producelarge, mucoid colonies on media that containcarbohydrates.

It is associated with severe respiratory infectionsand can cause urinary tract infections.

Klebsiella aerogenes

There has always been some confusion betweenthis organism and K. pneumoniae because the lat-ter name was used in the USA. It is now regarded asa subspecies of K. pneumoniae. K. aerogenes is glu-conate positive but K. pneumoniae is gluconatenegative.

Klebsiella oxytoca

This differs from K. pneumoniae in being indolepositive.

Klebsiella ozoenae

Differs from K. pneumoniae in failing to hydrolyseurea. It is also difficult to identify, but is notuncommonly found in the respiratory tract associ-ated with chronic destruction of the bronchi.

Klebsiella rhinoscleromatis

Differs from K. pneumoniae in that it does notgrow in citrate media, is β-galactosidase negativeand fails to decarboxylate lysine. It is difficult toidentify, is rare in clinical material and may befound in granulomatous lesions in the upperrespiratory tract.

Enterobacter cloacae

This is motile, indole negative, MR negative andVP positive, grows in citrate and liquefies gelatin(but this property may be latent, delayed and lost).It does not produce H2S and the lysine decarboxy-lase test is negative. It is found in sewage and inpolluted water.

288 Escherichia, Citrobacter, Klebsiella and Enterobacter

Enterobacter aerogenes

This resembles E. cloacae but the lysine decarboxy-lase test is positive. Gelatin liquefaction is late. It isoften confused with K. aerogenes. E. aerogenes ismotile and urease negative; K. aerogenes is non-motile and urease positive (see Table 26.1).

THE NON-LACTOSE-FERMENTINGENTEROBACTERIA

For more detailed information, especially about themany ‘new’ species not considered above, seeBarrow and Feltham (1993), Altwegg andBockemühl (1999) and Holmes (1999).

REFERENCES

Altwegg, M. and Bockemühl, J. (1999) Escherichia andShigella. In: Collier, L., Balows, A. and Sussman,M. (eds) Topley and Wilson’s Microbiology and

Microbial Infections, 9th edn. Vol 2, Systematic

Bacteriology, CD-ROM. London: Arnold, Chapter40.

Barrow, G. I. and Feltham, R .K. A. (eds) (1993)Cowan and Steel’s Manual for the Identification of


Donovan, T. J. (1966) A Klebsiella screening medium.Journal of Medical Laboratory Technology 23:194–196.

Gorbach, S. L. (1986) Infectious Diarrhoea. London:Blackwells.

Gorbach, S. L. (1987) Bacterial diarrhoea and itstreatment. Lancet ii: 1378–1382.

Holmes, B. (1999) The Enterobacteriaceae: generalcharacteristics. In: Collier, L., Balows, A. andSussman, M. (eds) Topley and Wilson’s

Microbiology and Microbial Infections, 9th edn.Vol. 2, Systematic Bacteriology, CD-ROM.London: Arnold, Chapter 39.

Perez, J. L., Berrocal, C. I. and Berrocal, L. (1986)Evaluation of a commercial beta-glucuronidase testfor the rapid and economical identification ofEscherichia coli. Journal of Applied Bacteriology

61: 541–545.

References 289

27

Edwardsiella, Hafnia and Serratia

Although most species in these genera do not usu-ally ferment lactose, some workers include them inthe coliforms.

They are Gram-negative, motile, oxidase-nega-tive, fermentative (O/F test), non-sporing rods thatare catalase and citrate positive, urease negative(with exceptions), and arginine dihydrolase andphenylalanine deaminase negative (see Table 25.1).

IDENTIFICATION

Test for indole, lysine decarboxylase, fermentationof mannitol and inositol, H2S (with TSI), gelatinliquefaction and ONPG (Table 27.1).

SPECIES

Edwardsiella tarda

This is the only species of medical interest in thegenus Edwardsiella. It is the only member of thegroup that produces indole and H2S (in TSI) and

that does not ferment mannitol. It is lysine decar-boxylase positive and ONPG negative, and doesnot liquefy gelatin.

It is a fish pathogen and a rare pathogen ofhumans, when it may cause wound infection.Although it is occasionally isolated from patientswith diarrhoea, its causative role is uncertain.

Hafnia alvei

This gives reliable biochemical reactions only at20–22°C. It ferments mannitol, decarboxylateslysine, is ONPG positive, but is indole and H2S neg-ative and does not liquefy gelatin. It is a commen-sal in the intestines of animals, and present inwater, sewage and soil although it is rarely, if ever,pathogenic.

Serratia marcescens

Once known as Chromobacterium prodigiosus,this is known for the red pigment it sometimes pro-

Table 27.1 Edwardsiella, Hafnia and Serratia species

Species Indole Lysine Acid from H2Sa Gelatin ONPG

production decarboxylaseMannitol Inositol

liquefaction hydrolysis

Edwardsiella tarda + + − − + − −Hafnia alvei − + + v − − +Serratia marcescens − v + + − + +a TSI, Triple Sugar Ironv, variable

References 291

duces on agar media depending on incubationtemperature. It ferments mannitol and inositol,liquefies gelatin and is ONPG positive, but does notproduce indole or H2S.

Originally thought to be a saprophyte, it was usedin aerosol and filter testing experiments; it is nowknown to be associated with sepsis, hospital-acquired infections such as meningitis and endo-carditis, and urinary tract infections. Several ‘new’species of Serratia have emerged recently (Holmesand Aucken, 1998).

For more information about the biochemical reac-tions, etc. of these organisms see Farmer et al.(1985), Barrow and Feltham (1993), and Holmesand Aucken (1999).

REFERENCES

Barrow, G. I. and Feltham R. K. A. (1993) Cowan and



Farmer, J. J., Davis, B. R., Hickman-Brenner, F. W. et

al. (1985) Biochemical identification of new speciesand biogroups of Enterobacteriaceae. Journal of

Clinical Microbiology 21: 46–76.Holmes, B. and Aucken, H. M. (1999) Citrobacter,

Enterobacter, Klebsiella, Serratia and othermembers of the Enterobacteriaceae. In: Collier, L.,Balows, A. and Sussman, M. (eds), Topley and

Wilson’s Microbiology and Microbial Infections,9th edn. Vol 2, Systematic Bacteriology,CD-ROM. London: Arnold, Chapter 42.

These are important intestinal pathogens of mam-mals and, although they are not related generically,they are considered together in this chapter becausethe methods for their isolation use the same culturemedia and methods, and their identification ismainly by agglutination tests using polyvalent andspecific antisera. An outline of their antigenic struc-tures is therefore presented first.

SALMONELLA ANTIGENS ANDNOMENCLATURE

The Kauffmann–White (see Threlfall et al., 1999) sys-tem is used for the identification of salmonellasaccording to their antigenic components and thetables that are used for identification bear their names.

There are over 60 somatic or O antigens amongthe salmonellas and these occur characteristicallyin groups. Antigens 1–50 are distributed amonggroups A–Z. Subsequent groups are labelled51–61. This enables more than 1700 Salmonella‘species’ or serotypes to be divided into about 40groups with the more common organisms in thefirst six groups, e.g. in Table 28.1 each of theorganisms in group B possesses the antigens 4 and12 but, although 12 occurs elsewhere, 4 does not.Similarly, in group C all the organisms possess anti-gen 6; some possess, in addition, antigen 7 and oth-ers antigen 8. In group D, 9 is the common antigen.Unwanted antigens may be absorbed from the seraproduced against these organisms and single-factorO sera are available that enable almost any salmo-nella to be placed by slide agglutination into one ofthe groups. Various polyvalent sera are also avail-able commercially.

To identify the individual organisms in each group,however, it is necessary to determine the H or fla-gella antigen. Most salmonellas have two kinds of Hantigen and an individual cell may possess one or theother. A culture may therefore be composed of organ-isms, all of which have the same antigens or may bea mixture of both. The alternative sets of antigensare called phases and a culture may therefore be inphase 1, phase 2 or both phases simultaneously.

The antigens in phase 1 are given lower-case let-ters. Unfortunately, after z was reached more anti-gens were found and so subsequent antigens werenamed z1, z2, z3, etc. It is important to note that z1

and z2 are as different as are a and b; they are notmerely subtypes of z.

Phase 2 antigens are given the arabic numerals1–7 but a few of the lettered antigens also occur inphase 2 cells, mostly in groups, e.g. as e, h; e, n, z15;e, n, x; l, y, etc. Lettered antigens in phase 2, apartfrom these, are uncommon.

In identification procedures it is usual to find theO group first. Various polyvalent and single-factorO antisera are available commercially for this. TheH antigens are then found with a commercialRapid Salmonella Diagnostic (RSD) sera. These aremixtures of antisera that enable the phase 1 anti-gens to be determined. The phase 2 antigens arefound with a polyvalent serum containing factors1–7 and then individual sera.

Another antigen is the Vi, found in S. typhi and afew other species. This is a surface antigen thatmasks the O antigen. If it is present the organismsmay not agglutinate with O sera unless the suspen-sion is boiled.

As ‘new’ salmonellas are not infrequentlyreported, the Kauffmann–White scheme is brought

28

Salmonella and Shigella

Salmonella antigens and nomenclature 293

Table 28.1 Antigenic structure of some of the common salmonellae (Kauffmann–White classification)

Absorbed O Group Name Somatic (O) Flagellar (H)antisera antigen antigenavailable for

Phase 1 Phase 2identification

Factor 2 A S. paratyphi A 1, 2, 12 a –

Factor 4 B S. paratyphi B 1, 4, 5, 12 b 1, 2S. stanley 4, 5, 12 d 1, 2S. schwarzengrund 4, 12, 27 d 1, 7S. saintpaul 1, 4, 5, 12 e, h 1, 2S. reading 4, 5, 12 e, h 1, 5S. chester 4, 5, 12 e, h e, n, xS. abortus equi 4, 12 – e, n, xS. abortus bovis 1, 4, 12, 27 b e, n, xS. agona 1, 4, 12 gs –S. typhimurium 1, 4, 5, 12 i 1, 2S. bredeney 1, 4, 12, 27 l, v 1, 7S. heidelberg 1, 4, 5, 12 r 1, 2S. brancaster 1, 4, 12, 27 z29 –

Factor 7 C1 S. paratyphi C 6, 7, Vi c 1, 5S. cholerae-suisa 6, 7 c 1, 5S. typhi-suisa 6, 7 c 1, 5S. braenderup 6, 7 e, h e, n, z15

S. montevideo 6, 7 g, m, s –S. oranienburg 6, 7 m, t –S. thompson 6, 7 k 1, 5S. infantis 6, 7, 14 r 1, 5S. virchow 6, 7 r 1, 2S. bareilly 6, 7 y 1, 5

Factor 8 C2 S. tennessee 6, 8 z –S. muenchen 6, 8 d 1, 2S. newport 6, 8 e, h 1, 2S. bovis morbificans 6, 8 r 1, 5S. hadar 6, 8 z10 e, n, z

Factor 9 D S. typhi 9, 12, Vi d –S. enteritidis 1, 9, 12 g, m –S. dublin 1, 9, 12 g, p –S. panama 1, 9, 12 lv 1, 5

Factors 3, 10 E1 S. anatum 3, 10 e, h 1, 6S. meleagridis 3, 10 e, h I, wS. london 3, 10 l, v 1, 6S. give 3, 10 l, v 1, 7

Factor 19 E4 S. senftenburg 1, 3, 19 g, s, t –

Factor 11 F S. aberdeen 11 i 1, 2Factors 13, 22 G S. poona 13, 22 z 1, 6

Reproduced by permission of Dr F. Kauffman, formerly Director of the International Salmonella Centre using the originalnomenclature. The complete tables, revised regularly, are too large for inclusion here. They are obtainable from Salmonella Centres.a Identical serologically but differ biochemically

up to date every few years by the InternationalSalmonella Centre.

For many years salmonellas were given speciesstatus, the specific epithet often being that of theplaces where they were first isolated. Although thisserved clinical bacteriologists and public healthofficers very well, it was not in accord with the rulesof the International Code of Nomenclature ofBacteria and proposals were made by Popoff and LeMinor (1992) for a single species, with a newlycoined name Salmonella enterica. Eventually, on thegrounds of DNA hybridization studies, theSalmonella ‘species’ were reduced to two: S. entericaand S. bongori, with six subspecies of S. enterica:enterica, which includes the mammalian pathogens,and salamae, arizonae, diarizonae, houtenae andindica, all five of which are found in cold-bloodedanimals and the environment, and are not consid-ered in this book.

The names of serotypes of subspecies of S. enter-ica are written in roman, not italic, and given uppercase initials. Serotypes of the other subspecies nolonger have names but are designated by their anti-genic formula.

Fortunately it has been generally agreed that cor-rect names such as S. enterica ssp. enterica serotypeTyphimurium may now be written simply as S.Typhimurium.

Threlfall et al. (1999) give detailed accounts ofthese changes. Only serotypes in subspecies enter-ica are considered in this book.

SHIGELLA ANTIGENS

The genus Shigella is divided into four antigenicand biochemical subgroups, A–D (Table 28.2).

Subgroup A

Shigella dysenteriaeThere are 10 serotypes that have distinct antigensand do not ferment mannitol.

Subgroup B

Shigella flexneriThis contains six serotypes (1–6) that can bedivided into subserotypes according to their posses-sion of some group factors designated: 3,4; 4; 6; 7;and 7,8.

The X variant is an organism that has lost its typeantigen and is left with the group factors 7,8. TheY variant is an organism that has lost its type anti-gen and is left with the group factors 3,4.

Subgroup C

Shigella boydiiThere are 15 serotypes with distinct antigens; allferment mannitol.

Subgroup D

Shigella sonneiThis subgroup has only one, distinct serotype; thisferments mannitol. It may occur in phase 1 orphase 2, sometimes referred to as ‘smooth’ and‘rough’. The change from phase 1 to phase 2 is aloss of variation and phase 2 organisms are often

294 Salmonella and Shigella

Table 28.2 Shigella classification (see also Table 28.4)

Subgroup Species Serotypes

A: mannitol not fermented S. dysenteriae 1–10 all distinctB: mannitol fermented S. flexneri 1–6 all related; 1–4 divided into subserotypesC: mannitol fermented S. boydii 1–15 all distinctD: mannitol fermented, lactose fermented late S. sonnei 1 serotype

reluctant to agglutinate or give a very fine, slowagglutination. Phase 2 organisms are rarelyencountered in clinical work but the sera suppliedcommercially agglutinate both phases.

ISOLATION OF SALMONELLASAND SHIGELLAS

Choice of media

Liquid mediaThese are generally inhibitory to coliforms but lessso to salmonellas. Selenite F media are commonlyused. If they are not overheated selenite F mediawill permit the growth, but not the enrichment, ofS. sonnei and S. flexneri 6. Mannitol selenite andcystine selenite broths are preferred by some work-ers for the isolation of salmonellas from foods.Tetrathionate media enrich only salmonellas;shigellas do not grow.

Cultures from these media are plated on solidmedia.

Solid mediaMany media exist for the isolation of salmonellasand shigellas but in our experience the Hynes mod-ification of deoxycholate citrate agar (DCA) andxylose lysine deoxycholate agar (XLD) give thebest results for faeces. For the isolation of salmo-nellas from foods, XLD and brilliant green agar(BGA) are superior to DCA.

Colony appearance

Deoxycholate citrate agar On the ideal DCA medium, colonies of salmonellasand shigellas are not sticky. Salmonella colonies arecreamy brown, 2–3 mm in diameter at 24 h andusually have black or brown centres.Unfortunately, colonies of Proteus spp. are similar.Shigella colonies are smaller, usually slightly pinkand do not have black centres. S. Cholerae-suisgrows poorly on this medium. White, opaque

colonies are not significant. Some strains ofKlebsiella grow.

Xylose lysine deoxycholate agar Salmonella colonies are red with black centres,3–5 mm in diameter. Shigella colonies are red,2–4 mm in diameter.

Brilliant Green Agar Salmonella colonies are pale pink with a pink halo.Other organisms have yellow–green colonies with ayellow halo.

Procedure

Faeces, rectal swabs and urinesPlate faeces and rectal swabs on DCA and XLDagars. Inoculate selenite F broth with a portion ofstool about the size of a pea or with 0.5–1.0 ml liq-uid faeces. Break off rectal swabs in the broth. Addabout 10 ml urine to an equal volume of double-strength selenite F broth.

Incubate plates and broths at 37°C for 18–24 h.Plate broth cultures on DCA, XLD or BGA andincubate at 37°C for 18 h.

Drains and sewersFold three or four pieces of cotton gauze (approxi-mately 15 × 20 cm) into pads of 10 × 4 cm. Tie withstring and enclose in small-mesh chicken wire toprevent sabotage by rats. Autoclave in bulk andtransfer to individual plastic bags or glass jars.Suspend in the drain or sewer by a length of wireand leave for several days. Squeeze out the fluidinto a jar and add an equal volume of double-strength selenite F broth. Place the pad in anotherjar containing 200 ml single-strength selenite Fbroth. Incubate both jars at 43°C for 24 h and pro-ceed as for faeces described by Moore (1948) (seealso Vassiliades et al., 1978).

Foods, feedingstuffs and fertilizersThe method of choice depends on the nature of thesample and on the total bacterial count. In general,

Isolation of salmonellas and shigellas 295

raw foods such as comminuted meats have veryhigh total counts and salmonellas are likely to bepresent in large numbers. On the other hand, heat-treated and deep-frozen foods should have lowercounts and if any salmonellas are present they maysuffer from heat shock or cold shock and requireresuscitation and pre-enrichment.

Pre-enrichmentMake a 1 : 10 suspension or solution of the samplein buffered peptone water. Use 25 g food if possi-ble. For dried milk make the suspension in 0.002%brilliant green in sterile water. Use a Stomacher ifnecessary. Incubate at 37°C for 16–20 h.

EnrichmentAdd 10 ml of the pre-enrichment to 100 mlMuller–Kauffman tetrathionate broth (MK–TB),mixing well so that the calcium carbonate stabilizesthe pH. Incubate at 43°C for up to 48 h.

Selective platingAt 18–24 h and 42–48 h subculture from the sur-face of the MK–TB to brilliant green phenol redagar. Incubate at 37°C for 22–24 h.

Salmonella colonies are pink, smooth, low andconvex. Subculture at least two colonies for identi-fication.

Additional proceduresAdd 0.1 ml of the pre-enrichment to 10 mlRappaport–Vasiliades (RV) enrichment medium.Subculture at intervals as with MK–TB. Thismethod may increase isolation rates, as will addi-tional cultures on DCA and XLD.

Many rapid and automated methods have beendeveloped for determining the presence of salmo-nellas in foods. There is a variety of commerciallyavailable equipment and instrumentation.

IDENTIFICATION

Kit methods, for screening materials for salmonel-las, have proliferated and are useful if all one needsto know is whether these organisms (any serotype)

are present, and in epidemic circumstances whenlarge numbers of specimens must be tested. Theyinclude: the Tecra (Tecra Diagnostics, USA) whichis an immunocapture method; Spectate (RhônePoulenc, France), coloured latex particles with spe-cific antibodies; Salmonella Rapid (Oxoid, UK),which uses a purpose-designed culture vessel;Rapid Salmonella Latex (Oxoid), latex agglutina-tion; Microscreen (Mercia, UK); and MUCAP,which uses a fluorogenic substrate for testingcolonies.

For full identification of salmonellas and shigellasto species/serotype, there are two approaches:agglutination tests supported by screening or bio-chemical tests; and biochemical tests followed byagglutination tests. The first of these requires con-siderable expertise in the recognition of coloniesand in agglutination procedures, but gives ananswer much earlier, often within 24 h from pri-mary plates but certainly by 48 h after enrichment.

Agglutination tests for salmonellas

Polyvalent, specific and RSD sera are availablecommercially. The last consist of three or four mix-tures of specific and non-specific sera; agglutinationby combinations indicates the presence of morecommon antigens or groups of antigens.

Test the suspected organism by slide agglutinationwith polyvalent O sera. If positive, do further slideagglutinations with the relevant single-factor Osera; only one should be positive. If in doubt, testagainst 1 : 500 acriflavine; if this is positive, theorganisms are unlikely to be salmonellas.

Subculture into glucose broth and incubate in awater bath at 37°C for 3–4 h, when there will besufficient growth for tube H agglutinations. TubeH agglutinations are more reliable than slide Hagglutinations because cultures on solid media maynot be motile. The best results are obtained whenthere are about 6–8 × 108 organisms/ml. Dilute, ifnecessary, with formol saline. Add 0.5 ml formalinto 5 ml broth culture and allow to stand for at least10 min to kill the organisms.


Place one drop of polyvalent (phase 1 and 2) sal-monella H serum in a 75 × 9 mm tube. Add 0.5 mlof the formolized suspension and place in a waterbath at 52°C for 30 min. If agglutination occurs,the organism is probably a salmonella.

Identify the H antigens as follows. Do tube agglu-tinations against the RSD sera, S. Typhimurium (i)if it is not included in the RSD set, phase 2 complex1–7 EN (e, n) complex and L (l) complex. The man-ufacturer’s guide will indicate which phase 1 anti-gens are present according to which sera or groupsof sera give agglutination. Check the result, if pos-sible, by doing tube agglutinations with the indi-cated monospecific serum. If the test indicates EN(e, n), G or L (l) complexes, send the cultures to apublic health or communicable diseases laboratory.

If agglutination occurs in the phase 2 1–7 serum,test each of the single factors, 2, 5, 6 and 7. Notethat, with some commercial sera, factor 1 is notabsorbed and it may be necessary to dilute this outby using one drop of 1 : 10 or weaker serum.

Changing the H phaseIf agglutination is obtained with only one phase, theorganism must be induced to change to the otherphase. Cut a 50 × 20 mm ditch in a well-dried nutri-ent agar plate. Soak a strip of previously sterilizedfilter paper (36 × 7 mm) in the H serum by whichthe organism is agglutinated, and place this stripacross the ditch at right angles. At one end, placeone drop of 0.5% thioglycollic acid to neutralize anypreservative in the serum. At the other end, place afilter-paper disc, about 7 mm in diameter, so thathalf of it is on the serum strip and the other half onthe agar. Inoculate the opposite end of the strip witha young broth culture of the organism and incubateovernight. Remove the disc with sterile forceps,place it in glucose broth and incubate it in a waterbath for about 4 h, when there should be enoughgrowth to repeat agglutination tests to find the alter-native phase. Organisms in the original phasedemonstrated will have been agglutinated on thestrip. Organisms in the alternate phase will not beagglutinated and will have travelled across the strip.

The phase may also be changed with a Craigietube. Place 0.1 ml serum and 0.1 ml 0.5% thiogly-

collic acid in the inner tube of a Craigie tube, andinoculate the inner tube with the culture. Incubateovernight and subculture from the outer tube intoglucose broth.

Some organisms, e.g. S. Typhi, have only one phase.These should be sent to a reference laboratory.

Antigenic formulaeList the O, phase 1 and phase 2 antigens in thatorder and consult the Kauffmann–White tables forthe identity of the organism. In the table, the anti-genic formula is given in full, but as single factorsera for identification it is usually written thus:

S. Typhimurium 4, i, 2S. Typhi 9, d, —S. Newport 6, 8, e, h, 2

(The — sign indicates that the organism ismonophasic.)

The Vi antigenIf this antigen is present and prevents the organismsagglutinating with O group sera, make a thick sus-pension in saline from an agar slope and boil it in awater bath for 1 h. This destroys the Vi and permitsthe O agglutination.

Biochemical screening tests

Pick representative colonies on a urea slope as earlyas possible in the morning. Incubate in a water bathat 35–37°C. In the afternoon, reject any urea-posi-tive cultures and from the others inoculate the fol-lowing: Triple Sugar Iron (TSI) medium byspreading on slope and stabbing the butt; broth forindole test; and lysine decarboxylase medium.Incubate overnight at 37°C (Table 28.3) and pro-ceed to serological identification if indicated.

Full biochemical characters

For salmonella serotypes and subgenera it is best touse kits such as the API 20E, Mast ID-15 (Mast,

Identification 297

UK), Cobas ID and Enterotube (Roche, UK). SeeHolmes and Costas (1992) for more details ofthese.

Salmonella species and serotypes

Salmonella Typhi, and in some countries S.Paratyphi A (see national lists) are in HazardGroup 3. If tests suggest that a suspect organism isone of these all further investigations should beconducted under Containment Biosafety Level 3conditions.

Salmonella TyphiThis produces acid but no gas from glucose andmannitol. It may produce acid from dulcitol butfails to grow in citrate media and does not liquefygelatin. It decarboxylates lysine but not ornithineand fails to hydrolyse ONPG. Freshly isolatedstrains may not be motile at first. This organismcauses enteric fever.

Other serotypesThese produce acid and usually gas from glucose,mannitol and dulcitol (anaerogenic strains occur),and rare strains ferment lactose. They grow incitrate and do not liquefy gelatin. They decar-boxylate lysine and ornithine (except S. Paratyphi

A) and fail to hydrolyse ONPG. S. Paratyphi A,B and C may also cause enteric fever. Strainswith the antigenic formula 4; b; 1, 2 may beS. Paratyphi B, usually associated with entericfever, or S. Java which is usually associated withfood poisoning. Many other serotypes cause foodpoisoning.

Reference laboratories

It is not possible to identify all of the hundreds ofsalmonella serotypes with the commercial seraavailable, or in the smaller laboratory. The follow-ing organisms should be sent to a reference or com-municable diseases laboratory:

• Salmonellas placed by RSD sera into group E,G or L.

• Salmonellas agglutinated by polyvalent H ormonospecific H sera but not by phase II sera.

• Salmonellas agglutinated by polyvalent H butnot by phase I or phase II sera.

• Salmonellas giving the antigenic formula 4; b;1, 2, which may be either S. Paratyphi B or asubtype associated with food poisoning.

• S. Typhi, S. Paratyphi B, S. Typhimurium,S. Thompson, S. Virchow, S. Hadar andS. Enteritidis should be sent for phage typing.


Table 28.3 Biochemical reactions of Salmonella, Shigella, etc.

Organism TSIa H2Sb Indole ONPG Lysine

Butt Slopeproduction production hydrolysis decarboxylase

S. typhi A − +b − − +S. paratyphi A AG − − − − −Other salmonellas AG − ± − − +Shigella A − − ± ± −Citrobacter AG − + − + −

A, acid; AG, acid and gasa In TSI medium acid in the butt indicates the fermentation of glucose and in the slope that of lactose and/or sucroseb H2S production by S. typhi may be minimal

Note that Brevibacterium spp., which are some-times found in foods, possess salmonella antigens.These organisms are Gram positive.

Agglutination tests for shigellas

Do slide agglutinations with S. sonnei (phase 1 and2), polyvalent Flexner and polyvalent Boyd antis-era. If these are negative, test with S. dysenteriaeantiserum. If there is agglutination with the polyva-lent antisera test with the individual antisera:Flexner 1–6 and Boyd 1–5.

Sometimes there is no agglutination although bio-chemical tests indicate shigellas. This may resultfrom masking by a surface (K) antigen. Boil a sus-pension of the organisms and repeat the tests.

Strains that cannot be identified locally, i.e. givethe correct biochemical reactions but are not agglu-tinated by available sera, should be sent to a refer-ence laboratory.

Note that the motile organism Plesiomonasshigelloides (see p. 279) may be agglutinated byS. sonnei antiserum.

Biochemical tests

Test for acid production from glucose, lactose,sucrose, dulcitol and mannitol, indole production,lysine and ornithine decarboxylases (Table 28.4).Alternatively use one of the kits for entericpathogens.

Species of Shigella

Shigella dysenteriae (S. shiga)This forms acid but no gas from glucose, and doesnot ferment mannitol. Types 1, 3, 4, 5, 6, 9 and 10are indole negative. Types 2, 7 and 8 are indolepositive and were known as S. schmitzii(S. ambigua). S. dysenteriae is responsible for theclassic bacillary dysentery of the Far East.

Shigella flexneriSerotypes 1–5 form acid but no gas from glucoseand mannitol and produce indole. Some type 6strains (Newcastle strains) may not ferment manni-tol and does not produce indole. A bubble of gasmay be formed in the glucose tube. This serotype isnot inhibited by selenite media. Serotypes 1–6 arewidely distributed, especially in the Mediterraneanarea, and often cause outbreaks in mental and geri-atric hospitals, nurseries and schools in temperateclimes.

Shigella flexneri subserotypesSome manufacturers do not absorb group 6 factorfrom their type 3 serum and they also provide Xand Y variant sera containing group factors 7, 8and 3, 4. These sera may therefore be used todetermine the Flexner subserotypes for epidemio-logical purposes (Table 28.4).

Shigella sonneiThis forms acid but no gas from glucose and man-nitol, is indole negative and decarboxylates

Identification 299

Table 28.4 Shigellas

Species Acid from Indole Lysine Ornithine

Glucose Lactose Sucrose Dulcitol Mannitolproduction decarboxylase decarboxylase

S. dysenteriae + − − − − − /+ − −S. flexneri 1–5 + − − − + v − −S. flexneri 6 (Boyd 88) + (G) − − − + /− − − −S. sonnei + (+ ) (+ ) − + − − −S. boydii 1–15 + − − − + + /− − −

+ (G), a small buble of gas may be formed; −/+, most strains negative; +/−, most strains positive

None of these organisms grow in citrate or ferment salicin; none produce H2S, liquefy gelatin or are motile

ornithine. It causes the most common and mildestform of dysentery, which mostly affects babies andyoung children, and spreads rapidly throughschools and nurseries. It survives in some selenitemedia.

Shigella boydiiThis forms acid but no gas from glucose and man-nitol; indole production is variable. The 15serotypes of this species are widely distributed butnot common and cause mild dysentery.

REFERENCES

Holmes, B. and Costas, M. (1992) Methods and typingof Enterobacteriacea by computerized methods. In:Board, R. G., Jones, D. and Skinner, F. A. (eds),

Identification Methods in Applied and

Environmental Microbiology. Society for AppliedBacteriology Technical Series No. 29. Oxford:Blackwells, pp. 127–150.

Moore, B. (1948) The detection of paratyphoid Bcarriers by means of sewage examination. Monthly

Bulletin Ministry of Health and PHLS 6: 241–251.Popoff, M. Y. and Le Minor, L. (1992) Antigenic

formulas of the salmonella serovars, 6th rev. WHOCollaborating Centre for Reference and Researchon Salmonella. Paris: Institut Pasteur.

Threlfall, J., Ward, L. and Old, D. (1999) Changing thenomenclature of salmonella. Communicable

Disease and Public Health 2: 156–157. Vassiliades, P., Trichopoulos, D., Kalandidi, A. and

Xirouchaki, E. (1978) Isolation of salmonellasfrom sewage with a new enrichment method.Journal of Applied Bacteriology 44: 233–239.


These organisms are widely distributed in nature.Some strains of Proteus spread over the surface ofordinary agar media (‘swarming’), obscuringcolonies of other organisms. This may be preventedby using chloral hydrate agar. Swarming does nottake place on cystine lactose electrolyte-deficient(CLED) media and media containing bile salts.

The important characteristic that distinguishesthese genera from other enterobacteria is their abil-ity to deaminate phenylalanine (positive PPA test).Not all species are described here. For informationabout others, see Senior (1999).

ISOLATION

Plate material on nutrient or blood agar to observeswarming, and on CLED and chloral hydrate agarto prevent swarming and reveal any other organ-isms. Incubate at 37°C overnight. Add heavily con-taminated material to tetrathionate broth, incubateovernight and plate as above.

IDENTIFICATION

Test PPA positive strains for motility, indole pro-duction, fermentation of mannitol and inositol,citrate utilization, H2S production, gelatin lique-faction and urease (Table 29.1).

Proteus species

Proteus vulgarisThis is indole positive, produces H2S (lead acetatepaper test) but does not ferment mannitol or inosi-tol. Many strains swarm on ordinary media (seeabove).

It is found in the intestines of humans and animalsand is an opportunist pathogen in the urinary tract,wounds and other lesions. It is a food spoilageorganism, widely distributed in soil and sewage-polluted water.

29

Proteus, Providencia and Morganella

Table 29.1 Proteus, Providencia and Morganella species

Species Motility Indole Acid from Citrate H2S Gelatin Ureaseproduction utilization production liquefaction

Mannitol Inositol

Proteus vulgaris v + − − v + v +P. mirabilis + − − − + + + +Providencia alcalifaciens v + − − + v − −P. rettgeri + + + + + + − +P. stuartii v + − + + − − −Morganella morganii + + − − v + − +

v, variable

Proteus mirabilisDiffers mainly from P. vulgaris in not producingindole. It is a doubtful agent of gastroenteritis,found in urinary tract and hospital-acquired infec-tions, and septic lesions. It is associated with putre-fying animal and vegetable matter.

Providencia species

Providencia alcalifaciensThis differs from Proteus spp. in not possessingurease. It is found in clinical material where it maybe an opportunist pathogen.

Providencia rettgeriThis ferments mannitol and inositol, produces H2S(on lead acetate paper but not in Triple Sugar Iron[TSI]) and is urease positive. It is associated with‘fowl typhoid’, and urinary tract and opportunistinfections in humans.

Providencia stuartiiFerments inositol but not mannitol, may produceH2S and is urease negative. It has been reported in

hospital infections, especially in catheterizedelderly people

Morganella species

Morganella morganiiThis is the only species so far described. It does notferment mannitol or inositol but fails to produceH2S and is urease positive. It is a commensal in theintestines of humans and other animals, found inhospital infections and may be associated with‘summer diarrhoea’ in children.

REFERENCE

Senior, B. W. (1999) Proteus, Morganella andProvidencia. In: Collier L, Balows, A. andSussman, M. (eds), Topley and Wilson’s


302 Proteus, Providencia and Morganella

The 13 genera considered here may be divided intofour groups, on the basis of common character-istics.

1. Brucella, Bordetella and Moraxella spp. arenon-motile, cannot grow anaerobically, arecatalase and oxidase positive, do not changeHugh and Leifson medium, and have nospecial growth requirements (see Chapter 31).

2. Haemophilus, Gardnerella and Streptobacillusspp. are non-motile, can grow anaerobically,are fermentative in Hugh and Leifsonmedium, and have special growthrequirements (see Chapter 32).

3. Campylobacter and Helicobacter spp., whichare motile, can grow anaerobically, do notchange Hugh and Leifson medium, and haveno special growth requirements, although theyare microaerophilic (see Chapter 33).

4. Actinobacillus, Pasteurella, Yersinia,Cardiobacterium and Francisella spp. arenon-motile, can grow anaerobically, arefermentative in Hugh and Leifson medium,and have no special growth requirements (seeChapter 34).

Table 30.1 shows the general characteristics.Those that assist in identification within the groupsare given in the appropriate chapters.

30Key to some miscellaneous aerobic,

non-sporing, Gram-negative bacilli of medicalimportance

Table 30.1 Gram-negative non-sporing bacilli

Genus Motility Facultative Catalase Oxidase O/Fa Growth factoranaerobe requirement

Brucella - - + +b - -Bordetella - - + v - -Moraxella - - + + - -

Haemophilus - + v + F +Gardnerella - + - - F +Streptobacillus - + - - F +

Campylobacter + vd v + - -Helicobacter + vd + + - -

Actinobacillus - + + +c F -Pasteurella - + + + F -Yersinia - + + - F -Cardiobacterium - + - + F -Francisella - + + v F -a Oxidation/fermentation test, Hugh and Leifson mediumb Usuallyc Except A. actinomecetemcomitansd micro-aerophilicv, variable

31

Brucella, Bordetella and Moraxella

BRUCELLA

The genus Brucella contains Hazard Group 3pathogens. All manipulations, especially thosethat might produce aerosols, should be done inmicrobiological safety cabinets in Biosafety/Containment Level 3 laboratories.

There are three important and several other speciesof small, Gram-negative bacilli in this genus. Theyare non-motile and reduce nitrates to nitrites, butcarbohydrates are not metabolized when normalcultural methods are used. There are several bio-types within some species.

Isolation

Human diseaseDo blood cultures. Any system may be used but theCasteñada double-phase method (see p. 157) inwhich the solid and liquid media are in the samebottle lessens the risk of contamination duringrepeated subculture, and also minimizes the haz-ards of infection of laboratory workers. UseBrucella agar plus growth and selective supple-ments.

Examine Casteñada bottles weekly for growth onthe solid phase. If none is seen, tilt the bottle toflood the agar with the liquid, restore it to a verti-cal position and reincubate. Do not discard until 6weeks have elapsed.

Treat bone marrow and liver biopsies in the sameway. Subculture on brucella-selective media and onserum glucose agar or chocolate blood agar.

Animal materialPlate uterine and cervical swabbings or homoge-nized fetal tissue on Brucella-selective medium

Milk

Dip a throat swab in the gravity cream and inocu-late brucella-selective medium. Incubate all cul-tures at 37°C under 10% CO2 and examine dailyfor up to 5 days.

Identification

Colonies on primary media are small, flat orslightly raised and translucent. Subculture onslopes of glucose tryptone agar with a moistenedlead acetate paper in the upper part of the tube totest for hydrogen sulphide production.

Do dye inhibition tests. In reference laboratoriesseveral concentrations of thionin and basic fuchsinare used to identify biotypes. For diagnostic pur-poses inoculate tubes of brucella-selective mediumor glucose tryptone serum agar containing (1)1 : 50 000 thionin and (2) 1 : 50 000 basic fuchsin(National Aniline Division, Allied Chemical andDye Corp.). Inoculate tubes or plates with a smallloopful of a 24-h culture.

It is advisable to control Brucella identificationwith reference strains, obtainable from TypeCulture Collections or CDCs: B. abortus 554,B. melitensis 16M and B. suis 1330 (Table 31.1).

Do slide agglutination tests with available com-mercial sera.

Species of Brucella

Brucella melitensisThis grows on blood agar and on glucose tryptoneserum agar aerobically in 3–4 days. It does notneed carbon dioxide to initiate growth. It does notproduce hydrogen sulphide and is inhibited by1 : 50 000 fuchsin or thionin. This organism causesbrucellosis in humans, the Mediterranean or Maltafever. The reservoirs are sheep and goats, and infec-tion occurs by drinking goats’ milk. There are threebiotypes.

Brucella abortusGrows on blood agar and on glucose tryptoneserum in 3–4 days. Most strains require 5–10%carbon dioxide to initiate growth. Strains producehydrogen sulphide, and vary in their susceptibilityto the presence of 1 : 50 000 thionin and fuchsin.There are eight biotypes, some of which resembleB. melitensis.

Brucella abortus causes contagious abortion incattle. Drinking infected milk can result in undu-lant fever in humans. Veterinarians and stockmenare frequently infected from aerosols released dur-ing birth or abortion of infected animals.Laboratory infections are usually acquired fromaerosols released by faulty techniques.

Brucella suisThe five biotypes of this species do not require car-bon dioxide for primary growth. Biotype 1 pro-

duces abundant hydrogen sulphide; the others donot. None is inhibited by 1 : 50 000 thionin butthey vary in their inhibition by 1 : 50 000 fuchsin.They cause contagious abortion in pigs and mayinfect humans, reindeer and hares.

Of the other three species (not considered here), B.ovis (sheep), B. canis (dogs) and B. neotomae (desertwood rats), only B. canis appears to have causeddisease in humans (Corbel and MacMillan, 1999).

Detection in milk

The milk ring test The antigen for this test may be obtained from aveterinary or commercial laboratory. It is a suspen-sion of B. abortus cells stained with haematoxylinor another dye

The test may be applied to bulk raw milk samplesor to those from individual animals. In the lattercase take samples from all four quarters of theudder. Store the milk samples overnight at 4°Cbefore testing.

Add one drop (0.03 ml) of the stained antigen to1 ml well-mixed raw milk in a narrow tube(75 × 9 mm). Mix immediately by inverting severaltimes and allow to stand for 1 h at 37°C.

If the milk contains antibodies, these will aggluti-nate the antigen and the stained aggregates ofbacilli will rise with the cream, giving a blue creamline above a white column of milk. Weak positivesgive a blue cream line and a blue colour in the milk.

Brucella 305

Table 31.1 Brucella species

Species Growth in 1 : 50 000 Needs CO2 H2S

Thionin Basic fuschsin

B. melitensis + + - -B. abortus -a +a +a +a

B. suis biogroup 1 + -a - +B. suis other biogroups + -a - -B. canis + -a - -a Most strains

306 Brucella, Bordetella and Moraxella

A white cream line above blue milk shows absenceof antibodies. It may be necessary to add knownnegative cream to low-fat milk.

False positives may be obtained with milk col-lected at the beginning and end of lactation, prob-ably as a result of leakage of serum antibodies intothe milk. Recent immunization with the S19 strainmay affect results.

The ring test does not give satisfactory resultswith pasteurized milk or with goats’ milk.

Whey agglutination testThis is a useful test when applied to milk from indi-vidual cows but is of questionable value for testingbulk milk.

Centrifuge quarter milk and remove the cream.Add a few drops of renin to the skimmed milk andincubate at 37°C for about 6 h. When coagulated,centrifuge and set up doubling dilutions of thewhey from 1 : 10 to 1 : 2560, using 1-ml amountsin 75 × 9 mm tubes. Add one drop of standard con-centrated B. abortus suspension and place in awater bath at 37°C for 24 h. Read the agglutina-tion titre. More than 1 : 40 is evidence of udderinfection unless the animal has been immunizedrecently.

Serological diagnosisApart from the standard agglutination test (above)there are others that are outside the scope of thisbook. They include complement fixation tests and

enzyme-linked immunosorbent assay (ELISA).Corbel and Hendry (1985) give useful informationabout these.

For general reviews on the laboratory diagnosis ofbrucellosis, see Corbel (1998) and Corbel andMacMillan (1999).

BORDETELLA

The bacilli in this genus are small, 1.5 × 0.3 μm andregular. Nitrates are not reduced and carbohy-drates are not attacked.


Pernasal swabs (preferably of calcium alginate) arebetter than cough plates, but the swabs should besent to the laboratory in one of the commercialtransport media. Plate on Bordetella, or CharcoalAgar medium containing cephalexin 40 mg/l(Oxoid Bordetella supplement).

Incubate under conditions of high humidity andat 37°C for 3 days. Examine daily and identify byslide agglutination, using commercially availablesera. FA reagents are also available.

Test for growth and pigment formation on nutri-ent agar, urease, nitrate reductase and motility(Table 31.2).

Table 31.2 Bordetella species

Species Growth on Brown Urease Nitrate Motilitynutrient coloration reduction agar

B. pertussis - - - - -B. parapertussis + + + - -B. bronchiseptica + - + + +

Species of Bordetella

Bordetella pertussisGrowth on the above media has been described aslooking like a ‘streak of aluminium paint’. Coloniesare small (about 1 mm in diameter) and pearlygrey. This organism cannot grow in primary cul-ture without heated blood, but may adapt togrowth on nutrient agar on subculture. B. pertussisis oxidase positive, nitrate and urease negative. Itdoes not grow on MacConkey agar and showsβ-haemolysis on blood agar.

Bordetella pertussis causes whooping cough. Fordetailed information about the bacteriologicaldiagnosis of this disease, see Wardlaw (1990) andParton (1998).

Bordetella parapertussis

Growth on blood agar may take 48 h and a brownpigment is formed under the colonies. It grows onnutrient and on MacConkey agar. On Bordetella orsimilar media, the pearly colonies (‘aluminiumpaint’) develop earlier than those of B. pertussis. Itdoes not change Hugh and Leifson medium anddoes not metabolize carbohydrates. It is oxidaseand nitrate negative but urease positive.

This organism is one of the causative organisms ofwhooping cough.

Bordetella bronchisepticaThis organism has been in the genera Brucella andHaemophilus. It forms small smooth colonies,occasionally haemolytic on blood agar, grows bestat 37°C, and is motile, oxidase, nitrate and ureasepositive.

It causes bronchopneumonia in dogs, often asso-ciated with distemper, bronchopneumonia inrodents and snuffles in rabbits. It has been associ-ated with whooping cough.

This genus is discussed in detail by Pittman andWardlaw (1981), Wardlaw (1990), and Parton(1998).

MORAXELLA

The bacilli are plump (2 × 1 μm), often in pairs endto end, and non-motile. They are oxidase positive anddo not attack sugars. They do not produce indole, donot produce hydrogen sulphide and are sensitive topenicillin. Some species require enriched media.

Plate exudate, conjunctival fluid, etc., on bloodagar and incubate at 37°C overnight. Subculturecolonies of plump, oxidase-positive, Gram-negativebacilli on blood agar and nutrient agar (notenriched), on gelatin agar (use the plate method)and on Loeffler medium and incubate at 37°C. Dothe nitrate reduction and catalase tests (Table 31.3).

Moraxella 307

Table 31.3 Moraxella species

Species Growth on Gelatin Nitrate Catalase Urease nutrient agar liquefaction reduction

M. lacunata v + + + −M. nonliquefaciens v − + + −M. bovis + + v + −M. osloensis + − v + −M. catarrhalisa + − v −K. kingiib + − − − −a Formerly Branhamellab Kingella, formerly Moraxellav, variable

Species of Moraxella

Moraxella lacunataColonies on blood agar are small and may behaemolytic. There is no or very poor growth onnon-enriched media. Colonies on Loeffler mediumare not visible but are indicated by pits of liquefac-tion (‘lacunae’). Gelatin is liquefied slowly. Nitratesare reduced.

This organism is associated with angular conjunc-tivitis and is known as the Morax–Axenfeld bacil-lus.

Moraxella liquefaciensThis is similar to M. lacunata and may be a biotypeof that species. It liquefies gelatin rapidly.

Moraxella nonliquefaciensThis is also similar to M. lacunata but fails to liq-uefy gelatin.

Moraxella bovisThis requires enriched medium, liquefies gelatinand may reduce nitrates. It causes pink eye in cattlebut has not been reported in human disease.

Moraxella osloensis (M. duplex, Mima polymorphavar. oxidans)An enriched medium is not required. Gelatin is notliquefied and nitrates may be reduced. It is foundon the skin, and in the eyes and respiratory tract ofhumans, but its pathogenicity is uncertain.

Moraxella kingii (now Kingella kingii)Colonies are haemolytic and may be mistaken forhaemolytic streptococci or haemolytichaemophilus. It does not require enriched media.This is the only member of the group that is cata-lase negative. It has been found in joint lesions andin the respiratory tract.

Moraxella catarrhalisFormerly known as Branhamella (and also asNeisseria) catarrhalis, this grows well on blood

agar, forming non-pigmented, non-haemolyticcolonies. It is β-lactamase and DNase positive(Buchanan, 1998). Carbohydrates are not fer-mented; oxidase and catalase tests are negative. Italso degrades tributyrin (a commercial butylasestrip test is available).

Moraxella catarrhalis is found in human mate-rial. Previously regarded as a commensal of therespiratory tract it is now considered to be anopportunist pathogen of the upper and lowerrespiratory system and is associated with lungabscesses. Middle-ear infections are not uncom-mon.

REFERENCES

Buchanan, B. K. (1998) Moraxella, Branhamella,

Kingella and Eikenella. In: Collier L, Balows, A.and Sussman, M. (eds), Topley and Wilson’s

Microbiology and Microbial Infections, 9th edn.Vol. 2, Systematic Bacteriology. London: Arnold,Chapter 48.

Corbel, M. J. (1998) Brucella. In: Collier L, Balows, A.and Sussman, M. (eds), Topley and Wilson’s


Corbel, M. J. and Hendry, D. (1985) Brucellas. In:Collins, C. H. and Grange, J. M. (eds), Isolation

and Identification of Micro-organisms of Medical

Importance. Society for Applied BacteriologyTechnical Series No. 21. London: Academic Press,pp. 53–82.

Corbel, M. J. and MacMillan, A. P. (1999) Brucellosis. In: Collier L, Balows, A. and Sussman,M. (eds), Topley and Wilson’s Microbiology and

Microbial Infections, 9th edn. Vol. 3, Bacterial

Infections, CD-ROM. London: Arnold, Chapter41.

Parton, R. (1998) Bordetella. In: Collier L, Balows, A.and Sussman, M. (eds), Topley and Wilson’s


308 Brucella, Bordetella and Moraxella

Pittman, M. and Wardlaw, A. C. (1981) The genusBordetella. In: Starr, M. P., Stolp, H., Truper, G.,Balows, A. and Schlegel, A. (eds), The Prokaryotes:

A handbook of habitats, isolation and

identification of bacteria. New York: Springer-Verlag, pp. 1506–1529.

Wardlaw, A. C. (1990) Bordetella. In: Parker, M. T.and Duerden, B. I. (eds), Topley and Wilson’s

Principles of Bacteriology, Virology and Immunity,8th edn, vol. 2. Edward Arnold: London, pp.321–338.

References 309

32

Haemophilus, Gardnerella and Streptobacillus

HAEMOPHILUS

Members of this genus are in general small coc-cobacilli (1.5 × 0.3 μm), although they may bepleomorphic and sometimes filamentous. They areGram negative and non-motile, and reduce nitrateto nitrite.

They fail to grow on ordinary media, requiringeither or both of two factors that are present inblood: X factor, which is haematin, and V factor,which is nicotinamide adenine dinucleotide(NAD) and can be replaced by coenzymes I or II.The X factor is heat stable and the V factor isheat labile. V factor is synthesized byStaphylococcus aureus.


Plate sputum, cerebrospinal fluid (CSF), eyeswabs, etc., on chocolate agar, GC agar base plus1% yeast autolysate and 5% sheep bloodprepared as chocolate agar (Rennie et al., 1992).Incubate at 37°C for 18–24 h. Colonies areusually 1–2 mm, grey and translucent. Gramstaining may show filamentous forms in culturesfrom spinal fluids.

‘Satellitism’, i.e. large colonies around colonies ofS. aureus and other organisms synthesizing V fac-tor, may be observed.

Test for X and V factor requirements as follows:subculture several colonies and spread on nutrientagar to make a lawn. Place X, V and X + V factordiscs on the medium and incubate at 37°Covernight. Observe growth around the discs, i.e.

whether the organisms require both X and V or Xor V or neither (Table 32.1).

Agglutination and capsular swelling testsDo slide agglutination tests using commercial serato confirm identity. These are not satisfactory withcapsulated strains because agglutination can occurwith more than one serum. To test for capsuleswelling with homologous sera, use a very light sus-pension of the organism in saline, coloured with fil-tered methylene blue solution. Mix a drop of thiswith a drop of serum on a slide and cover with acover slip. Examine with an oil immersion lenswith reduced light. Swollen capsules should beobvious, compared with non-capsulated organ-isms. More information about these tests is givenby Turk (1982).

Antigen detectionHaemophilus antigen may be directly detected inCSF by countercurrent immunoelectrophoresis(McIntyre, 1987)

Species of Haemophilus

Haemophilus influenzaeThis requires both X and V factors, produces acidfrom glucose but not lactose or maltose, and variesin indole production. It occurs naturally in thenasopharynx and is associated with upper respira-tory tract infections, including sinusitis and life-threatening acute epiglottitis. It also causespneumonia, particularly post-influenzal. In eye

infections (‘pink eye’), it is known as theKoch–Weekes bacillus. It is also found in the nor-mal vagina and is one of the causative organisms ofpurulent meningitis and purulent otitis media.There are several serological types and growth‘phases’

Haemophilus parainfluenzaeThis differs only in not requiring X factor. It pro-duces porphyrins from 6-aminolaevulinic acid. It isnormally present in the throat, but may be patho-genic.

Haemophilus haemolyticus and parahaemolyticusColonies of these are haemolytic on blood agar andotherwise have the same properties as H. influenzaeand H. parainfluenzae, respectively.

Haemophilus haemoglobinophilus (H. canis)This requires X but not V factor. It is found inpreputial infections in dogs and in the respiratorytract of humans.

Haemophilus ducreyiDucrey’s bacillus is associated with chancroid orsoft sore. In direct films of clinical material, the

organisms appear in a ‘school of fish’ arrangement.They are difficult to grow but may be obtained inpure culture by withdrawing pus from a bubo andinoculating inspissated whole rabbit blood slopesor 30% rabbit blood nutrient agar orMueller–Hinton medium containing Isovitalex.Culture at 30–34°C but no higher, in 10% carbondioxide. Colonies at 48 h are green, grey or brown,intact and easily pushed along the surface of themedia. It requires X but not V factor.

Haemophilus aphrophilus

Colonies on chocolate agar are small (0.5 mm) at24 h, smooth and translucent. Better growth isobtained in a 10% carbon dioxide atmosphere.Some strains require X factor. There is no growthon MacConkey agar. Acid is produced from glu-cose, maltose, lactose and sucrose but not frommannitol. Fermentation media should be enrichedwith Fildes’ extract. It is oxidase and catalase neg-ative. This organism closely resemblesActinobacillus actinomycetemcomitans (see p. 318)but it does not produce acid from lactose andsucrose (see Table 34.1).

Human infections, including endocarditis, havebeen reported. For more information about thegenus Haemophilus, see Slack and Jordans (1999).

Haemophilus 311

Table 32.1 Haemophilus species

Species Haemolysis Factors required Growth on blood enhanced byagar X + V V only X only 10% CO2

H. influenzae − + −H. parainfluenzae − + −H. haemolyticus + + −H. parahaemolyticus + + −H. haemoglobinophilus − + −H. ducreyi v + vH. aphrophilus − v +

v, variable

GARDNERELLA

The species Gardnerella vaginalis was previouslyknown as Haemophilus vaginalis orCorynebacterium vaginale. It is Gram variable,pleomorphic, non-motile and facultatively anaero-bic.

Direct examination

Giemsa-stained films made from vaginal secretionsusually show many squamous epithelial cells cov-ered with large numbers of organisms (‘clue cells’).Gram-stained films show large numbers of Gram-indifferent bacilli rather than the usual Gram-posi-tive lactobacilli.

Culture

Place specimens in transport medium (Stuart’s orAmies’). Plate on Columbia blood agar plus sup-plement (Oxoid) and incubate under 5–10% car-bon dioxide, e.g. in a candle jar. Colonies at 48 hare very small (1 mm), glistening ‘dew drops’ andβ-haemolytic on human blood agar. Gram-stainedfilms show thin, poorly stained bacilli (unlike thesolidly stained diphtheroids). They are Grampositive when young but become Gram negativelater.

Make lawns on two plates of Columbia bloodagar. On one place a drop of 3% hydrogenperoxide. On the other place discs of trimethoprim(5 μg). Incubate for 48 h. Gardnerellas areinhibited by the peroxide and are sensitive totrimethoprim. Lactobacilli and diphtheroids areresistant. Neither X nor V factor is required. Thereis no growth on MacConkey agar and, apart fromacid from glucose (add Fildes’ extract), biochemicaltests all seem to be negative.

Subculture on blood agar and apply discs ofmetronidazole (50 μg) and sulphonamide (100 μg)(Oxoid). G. vaginalis is sensitive to metronidazolebut resistant to sulphonamide.

Two other tests are useful: SPS sensitivity and hip-purate hydrolysis. Place a disc (commercially avail-able) of sodium polyethanol sulphate (SPS) on theblood plate and incubate overnight. Gardnerellasare sensitive and show β haemolysis. For the hip-purate test make a suspension of the organism in1 ml hippurate broth, or saline containing a (com-mercial) hippurate disc. Incubate for 4 h and thenadd a few drops of ninhydrin reagent. Hippuratehydrolysis is indicated by a deep blue–purplecolour (Forbes et al., 1998).

The AP120 kit is useful. G. vaginalis is found inthe human vagina and may be associated with non-specific vaginitis (see Easmon and Ison, 1985).

STREPTOBACILLUS

There is only one species, Streptobacillus monili-formis, which may be filamentous with or withoutswelling. It is fastidious, requiring blood or serumfor growth. It is a commensal of rodents and apathogen of mice, and is one of the agents of rat-bite fever in humans. Milk-borne disease (Haverhillfever) has been reported.


Blood culture is the usual method of isolation, butmedia containing Liquoid should be avoidedbecause this inhibits the growth of the organism.Inoculate enriched broth medium with aspiratedfluids from joints, etc.

The organism grows slowly in liquid media, form-ing fluffy balls or colonies that resemble bread-crumbs. Remove these with a Pasteur pipette forGram staining and subculture on blood agar.Incubate at 37°C for 3 days, when colonies will beraised, granular and 1–4 mm in diameter. L-formcolonies may be seen (Barrow and Feltham, 1993).

Streptobacillus moniliformis is non-motile, a fac-ultative anaerobe, catalase and oxidase negativeand fermentative in Hugh and Leifson medium (seeTable 30.1).

312 Haemophilus, Gardnerella and Streptobacillus

References 313

REFERENCES

Barrow, G. I. and Feltham, R. K. A. (1993) Cowan and



Easmon, C. S. F. and Ison, C. A. (1985) Gardnerella

vaginalis. In: Collins, C. H. and Grange, J. M.(eds), Isolation and Identification of Micro-

organisms of Medical and Veterinary Importance.Society for Applied Bacteriology Technical SeriesNo. 21. London: Academic Press, pp. 115–122.

Forbes, B. A., Sahm, D. F. and Weissfeld, A. S. (1998)Bailey and Scott’s Diagnostic Microbiology, 10thedn. St Louis, MO: Mosby.

McIntyre, M. (1987) Counter-currentimmunoelectrophoresis for the rapid detection ofbacterial polysaccharide antigen in body fluids. In:

Immunological Techniques in Microbiology

Grange, J. M., Fox, A. and Morgan, N. L. (eds),Society for Applied Bacteriology Technical SeriesNo. 24. Oxford: Blackwell, pp. 137–143.

Rennie, R., Gordon, T., Yaschak, Y. et al. (1992)Laboratory and clinical evaluations of media forthe primary isolation of Haemophilus species.Journal of Clinical Microbiology 30: 1917–1921.

Slack, M. P. E. and Jordens, J. Z. (1999) Haemophilus.In: Collier L, Balows, A. and Sussman, M. (eds),Topley and Wilson’s Microbiology and Microbial



Turk, D. C. (1982) Haemophilus influenzae. PublicHealth Laboratory Service Monograph. London:HMSO.

These organisms were originally in the genusVibrio, then in Campylobacter, from whichHelicobacter was recently separated because of anumber of taxonomic differences.

CAMPYLOBACTER

These small, curved, actively motile rods aremicroaerophilic, reduce nitrates to nitrites, but donot metabolize carbohydrates. They have becomevery important in recent years as a cause of foodpoisoning (see Chapter 13). There are about 20species but only those that are frequently encoun-tered are considered here.

Isolation

FaecesPlate emulsions of faeces on blood agar containing(commercially available) campylobacter growthand antibiotic selective supplements. Incubateduplicate cultures at 37 and 42°C for 24–48 h in anatmosphere of approximately 5% oxygen, 10%carbon dioxide and 85% nitrogen, preferably usinga gas-generating kit, but otherwise a candle jar.

FoodsThese may have been frozen, chilled or heated, allof which may cause sublethal damage to the organ-isms.

MilkFilter 200 ml through a cottonwool plug. Place theplug in a sterile jar containing 100 ml campylobac-

ter enrichment medium without antibiotics.Incubate at 37°C for 2 h. Then add selective antibi-otics and incubate at 43°C for 36 h. Subculturefrom the surface layer to selective campylobacteragar and incubate plates at 43°C for 36–48 h undermicroaerophilic conditions.

WaterFilter 100 ml through a 0.45 μm membrane andplace the membrane face down on selective campy-lobacter agar. Incubate at 43°C for 24 h undermicroaerophilic conditions. Remove the filter andreincubate plates at 43°C for 36–48 h.

Place the filter in 100 ml enrichment broth andproceed as for milk.

Meat, poultry, etc.Add 10 g to 100 ml campylobacter-selective brothplus antibiotic supplements and incubate at 37°Cfor 2 h. Then continue incubation at 43°C and pro-ceed as for milk.

Enzyme-linked immunosorbent assay (ELISA)techniques are now available for the detection ofcampylobacters in food (Fricker and Park, 1987).

Identification

Campylobacter colonies are about 1 mm diameterat 24 h, grey, watery and flat. Examine Gram-stained films and test for growth at 43°C, hippu-rate hydrolysis, H2S production in triple sugar iron(TSI) medium and growth in the presence of 3.5%NaCl. Use media that are known to supportgrowth of the organisms and include controls withknown species (Table 33.1).

33

Campylobacter and Helicobacter

The CAMPY kit (BioMerieux) and Microscreen(Microgen) are useful.

Campylobacter species

The species that grow at 43°C are called the ‘ther-mophilic campylobacters’ and are those most oftenisolated from humans.

Campylobacter jejuniThis is an important agent of acute enterocolitis inhumans (see ‘Campylobacter enteritis in food poi-soning’, Chapter 13). It is also a commensal in theintestines of many wild and domestic animals.

Campylobacter coliAlso responsible for acute gastroenteritis inhumans, this is less common than C. jejuni.

Campylobacter lariThis is the least common cause of campylobacterenteritis.

Campylobacter fetusThe subspecies fetus is an agent of infectious abor-tion in cattle and sheep and occasionally infectshumans.

Campylobacter hyointestinalisThis is a pathogen of pigs and cattle and is some-times responsible for diarrhoea in humans.

Campylobacter sputorumThis species is a commensal in the mouth of humans.

There are a number of subspecies and biotypes ofcampylobacters. For information about biotyping,etc., see Bolton et al. (1992).

Campylobacters have received much attention inthe last few years (see Griffiths and Park, 1990;Skirrow, 1990; Bolton et al., 1992; Nachamkin andSkirrow, 1999). See also the review in relation tothe aquatic environment by Thomas et al. (1999).

HELICOBACTER

Helicobacters are motile, curved, Gram-negative,microaerophlic bacilli. There are about 14 speciesbut only one, H. pylori, is considered here, becauseof its association with gastric cancer and pepticulcers.

Direct microscopy

Stain smears with haematoxylin and eosin or by theGiemsa method and look for characteristic curvedor S-shaped rods.


Use freshly collected biopsy material (otherwise usea transport medium). Culture on one of the

Helicobacter 315

Table 33.1 Campylobacter species

Species Growth at Hippurate hydrolysis H2S in TSI Growth in 3.5%

25°C 43°CNaCl medium

C. jejuni − + + − −C. coli − + − + −C. lari − + − + −C. fetus + v − − −C. hyointestinalis v + − v −C. sputorum − v − + +

v, variable

316 Campylobacter and Helicobacter

commercial campylobacter media plus supple-ments. Incubate for 3–5 days under microaerobicconditions (5–20% CO2) and high humidity as forcampylobacters. Colonies are circular, convex andtranslucent.

Test for oxidase, catalase and urease. H. pylorigives positive reactions for all three (campylobac-ters are urease negative).

Other tests

The ClotestThis a commercial test (CLO = campylobacter-likeorganisms; Tri-Med Specialities, Utah, USA). It is aplastic tray containing a urea agar media and anindicator. A small piece of biopsy material isembedded in the medium. After 1–24 h, a colourchange from yellow to magenta indicates the pres-ence of a helicobacter (Murray et al., 1999).

The urea breath testThe patient is given urea labelled with a carbon iso-tope. This is hydrolysed to ammonia and carbondioxide if H. pylori is present in the stomach andthe latter is exhaled. This is measured with a scin-tillation counter or mass spectrometer, dependingon which carbon isotope was used, indicating theamount of urease activity of the organism in thestomach.

Molecular and ELISA methods are also available.There are at least two useful diagnostic kits –CAMPY, RAPIDEC Pylori (Biomerieux) andSigma EIA. For more information, see Skirrow(1990), Blaser, (1998) and Glupczynski (1999).

REFERENCES

Blaser, M. J. (1998) Helicobacter pylori and gastricdisease. British Medical Journal 316: 1507–1510.

Bolton, F. J., Waring, D. R. A., Skirrow, M. B. andHutchinson, D. N. (1992) Identification and

biotyping of campylobacters. In: Board, R. G.,Jones, D. and Skinner, F. A. (eds), Identification

Methods in Applied and Environmental

Microbiology. Society for Applied MicrobiologyTechnical Series No. 29. Oxford: Blackwell, pp.151–162.

Fricker, C. R. and Park, R. W. A. (1987) CompetitiveELISA, co-agglutination and haemagglutination forthe detection and serotyping of campylobacters. In:Grange , J. M., Fox, A. and Morgan, N. L. (eds),Immunological Techniques in Microbiology.Society for Applied Bacteriology Technical SeriesNo. 24. Oxford: Blackwells, pp. 195–210.

Glupczynski, Y. (1999) Infections with Helicobacter.

In: Collier L, Balows, A. and Sussman, M. (eds),Topley and Wilson’s Microbiology and Microbial


Bacteriology, CD/ROM. London: Arnold, Chapter30.

Griffiths, P. L. and Park, R. W. A. (1990)Campylobacters associated with human diarrhoealdisease. Journal of Applied Bacteriology 69:281–301.

Nachamkin, I. and Skirrow, M. B. (1999)Campylobacter, Arcobacter and Helicobacter. In:Collier L, Balows, A. and Sussman, M. (eds),Topley and Wilson’s Microbiology and Microbial


Bacteriology, CD/ROM. London: Arnold, Chapter54.

Murray, P. R., Baron, E. J., Pfaller, M.A. et al. (1999)Manual of Clinical Microbiology, 7th edn.Washington: American Society of Microbiologists.

Skirrow, M. B. (1990) Campylobacter, Helicobacter

and other motile, curved Gram-negative rods. In:Parker, M. Y. and Duerden, B. I. (eds), Topley and

Wilson’s Principles of Bacteriology, Virology and

Immunity, 8th edn. London: Edward Arnold, pp.531–549.

Thomas, C., Gibson, H., Hill, D. J. and Mabey, M.(1999) Campylobacter epidemiology: an aquaticperspective. Journal of Applied Bacteriology

Symposium Supplement 85: 168S–177S.

The general properties of the organisms describedin this chapter are shown in Table 30.1.

ACTINOBACILLUS

This genus contains non-motile, Gram-negativerods that are fermentative but do not produce gas,and are indole negative. Most strains grow onMacConkey agar and are ONPG positive. Someare human and animal pathogens; others are com-mensals.


Plate pus, which may contain small white granules,or macerated tissue on blood agar and incubate at37°C for 48–72 h under a 10% carbon dioxideatmosphere. Colonies are small, sticky andadherent. Subculture small flat colonies on to

MacConkey agar, in Hugh and Leifson medium,and test for motility, catalase, oxidase, acidproduction from lactose, mannitol and sucrose,and aesculin hydrolysis (Table 34.1, and see Table30.1).

Species of Actinobacillus

Actinobacillus lignieresiiThis species grows on MacConkey agar, is fermen-tative, and produces acid from lactose (late), man-nitol and sucrose.

It is oxidase, catalase and urease positive, butdoes not hydrolyse aesculin. It is associated withwoody tongue in cattle and human infections havebeen reported.

Actinobacillus equuliThis species resembles A. lignieresii but fermentslactose rapidly. Lactose, sucrose and mannitol are

34

Actinobacillus, Pasteurella, Yersinia,Cardiobacterium and Francisella

Table 34.1 Actinobacillus species

Species Oxidase Growth on Acid from Aesculin UreaseMacConkey

Lactose Mannitol Sucrosehydrolysis

A. lignieresii + + v + + − +A. equuli v + + + + − +A. suis v + + − − + +A. actinomycetecomitans + v − v − −

v, variable

fermented. It is urease positive but aesculin is nothydrolysed. It is associated with joint ill and sleepydisease of foals.

Actinobacillus actinomycetemcomitansThis organism requires carbon dioxide for pri-mary isolation and some strains require the Xfactor. Use an enriched medium and place an Xfactor disc on the heavy part of the inoculum.There is no growth on MacConkey agar and theoxidase test is positive. Lactose and sucrose arenot fermented but some strains may produceacid from mannitol. It is aesculin and urease neg-ative.

This organism is difficult to distinguish fromHaemophilus aphrophilus (see Table 32.1). It issometimes associated with infections byActinomyces israelii and it has been recovered fromblood cultures of patients with endocarditis.

Actinobacillus suisThis hydrolyses aesculin and does not fermentmannitol. It has been isolated from pigs.

For further information about the identificationof these organisms, see Barrow and Feltham (1993)and Holmes (1999).

YERSINIA

Yersinias cause plague, pseudotuberculosis andgastroenteritis in humans and animals. They arenow classified among the enterobacteria but areretained here for pragmatic reasons. They are cata-lase positive, fermentative in Hugh and Leifsonmedium (slow reaction), reduce nitrates to nitritesand fail to liquefy gelatin.

The plague bacillus, Yersinia pestis, is in HazardGroup 3 and all work should be done in a microbio-logical safety cabinet in a Biosafety/ContainmentLevel 3 laboratory. After isolation and presumptiveidentification cultures should be sent to a referencelaboratory for any further tests.


Yersinia pestisIn suspected bubonic plague examine pus frombuboes, in pneumonic plague, sputum, in septi-caemic plague, blood, and in rats the heart blood,enlarged lymph nodes and spleen. Before examin-ing rats immerse them in disinfectant for severalhours to kill fleas that might be infected.

Make Gram- and methylene blue-stained filmsand look for small oval Gram-negative bacilli withcapsules. Bipolar staining is often seen in films offresh isolates stained with methylene blue. Plate onblood agar containing 0.025% sodium sulphite toreduce the oxygen tension, on 3% salt agar and onMacConkey agar. Incubate for 24 h.

Colonies of plague bacilli are flat or convex, grey-ish white and about 1 mm in diameter at 24 h.Subculture into nutrient broth and cover with liq-uid paraffin. Test for motility at 22°C, fermenta-tion of salicin and sucrose, aesculin hydrolysis andurease.

Films of colonies on salt agar show pear-shapedand globular forms. Y. pestis grows on MacConkeyagar and usually produces stalactite growth inbroth covered with paraffin. It is non-motile at 37and 22°C. It produces acid from salicin but notsucrose and hydrolyses aesculin, but is urease neg-ative (Table 34.2).

Reference and communicable diseases laborato-ries use fluorescence antibody (FA) methods, bacte-riophage, specific agglutination tests, precipitintests and animal inoculations.

Yersinia enterocolitica

Culture faeces on yersinia-selective medium (cefsu-lodin–irgasan–novobiocin agar, CIN). Incubate at30°C for 24–48 h and look for ‘bulls’ eye’ colonies.Enrichment is not usually recommended for isolat-ing this organism from faeces. Some strains growon deoxycholate agar (DCA) as small non-lactosefermenting colonies, even after incubation at 37°C.

Make a 10% suspension of food in peptone waterdiluent. Incubate some at 32°C for 24 h.Refrigerate the remaining suspension at 4°C for up

318 Actinobacillus, Pasteurella, Yersinia, Cardiobacterium and Francisella

to 21 days and subculture at intervals on yersinia-selective agar. Media used for other entericpathogens can be reincubated at 28–30°C for 24 hand examined for Y. enterocolitica but some strainsof this organism are inhibited by bile salts anddeoxycholate.

Gram-stained films show small coccoid bacilli,unlike the longer bacilli of other enterobacteria.

Test for growth on nutrient and MacConkeyagar, motility at 22°C, acid from sucrose, aesculinhydrolysis and urease. Kits may be used for all ormost of these. Incubate at 30°C rather than at 37°C(see Table 34.2).

There are several serotypes and biotypes. Someare pathogenic for humans (O:3, O:5, O:8, O:9),others for animals. Serological tests for specificantibodies may be of value in diagnosis.

Yersinia enterocolitica causes enteritis in humansand animals. In humans there may also be lym-phadenitis, septicaemia, arthritis and erythemanodosum. The reservoirs are pigs, cattle, poultry,rats, cats, dogs and chinchillas. The organism hasbeen found in milk and milk products, water, oys-ters and mussels. It grows at 4°C and may thereforemultiply in cold storage of food. Person-to-personspread occurs, mainly in families.

Yersinia fredericksenii, Y. kristensenii and Y. inter-media resemble Y. enterocolitica but their roles indisease are uncertain. For further information, seeBrewer and Corbel (1985), Mair and Fox (1986),Doyle and Cliver (1990), and Holmes (1999).

Yersinia pseudotuberculosisHomogenize lymph nodes, etc. in tryptone broth.Inoculate blood agar with some of the suspension.

Retain the remainder at 4°C and subculture toblood agar every 2–3 days for up to 3 weeks.

Emulsify faeces in peptone water diluent andinoculate yersinia-selective medium (CIN).Colonies on blood agar are raised, sometimesumbonate, granular and about 1–2 mm in diame-ter. Rough variants occur. Growth on MacConkeyagar is obvious but poor. Y. pseudotuberculosis ismotile at 22°C, ferments salicin but not sucrose,hydrolyses aesculin and is urease positive (seeTable 34.2).

This organism causes pseudotuberculosis inrodents, especially in guinea-pigs. Human infec-tions occur and symptoms resemble those of infec-tion with Y. enterocolitica. Y. pseudotuberculosisis rarely isolated from faeces, however, and sero-logical tests for specific antibodies are useful. Formore information about yersinias, see Wanger(1999).

PASTEURELLA

Pasteurella multocida

Bipolar staining and pleomorphism are less obvi-ous than in Yersinia species. The bacilli are verysmall (about 1.5 × 0.3 μm) and non-motile.Colonies on nutrient agar (37°C) are translucent,slightly raised, and about 1 mm in diameter at 24 h;on blood agar they are slightly larger, more opaqueand non-haemolytic. There is no growth onMacConkey agar. Most strains produce acid frommannitol, indole and urease produce. They do nothydrolyse aesculin (Table 34.3).

Pasteurella 319

Table 34.2 Yersinia species

Species Motility at 22°C Acid from sucrose Aesculin hydrolysis Urease

Y. pestis − − + −Y. pseudotuberculosis + − + +Y. enterocolitica + + − +

This organism causes haemorrhagic septicaemiain domestic and wild animals and birds, e.g. fowlcholera, swine plague, transit fever. Humans maybe infected by animal bites or by inhaling dropletsfrom animal sneezes. It has also been isolated fromseptic fingers.

It is often given specific names according to itsanimal host, e.g. avicida, aviseptica, suilla, suisep-tica, bovicida, boviseptica, ovicida, oviseptica,cuniculocida, lepiseptica, muricida, muriseptica.There is some host interspecificity: cattle strains arealso pathogenic for mice but not for fowls; fowlcholera strains are pathogenic for cattle and mice;lamb septicaemia strains are not pathogenic forrodents.

Pasteurella pneumotropica, P. haemolyticaand P. gallinarum

These species are not infrequently isolated fromveterinary material.

For further information about the genusPasteurella, see Carter (1984), Curtis (1985),Barrow and Feltham (1993), and Holmes (1999).

CARDIOBACTERIUM

Cardiobacterium hominis

This is a facultative anaerobe and requires anenriched medium and high humidity. Growth isbest under 7–10% carbon dioxide. Colonies on

blood agar at 37°C are minute at 24 h and about1 mm in diameter after 48 h, and are convex, glossyand butyrous. There is no growth on MacConkeymedium, the oxidase test is positive and glucoseand sucrose, but not lactose, are fermented withoutgas production. The catalase, urease and nitratereduction tests are negative. Hydrogen sulphide isproduced but gelatin is not liquefied.

This organism has been isolated from blood cul-tures of patients with endocarditis but may also befound in the upper respiratory tract.

FRANCISELLA

Francisella tularensis is in Hazard Group 3. It ishighly infectious and has caused many laboratoryinfections. It should be handled with great care inmicrobiological safety cabinets in Biosafety/Containment Level 3 laboratory.

Culture blood, exudate, pus or homogenized tissueon several slopes of blood agar (enriched), cystineglucose agar and inspissated egg yolk medium.Also inoculate tubes of plain nutrient agar.Incubate for 3–6 days and examine for very smalldrop-like colonies. If the material is heavily con-taminated, add 100 μg/ml cycloheximide or200 units/ml nystatin, and 2.5–5 μg/ml neomycin.

Subculture colonies of small, swollen or pleomor-phic Gram-negative bacilli on blood and nutrientagar, on MacConkey agar and in enriched nutrientbroth. Test for acid production from glucose andmaltose, motility at 22°C and urease activity.

320 Actinobacillus, Pasteurella, Yersinia, Cardiobacterium and Francisella

Table 34.3 Pasteurella species

Species Growth on Acid from Aesculin hydrolysis Indole UreaseMacConkey mannitol production

P. multocida − (+) − (+) −P. pneumotropica v − − + +P. haemolytica + − v − −

v, variable; (+) usually positive

The bacilli are very small, show bipolar stainingand are non-motile. There is no growth on nutrientor MacConkey agar, and poor growth on bloodagar with very small, grey colonies; on inspissatedegg, at 3–4 days, colonies are minute and drop like.It is a strict aerobe. Acid is formed in glucose andsucrose. It is oxidase positive and produces indole.

F. tularensis is responsible for a plague-like dis-ease (tularaemia) in ground squirrels and otherrodents in western USA and Scandinavia.

Cultures and materials in cases of suspectedtularaemia should be sent to a reference laboratory.Fluorescent antibody procedures are used for rapiddiagnosis.

The genus Francisella is described in detail byEigelsbach and McGann (1984) and Nano (1992).

REFERENCES

Barrow, G. I. and Feltham, R .K. A. (eds) (1993)Cowan and Steel’s Manual for the Identification of


Brewer, R. A. and Corbel, M. J. (1985) Yersiniaenterocolitica and related species. In: Collins, C. H.and Grange, J. M. (eds), Isolation and



Carter, G. R. (1984) Pasteurella. In: Krieg, N. R. andHolt, J. G. (eds), Bergey’s Manual of Systematic

Bacteriology, Vol. 1. Baltimore, MA: Williams &Wilkins, pp. 552–558.

Curtis, P. E. (1985) Pasteurella multocida. In: Collins,C. H. and Grange, J. M. (eds), Isolation and



Doyle, M. P. and Cliver, D. O. (1990) Yersinia

enterocolitica. In: Cliver D. O. (ed), Foodborne

Diseases. New York: Academic Press, pp.224–229.

Eigelsbach, H. T. and McGann, V. G. (1984) GenusFrancisella. In: Krieg, N. R. and Holt, J. G. (eds),Bergey’s Manual of Systematic Bacteriology, Vol.1. Baltimore, MA: Williams & Wilkins, p. 394.

Holmes, B. (1999) Actinobacillus, Pasteurella andEikenella. In: Collier L, Balows, A. and Sussman,M. (eds), Topley and Wilson’s Microbiology and

Microbial Infections, 9th edn. Vol. 2, Systematic


Mair, N. S. and Fox, E. (1986) Yersiniosis: Laboratory

diagnosis, clinical features and epidemiology.London: Public Health Laboratory Service.

Nano, F. E. (1992) The genus Francisella. In: Balows,A., Truper, H. G., Dworkin, M. et al. (eds), The

Prokaryotes, 2nd edn. New York: Springer-Verlag.Wanger, A. R. (1999) Yersinia. In: Collier L, Balows,

A. and Sussman, M. (eds), Topley and Wilson’s


References 321

These two genera are considered together herebecause they are both associated with atypicalpneumonia.

LEGIONELLA

This genus of aerobic Gram-negative bacteriaincludes 42 species, 18 of which are responsible forhuman diseases such as legionellosis (legionnaires’disease) and Pontiac fever. The organisms arewidely distributed in aquatic habitats and soil. Forfurther details on this genus and the diseases that itcauses, see Harrison and Taylor (1988), Stout andYu (1997), and Winn (1999).

Isolation

Culture is the preferred method for laboratorydiagnosis. Legionellas have fastidious growthrequirements and do not grow on ordinary bloodagar.

Homogenize the material (e.g. sputum, bronchialaspirates) in peptone water diluent and plate onbuffered charcoal yeast extract agar (BCYE) con-taining 2-oxoglutarate (α-ketoglutarate) (BCYE-α)to which various antibiotics may be added to sup-press the growth of other micro-organisms (com-mercial legionella-selective medium plus antibioticsupplements).

Incubate under 5% CO2 and high humidity andexamine plates after 3, 5, 7 and 10 days before dis-carding them. Most strains are recovered after 2–3days of incubation. Colonies of legionellas are small,

round, up to 3 mm in diameter and grey or green-ish brown, and present a ‘ground-glass’ appearance.

Legionellas may also be isolated by modern bloodculture systems (e.g. Oxoid Signal, Difco Sentinel)but additional processing is required, including theinoculation of selective media, and the sensitivity islow.

For the isolation of legionellas from watersamples, see Chapter 20.

Identification

Examine Gram-stained films of suspect colonies,test those that are Gram negative with fluorescenceantibody (FA) sera, and do agglutination tests withpolyvalent antisera. Proceed with specific sera ifavailable.

Subculture on BCYE medium for catalase, oxi-dase and hippurate hydrolysis tests. Test also forgelatin liquefaction but ensure that the mediumcontains cysteine and iron salts (growth supple-ments are available commercially). Legionellas arecatalase positive. Other reactions vary with species(see below).

Kits for screening and identifying legionellasinclude those of Sigma and Microscreen.

It is advisable to send cultures of legionellas to areference laboratory for full identification andserogrouping.

Species of Legionella

Only the relatively common species are listedbelow. Biochemical properties are of limited use.Identification must rely on agglutination tests.

35

Legionella and Mycoplasma

Legionella 323

Legionella pneumophilaThis is the most commonly encountered species. Itis oxidase positive and the only one that hydrolyseshippurate. It liquefies gelatin.

Legionella bozemanii, L. dumoffii and L. gormaniiThese are oxidase negative, do not hydrolyse hip-purate but liquefy gelatin.

Legionella micdadeiThis is oxidase positive, does not hydrolyse hippu-rate and does not liquefy gelatin.

Legionella longbeachii and L. jordaniiThese are also oxidase positive and do not hydro-lyse hippurate, but they do liquefy gelatin.

Direct immunofluorescence

This may be used to detect legionella antigens inclinical material. Although results are availablerapidly, the sensitivity of this method is oftenpoor and cross-reactions may occur with otherbacteria, giving false positives. This test has a lim-ited role in areas with a low prevalence oflegionella infections because of the high frequencyof cross-reaction.

Urinary antigen

The detection of legionella antigens in urine islikely to become the preferred diagnostic method astests continue to improve. Enzyme immunoassay(EIA) kits are now available commercially. Bothsensitivity and specificity are high and testing maybe performed rapidly. The major limitation of thesetests is their unreliability in the diagnosis oflegionella infections other than L. pneumophilaserogroup 1. In many parts of the world other

species and serogroups cause a sizeable proportionof cases of legionella pneumonia. Urinary antigentests may remain positive for weeks to months. Apositive result does not, therefore, necessarilyreflect acute infection.

Serology

An acute serum sample should be collected andstored. If the results of other tests are negative anda legionella infection is still suspected, a convales-cent serum sample should be collected at least3 weeks later and both samples should be testedin parallel for antibodies. If seroconversion hasnot occurred at this stage, another serumsample should be collected and tested after afurther 3 weeks.

The detection of legionella antibodies in serum isa sensitive method for diagnosing legionella infec-tion, especially when caused by L. pneumophilaserogroup 1. The main disadvantages of serologicaltests are that they may provide only a retrospectivediagnosis, and cross-reactions with other bacterialspecies may cause false-positive results. Such cross-reacting bacteria include Pseudomonas aeruginosa,Stenotrophomonas maltophilia, Bacteroides spp.,Campylobacter jejuni and Bordetella pertussis.Many populations have relatively high backgroundseropositivity rates to legionella antigens, presum-ably as a result of previous (often asymptomatic)infection. Consequently, it is necessary to docu-ment a fourfold or greater rise in antibody titre (to> 1 : 128) before legionella infection may be diag-nosed. As seroconversion is frequently delayedafter legionella infection, it is recommended that aconvalescent serum sample be collected at least3 weeks after an acute sample. There are very fewsituations where testing of a single serum sample(acute or convalescent) is justified.

Table 35.1 summarizes the laboratory tests forlegionellosis.

MYCOPLASMA

Mycoplasmas are the smallest free-living bacteriaand are usually 0.2–0.3 μm in diameter. They lackcell walls and are therefore resistant to penicillin.They can pass through bacteria-excluding filters.Their morphology is variable: cells may be coccoid,filamentous or star shaped. Individual cells may notbe stained by Gram’s method. Mycoplasmas areusually recognized by their characteristic colonialmorphology as observed by low-power microscopy.

Isolation

SpecimensThese include sputum, throat swabs, bronchiallavage fluid, lung biopsies and cerebrospinal fluid.Homogenize sputum by shaking it with an equalvolume of mycoplasma broth in a bottle containingglass beads. Homogenize tissue in mycoplasmabroth. Transport swabs to the laboratory inmycoplasma broth; squeeze the swabs into thebroth to express material. Culture other specimens

(e.g. cerebrospinal fluid, urine) without pre-treat-ment or centrifuge them and use the deposit.

Culture mediaMycoplasma culture media are available commer-cially as agar or broth, or in diphasic form. Theyinclude thallous acetate and penicillin to inhibit thegrowth of other bacteria and amphotericin B tosuppress fungi. They may be supplemented withglucose, urea or arginine as indicated in Table 35.2.

Inoculate mycoplasma agar plates, mycoplasmabroth and either SP4 broth (mycoplasma broth con-taining fetal calf serum) or selective media in dupli-cate, with 0.1 ml of the specimen. Incubate at 37°Cin anaerobic-type jars under the atmospheric condi-tions shown in Table 35.2. Examine agar media forcolonies daily for up to 20 days. M. pneumoniae orM. genitalium colonies develop between 5 and 20days. Subculture broth media to solid media whenthe appropriate colour changes have occurred.

Colonies of some species have a dark central zoneand a lighter peripheral zone, giving them a ‘friedegg’ appearance; others, notably Mycoplasmapneumoniae, lack this zone.

324 Legionella and Mycoplasma

Table 35.1 Summary of laboratory diagnosis procedures for legionellosis

Test Sample Sensitivity (%) Specificity (%) Cost Comment

Culture Sputum, ++ +++ ++ All species BAL

Direct IF Sputum, ++ ++ + L. pneumophilaBAL serogroups only

Urinary antigen Urine ++ +++a ++ L. pneumophila serogroup 1 only

Serology:Fourfold change Blood ++ ++ ++Single titre > 1 : 256 + ++ ++ L. pneumophila

serogroups onlyPCR Sputum, +++ +++ +++ All species but not

BAL routinely available

BAL, bronchoalveolar lavage fluid; IF, immunofluorescence; PCR, polymerase chain reaction+ < 30%; ++ 30–90%; +++ > 90%; a but see p. 323

Identification

Mycoplasmas cannot be removed from agar mediafor microscopy and subcultured in the usual waywith inoculating wires and loops. With a sterilescalpel, cut out a block of agar about 5 mm squareand containing one or more colonies. Proceed asindicated below.

Microscopy

Place an agar block, colonies uppermost, on a slide.Place a drop of Diene’s stain on a cover glass andinvert it over the agar block. Examine under a low-power microscope.

Imprints of mycoplasma colonies are stained darkblue and those of ureaplasma greenish blue.Bacteria are also stained blue, but they lose thecolour after 30 min, whereas the mycoplasmasretain it for several hours. This stain differentiatesmycoplasmas from artefacts such as crystals whichmay have a similar appearance.

Subculture

Place an agar block, colonies down, on the surfaceof mycoplasma agar and move it around. Newcolonies will develop along the track of the block.

Inhibition by hyperimmune serumCut out agar blocks containing colonies and placethem in mycoplasma broth. Incubate for 4–10 daysand then flood mycoplasma agar plates with theculture. Allow to dry and apply filter-paper discsimpregnated with hyperimmune sera. Incubateuntil colonies are visible. Zones of growth inhibi-tion will be seen around discs that are impregnatedwith the homologous antibody to the isolate.

Haemadsorption test

Pour a 0.5% suspension of washed guinea-pig ery-throcytes in phosphate-buffered saline (PBS) overan agar plate bearing about 100 colonies. Leave atroom temperature for 30 min, remove the cellsuspension, wash the agar surface with PBS and

Mycoplasma 325

Table 35.2 Isolation of mycoplasmas causing human disease

Species Clinical Culture media Substrate pH Culture Colony Growth specimensa atmosphereb form rate

M. pneumoniae BAL fluid, Diphasic medium; Glucose 7.8 1 + 2 Mulberry Slow throat swabs, SP4 medium; horse (over sputum serum agar and 5 days)

broth; SP4 broth or agar

M. genitalium Urethral swabs, SP4 broth or agar Glucose 7.8 1 + 2 Mulberry Slow throat swabs, (oversputum 5 days)

Ureaplasma Vaginal swabs, Urea broth or Urea 6.0 3 Small fried egg Medium urealyticum cervical swabs, agar supplemented (10–15 μm) (over

urethral swabs, with MnCl2 which 2 days)urine will stain colonies

M. hominis Vaginal swabs, Horse serum Arginine 7.0 1 + 2 Fried egg Rapid cervical swabs, broth or agar (50–500 μm) (occasionallyurethral swabs by 24 h)

a Swabs should be transported to the laboratory in a suitable medium (see text)b Atmosphere: 1 = 5% CO2/95% N2; 2 = air; 3 = 10–20% CO2/80–90% N2

BAL, bronchoalveolar lavage

examine under a low-power microscope foradsorption of erythrocytes, which occurs withcolonies of M. pneumoniae.

Haemolysis test

Prepare an agar overlay containing 1 ml of a 20%suspension of washed guinea-pig or sheep erythro-cytes in PBS and 3 ml mycoplasma agar base (at45°C). Pour it gently over a plate on which thereare colonies of mycoplasmas, allow to solidify andincubate aerobically at 37°C. After 24 h and againat 48 h refrigerate the plates at 4°C for 30 min andexamine for haemolysis. Colonies of M. pneumo-niae are surrounded by a zone of β-haemolysis.Other mycoplasmas are surrounded by zones ofα-haemolysis, are smaller and take longer todevelop the zone.

Immunofluorescent staining

This is a reliable method for identifying myco-plasma colonies but the specific antibodies raised inrabbits are currently not commercially available.

Species of mycoplasmas

Table 35.3 lists the mycoplasmas responsible forhuman and animal disease. Those associated withhuman disease are described briefly below. Fordetails of the clinical aspects of diseases caused bymycoplasmas, see Mårdh (1999) and Taylor-Robinson (2002).

Mycoplasma pneumoniaeThis forms ‘lumpy’ colonies in 4–5 days in mediacontaining glucose, incubated in an atmosphere of5% CO2 and 95% N2 as well as aerobically.


Table 35.3 Mycoplasmas causing disease in humans and animals

Host Mycoplasma sp. Primary isolation site Disease association

Human M. pneumoniae Respiratory tract Atypical pneumoniaM. genitalium Genital and respiratory tract Non-specific urethritisM. hominis Genital, occasionally respiratory Septic wound infectionsM. fermentans Genital tract Genital (and other?) infections Ureaplasma urealyticum Genital tract and oropharynx Non-gonococcal urethritis and

chronic lung diseaseCattle M. mycoides subsp. mycoides Respiratory tract Contagious bovine pleuropneumonia

M. bovis Joints, milk, genital tract Mastitis and respiratory diseaseMycoplasma spp.(bovine group 7) Contagious agalactia

Sheep/goats M. agalactiae Joints, milk, blood Contagious agalactiaM. mycoides subsp apr 1 Respiratory tract Contagious caprine

pleuropneumoniaM. capricolum Joints, udder, blood Septicaemic arthritis

Poultry M. gallisepticum Respiratory tract Air sacculitis, sinusitis and arthritisM. synoviae Joints, respiratory tract Air sacculitis and arthritis

Pigs M. hypopneumoniae Respiratory tract Enzootic pneumonia Rats/mice M. artthritidis Joints Arthritis

M. pulmonis Respiratory tract Murine respiratory mycoplasmosis

It is a major cause of primary atypical pneumonia,accounting for up to 15% of hospital-admittedpneumonias. Epidemics occur over a 4-year cycle.During periods of peak infection all available labo-ratory tests should be used to ensure adequatediagnosis and prompt and appropriate chemother-apy, usually with erythromycin or a tetracycline.

Mycoplasma genitaliumColonies and growth conditions are the same as forM. pneumoniae. Colonies may take 5 days todevelop. M. genitalium was originally isolatedfrom the urethras of men with non-gonococcal ure-thritis. Subsequently, it has been isolated from therespiratory tract in association with M. pneumo-niae with which it has several properties in com-mon, including the ability to metabolize glucoseand some antigenic cross-reactions. Its frequency inthe urogenital and respiratory tracts has yet to beestablished.

Mycoplasma hominisThis forms ‘fried egg’ colonies on media containingarginine under the same atmospheric conditions asM. pneumoniae. Growth is rapid, occasionally vis-ible in 24 h.

It is present in the genital tract of about 20% ofwomen and may be regarded as an opportunistpathogen. It is sometimes isolated from bloodcultures of women with mild postpartum fever andmay also cause pelvic inflammation and infertility.

Ureaplasma urealyticumSmall ‘fried egg’ colonies appear on media contain-ing urea and incubated in an atmosphere contain-ing 10–20% CO2 and 80–90% N2. Colonies maytake 2 or more days to develop.

About 60% of healthy women carry thisorganism in their genital tracts. Like M. hominis itmay be regarded as an opportunist pathogenbecause it is also occasionally isolated from bloodcultures of women with mild postpartum fever andmay cause pelvic inflammation and infertility. It isassociated with non-specific urethritis in men andchronic lung disease in infants with low birth-weights.

Serological diagnosis

Serological tests have been described for a numberof mycoplasma infections, notably those caused byM. pneumoniae. Culture, however, remains themost useful diagnostic method at present.

Historical methods, such as the cold agglutinationtest are relatively insensitive and should be reservedfor rapid diagnosis during epidemics. The comple-ment fixation test for M. pneumoniae is also of lowsensitivity. More sensitive tests include aggluti-nation of antigen-coated gelatin particles andμ-capture enzyme-linked immunosobent assay(ELISA) methods for specific IgM antibodies.During re-infection with M. pneumoniae, specificIgM production is poor and consequently, for thehighest rates of detection in such patients, IgMassays should be supplemented with those for aspecific IgG or IgA.

Polymerase chain reaction

There are several very sensitive commercialmolecular assay kits for detecting M. pneumoniaenucleic acid by the PCR method. As the organismmay reside in the respiratory tract for severalweeks after infection interpretation of these assaysmay be difficult. Methods to assess the quantita-tive level in secretions are awaited. The diagnostictests for M. pneumoniae are summarized in Table35.4.

Mycoplasmas as contaminants

Mycoplasmas are not infrequent contaminants oftissue cultures. They may be detected by the cul-tural methods described above or by a DNA probe(e.g. Gen-Probe, San Diego). If they are presentantimicrobial susceptibility tests will indicate theappropriate addition to the tissue culture medium.For further information about mycoplasamas, etc.,see Taylor-Robinson and Tully (1999).

Mycoplasma 327

REFERENCES

Harrison, T. G. and Taylor, A. G. (1988) A Laboratory

Manual for Legionella. London: Wiley. Mårdh, P-A. (1999) Mycoplasma and ureaplasma. In:

Armstrong, D. and Cohen, J. (eds), Infectious

Diseases, Vol. 2, Section 8. London: Mosby, pp. 1–6.Stout, J. E. and Yu, V. L. (1997) Legionellosis. New

England Journal of Medicine 337: 682–687.Taylor-Robinson, D. (2002) Mycoplasmas. Atypical

pneumonia: genital tract infection. In: Greenwood,D., Slack, R., Peutherer, J. (eds), Medical

Microbiology, 16th edn. Edinburgh: ChurchillLivingstone, pp. 379–389.

Taylor-Robinson, D. and Tully, J. G. (1999)Mycoplasmas, ureaplasmas, spiroplasmas andrelated organisms. In: Collier, L., Balows, S. andSussman, M. (eds), Topley and Wilson’s

Microbiology and Microbial Infections, 9th edn,Vol. 2, Systematic Bacteriology, CD-ROM.London: Arnold, Chapter 34.

Winn, W. C. (1999) Legionella. In: Murray, P. R.,Baron, E. J., Pfaller, M. A. et al. (eds), Manual of

Clinical Microbiology, 7th edn. Washington DC:ASM, pp. 572–585.


Table 35.4 Summary of diagnostic tests for Mycoplasma pneumoniae

Test Sample Sensitivity (%) Specificity (%) Cost Comment

Culture Throat swabs, ++ +++ ++ 2 weeks for colony sputum, BAL development, not

widely available

Serology: CFT Blood ++ ++ + Paired samples required

Serology: specific Blood ++(+) ++ ++ Re-infections remain

IgM undetected

Serology: IgM and Blood +++ ++ ++(+) Current and re-infections IgG/A detected

PCR Throat swabs, +++ +++ +++ Not widely availablesputum, BAL

BAL, bronchoalveolar lavage fluid; CFT, complement fixation test; Ig, immunoglobulin; PCR, polymerase chain reaction++ 30–90%; +++ ≥ 90%

These two genera are considered together,although they are unrelated. They are frequentlyisolated from pathological material and foods.Distinguishing between them is important. Somestaphylococci are pathogens, some are doubtful oropportunist pathogens, and others, and micro-cocci, appear to be harmless but are useful indica-tors of pollution. Staphylococci are fermentative,and capable of producing acid from glucose anaer-obically; micrococci are oxidative and produce acidfrom glucose only in the presence of oxygen.Staphylococci divide in more than one plane, givingrise to irregular clusters, often resembling bunchesof grapes.

As with other groups of micro-organisms, recenttaxonomic and nomenclatural changes have notexactly facilitated the identification of the Gram-positive cocci that are isolated from clinical mate-rial and foods. The term ‘micrococci’ is still looselybut widely used to include many that cannot beeasily identified (see also under Aerococcus, p. 342and Pediococcus, p. 344).

ISOLATION

Pathological material

Plate pus, urine, swabs, etc., on blood agar orblood agar containing 10 mg/l each of colistin andnalidixic acid to prevent the spread of Proteus spp.;also inoculate salt meat broth. Incubate overnightat 37°C. Plate the salt meat broth on blood agar.

Foodstuffs

Prepare 10% suspensions in 0.1% peptone waterin a Stomacher. If heat stress is suspected add 0.1-ml amounts to several tubes containing 10 mlbrain–heart infusion broth. Incubate for 3–4 h andthen subculture. Use one or more of the followingmedia: milk salt agar (MSA), phenolphthaleinphosphate polymyxin agar (PPP), Baird-Parkermedium (BP) and tellurite polymyxin egg yolk(TPEY). Incubate for 24–48 h. Test the PPPmedium with ammonia for phosphatase-positivecolonies (see below). There are also commercialtests for the detection of enterotoxins (Berry et al.,1987).

MSA agar relies on the high salt content toselect staphylococci. In PPP medium, thepolymyxin suppresses many other organisms. BPmedium is very selective but may be overgrownby Proteus spp. This may be suppressed byadding sulphamethazine 50 μg/ml to themedia.

For enrichment or to assess the load of staphylo-cocci, use salt meat broth or mannitol salt broth.Add 0.1 and 1.0 ml of the emulsion to 10 ml brothand 10 ml to 50 ml. Incubate overnight and plateon one of the solid media. If staphylococci are pres-ent in large numbers in the food, there will be aheavy growth from the 0.1-ml inoculum; very smallnumbers may give growth only from the 10-mlsample.

If counts are required, use the Miles andMisra method (see p. 148) and one of the solidmedia.

36

Staphylococcus and Micrococcus

STAPHYLOCOCCAL FOODPOISONING

There is no need to examine the stools of all thepatients; a sample of 10–25% is sufficient.Inoculate Robertson’s cooked meat medium con-taining 7–10% sodium chloride (salt meatmedium) with 2–5 g faeces or vomit and incubateovernight, and plate on blood agar or one of thespecial staphylococcal media. Examine all foodhandlers for lesions on exposed parts of the bodyand swab these, as well as the noses, hands and fin-gernails of all kitchen staff. Swab chopping boardsand utensils that have crevices and cracks likely toharbour staphylococci. Use ordinary pathologicallaboratory swabs: cottonwool wound on a thinwire or stick, sterilized in a test tube (these swabscan be obtained from most laboratory suppliers).Plate on the solid medium and break off the cot-tonwool swab in cooked meat medium.

All strains of S. aureus isolated from patients,food, implements and food handlers in incidents ofstaphylococcal food poisoning should be sent to areference laboratory. Not all strains can cause thedisease and others are usually encountered duringthe investigation (see Chapter 13).

Identification

Colonies of staphylococci and micrococci on ordi-nary media are golden, brown, white, yellow orpink, opaque, domed, 1–3 mm in diameter after24 h on blood agar and are usually easily emulsi-fied. There may be β-haemolysis on blood agar.Aerococci show α-haemolysis.

On Baird-Parker medium after 24 h, S. aureusgives black, shiny, convex colonies, 1–1.5 mm indiameter; there is a narrow white margin and thecolonies are surrounded by a zone of clearing 2–5mm in diameter. This clearing may be evident onlyat 36 h.

Other staphylococci, micrococci, some entero-cocci, coryneforms and enterobacteria may growand produce black colonies, but do not produce theclear zone. Some strains of S. epidermidis have a

wide opaque zone surrounded by a narrow clearzone. Any grey or white colonies can be ignored.Most other organisms are inhibited (but notProteus spp. – see above).

On TPEY medium, colonies of S. aureus are blackor grey and give a zone of precipitation aroundand/or beneath the colonies.

Examine Gram-stained films. Do clumping factorand coagulase tests on Gram-positive cocci isolatedfrom clinical material and growing in clusters. Thisis a short cut: strains that are positive by both testsare probably S. aureus.

Clumping factor and coagulase tests

Possession of the enzyme coagulase, which coagu-lates plasma, is an almost exclusive property of S.aureus. The slide test detects ‘bound’ coagulase(‘clumping factor’), which acts on fibrinogendirectly. The tube test detects ‘free’ coagulase,which acts on fibrinogen in conjunction with otherfactors in the plasma. Either or both coagulasesmay be present.

Slide, clumping factor testEmulsify one or two colonies in a drop of water ona slide. If no clumping occurs in 10–20 s dip astraight wire into human or rabbit plasma (EDTA)and stir the bacterial suspension with it. S. aureusagglutinates, causing visible clumping in 10 s. Usewater instead of saline because some staphylococciare salt sensitive, particularly if they have been cul-tured in salt media. Avoid excess (e.g. a loopful)plasma because this may give false positives. Checkthe plasma with a known coagulase-positivestaphylococcus.

Tube coagulase test Do this to confirm the clumping factor test or if it isnegative. Add 0.2 ml plasma to 0.8 ml nutrient (notglucose) broth in a small tube. Inoculate with thesuspected staphylococcus and incubate at 37°C in awater bath. Examine at 3 h and if negative leaveovernight at room temperature and examine again.

330 Staphylococcus and Micrococcus

Staphylococcus aureus produces a clot, gellingeither the whole contents of the tube or forming aloose web of fibrin. Longer incubation may resultin disappearance of the clot as a result of digestion(fibrinolysis).

Include known positive and negative controls. Inthe tube test, citrated plasma may be clotted by anyorganism that can use citrate, e.g. by faecal strepto-cocci (although these are catalase negative),Pseudomonas and Serratia spp. It is advisabletherefore to use EDTA plasma (available commer-cially) or oxalate or heparin plasma. Check Gramfilms of all tube coagulase-positive organisms.

There are several commercial kits for identifyingS. aureus by latex agglutination, e.g. Prolex(Prolab), Microscreen (Microgen) and Staphytect(Oxoid). Such kits remove the potential hazard ofusing human plasma. Some combine tests forclumping factor and protein A. Others employhaemagglutination.

Phosphatase test

Inoculate phenolphthalein phosphate agar andincubate overnight. Expose to ammonia vapour.Colonies of phosphatase-positive staphylococciwill turn pink. S. aureus gives a positive test (butnegative strains have been reported).

If identification to species (other than S. aureus) isrequired use the API STAPH-IDENT system or test

for nitrate reduction, susceptibility to novobiocin(5-μg disc), and fermentation of mannitol, tre-halose and sucrose (Baird-Parker sugar medium)(Table 36.1 and see below). (For further informa-tion, see Jones et al., 1990 and Kloos, 1999.)

STAPHYLOCOCCUS

There are now at least 30 species of Staphylococcusbut some are of little interest. They may be dividedinto three groups: (1) coagulase positive; (2) coagu-lase negative and novobiocin susceptible; and (3)coagulase negative and novobiocin resistant. Onlyknown and opportunist pathogens are mentionedbelow.

Staphylococcus aureus

This species is coagulase and phosphatase positive,forms acid from mannitol, trehalose and sucrose,and is sensitive to novobiocin. Some strains arehaemolytic on horse blood agar, but the zone ofhaemolysis is relatively small compared with thediameter of the colony (differing from thehaemolytic streptococcus).

Production of the golden-yellow pigment is prob-ably the most variable characteristic. Young cul-tures may show no pigment at all. The colour maydevelop if cultures are left for 1 or 2 days on the

Staphylococcus 331

Table 36.1 Properties of some Staphylococcus species

Species Pigment Coagulase Phosphatase Novobiocin Acid from5-μg disc

Mannitol Trehalose Sucrose

S. aureus + + + S + + +S. chromogenes + − + S v + +S. hyicus − − + S − + ++S. intermedius − v + S + + +S. epidermidis − − + S − − +S. warneri − − − S v + +S. saprophyticus − − − R + + +

v, variable; S, sensitive; R, resistant

bench at room temperature. Pigment production isenhanced by the presence in the medium of lactoseor other carbohydrates and their breakdown prod-ucts.

Staphylococcus aureus is a common cause of pyo-genic infections and food poisoning (see Chapter13). Staphylococci are disseminated by commondomestic and ward activities such as bedmaking,dressing or undressing. They are present in thenose, on the skin and in the hair of a large propor-tion of the population.

Methicillin-resistant staphylococci

Methicillin-resistant S. aureus (MRSA) is involvedin serious hospital infections and requires earlyidentification. Cultures are usually a mixture ofresistant and sensitive organisms.

Inoculate mannitol salt agar containing 4 mg/lmethicillin and incubate overnight. Most strains ofS. aureus that ferment mannitol will grow, but somay some coagulase-negative staphylococci. Asdirect agglutination tests may not be satisfactoryon this medium, subculture several colonies onblood agar and test with the growth as describedabove or with one the kits mentioned. Othermedia are available: see Davies (1997) and Davieset al. (2000); see also Cookson and Phillips(1990).

Coagulase-negative staphylococci

Staphylococcus epidermidis (S. albus)This produces no pigment; the colonies are ‘chinawhite’. Antibiotic-resistant strains are not uncom-mon and are often isolated from clinical material,including urine, where they may be opportunistpathogens.

Staphylococcus epidermidis is associated withinfections with implanted material, e.g. prostheticheart valves and joints, ventricular shunts andcannulae. Strains resistant to many antibioticsoccur.

Staphylococcus saprophyticusThis causes urinary tract infections in young sexu-ally active women. There is a diagnostic kit(Dermaci, Sweden).

Other speciesStaphylococcus chromogenes produces pigmentlike that of S. aureus and ferments sucrose. It is anopportunist pathogen and has been found in pigsand in cows’ milk. S. hyicus is an opportunistpathogen and has been found in dermatitis of pigs,in poultry and cows’ milk. S. intermedius is knownto cause infections in dogs. S. warneri and S. cohniiare opportunist pathogens.

Staphylococcal toxinsThere are kits for testing for the presence of these infoods and also for investigating toxic shock syn-drome which is associated with the use of tampons(see Berry et al., 1987; Arbuthnot et al., 1990).

Staphylococcal typingStrains isolated from hospital infections and foodpoisoning incidents should be typed for epidemio-logical purposes. This is best done at a referencelaboratory (see Richardson et al., 1992).

The genus Staphylococcus is reviewed in aSociety for Applied Bacteriology Symposium (Joneset al., 1990), and by Kloos and Bannerman (1995)and Kloos (1999).

MICROCOCCUS

These are Gram-positive, oxidase- and catalase-positive cocci that differ from the staphylococci inthat they use glucose oxidatively or do not produceenough acid to change the colour of the indicator inthe medium. They are common saprophytes of air,water and soil, and are often found in foods.

The classification is at present confused: the genuscontains the organisms formerly called Gaffkyaand Sarcina, tetrad- and packet-forming cocci.These morphological characteristics vary with cul-tural conditions and are not considered constant

332 Staphylococcus and Micrococcus

enough for taxonomic purposes. The genus Sarcinanow includes only anaerobic cocci.

At present, it does not seem advisable to describenewly isolated strains by any of the specific namesthat abound in earlier textbooks.

REFERENCES

Arbuthnott, J. P., Coleman, D. C. and De Azavedo, J.S. (1990) Staphylococcal toxins in human disease.In: Jones, D., Board, R. G. and Sussman, M. (eds),Staphylococci. Society for Applied BacteriologySymposium Series No. 19. Oxford: Blackwells, pp.101S–108S.

Berry, P. R., Weinecke, A. A., Rodhouse, J. C. andGilbert, R. J. (1987) Use of commercial tests forthe detection of Clostridium perfringens andStaphylococcus aureus enterotoxins. In: Grange, J.M., Fox, A. and Morgan, N. L. (eds),Immunological Techniques in Microbiology.Society for Applied Bacteriology Technical SeriesNo. 24. London: Blackwells, pp. 245–250.

Cookson, B. and Phillips, I. (1990) Methicillin-resistantstaphylococci. In: Jones, D., Board, R. G. andSussman, M. (eds), Staphylococci. Society forApplied Bacteriology Symposium Series No. 19.Oxford: Blackwells, pp. 55S–70S.

Davies, S. (1997) Detection of methicillin-resistantStaphylococcus aureus: then evaluation of rapidagglutination methods. British Journal of

Biomedical Science 54: 13–15.Davies, S., Zadik, P. M., Mason, C. M. et al. (2000)

Methicillin-resistant Staphylococcus aureus:evaluation of five selective media. British Journal

of Biomedical Science 57: 269–272.Jones, D., Board, R. G. and Sussman, M. (eds) (1990)

Staphylococci. Journal of Applied Bacteriology

Symposium No. 19, 69: 1S–188S.Kloos, W. E. (1999) Staphylococcus. In: Collier, L.,

Balows, A. and Sussman, M. (eds), Topley and

Wilson’s Microbiology and Microbial Infections,

9th edn. Vol. 2, Systematic Bacteriology. London:Arnold, Chapter 27, CD-ROM.

Kloos, W. E. and Bannerman, T. L. (1995)Staphylococcus and Micrococcus. In: Murrau, P.R., Baron, E. J., Pfaller, M. A. et al. (eds), Manual

of Clinical Microbiology, 6th edn. Washington:ASM Press.

Richardson, J. F., Noble, W. C. and Marples, R. M.(1992) Species identification and epidemiologicaltyping of the staphylococci. In: Board, R. G., Jones,D. and Skinner, F. A. (eds), Identification Methods

in Applied and Environmental Microbiology Societyfor Applied Bacteriology Technical Series No. 29.Oxford: Blackwells, pp. 193–220.

References 333

37

Streptococcus, Enterococcus, Lactococcus,Aerococcus, Leuconostoc and Pediococcus

This group includes organisms of medical, dentaland veterinary importance as well as starters usedin the food and dairy industries, spoilage agentsand saprophytes. The Gram-positive, catalase-neg-ative cocci have undergone extensive taxonomicrevision; several new genera and species have beendescribed since the last edition of this book(Facklam and Elliott, 1995; Hardie and Whiley,1997; Facklam, 2002).

The important characters that distinguishStreptococcus, Enterococcus, Lactococcus,Leuconostoc, Abiotrophia and Aerococcus spp. areshown in Table 37.1.

STREPTOCOCCUS

Streptococci are Gram-positive cocci that alwaysdivide in the same plane, forming pairs or chains;they are catalase and oxidase negative, non-sporingand non-motile, and some are capsulated. They are

facultatively anaerobic and some require increasedCO2 concentration.

Isolation

From clinical materialBlood agar is the usual primary medium. If incu-bated anaerobically, important streptococci willgrow and many other organisms will be sup-pressed. If the material is known to contain manyother organisms, place a 30-μg neomycin disc onthe heavy part of the inoculum. Alternatively useblood agar containing 10 μg/ml colistin and5 μg/ml oxolinic acid (COBA medium: Petts, 1984)which is selective for streptococci. (Crystal violetblood agar is not very satisfactory because batchvariation in the dye affects colony size and amountof haemolysis.)

Islam’s medium (Islam, 1977; available commer-cially) facilitates the recognition of group B strep-

Table 37.1 General properties

Genus Haemolysis Growth at Growth in Aesculin Gas Vancomycin PYR45°C 6.5% NaCl hydrolysis (30-μg disc)

Streptococcus α β n − − v − S −a

Enterococcus α β n + + + − Sb +Lactococcus α n − v − − S +Aerococcus α − + v − S ±Leuconostoc α n − v v + R −Abiotrophia α − − − − S +

PYR, pyrrolidonyl-β-naphthylamide reactionn, none; v, variable; S, sensitive; R, resistanta S. pyogenes, S. iniae and S. porcinus are positiveb Resistant strains occur

tococci, e.g. in antenatal screening. Incubate anaer-obically at 37°C.

Dental plaquePlate on blood agar and trypticase yeast extractcystine agar or mitis salivarius agar.

Bovine mastitisUse Edwards’ medium. The crystal violet and thal-lous sulphate inhibit most saprophytic organisms;aesculin-fermenting saprophytic streptococci giveblack colonies and mastitis streptococci pale-greycolonies.

Dairy productsUse yeast glucose agar for mesophiles and yeast lac-tose agar for thermophiles.

AirFor β-haemolytic streptococci in hospital cross-infection investigations, use crystal violet agar con-taining 1 : 500 000 crystal violet (satisfactory here)with slit samplers. For evidence of vitiation by oralstreptococci use mitis salivarius agar.

Identification of streptococci

Colonies on blood agar are usually small, 1–2 mmin diameter, and convex with an entire edge. Theymay be ‘glossy’, ‘matt’ or ‘mucoid’. Growth inbroth is often granular, with a deposit at the bot-tom of the tube.

Primary classification is made on the basis ofhaemolysis on sheep or horse blood agar. α-Haemolytic streptococci, often referred to as‘viridans’ streptococci, produce a small greenishzone around the colonies. This is best seen onchocolate blood agar.

β-Haemolytic streptococci give small colonies sur-rounded by a much larger, clear haemolysed zonein which all the red cells have been destroyed.

Haemolysis on blood agar is only a rough guideto pathogenicity. The β-haemolytic streptococciinclude those strains that are pathogenic for

humans and animals, but the type of haemolysismay depend on conditions of incubation and themedium used as a base for the blood agar. Some α-haemolytic streptococci show β-haemolysis onColumbia-based blood agar.

Some saprophytic streptococci are β-haemolytic,and so are organisms in other genera having similarcolonial morphology. Haemolytic Haemophilusspp. are often reported as haemolytic streptococciin throat swabs because Gram-stained films are notmade.

Clinical strainsβ-Haemolytic streptococci from clinical materialare usually identified by determining theirLancefield group (see below) or as S. pyogenes(group A) by their sensitivity to bacitracin (seebelow). S. pneumoniae (the pneumococcus) is iden-tified by its sensitivity to optochin (see p. 99) or bilesolubility. Other streptococci require further tests.

Streptococcal antigens

Species and strains of streptococci are usually iden-tified by their serological group and type. There areLancefield groups (A–W, excluding I) characterizedby a series of carbohydrate antigens contained inthe cell wall. Rabbits immunized with knownstrains of each group produce serum, which willreact specifically in vitro by a precipitin reactionwith an extract of the homologous organism. Thecarbohydrate is known as the C substance. Thestreptococci within S. pyogenes (group A) can bedivided into serological (Griffith) types by means oftwo classes of protein antigens: M and T.

M is a type-specific antigen near the surface of theorganism that can be removed by trypsinization; it ispresent in matt and mucoid types of colonies but notin glossy types. It is demonstrated by a precipitintest. T is not type specific, may be present with orindependent of the M antigen, and is demonstratedby agglutination tests with appropriate antisera.

Lancefield grouping is usually carried out in thelaboratory where the organisms are isolated, using

Streptococcus 335

sera or agglutination reagents obtained commer-cially. Only reagents for groups A, B, C, D and Gneed be used for routine purposes, although mostagglutination kits also contain group F.

Workers should be aware that the groups are notalways species specific. Some enterococci possessthe G antigen in addition to their D antigen. Thespecies in the anginosus group of streptococci (seebelow) may react as A, C, F or G and, whenhaemolytic, may be confused with pyogenic strep-tococci. Some ‘viridans’ streptococci may alsogroup as A or C and under certain conditions mayappear to be β-haemolytic.

Griffith typing of haemolytic (group A, etc.)strains is necessary for epidemiological purposes.Epidemiological typing schemes exist for most ofthe major pathogens. These are best done by refer-ence laboratories.

Serological grouping of streptococci

There are two approaches. Commercially availablekits for latex agglutination are rapid and giveresults in less than 1 h of obtaining a satisfactorygrowth on the primary culture. Precipitin tests takelonger but allow a larger number of groups to beidentified.

Latex agglutination kit tests

Several kits are available from various suppliers,including Oxoid, Murex, Pharmacia, Prolab andDiagnostic Products. The streptococcal antibody isattached to latex particles. These antibody-coatedparticles are agglutinated when mixed on a slidewith suspensions or extracts of streptococci of thesame group. In the latex agglutination methods anenzyme or nitrous acid extract of the organism ismixed with the antibody-coated latex reagent on aslide. Groups, A, B, C, D, F and G may be identifiedby these methods, but it is recommended that bio-chemical tests are done on group D streptococci toidentify enterococci and S. bovis, to identify the

species of strains of group C or G and to identify‘viridans’ streptococci.

Precipitin tube methods

These have largely been superseded by the aggluti-nation (kit) methods but the following threemethods are still in use.

Centrifuge 50 ml of an overnight culture of thestreptococcus in 0.1% glucose broth or use a sus-pension prepared by scraping the overnight growthfrom a heavily inoculated blood agar plate.

For Lancefield’s method suspend the deposit in0.4 ml 0.2 mol/l hydrochloric acid and place in aboiling water bath for 10 min. Cool, add 1 drop0.02% phenol red and loopfuls of 0.5 mol/l sodiumhydroxide solution until the colour changes to faintpink (pH 7.0). Centrifuge; the supernatant is theextract.

For Fuller’s formamide method suspend thedeposit in 0.1 ml formamide and place in an oilbath at 160°C for 15 min. Cool and add 0.25 mlacid–alcohol (95.5 parts ethanol : 0.5 parts 1 mol/lhydrochloric acid). Mix and centrifuge. Removethe supernatant fluid and add to it 0.5 ml acetone.

Acetone may explode during centrifugation.Mix, centrifuge and discard the supernatant. To

the deposit add 0.4 ml saline, 1 drop 0.02% phenolred and loopfuls of 0.2 mol/l sodium hydroxidesolution until neutral (pH 7.0). This is the extract. Itis not a very good method for group D streptococci.

In Maxted’s method the C substance is extractedfrom streptococcal suspension by incubation with anenzyme prepared from a strain of Streptomyces. Toprepare this enzyme, obtain Streptomyces sp. No.787 from the National Collection of Type Culturesand grow it for several days at 37°C on buffered yeastextract agar. The medium is best sloped in flat bot-tles (120-ml ‘medical flats’). When there is goodgrowth, place the cultures in a freezer or a bowl con-taining broken pieces of solid carbon dioxide. After24 h allow the medium to thaw and remove the fluid;this contains the enzyme. Bottle it in small amountsand store in a refrigerator. To prepare the strepto-coccal extract, suspend a large loopful of growth from

336 Streptococcus, Enterococcus, Lactococcus, Aerococcus, Leuconostoc and Pediococcus

a blood agar culture of the organisms into 0.5 ml ofenzyme solution in a small tube and place in a waterbath at 37°C for 2 h. Centrifuge and use the super-natant to do precipitin tests as described below. Thismethod does not work well with group D strains

Prepare capillary tubes from Pasteur pipettes. Dipthe narrow end in the grouping serum so that a col-umn a few millimetres long enters the tube. Place it ina block of Plasticine and, with a very fine Pasteurpipette, layer extract on the serum so that the two donot mix but a clear interface is preserved. Some prac-tice is necessary in controlling the pipette. If an airbubble develops between the two liquids, introduce avery fine wire, when an interface is usually produced.

A positive result is indicated by a white precipitatethat develops at the interface. It is sometimes nec-essary to dilute the extract 1 : 2 or 1 : 5 with salineto obtain a good precipitate.

The bacitracin disc method

Inoculate a blood agar plate heavily and place acommercial bacitracin disc on the surface and incu-bate overnight. A zone of inhibition appearsaround the disc if the streptococci are group A.This test is not wholly reliable and is declining inpopularity except as a screening method.

Biochemical tests

There are now so many species that conventionaltests become very time-consuming. It is best, there-fore, to use commercial identification systems suchas the API20 Strep (bioMérieux) or ID 32 STREP(bioMérieux) or RapidID STR (InnovativeDiagnostic Systems), and refer to their databases.Tables 37.2 and 37.3 show the characteristics ofsome of the more common species.

Species and groups of streptococci

Lancefield grouping gives a useful indication of theidentity of a streptococcus but it is not always

species specific. Taxonomic studies have shownthat the streptococci may be divided into six clus-ters, which reflect their ecological and pathogenicproperties (Hardie and Whiley, 1997):

1. Pyogenic: mainly β-haemolytic speciespathogenic for humans or animals.

2. Anginosus: found in the human mouth, gutand genital tract, and may be isolated fromwound infections and abscesses.

3. Mitis: oral streptococci including S.pneumoniae.

4. Salivarius: some dairy streptococci and speciesfound in the human mouth.

5. Bovis: inhabit the gut in humans and animals.6. Mutans: colonize the surfaces of teeth of

humans and some animals.

There are some species that are not included inany group.

Pyogenic groupStreptococcus pyogenes (group A)These are β-haemolytic and are the so-calledhaemolytic streptococci of scarlet fever, tonsillitis,puerperal sepsis and other infections of humans.Some strains are capsulated and form large (3-mm)colonies like water drops on the surface of themedium. They are differentiated from other speciesthat are grouped as A by a positive PYR (pyrroli-donyl-β-naphthylamide) reaction for which thereare commercial kits.

Streptococcus agalactiae (group B)This is the causative organism of chronic bovinemastitis and an important cause of neonatal menin-gitis and septicaemia. It is found in the female gen-ital tract and gut. It has been reported as a cause ofurinary tract infections. It is usually β-haemolyticbut non-haemolytic strains occur. It grows onmedia containing 10% bile salt and is CAMP(Christie, Atkins and Munch-Petersen) test positive(see p. 93).

Streptococcus dysgalactiae (group C, G or L)The group C β-haemolytic streptococcus isolatedfrom humans, ‘S. equisimilis’, the α- or β-haemolytic

Streptococcus 337

Table 37.2 Pyogenic streptococci and S. suis

Species Lancefield Haemolysis CAMP PYR VP Hydrolysis of Growth Acid fromgroup

Aesculin Hippurate Arginineon bile

Mannitol Sorbitol Lactose Riboseagar

S. pyogenes A β − + − v − + − v − + −S. agalactiae B β + − + − + + + − − v +S. equi ssp. equi C β − − − − − + − − − − −S. equi ssp. zooepidemicus C β − − − v − + − − + + v

S. dysgalactiae ssp. dysgalactiae C α, β − − − − − + − − − v +S. dysgalactiae ssp. equisimilis C, G L β − − − v − + − − − − +S. canis G β ± − − v − + − − − − +S. inaie a αβ + + − + − − + + − −S. porcinus E, P, U, Va β + + + + v + − + + v +S. uberis Ea α − + + + + + − + + + +S. suis D α β n − − − + − + + − − + −

a No group or not A, B, C, D, F, Gn, none; v, variable

Streptococcus 339

group C from bovine mastitis, S. dysgalactiae, thelarge colony group G β-haemolytic streptococcusfrom humans, and the group L streptococcus fromanimals have been shown to be genetically identicaland one species – S. dysgalactiae. There are two sub-species, S. dysgalactiae ssp. equisimilis, β-haemolytichuman strains of group C or G, and S. dysgalactiaessp. dysgalactiae, α-, β- or non-haemolytic strains ofgroup C or L from animals. These can be differenti-ated from each other by their effect on human oranimal fibrin and plasminogen.

Streptococcus equi and S. zooepidemicus (group C)These β-haemolytic group C streptococci have alsobeen shown to be genetically identical and are nowS. equi with two subspecies. These are S. equi ssp.equi which causes strangles in horses and donkeys,and S. equi ssp. zooepidemicus which comprisesgroup C strains causing infections and outbreaks inanimals and some reported human infections.

Streptococcus canis (group G)A pathogen of dogs and other animals and also anagent of bovine mastitis.

Streptococcus inae A pathogen of dolphins and of wild and farmedfish.

Streptococcus porcinus This is found in the respiratory tract and cervicalabscesses of pigs

Streptococcus uberis This is an animal species and one of the agents ofbovine mastitis.

Anginosus groupPreviouslyS. milleri, this now consists of three species:S. anginosus, S. constellatus and S. intermedius.

Table 37.3 Examples of the Anginosus, Salivarius, Mitis, Bovis and Mutans groups and other streptococci***

Species Lancefield Haemolysis Hydrolysis of VP Growth Acid fromgroup

Aesculin Arginineon bile

Mannitol Sorbitol Lactoseagar

Anginosus groupS. aginosus A, C, F, G, * β, n + + + − − − +S. constellatus C, F, * β, n + + + − − − vS. intermedius * β, n + + + − − − +

Salivarius groupS. salivarius * α + − + − − − −S. thermophilus * α, n − − v + − − v

Mitis groupS. pneumoniae** * α − − − − − − −S. mitis * α, β − − − + − − vS. oralis * α, β − − − + + − v

Bovis groupS. bovis D α + − + + v − +S. equinus D α + − + + − − −

Mutans groupS. mutans * α + − + − + + +

Other streptococciS. acidominus * α + − − − + + +

* No group or not A, B, C, D, F, G** Optochin sensitive and bile solubility test positive*** For further information consult Facklam RR (2002)v, variable; n, no haemolysis


Salivarius groupStreptococcus salivarius is part of the normal floraof the mucous membranes of the human mouth.S. thermophilus resembles group D streptococci bio-chemically but has no D antigen; its optimum growthtemperature is 50°C. It is used (along withLactobacillus bulgaricus) in the manufacture ofyoghurt.

Mitis groupThe most important member of this cluster isS. pneumoniae, commonly termed the pneumococ-cus. It is a causative organism of lobar pneumonia,otitis media and meningitis in humans, and of var-ious other infections in humans and animals,including (rarely) mastitis. In clinical specimens theorganism appears as a capsulated diplococcus butit usually grows on laboratory media in chains andthen shows no capsule. There are several serologi-cal types, but serological identification is rarelyattempted nowadays. Strains of serotype III usuallyproduce highly mucoid colonies but most otherstrains give flat ’draughtsman’-type colonies1–2 mm in diameter with a greenish haemolysis.

Pneumococci often resemble ‘viridans’ strepto-cocci on culture. The following tests allow rapiddifferentiation. Pneumococci are sensitive tooptochin (ethylhydrocupreine hydrochloride).Most media manufacturers supply optochin discs.A disc placed on a plate inoculated with pneumo-cocci will give a zone of inhibition of at least10 mm when incubated aerobically (not in carbondioxide). Pneumococci are lysed by bile; otherstreptococci are not. Add 0.2 ml 10% sodiumdeoxycholate in saline to 5 ml of an overnightbroth culture (do not use glucose broth). Incubateat 37°C. Clearing should be complete in 30 min.

Pneumococci are alone among the streptococci infermenting inulin and are not heat resistant as aremany ‘viridans’ streptococci. These two criteriashould not be used alone for differentiation.

Streptococcus sanguis This is associated with endocarditis in humans asare other members of this cluster.

Streptococcus parasanguis This has been isolated form humans and is reportedas a cause of asymptomatic mastitis in sheep.

Bovis group

Streptococcus bovis (group D)This is present in the human bowel and animal intes-tines. In bowel disorders of humans it may enter thebloodstream, leading to endocarditis. A blood cul-ture positive for this species is often associated withintestinal malignancy. It may be differentiated fromenterococci by a negative PYR reaction.

Streptococcus equinus (group D)Inhabits animal intestines (especially horses) and isfound in milk. Human infections have been reported.

Mutans group

Streptococcus mutansThis is found in dental plaque and is associatedwith caries.

Other species

Streptococcus suis This is associated with meningitis in pigs and mayinfect humans.

Streptococcus acidominimus This is a commensal in the genital tract of cows.

ENTEROCOCCUS

This genus contains aerobic Gram-positive coccithat form pairs or short chains. As with strepto-cocci, they are catalase negative, but grow at 45°C,in the presence of 40% bile and 6.5% NaCl andhydrolyse aesculin. Haemolysis on blood agar isvariable (α, β or none). They are Lancefield groupD and most hydrolyse PYR. There are 17 species,some of which are motile. They are found in theintestines of a wide range of animals and are usedas indicators of faecal pollution (Leclerc et al.,1996; Goodfree et al., 1997).

Isolation

Clinical materialPlate on blood agar, MacConkey and Edwards’medium. This contains aesculin which, whenhydrolysed by enterococci, gives black colonies.

Identification

Test for growth at 10°C and 45°C, aesculin hydrol-ysis, arginine dihydrolase, motility, production of ayellow pigment, and acid production from manni-tol, sorbitol, sucrose, arabinose and adonitol(Table 37.4).

The API20 STREP system identifies most of thespecies. Some species may be difficult to identify bytraditional methods and polymerase chain reaction(PCR) methods are used (Woodford et al., 1997).For further information on identification of thisgenus see Facklam et al. (1999).

Common Enterococcus species

The important biochemical reactions are shown inTable 37.4.

Enterococcus faecalisThis is normally present in the human gut, hence itsuse as an indicator of faecal pollution. It is alsofound in the gut of chickens but less often in that ofother animals. It can be isolated from wound andurinary tract infections. It is the only enterococcusthat reduces 2,3,5-triphenyltetrazolium chloride(TTC; in Mead’s medium). It grows in the presenceof 0.03% potassium tellurite.

Enterococcus faeciumThis is present in the intestines of pigs and otheranimals and has caused spoilage of ‘commerciallysterilized’ canned ham. TTC is not reduced and itdoes not grow in media containing 0.03% potas-sium tellurite. It is more resistant to antibioticsthan other enterococci and can be isolated fromhospital-associated infections.

Enterococcus duransThis is found in milk and dairy products but isuncommon in clinical material.

Enterococcus aviumThis is present in the gut of chickens and some otheranimals, but is not often found in clinical material.

Enterococcus 341

Table 37.4 Enterococcus species

Species Growth at Arginine Motility Yellow Acid from

10°C 45°C dehydrolase pigmentMannitol Sorbitol Sucrose Arabinose Adonitol

E. faecalis + + + − − + + + − −E. faecium + + + − − + v + + −E. avium + + − − − + + + v +E. durans + + + − − − − − − −E. casseliflavus + + + + + + v + + −E. gallinarum + + + + − + − + + −E. malodoratus + + − − − + + + − +E. mundtii + + + − + + − + + −

v, variable

Enterococcus casseliflavus This is associated with plant material but has beenisolated from humans. It is motile and if coloniesare touched with a swab, the swab will be stainedyellow. It has a natural low-level resistance tovancomycin.

Enterococcus gallinarum This is found in the gut of chickens, it is motile, butdoes not produce a yellow pigment and also hasnatural low-level resistance to vancomycin.

Enterococcus mundtii This produces yellow pigment but is non-motile.

Enterococcus malodoratus This is found in cheese.

LACTOCOCCUS

Formerly in Streptococcus, these species are associ-ated with milk, dairy products, vegetables and fish.


Use blood agar or milk agar and incubate at 10, 25and 45°C. Test for arginine hydrolysis, fermenta-tion of mannitol and sorbitol, and do PYR andVoges–Proskauer tests (Table 37.5).

Lactococcus species

Lactococcus garveiae (previously Enterococcusseriolicida)This is a cause of mastitis in buffalos and cows; ithas been isolated from human blood and urine.

Lactococcus lactis ssp. lactisThis is present in souring milk, to which itimparts a distinctive flavour; it is used as a cheesestarter.

Lactococcus lactis ssp. cremorisThis is used as a starter in the dairy industry.

Lactococcus plantarum This has been implicated in the spoilage of frozenvegetables.

AEROCOCCUS

These species usually occur in clusters, pairs orshort chains.

Isolation

Use blood agar for food homogenates and for airsampling. Incubate at 30–35°C.


Table 37.5 Lactococcus species and S. thermophilus

Species Growth at Arginine hydrolysis Acid from PYR VP

10°C 45°C Mannitol Sorbitol

L. lactis ssp. lactis + − + + − v vL. lactis ssp. cremoris + − − − − − +L. plantarum + − − + + − +S. thermophilus − + − − − − +

v, variable

Identification

Aerococci give α-haemolysis on blood agar andresemble other ‘viridans’ streptococci. They growon bile agar but, unlike the enterococci, they donot grow at 45°C or possess the D antigen.

Aerococcus species

Aerococcus viridans This is a common airborne contaminant; it is alsofound in curing brines. In humans it occurs on theskin, and may infect wounds and be responsible forendocarditis. Lobsters infected with this organismdevelop a pink discoloration on the ventral surfaceand the blood loses its characteristic bluish-greencolour, becoming pink.

Aerococcus urinae This has been isolated from urine and cases ofendocarditis.

LEUCONOSTOC

This genus contains several species of microaerophilicorganisms of economic importance. Some produce adextran slime on frozen vegetables. Leuconostocs areused as cheese starters. When isolated from humaninfections, because they are resistant to vancomycin,they may be confused with vancomycin-resistant

enterococci (VRE), although they grow at 10°C butnot at 45°C and are PYR negative.

Isolation

Culture material on semi-solid yeast glucose agar atpH 6.7–7.0. Incubate at 20–25°C under 5% car-bon dioxide.

Identification

Test for acid production from sugars – glucose, lac-tose, sucrose, mannose, arabinose and xylose – inan inoculate MRS medium (see p. 347) and lookfor dextran slime on solid medium containing glu-cose (Table 37.6).

Leuconostoc species

Leuconostoc mesenteroidesThis is the most important species. It produces gasand a large amount of dextran slime on media andvegetable matter containing glucose. Colonies onagar media without the sugar are small and grey.Growth is very poor on media without yeast extract.It takes part in sauerkraut fermentation and silageproduction and is responsible for slime disease ofpickles (unless Lactobacillus plantarum is encour-aged by high salinity). It is also responsible for ‘slimysugar’ or ‘sugar sickness’ in peas and fruit juices.

Leuconostoc 343

Table 37.6 Leuconostoc species

Species Acid from Slime

Arabinose Xylose Glucose Mannose Lactose Sucrose

L. cremoris − − + − + − −L. dextranicum − v + v + + +L. lactis − − + v + + −L. mesenteroides + v + + v + + +L. paramesenteroides v v + + v + −

v, variable

Leuconostoc paramesenteroidesThis is similar to L. mesenteroides but it does notproduce a dextran slime. Widely distributed onvegetation and in milk and dairy products.

Leuconostoc dextranicumThis produces rather less slime than L. mesen-teroides and is less active biochemically. It is widelydistributed in fermenting vegetables and in milkand dairy products.

Leuconostoc cremorisThis is rare in nature but extensively used as astarter in dairy products.

Leuconostoc lactisThis is not common. It is found in milk and dairyproducts

PEDIOCOCCUS

Pediococci are non-capsulated microaerophiliccocci that occur singly and in pairs. They are nutri-tionally exacting and are of economic importancein the brewing, fermentation and food-processingindustries.

Isolation

Use enriched media and wort agar for beer spoilagepediococci. Tomato juice media at pH 5.5 is best.

Incubate at temperatures according to speciessought under 5–10% carbon dioxide.

Pediococci differ from leuconostocs inhydrolysing arginine and not producing slimy ormucoid colonies. They grow in acetate or aceticacid agar.

Identification

If it is necessary to proceed to species identification,inoculate MRS sugar media: lactose, sucrose,galactose, salicin, maltose, mannitol, arabinose andxylose. Incubate at appropriate temperature. Gas isnot produced by any species (Table 37.7).

Pediococcus species

Pediococcus damnosus (P. cerevisiae)This is found in yeasts and wort. Contamination ofbeer results in a cloudy, sour product with a pecu-liar odour – ‘sarcina sickness’. P. damnosus isresistant to the antibacterial agents in hops.

Pediococcus acidi-lacticiThis is found in sauerkraut, wort and fermentedcereal mashes. It is sensitive to the antibacterialactivity of hops.

Pediococcus pentosaceusIs also found in sauerkraut as well as in pickles,silage and cereal mashes, but not in hopped beer.


Table 37.7 Pediococcus species

Species Growth at Acid from

37°C 45°C Lactose Sucrose Maltose Mannitol Galactose Salicin Arabinose Xylose

P. damnosus − − − − + − − − − −P. acidi-lactici + + v v − − + − − −P. pentosaceus + + + v + − + + + +P. urinae-equi + − + + + − + + v v

v, variable

Pediococcus urinae-equiThis is a contaminant of brewers’ yeasts. (Thename indicates that it was originally isolated fromurine of a horse.)

ABIOTROPHIA

Formerly called nutritionally variant streptococci,these require blood agar containing 0.001% pyri-doxal hydrochloride or 0.01% L-cysteine. Theyshow satellitism when cross-streaked withStaphylococcus aureus. They are found in thehuman mouth and have been isolated from endo-carditis, ophthalmic infection and brain abscesses.

REFERENCES

Facklam, R. R. (2002) What happened to thestreptococci?: overview of taxonomic andnomenclature changes. Clinical Microbiology

Reviews 15: 613–630.Facklam, R. R. and Elliott, J. A. (1995) Identification,

classification, and clinical relevance of catalase-negative gram-positive cocci; excluding thestreptococci and enterococci. Clinical Microbiology

Reviews 8: 479–495.

Facklam, R. R., Sahm, D. F, Tiexeira, L. M. (1999)Enterococcus. In: Murray, R.M., Baron, E.J.,Pfaller, M. A. et al. (eds), Manual of Clinical

Microbiology, 7th edn. Washington DC: ASMPress, pp. 297–305.

Goodfree, A. F., Kay, D. and Wyer, M. D. (1997)Faecal streptococci as indicators of faecalcontamination in water. Society for Applied

Bacteriology Symposium Series no. 26 83:110S–119S. London: Blackwells.

Hardie, J. M. and Whiley, R. A. (1997) Classificationand overview of the genera Streptococcus andEnterococcus. Society for Applied Bacteriology

Symposium Series no.26 83: 1S–11S.Islam, A. K. (1977) Rapid recognition of Group B

streptococci. Lancet i: 256–257.Leclerc, H., Devriese, L. A. and Mossel, D. A. A.

(1996) Taxonomical changes in intestinal (faecal)enterococci and streptococci: consequences on theiruse as indicators of faecal contamination indrinking water. Journal of Applied Bacteriology

81: 459–466.Petts, D. N. (1984) Colistin-oxolinic acid blood agar: a

new selective medium for streptococci. Journal of

Clinical Microbiology 19: 4–7.Woodford, N., Egelton, C. M. and Morrison, D. (1997)

Comparison of PCR with phenotypic methods forthe speciation of enterococci. Advances in

Experimental Medicine and Biology 418: 405.

References 345

38

Lactobacillus and Erysipelothrix

These two genera contain Gram-positive, non-motile rods that are microaerophilic and faculta-tively anaerobic. Tables 38.1 and 38.2 show theprincipal characteristics that distinguish them fromone another and from Listeria.

LACTOBACILLUS

These Gram-positive bacilli may form chains andmay resemble corynebacteria. They are non-motile,catalase negative and fermentative, and havespecific growth requirements.

Table 38.1 Some Lactobacillus species

Species Growth at Acid from Arginine Aesculin

15°C 45°C Lactose Salicin Mannitol Sorbitoldihydrolase hydrolysis

L . casei + v v + + + − +L . plantarum + − + + + − − +L . brevis + − v − − − + vL . leichmanhii − + v + − − v +L . acidophilus − + + + − − − +L . delbrueckii − + v v − − v −L . salivarius − + + v + + − v

v, variable

Table 38.2 Lactobacillus, Erysipelothrix and Listeria spp.a

Genus Motility Catalase Growth Acid from H2S Growth at 5°C salicin production requirement b

Lactobacillus − − − + − +Erysipelothrix − − + − + −Listeria + + + + − −a Added for comparisonb E.g. MRS medium

They are important in the food, dairy and brew-ing industries, commensals of animals, and foundin the human gut and vagina. Some species arefound in plant material. Although generally consid-ered to be non-pathogenic, some strains are associ-ated with caries and others have been recoveredfrom blood cultures.

Isolation

Plate in duplicate on MRS (deMann, Rogosa andSharp), Rogosa and similar media at pH 5.0–5.8.LS Differential Medium is particularly useful in theexamination of yoghurt. Use Raka-Ray agar forbeer. Many strains grow poorly, if at all, at pH 7.0,but plate human pathological material on bloodagar (see below). Incubate one set of plates aerobi-cally and the other in a 5 : 95 carbon dioxide–hydrogen atmosphere. Incubate cultures from foodat 28–30°C and from human or animal material at35–37°C for 48–72 h. Examine for very small,white colonies. Few other organisms, apart frommoulds, will grow in the acid media.

For provisional identification of L. acidophilus,which is the most common species in human mate-rial, place the following discs on seeded blood agarplates: (1) a sulphonamide sensitivity disc that hasbeen dipped in saturated sucrose solution, and (2) apenicillin (5 units) disc.

Lactobacillus acidophilus gives a green haemoly-sis. Growth is stimulated by sucrose; it is resistantto sulphonamides and sensitive to penicillin.

Identification

Identification to species is rarely required. It is noteasy; reactions vary according to technique and thetaxonomic position of some species is still uncer-tain.

Subculture if necessary to obtain pure growth andheavily inoculate the modified MRS broth contain-ing the following sugars: lactose, salicin, mannitol(for arginine hydrolysis test) and sorbitol. Incubate

28–30°C for 3–4 days. Inoculate tubes of MRSbroth and incubate at 15°C and at 45°C (Table38.1). Test for arginine dihydrolase and aesculinhydrolysis.

Culture on solid medium low in carbohydrate totest for catalase (high-carbohydrate medium maygive false positives). The catalase test is negative(corynebacteria and listeria, which may be con-fused with lactobacilli, are catalase positive).

Table 38.1 lists the properties of some of the morecommon lactobacilli, but there are many morespecies. The API 50CHL system is recommendedfor full identification of members of this genus.

Species of lactobacilli

Lactobacillus acidophilus and L. casei

These are widely distributed and found in milk anddairy products and as commensals in the alimen-tary tract of mammals. They are used, along withother organisms, to make yoghurt and are alsoassociated with dental caries in humans as a sec-ondary invader. L. acidophilus is probably theorganism described as the Boas–Oppler bacillus,observed in cases of carcinoma of the stomach inhumans, and also Doderlein’s bacillus, found in thehuman vagina.

Colonies of this organism on agar media aredescribed as either ‘feathery’ or ‘crab like’.

Lactobacillus plantarum

This species is widely distributed on plants, aliveand dead. It is one of the organisms responsible forfermentation in pickles and is used in the manufac-ture of sauerkraut and silage.

Lactobacillus delbrueckii

This is common in vegetation and grain. It is usedas a ‘starter’ to initiate acid conditions in yeast fer-mentation of some grains. This organism grows onagar medium as small, flat colonies with crenatededges. This species now includes the former L. lac-tis and L. bulgaricus.

Lactobacillus 347

Lactobacillus leichmannii

This is found in most dairy products. It is used inthe commercial production of lactic acid and growson agar as small white colonies.

Other species

For these, see Sharpe (1981) and Kandler andWeiss (1986). Some lactobacilli, isolated frommeat, are now placed in the genus Carnobacterium(see Collins et al., 1987).

Counting lactobacilli

Enumeration of lactobacilli is a useful guide tohygiene and sterilization procedures in dairy indus-tries. Prepare suitable dilutions of bottle or churnrinsings or of swabbings steeped in 0.1% peptonewater. Do pour plates (see ‘Plate counts’, p. 146) inRogosa medium or one of the other media recom-mended for lactobacilli. Incubate plates at 37°C for3 days or at 30°C for 5 days in an atmosphere of5% carbon dioxide.

Dental surgeons and oral hygienists sometimesrequest lactobacillus counts on saliva. The patientchews paraffin wax to encourage salivation, and10 ml saliva are collected and diluted for platecounts. Usually dilutions of 1 : 10, 1 : 100 and1 : 1000 are sufficient.

Counts of 105 lactobacilli/ml of saliva are thoughtto indicate the likelihood of caries. There is a sim-ple colorimetric test in which 0.2 ml saliva is addedto medium, as a shake tube culture, and a colourchange after incubation suggests a predispositionto caries.

For more information about this genus, seeSharpe (1981), and Kandler and Weiss (1986).

ERYSIPELOTHRIX

This genus contains small (2 × 0.3 μm), Gram-pos-itive rods that are found in and on healthy animalsand on the scales of fish. Humans may be infectedthrough abrasions when handling such animals,

particularly pigs and fish. The resulting skin lesionsare called erysipeloid. Epizootic septicaemia ofmice has also been reported. Table 38.2 shows thecharacteristics that distinguish Erysipelothrix fromLactobacillus and Listeria spp.

The two species in this genus are aerobic, faculta-tively anaerobic, non-motile, and produce acidfrom glucose, lactose and maltose but not fromxylose or mannitol. They produce H2S, do nothydrolyse aesculin, and are catalase negative andresistant to neomycin. They will grow at 5°C(Table 38.2).


Culture tissue or fluid from the edge of a lesion.Emulsified biopsy material is better than swabs oflesions. Plate on blood agar and on crystal violet orazide blood agar and incubate at 37°C in 5–10%carbon dioxide for 24–48 h.

Colonies are either small (0.5–1.0 mm) and ‘dew-drop’ or larger and granular and show α-haemoly-sis. Young cultures are Gram variable; oldercultures show filaments. Test for motility with aCraigie tube. Test for fermentation of glucose, lac-tose, maltose, sucrose and mannitol using enrichedmedium, e.g. broth containing Fildes’ extract.Inoculate aesculin broth. Inoculate two tubes ofenriched agar and incubate one each at 5 and 37°C.Do a catalase test and test for neomycin sensitivityby the disc method. Inoculate gelatin stab tubes,incubate at 22°C for 2–3 days and look for the‘test-tube brush’ growth that is characteristic of thetwo species.

Species of Erysipelothrix

There are two species – E. rhusiopathiae andE. tonsillarum – which have very similar culturaland biochemical properties, except that the latterproduces acid from sucrose and the formerdoes not. E. rhusiopathiae is a pathogen of pigs;E. tonsillarum may be found in their tonsils but isnot pathogenic.

348 Lactobacillus and Erysipelothrix

For more information about the genusErysipelothrix, see Jones (1986) and McLauchlinand Jones (1999).

REFERENCES

Collins, M. D., Farrow, J. A., Phillips, B. A., Feresu, S.B. and Jones, D. (1987) Classification ofLactobacillus divergens, Lactobacillus piscicola

and some catalase-negative, asporogenous, rod-shaped bacteria from poultry in a new genusCarnobacterium. International Journal of

Systematic Bacteriology 37: 310.Jones, D. (1986) The genus Erysipelothrix. In: Sneath,

P. H. A. et al. (eds), Bergey’s Manual of Systematic

Bacteriology, Vol. 2. Baltimore, MA: Williams &Wilkins, p. 1245.

Kandler, O. and Weiss, N. (1986) The genusLactobacillus. In: Sneath, P. H. A., Mair, N. H. S.,Sharpe, M. E. et al. (eds), Bergey’s Manual of

Systematic Bacteriology, Vol. 2. Baltimore, MA:Williams & Wilkins, p. 1245.

McLauchlin, J. and Jones, D. (1999) Erysipelothrix andListeria. In: Collier, L., Balows, A. and Sussman,M. (eds), Topley and Wilson’s Microbiology and

Microbial Infections, 9th edn. Vol. 2, Systematic


Sharpe, M. E. (1981) The genus Lactobacillus. In:Starr, N. P., Stolph, H., Trüper, H. G. et al. (eds),The Prokaryotes: a Handbook on Habitats,

Isolation and Identification of Bacteria. New York:Springer, p. 1653.

References 349

These organisms are frequently club shaped, thin inthe middle with swollen ends that containmetachromatic granules. These are best seen whenAlbert’s or Neisser’s stain is used. The stainedbacilli may also be barred, with a ‘palisade’ or‘Chinese letter’ arrangement, as a result of a snap-ping action in cell division.

THE DIPHTHERIA GROUP ANDRELATED ORGANISMS

Diphtheria is now a very rare disease in developedcountries and may be clinically atypical.

If an organism is suspected of being a diphtheriabacillus because of its colonial and microscopicappearance, inform the physician at once andbefore proceeding to identify the bacillus. Ifpossible send a culture to an appropriate referencelaboratory.

It is in the best possible interests of the patient thatthis is done. No harm will result if the final result isnegative, but a delay of 24 h or more to confirm apositive case may have serious consequences, espe-cially in an unimmunized child, apart from the pos-sibility of an outbreak of the disease. A case ofdiphtheria will usually generate large numbers ofswabs from contacts. For the logistics of mass swab-bing and the examination of large numbers of swabsin the shortest possible time, see Collins and Dulake(1983).

‘Diphtheroids’ is an informal name traditionallygiven by clinical microbiologists to micro-organisms which, on primary culture, superficiallyresemble the diphtheria bacillus.

Isolation and identification of thediphtheria organism and related species

Plate throat, nasal swabs, etc., on Hoyle’s or othertellurite media and on blood agar. Incubate at 37°Cand examine at 24 and 48 h. For colonial charac-teristics, see species descriptions.

Make Gram-stained films of suspicious colonies.This is better than Albert’s or other stains for theexamination of colonies from tellurite media. C.diphtheriae tends to decolourize easily and appearsGram negative compared with diphtheroids thatstain solidly Gram positive. Stain a control film ofstaphylococci on the same slide.

Subculture a colony from the tellurite to aLoeffler medium/slope as soon as possible.Incubate at 37°C for 4–6 h. The best results areobtained with Loeffler medium containing anappreciable amount of water of condensation andwhen the tube has a cottonwool plug or the cap isleft loose. Examine Gram- and Albert-stainedfilms for typical beaded bacilli, which may showmetachromic granules at their extremities.Inoculate Robinson’s serum water sugars (glucose,sucrose and starch) and a urea slope. But do notwait for the result before doing the plate toxi-genicity test.

Note that Robinson’s sugar medium is bufferedand contains horse serum. Unbuffered horse serummedium may give false-positive results because offermentation of the small amount of glycogen itcontains, which is metabolized by many corynebac-teria. Similarly, media that contain unheated rabbitserum may give false-positive starch fermentationreactions. Natural amylases hydrolyse starch,forming glucose, which is fermented by the

39

Corynebacteria

organism. The biochemical reactions are given inTable 39.1. The API CORYNE kit is useful.

Plate toxigenicity testElek’s method, as modified by Davies (1974) givesexcellent results. This requires control strains, onetoxigenic (e.g. NCTC 10648) and one non-toxigenic(e.g. NCTC 10356) and commercial diphtheria anti-toxin. These may be maintained on Dorset eggmedium slopes. The titres of available antisera vary,so laboratories should titrate each new batch againststock cultures, using concentrations between 500 and1000 units/ml. This should be done in anticipation,not when a diphtheria investigation is imminent.

For reasons not yet understood, certain batches ofmedium and serum give poor results. To test themedium and serum, use the control strains notedabove and also NCTC 3894, which is weakly toxi-genic. The strength of the antitoxin used is critical.

To be of value to the clinician, the plate toxi-genicity test must give clear results in 18–24 h. Ifthe toxigenic control does not show lines at 24 h,the medium or serum is unsatisfactory. The testrequires a certain amount of experience, and themedium and serum used must be tested frequently.

Inoculate a moist slope of Loeffler’s medium witha colony from the primary plate as early as possiblein the morning. Also inoculate Loeffler slopes withthe stock control cultures. Incubate at 37°C for 4–6h.

Melt two 15-ml tubes of Elek’s medium and coolto 50°C. Add 3 ml sterile horse serum to each and

pour into plastic or very clear glass Petri dishes.Place immediately, in the still liquid medium ineach plate, a strip of filter paper (60 × 15 mm) thathas been soaked in diphtheria antitoxin. Place thestrip along a diameter of the plate. Dry the plates.These may be stored in a refrigerator for severaldays.

On each plate, streak the 4–6 h Loeffler culturesat right-angles to the paper strip so that theunknown strain is between the toxigenic and non-toxigenic controls as in Figure 39.1, approximately10 mm apart. Incubate at 37°C for 18–24 h. If nolines are visible re-incubate for a further 12 h.Examine by transmitted light against a black back-ground. If the unknown strain is toxigenic, its pre-cipitation lines should join those of the toxigeniccontrol, i.e. a reaction of identity. Strains that pro-duce little toxin may not show lines but turn thelines of adjacent toxigenic strains.

An alternative method is that of Jamieson (1965).Prepare the Loeffler cultures as above. Pour theplates but do not add the antitoxin strip. Dry theplates and place along the diameter of each a filterpaper strip (75 × 5 mm) that has been soaked indiphtheria antitoxin (for concentration see above).Place the plates over a cardboard template markedout as in Figure 39.2 and heavily inoculate themfrom the Loeffler cultures in the shape of thearrowheads. Place the unknown strain between thetwo controls. Incubate at 37°C for 18–24 h andexamine for reactions of identify.

The diphtheria group and related organisms 351

Table 39.1 Corynebacteria from human sources

Species Acid from β-Haemolysis Urease

Glucose Sucrose Starch

C. diphtheriaegravis + − + − −mitis + − − + −intermedius + − − − −

C. ulcerans + − + − +C. pseudodiphtheriticum − − − −C. xerosis + + + − −C. jeikanum + − − −

Report the organism as toxigenic or non-toxi-genic C. diphtheriae cultural type gravis, mitis orintermedius.

Note that, in any outbreak of diphtheria causedby a toxigenic strain, a proportion of contacts maybe carrying non-toxigenic C. diphtheriae.

Species of corynebacteria fromhuman sources

The following general descriptions of colonyappearance and microscopic morphology of diph-theria bacilli and diphtheroids is a guide only. Theappearance of the colony varies considerably withthe medium and it is advisable to check this period-

ically with stock (NCTC and ATCC) organismsand, if possible, strains from recent cases of the dis-ease. Even then, strains may be encountered thatpresent new or unusual colony appearances.

Corynebacterium diphtheriaeThree biotypes, gravis, mitis and intermedius, wereoriginally given these names because of theirrespective association with the severe, mild andintermediate clinical manifestations of the disease.These names have become attached to the colonialforms and related to starch fermentation. Theseproperties do not always agree and it is moreimportant to carry out the toxigenicity test than toattempt to interpret the colonial and biochemicalproperties, which should be used only to establishthat the organism belongs to the species. The

352 Corynebacteria

No reaction Non-toxigenic control

Test strain (toxigenic)

Toxigenic control

Weakly toxigeniccontrol

Test strain

Reaction of identity

Turns lines oftoxigenic strains

Reaction ofidentity

No reaction

Non-toxigenic control

Test strain (toxigenic)

Toxigenic control

Test strain (non-toxigenic)

Weakly toxigenic control

Figure 39.1 Elek plate toxigenicity test

Figure 39.2 Jamieson’s method for plate toxigenicity test

following colony appearances relate to telluritemedium of the Hoyle type.

Corynebacterium diphtheriae, gravis typeAt 18–24 h, the colony is 1–2 mm in diameter,pearl grey with a darker centre; the edge is slightlycrenated. It fractures easily when touched with awire but is not buttery. At 48 h, it has enlarged to3–4 mm and is much darker grey, with the edgemarkedly crenated and with radial striations. Thisis the ‘daisy head’ type of colony. The organismdoes not emulsify in saline. Films stained withmethylene blue show short, often barred or beaded,bacilli arranged in irregular ‘palisade’ form or‘Chinese letters’. Well-decolourized Gram filmscompared on the same slide with staphylococci ordiphtheroids appear relatively Gram negative.Glucose and starch are fermented, but not sucrose.There is no haemolysis on blood agar.

Corynebacterium diphtheriae, mitis typeAt 18–24 h, colonies are 1–2 mm across and aredark-grey, smooth and shining. The colonies frac-ture when touched but are much more buttery thanthe gravis type. At 48 h, they are larger (2–3 mmdiameter), less shiny and darker in the centre, whichmay be raised, giving the ‘poached egg’ appearance.The organisms emulsify easily in saline. Films showlong, thin bacilli, usually beaded and with granules,which may not be very Gram positive (see above).Glucose is fermented but not starch or sucrose.Colonies on blood agar are haemolytic.

C. diphtheriae, intermedius typeColonies at 18–24 h are small (1 mm diameter), flatwith sharp edges, black and not shining or glossy.At 48 h, there is little change. They are easily emul-sified and films show beaded bacilli, intermediate insize between gravis and mitis types. Glucose is fer-mented but not starch or sucrose. Colonies onblood agar are non-haemolytic.

Corynebacterium ulceransThis organism resembles the gravis type of diphthe-ria bacillus and is often mistaken for it. It stainsmore evenly, however, and the bacilli are usuallyshorter than those of C. diphtheriae. Colonies

tends to be more granular in the centre. Glucoseand starch are fermented but not sucrose. C. ulcer-ans splits urea; C. diphtheriae does not. C. ulceransalso grows quite well at 25–30°C. It gives a reac-tion of incomplete identity with the plate toxigenic-ity test and C. diphtheriae antitoxin. This organismdoes not cause diphtheria and is not readily com-municable, but can cause ulcerated tonsils.

Corynebacterium pseudodiphtheriticum Formerly known as Hoffman’s bacillus, colonies ofthis species at 18–24 h are 2–3 mm in diameter,domed, shiny, and dark-grey or black. They arebuttery (occasionally sticky) and easily emulsified,and films show intensely Gram-positive bacilli thatare smaller and much more regular in shape, sizeand arrangement than diphtheria bacilli.Carbohydrates are not fermented. These organismsare common and harmless commensals.

Corynebacterium xerosisAt 18–24 h, colonies are 1–2 mm in diameter, flat,grey and rough, with serrated edges. At 48 h, theyare 2–3 mm in diameter. Films show beaded bacilli,very Gram positive. This is a commensal of theconjunctivae and often found in the femalegenitalia. It is not a pathogen but resembles veryclosely the diphtheria bacillus in microscopic (butnot colonial) morphology. It is, however, muchmore strongly Gram positive than C. diphtheriaeand produces acid from glucose, usually sucrosebut not starch.

Corynebacterium jeikeiumFormerly the ‘JK diphtheroids’, these have beenisolated from blood and ‘sterile’ body fluids. Theymay colonize prostheses and are resistant to manyantibiotics (see Jackman et al., 1987).

CORYNEBACTERIA FROM ANIMALSOURCES


Culture, pus, etc., on blood agar and telluritemedium, incubate at 37°C and examine at 24 and

Corynebacteria from animal sources 353

48 h for colonies of beaded Gram-positive bacilli.Some corynebacteria are haemolytic on blood agar.Small grey–black colonies are usually found on tel-lurite medium.

Subculture on Loeffler medium and examineAlbert-stained films at 6–18 h for clubbed orbeaded bacilli which may have metachromaticgranules. From the Loeffler medium inoculate gela-tin medium, nutrient agar, Robinson’s serum waterwith glucose, sucrose, mannitol, maltose, lactoseand urea medium. Incubate at 37°C for 24–48 h(Table 39.2). The API CORYNE kit is useful.

Corynebacterium pseudotuberculosis (C. ovis)Colonies are small, usually grey but may be yellow-ish and are haemolytic, especially in anaerobic cul-tures. Loeffler serum may be liquefied. The bacilliare slender and clubbed, very like C. diphtheriae. Itis responsible for epizootics in rodents and is a nui-sance in laboratory animal houses.

This is the Preisz–Nocard bacillus that causespseudotuberculosis in sheep, cattle and swine andlymphangitis in horses.

Corynebacterium kutscheri (C. murium)Colonies on blood agar are flat, grey and non-haemolytic. The bacilli are slender, often filamen-tous. Loeffler medium is not liquefied. It isresponsible for fatal septicaemia in mice and rats.

Corynebacterium renaleThis is now believed to be three separate species –C. renale, C. pilosum and C. cystitidis – all associ-ated with urinary tract infections of cattle.

Corynebacterium bovisThis is found in cows’ udders and may cause mas-titis.Two organisms formerly regarded as corynebacte-ria have been transferred to other genera. ‘C. equi’is now Rhodococcus equi (see p. 402) and ‘C. pyo-genes’ is now Actinomyces pyogenes (see p. 406).

‘CORYNEFORM BACILLI’

This informal name is given to various bacilli thatresemble corynebacteria in morphology and arefound in association with animals and dairy prod-ucts. They are relatively heat resistant (70°C for15 min). One such is Microbacterium lacticum,which is thermoresistant and a strict aerobe. It isnon-motile, grows slowly at 30°C on milk agar,forms acid from glucose and lactose, is catalasepositive, hydrolyses starch, is lipolytic and does notliquefy gelatin in 7 days. A pale-yellow pigmentmay be formed. These organisms may causespoilage in milk and indicate unsatisfactory dairyhygiene. They are also found in deep litter in henhouses.

The genus Corynebacterium is reviewed by vonGraevenitz et al. (1998). See also Clarridge et al.(1999).

REFERENCES

Clarridge, J. E., Popovic, T. and Inzana, T. J. (1999)Diphtheria and other corynebacterial and

354 Corynebacteria

Table 39.2 Corynebacteria from animal sources

Species Gelatin Acid from β-Haemolysis Ureaseliquefaction

Glucose Sucrose Mannitol Maltose Lactose

C. pseudotuberculosis v + v − + v v +C. kutscheri − + + − + − − vC. renale − + − − v − − +C. bovis − + − v + v − −

v, variable

References 355

coryneform infections. In: Collier, L., Balows, A.and Sussman, M. (eds), Topley and Wilson’s

Microbiology and Microbial Infections, 9th edn.Vol. 3, Bacterial Infections, CD-ROM. London:Arnold, Chapter 19.

Collins, C. H. and Dulake, C. (1983) Diphtheria: thelogistics of mass swabbing. Journal of Infection 6:227–230.

Davies, J. R. (1974) Identification of diphtheria bacilli.In: Collins, C. H. and Willis, A. T. (eds),Laboratory Methods 1. Public Health LaboratoryService Monograph No. 4. London: HMSO.

Jackman, P. J. H., Pitcher, D. G., Pelcznska, S. and

Borman, P. (1987) Classification of corynebacteriaassociated with endocarditis (Group JK) asCorynebacterium jeikeium sp. nov. Systematic and

Applied Microbiology 9: 83–90.Jamieson, J. E. (1965) A modified Elek test for

toxigenic Corynebacterium diphtheriae. Monthly

Bulletin of the Ministry of Health 24: 55–58.von Graevenitz, A. W. C., Coyle, M. B. and Funke, G.

(1998) Corynebacteria and rare corynefoms. In:Collier, L., Balows, A. and Sussman, M. (eds),Topley and Wilson’s Microbiology and Microbial


Bacteriology. London: Arnold, Chapter 25.

These are aerobic, Gram-positive, catalase-posi-tive, non-sporing rods. Both are of interest to thefood industry. Listeria is associated with food-borne disease (see Chapter 13) and Brochothrix isa food-spoilage organism. Table 40.1 shows somedistinguishing characteristics.

LISTERIA

These are aerobic, facultative anaerobic, Gram-positive rods that are catalase positive and fermentcarbohydrates. They do not grow on Gardner’sSTAA medium.

They are found in grass, silage, soil, sewage andwater. There are seven species, two of which,L. monocytogenes and L. ivanovii, are pathogenicfor humans and animals. Listerias have becomeimportant food-borne pathogens (see Chapter 13).They survive in cold storage and may multiply atlow temperatures.

Isolation

Clinical materialPlate cerebrospinal fluid (CSF), centrifugeddeposits and blood cultures on blood agar and

(Oxford) listeria-selective agar. There are otherselective commercially available media, which con-tain various antibiotics (e.g. cycloheximide colistinsulphate, cefotetan, fosfomycin) that suppress thegrowth of other organisms.

Homogenize tissues in peptone water diluent andplate on blood agar and listeria-selective agar.Treat the remainder of the homogenate as for fae-ces (see below). Incubate primary plates at 37°C for24–48 h.

Homogenize faeces in peptone water. Add l vol-ume of homogenate to 9 volumes of listeria selec-tive broth. Subculture at 4 and 24 h to selectivemedia. At 24 h add 0.1 ml to secondary listeria-selective broth. Incubate at 37°C for 24–48 h andplate on selective media.

Food

Homogenize 25 g of the material in 225 ml com-mercial listeria-enrichment broth. Incubate at 30°Cfor 48 h. Subculture to listeria-selective (ModifiedOxford – MOX – or PALCAM) agar and incubateat 37°C for 48 h under microaerophilic conditions(e.g. with campylobacter gas-generating kit,Oxoid). Colonies on MOX are black, and those onPALCAM are grey–green or black, with shrunkencentres and a black halo. Neither medium is whollyselective for listerias.

Identification

Colonies on blood agar are small (1 mm diameter),smooth and surrounded by a narrow zone of

40

Listeria and Brochothrix

Table 40.1 Listeria and Brochothrix species

Genus Growth

At 37°C On Gardner’s STAA

Listeria + –Brochothrix – +

β-haemolysis. On (Oxford) selective agar they are thesame size but are black, with a black halo and a sunkencentre. Colonies on translucent media, viewed by lightreflected at 45° from below (Henry method, seePrentice and Neaves, 1992), are grey–blue in colourand finely textured. Colonies of most other organismsare opaque and/or otherwise coloured.

Examine for typical ‘tumbling’ motility at20–22°C (no higher, because listerias are non-motile above 25°C). Test for acid production fromglucose, rhamnose, xylose and mannitol (10% car-bohydrate in purple broth) and do the CAMP(Christie, Atkins and Munch-Petersen) test.

CAMP testUse blood agar prepared from fresh, washed, defib-rinated sheep blood. Make dense suspensions fromcolonies of the suspected listerias and ofStaphylococcal aureus (NCTC 1803). Makestreaks of the S. aureus across the blood agar,about 2–3 cm apart. Streak the suspect listerias atright angles but do not touch the other streaks.Incubate at 37°C for 12–18 h (not longer).

Listeria monocytogenes and L. seeligeri give nar-row zones of β-haemolysis near the staphylococcalcross steaks. Other species give no reactions (Table40.2).

Kit and commercial testsThere are several commercial kits that facilitate thetesting of food samples. These include the Rosco

Listeria ZYM (Lab M), Pathalert (Merck),Microscreen (Microbact), Mastalist (Mast), Micro-ID and the API listeria strip. There is an enzyme-linked immunosorbent assay (ELISA) kit that usesmonoclonal antibodies (Listeria-TEC, Organon),and gene probe systems (Gene-Trak and Gen-Probe) For a review of some of these, see Beumer etal. (1996).

Listeria species

Listeria monocytogenesThis is responsible for human listeriosis, usuallyfood borne. Symptoms include meningitis (espe-cially in neonates) and septicaemia.

Listeria ivanoviiThis causes septicaemia, encephalitis and abortionin farm animals and also disease in humans.

The other species, L. innocua, L. seeligeri,L. welshimeri, L. grayi and L. murrayi, are notclinically significant but are included in Table 40.2because it is important to distinguish them from thepathogens.

For a review of listeriosis, see Bahk and Marth(1990), and for more detailed information aboutisolation and identification, see Prentice andNeaves (1992).

Listeria 357

Table 40.2 Listeria species

Species β-Haemolysis CAMP test (S. aureus) Acid from

Rhamnose Xylose Mannitol

L. monocytogenes + + + − −L. ivanovii + − − + −L. innocua + − v − −L. seeligeri w w − v −L. welshimeri − − v + −L. grayi − − − − +L. murrayi − − − − +

w, weak; v, variable

358 Listeria and Brochothrix

BROCHOTHRIX

These are aerobic, facultative anaerobic, Gram-positive, non-motile rods that occur singly or inshort chains or filaments, often with pleomorphicforms. They are catalase positive, ferment carbohy-drates, and grow at 4°C and 25°C, but not at 37°C.They grow poorly on MRS but well on STAAmedia.

There are two species: B. thermosphacta is a meatspoilage organism, found in souring (‘British fresh’)sausages at low temperatures, in film-packagedproducts, milk and cheese. B. campestris is foundin grass and soil.

Isolation

Culture on Gardner’s STAA medium and incubateat 20–25°C.

Identification

The most important characteristic is growth onSTAA medium. Test for growth in the presence of8% NaCl and for hippurate hydrolysis. B. thermos-phacta grows in the NaCl medium but is hippuratenegative. B. campestris fails to grow in NaClmedium but hydrolyses hippurate.

For more information, see Gardner (1966) andDodd and Dainty (1992).

REFERENCES

Bahk, J. and Marth, E. H. (1990) Listeriosis andListeria monocytogenes. In: Cliver, D. O. (ed.),Foodborne Diseases. New York: Academic Press,pp. 248–259.

Beumer, R. R., te Giffel, M. C., Kok, M. T. C. et al.(1996) Confirmation and identification of Listeria

species. Letters in Applied Microbiology 22:448–452.

Dodd, C. E. R. and Dainty, R. H. (1992) Identificationof Brochothrix by intracellular and surfacebiochemical composition. In: Board, R. G., Jones,D. and Skinner, F. A. (eds), Identification Methods

in Applied and Environmental Microbiology.Society for Applied Bacteriology Technical SeriesNo. 29. Oxford: Blackwells, pp. 297–310.

Gardner, G. A. (1966) A selective medium for theenumeration of Microbacterium thermosphactum

in meat and meat products. Journal of Applied

Bacteriology 29: 455–457.Prentice, G. A. and Neaves, P. (1992) The identification

of Listeria species. In: Board, R.G., Jones, D. andSkinner, F.A. (eds), Identification Methods in

Applied and Environmental Microbiology. Societyfor Applied Bacteriology Technical Series No. 29.Oxford: Blackwells, pp. 283–296.

Members of Neisseria species are aerobic, oxidaseand catalase positive, Gram-negative cocci. Theyoccur mostly as oval or kidney-shaped cocciarranged in pairs, with their long axes parallel.They are non-motile, non-sporing and oxidase pos-itive, and reduce nitrates to nitrites. The twoimportant pathogens are N. gonorrhoeae (thegonococcus), causative organism of gonorrhoea,and N. meningitidis (the meningococcus), one ofthe organisms causing meningitis. Both requiremedia containing blood or serum. Colonies arenon-pigmented and translucent.

There are about 17 other species that are com-mensals or opportunist pathogens. Most of themgrow at 22°C on nutrient agar with opaque andoften yellow-pigmented colonies.

THE TWO IMPORTANTPATHOGENS

Neisseria gonorrhoeae

Examine Gram-stained films of exudates (urethra,cervix, conjunctivae, etc.) for intracellular Gram-negative diplococci. Exercise caution in reportingthe presence of gonococci in vaginal and conjuncti-val material and in specimens from children.

Culture at once or use one of the transport media.Culture on deep plates (20–25 ml of medium) ofNew York City (NYC) or Thayer Martin (TM)media with commercial selective supplements.Incubate all cultures 35–36°C (better than 37°C) in5–10% CO2 and 70% humidity. Examine at 24 and48 h. Colonies of N. gonorrhoeae are transparentdiscs about 1 mm in diameter, later increasing in size

and opacity, when the edge becomes irregular. Testsuspicious colonies by the Gram film and oxidasetest. Typical morphology and a positive oxidase testare presumptive evidence of the gonococcus in mate-rial from the male urethra, but not from other sites.

For rapid results test colonies by FA method, co-agglutination (Phadebact) or rapid biochemicaltests (Gonocheck II, Rapid IDNH).

Subculture oxidase positive colonies on chocolateagar and incubate overnight in a 10% carbon diox-ide atmosphere. Immediate subculture of colonieson primary media into carbohydrate test mediamay not be satisfactory because such media containantibiotics; other organisms may grow. Repeat theoxidase test on the subculture and emulsify positivecolonies in about 1 ml serum broth. Use this to testfor acid production from glucose, maltose, sucroseand lactose in a serum-free medium. Some serummedia may contain a maltase that may give a false-positive reaction with maltose. The best results areobtained with a semisolid medium, as gonococci donot like liquid media (Table 41.1). The API QUAD-FERM and API WH are useful.

Confirm fermentation results by agglutination orco-agglutination tests (Phadebact).

Exercise caution in reporting these organisms with-out adequate experience, especially if they are fromchildren, eye swabs, anal swabs or other sites. Theymay be meningococci or other Neisseria that are gen-ital commensals, which do not cause sexually trans-mitted disease. Sugar reactions and colonialmorphology are not entirely reliable. Certain otherorganisms, Moraxella (see p. 307) and Acinetobactercalcoaceticus, may resemble gonococci in direct filmsand on primary isolation. Moraxella, like Neisseria, isoxidase positive but Acinetobacter is oxidase negative.

41

Neisseria

For further information on gonococci, seeJephcott and Egglestone (1985), Jephcott (1987)and Morse and Genco (1999).

Neisseria meningitidis

Examine films of spinal fluid for intracellularGram-negative diplococci. Inoculate blood agarand chocolate agar with cerebrospinal fluid (CSF),or its centrifuged deposit. Incubate, preferably in a5% carbon dioxide atmosphere, at 37°C overnight.Incubate the remaining CSF overnight and repeatculture on chocolate agar.

Colonies of meningococci colonies are transpar-ent, raised discs about 2 mm in diameter at18–24 h. In CSF cultures, the growth will be purewhereas, in eye discharges and vaginal swabbingsof young children, etc., other organisms will bepresent. Subculture oxidase-positive colonies ofGram-negative diplococci in semisolid serum sugarmedia (glucose, maltose, sucrose). Meningococcigive acid in glucose and maltose (see Table 41.1).

Agglutinating sera are available but should beused with caution. There are at least four serologi-cal groups. Slide agglutinations may be unreliable.It is best to prepare suspensions in formol saline fortube agglutination tests.

OTHER SPECIES AND SIMILARORGANISMS

These grow on nutrient agar. Colonies are variable:1–3 mm, opaque, glossy or sticky, fragile or coher-ent; others may be rough and granular.

Neisseria lactamica is important in that it mightbe confused with pathogenic species because it isoxidase positive and does not grow at 22°C. It isfermentative and produces acid from lactose, butthis may be delayed. The pathogens do not changelactose and are oxidative.

Other species include N. flavescens, which pro-duces a yellow pigment, N. mucosa, N. elongataand N. subflava. For the properties of these seeCowan and Steel’s Manual (Barrow and Feltham,1993) and Johnson (1983) for potential patho-genicity.

Gemella haemolysans is sometimes confused withneisserias because, although it is Gram positive, itis easily decolourized. It differs from the neisseriasin being β-haemolytic, oxidase and catalase nega-tive.

REFERENCES

Barrow, G. I. and Feltham, R. K. A. (eds) (1993)Cowan and Steel’s Manual for the Identification of


Jephcott, A. E. (1987) Gonorrhoea. In: Sexually

Transmitted Diseases. London: Public HealthLaboratory Service, pp. 24–40.

Jephcott, A. E. and Egglestone, S. I. (1985) Neisseria

gonorrhoeae. In: Collins, C. H. and Grange, J. M.(eds), Isolation and Identification of Micro-

organisms of Medical and Veterinary Importance.Society for Applied Bacteriology Technical SeriesNo. 21. London: Academic Press, pp. 143–160.

Johnson, A. P. (1983) The pathogenic potential of

360 Neisseria

Table 41.1 Neisseria species

Species Growth on Growth at 22°C Acid fromnutrient agar

Glucose Maltose Sucrose Lactose

N. gonorrhoeae − − + − − −N. meningitidis − − + + − −N. lactamica + − + + − +

commensal species of Neisseria. Journal of Clinical

Pathology 36: 213–223.Morse, S. A. and Genco, C. A. (1999) Neisseria. In:

Collier, L., Balows, S. and Sussman, M. (eds),

Topley and Wilson’s Microbiology and Microbial

Infections, 9th edn, Vol. 2, Systematic

Bacteriology, CD-ROM. London: Arnold,Chapter 37.

References 361

This is a large genus of Gram-positive, spore-bearing bacilli that are aerobic, facultativelyanaerobic and catalase positive. Many species arenormally present in soil and in decaying animaland vegetable matter. One species, Bacillusanthracis, is responsible for anthrax in humansand animals. Several other species are now knownto cause disease in humans (e.g. food poisoning)and animals. Some are responsible for foodspoilage.

The bacilli are large (up to 10 × 1 μm) and com-monly adhere in chains. Most species are motile,some are capsulated, and some produce a stickylevan on media that contain sucrose. Spores may beround or oval, central or subterminal.

Physiological characters vary. There are strictaerobes and species that are facultative anaerobes.A few are thermophiles. In old cultures, Gram-negative forms may be seen and some species arebest described as ‘Gram indifferent’.

ANTHRAX

Bacillus anthracis causes a fatal septicaemia in ani-mals. It is transmissible to humans, producing alocalized cutaneous necrosis (‘malignant pustule’)or pneumonia (once known as woolsorters’ dis-ease) as a result of the inhalation of spores. Suchinhalation may also result in a ‘flu-like’ illness, ashas happened when the organisms were used as aterrorist weapon.

Imported bone meal, meat meal and other fertiliz-ers, and sometimes feeding-stuffs may be infected.

These are often prepared from the remains ofanimals that died of anthrax. In most developedcountries, an animal dying of anthrax must beburied in quicklime under the supervision of a vet-erinarian and below the depth at which earth-worms are active.

There are regulations governing the importationof hairs and hides from foreign countries whereanthrax is endemic.

Isolation of B. anthracis frompathological material

Veterinarians often diagnose anthrax in animals onclinical grounds and by the examination of bloodfilms stained with polychrome methylene blue.These show chains of large, square-ended bacilliwith the remains of their capsules forming pink-coloured debris between the ends of adjacentorganisms. This is M’Fadyean’s reaction.

Culture animal blood, spleen or other tissueground in a Griffith’s tube or macerator. Inhumans, culture material from the cutaneouslesions, faeces, urine and sputum.

Plate on blood agar and on PLET medium: i.e.brain–heart infusion agar containing (per milli-litre): polymyxin, 30 units; lysozyme, 40 μg;EDTA, 300 μg; thallous acetate, 40 μg. Incubateovernight. Subculture woolly or waxy ‘medusahead’ colonies of Gram-positive rods into Craigietubes to test for motility and on chloral hydrateblood agar. It may be difficult to obtain puregrowths without several subcultures. For identifi-cation, see below.

42

Bacillus

Isolation of B. anthracis from hairs,hides, feeding-stuffs and fertilizers

The sample should be in a 200- to 300-ml screw-capped jar, and should occupy about 110 ml.

Add sufficient warm 0.1% peptone solution tocover it. Shake, stand at 37°C for 2 h, decant andheat the fluid at 60°C for 15–30 min. If the suspen-sion is very turbid, centrifuge it, retain the depositand make 10−1, 10−2 and 10−3dilutions in sterile water.Plate these and the deposit on PLET medium (seeabove). Incubate at 37°C for 36–48 h. Subculturecharacteristic colonies as described above.

In addition, place 0.1- to 0.5-ml amounts of thedilutions and a few loopfuls of the deposit in a Petridish, pour on 15–20 ml melted PLET medium at40–50°C and incubate as above. Examine under alow-power binocular (plate) microscope. Ignoresurface colonies and look for deep colonies thatresemble dahlia tubers or Chinese artichokes.Subculture these as above.

Identification of B. anthracis

The growth of B. anthracis on agar media has acharacteristic woolly or waxy nature when touchedwith a wire (the ‘tenacity test’).

Bacillus anthracis is non-motile. The Craigie tubeis the best way of demonstrating this. Most otherBacillus species are motile. There is no haemolysison standard blood agar media. It may be readilyidentified by immunofluorescence. Sera are avail-able commercially. If the anthrax w (or γ) bacterio-phage is available (e.g. from a research orcommercial institution), make a lawn of the suspectculture on blood agar, place one drop (0.02 ml) ofthe phage (titre about 106 plaque-forming units[pfu]/ml) on the surface and incubate at 37°Covernight. B. anthracis gives a clear zone of lysis(so do some other Bacillus spp.). Some researchand commercial institutions keep stocks of thephage but it is not available commercially.

Bacillus anthracis is sensitive to penicillin (10-unitdisc on nutrient agar) and gives a positive ‘string-

of-pearls’ appearance in a wet preparation aftergrowing in nutrient broth containing 0.5 unit peni-cillin per ml for 3–6 h at 37°C.

The biochemical properties given in Table 42.1may be helpful but not altogether reliable becauseof much variation within and between species. TheAPI 50 CHB kit is probably better.

For further information on B. anthracis, seeTurnbull (1990), Turnbull and Kramer (1995), andQuinn and Turnbull (1999).

FOOD POISONING

Several species of Bacillus are associated with foodpoisoning (see Chapter 13). They are B. cereus,and, less frequent, B. subtilis, B. lichenifornis, B.pumilus and B. sphaericus (Turnbull, 1997; seealso Chapter 13).

Isolation from food, etc.

Make 10% suspensions of the food, faeces andvomit in 0.1% peptone water, using a Stomacher.Inoculate glucose tryptone agar and Bacilluscereus-Selective Agar. Incubate at 30°C for24–48 h. This medium shows the egg yolk reactionand mannitol fermentation, and encourages sporu-lation (see below). Colonies of B. cereus are yellow,whereas other species, which ferment mannitol, arepink.

Prepare 10−1, 10−2 and 10−3 dilutions of these andplate 0.1-ml amounts on B. cereus-selective agar.Incubate at 37°C for 24 h and then at 25–30°C fora further 24 h. Count colonies of presumptiveB. cereus; more than 105 cfu/g is suggestive.

Rapid staining method for B. cereus

Make films from colonies on B. cereus-selectivemedium, which encourages sporulation, and dry inair. Place the slide over boiling water and stain with5% malachite green for 2 min. Wash and blot dry.

Food poisoning 363

Table 42.1 Some Bacillus species

Species Pathogenicitya Food Motility VPb Anaerobic Growth at Acid from Starch Sporesspoilage growth 5°C 60°C glucosec hydrolysis

B. anthracis + − − + + − − + + COB. cereus + FP + + + + − − − + COB. subtilis (+) FP + + + − + − − + COB. pumilus (+) FP − + + − + − − − COB. coagulans + − + + + + − − + C(T)OB. macerans + − + − + + − + + COB. circulans + − + − v + − − + C(T)OB. brevis + − + − − + − − − COB. licheniformis + FP − + + + + − − + COB. sphaericus + FP − + − − − − − − TRB. stearothermophilus − + + − − + + − + C(T)OB. megaterium − − + − − − − − + COB. polymyxa − + + + + − − + + CO

a From Turnbull (1997)b In 1% NaCl glucose brothc In ammonium salt sugarsFP, food poisoning; CO, central, oval; TR, terminal, round; C(T)O, central, occasionally terminal, oval

Stain with 0.3% Sudan black in 70% alcohol for15 min. Wash with xylol and blot dry.Counterstain with 0.5% safranin for 20 s. Washand dry. B. cereus spores are oval, central or sub-terminal, do not swell the cells and are stainedgreen. The vegetative cells stain red and containblack granules.

Food spoilage

Examine Gram-stained films for spore-bearing,Gram-positive bacilli. Make 10% suspensions offood in 0.1% peptone water, and pasteurize someof the suspension at 75–80°C for 10 min to killvegetative forms.

Inoculate glucose tryptone agar and B. cereus-selective media with both unheated and pasteurizedmaterial.

Incubate at 25–30°C overnight. If the food wascanned, incubate replicate cultures at 60°C.Identify colonies of Gram-positive bacilli.

Cold-tolerant spore-formers are importantspoilage organisms. Incubate at 5°C for 7 days.

Identification

Test colonies for VP reaction (use 1% NaCl glu-cose broth), anaerobic growth, growth at 5 and60°C, production of acid from glucose (ammoniumsalt sugar media) and starch hydrolysis (see Table42.1). The API 20E and API50 CHB will identifymore species.

BACILLUS SPECIES

Bacillus cereus

General properties are described above. Thisorganism is responsible for ‘bitty cream’, particu-larly in warm weather. It may cause food poisoning

(see above). Bacteraemia and pneumonia associ-ated with B. cereus have been reported. It is a com-mon contaminant on shell eggs. It does not fermentmannitol, unlike B. megaterium, which it may oth-erwise resemble.

Bacillus subtilis (‘B. mesentericus’)

Colonies on glucose tryptone agar are wrinkled orsmooth and folded colonies, 4–5 mm across, withyellow halo (acid production). It is responsible forspoilage in dried milk and in some fruit and veg-etable products. It causes ‘ropey’ bread. B. subtilisis used in industry to manufacture enzymes for bio-logical washing products.

Bacillus stearothermophilus

This and associated thermophiles form largecolonies (4 mm in diameter) on glucose tryptoneagar with a yellow halo caused by acid productionat 60°C. They grow poorly at 37°C and not at20°C. This organism is responsible for ‘flat-sour’(i.e. acid but no gas) spoilage in canned foods. Formore detailed investigation, see Chapter 16.

Bacillus licheniformis

This may cause food poisoning.

Bacillus megaterium

This is a very large bacillus, widely distributed. Itmay cause B. cereus-like food poisoning.

Bacillus pumilus

This is found on plants and is a common contami-nant of culture media.

Bacillus species 365

366 Bacillus

Bacillus coagulans

This is widely distributed, and found in cannedfoods.

Bacillus anthracis

See above (p. 362).

Bacillus polymyxa

This is widely distributed in soil and decaying veg-etables (possesses a pectinase). It is the source ofthe antibiotic polymyxin.

Bacillus macerans

This is common in soil and found in rotting flax.

Bacillus circulans

This forms motile colonies with non-motile vari-ants. It is found in soil.

Bacillus sphaericus

This has large spores, and resembles C. tetani inappearance. It forms motile colonies and is a soilorganism.

There are many other species (see Claus andBerkeley, 1986; Quinn and Turnbull, 1999).

REFERENCES

Claus, D. and Berkeley, R. C. W. (1986) The genusBacillus. In: Sneath, P. H. A., Mair, N. S. andSharpe, M. E. (eds), Bergey’s Manual of

Determinative Bacteriology. Baltimore, MA:Williams & Wilkins, pp. 1105–1139.

Quinn, C. P. and Turnbull, P. C. B. (1999) Anthrax.In: Collier, L., Balows, A. and Sussman, M. (eds),Topley and Wilson’s Microbiology and Microbial

Infections, 9th edn. Vol. 3, Bacterial Infections,CD-ROM. London: Arnold, Chapter 40.

Turnbull, P. C. B. (ed.) (1990) Proceedings of an

International Workshop on Anthrax, Winchester,

1989. Salisbury Medical Bulletin No. 68. SpecialSupplement. Porton Down, Salisbury: PHLS.

Turnbull, P. C. B. (1997) The role of the Bacillus genusin infection. Culture 18: 5–8.

Turnbull, P. C. B. and Kramer, J. M. (1995) Intestinalcarriage of Bacillus cereus: faecal isolation studiesin three population groups. Journal of Hygiene,

Cambridge 95: 629–638.

GRAM-NEGATIVE ANAEROBICBACILLI

Gram-negative anaerobic bacilli are found in thealimentary and genitourinary tracts of humans andother animals and in necrotic lesions, often in asso-ciation with other organisms. They are pleomor-phic, often difficult to grow and die easily incultures. Identification is often difficult. Referenceexperts use gas–liquid chromatography.

There have been changes in the classification ofthese organisms. The genus Bacteroides now con-tains only the B. fragilis group. The originalB. melaninogenicus group now contains two newgenera: Prevotella (saccharolytic) andPorphyromonas (asaccharolytic). Other new gen-era include Tissierella, Sebaldella, Rikenella,Fibrobacter, Megamonas and Mitsuokella. Anothernew genus, Bilophila, has been found in gangrenousappendices. Eikenella species are not strict anae-robes and will grow in 10% CO2 in air. The organ-ism known as E. corrodens may be confused withanother corroder – Bacteroides urealyticus.

In pathological material they are not uncom-monly mixed with other anaerobes and capnophilicorganisms. In general, primary infections above thediaphragm are caused by Fusobacterium species,but Bacteroides species may occur anywhere andare frequently recovered from blood cultures.

Motile, anaerobic, Gram-negative bacilli prob-ably belong to newly described genera such asSelenomonas or Wolinella, or to Mobiluncus.Capnocytophaga spp. are CO2 dependent but growanaerobically; they are found in the human andanimal mouth.

Isolation

Examine Gram films of pathological material.Diagnosis of Vincent’s angina can be made fromfilms counterstained with dilute fuchsin, whenfusiform bacilli can be seen in large numbers,associated with spirochaetes (B. vincenti – seeChapter 49). In pus from abdominal, brain, lungor genital tract lesions, large numbers of Gram-negative bacilli of various shapes and sizes, withpointed or rounded ends, suggest infection withBacteroides spp. Such material often has a foulodour.

Infections involving Prevotella melaniniogenicaand other members of the genus Prevotella are rap-idly diagnosed by viewing the pus under long-waveultraviolet (UV, 365 nm) light. These andPorphyromonas spp. produce porphyrins whichgive a bright red fluorescence.

Culture pus and other material anaerobically,aerobically, and aerobically under 5–10% carbondioxide. For anaerobic cultures use both non-selective and enriched selective media, e.g.haemin, menadione, sodium pyruvate, cysteine-HCl, or use commercial fastidious anaerobe (FA)agar. The usual selective agents are neomycin75 μg/ml, nalidixic acid 10 μg/ml and vancomycin2.5 μg/ml. Various combinations may be used torestrict contaminating facultative flora from cer-tain specimens, e.g. neomycin and vancomycin forpus from the upper respiratory tract.

A selective medium for fusobacteria containingjosamycin, vancomycin and norfloxacin at 3, 4 and1 μg/ml, respectively, is promising (Brazier et al.,1991).

43

Gram-negative anaerobic bacilli and cocci

Place a 5-μg metronidazole disc on the streaked-out inoculum for the rapid recognition of obligateanaerobes, all of which are sensitive. Incubate at37°C for 48 h.

Compare growths on selective and non-selectivemedia and compare the anaerobic with aerobiccultures.

Identification

The Gram stain is important but may give equivo-cal results. Full identification of these organisms isdifficult and rarely necessary. All that is required ina clinical laboratory is confirmation that anaerobesare involved. A simple report that ‘mixed anaer-obes are present – sensitive to metronidazole’usually suffices.

Identification is more important when anaerobesare present in blood cultures and other normallysterile sites. These simple presumptive tests areuseful.

Potassium hydroxide testEmulsify a few colonies in a drop of 3% KOH on aslide. Bacteroides spp. tend to become ‘stringy’ andform long strands when the loop is moved awayfrom the drop.

UV fluorescenceExamine under long-wave UV light. Red fluores-cence suggests Prevotella or Porphyromonas spp.and develops much earlier than the characteristicblack pigment. Yellow fluorescent colonies ofGram-negative rods are probably fusobacteria(Brazier, 1986).

Phosphomycin testPlace a disc, soaked in phosphomycin 300 μg/ml,on a seeded plate and incubate. Fusobacteria areusually sensitive and Bacteroides resistant (Bennettand Duerden, 1985).

Carbohydrate fermentationFor information on this, see Phillips (1976) andHoldeman et al. (1977).

Antibiogram and commercial identification kitsAntibiogram patterns give a low level of accuracyand offer only a limited range of identifications.Commercial kits are an improvement but resultsbased on a generated number may be misleading.Much depends on the database on which thescheme is based. The API 32 ATB kit is one of themost useful.

ANAEROBIC COCCI

These may occur in cultures from a wide variety ofhuman and animal material. They have undergonerecent taxonomic changes and are now described asPeptostreptococcus, Peptoniphilus, Anaerococcus,Finegoldia and Peptococcus.

It is difficult to separate these genera. Cocci occursingly, in pairs, clumps or short chains. They growon blood agar at 37°C and are non-haemolytic, butgrow better on Wilkins–Chalgren agar containing asupplement for non-sporing anaerobes.

These organisms are sensitive to metronidazole(5-μg disc) and grow slowly. There is no need todistinguish between them.

Peptostreptococci may be pathogenic for humansand animals, and associated with gangrenouslesions. They are commensals in the intestine andhave been isolated from the vagina in health and inpuerperal fever. They produce large amounts ofhydrogen sulphide from high-protein media, e.g.blood broth, and a putrid odour in ordinary media.The API system is useful for identifying theseorganisms.

The organism formerly known as Gaffkya tetra-genus is now called Peptostreptococcus tetradius.The genus Peptococcus now contains only onespecies, P. niger.

Sarcina

These cocci are in packets of eight. They are obli-gate anaerobes, requiring incubation at 20–30°C

368 Gram-negative anaerobic bacilli and cocci

and carbohydrate media. They are found in air,water and soil, and occasionally in clinicalmaterial.

Sarcina ventriculi is a large coccus arranged inclumps of four to eight. It is found in the humangut, particularly in vegetarians.

Veillonella

The members of this genus are obligate anaerobesand occur as irregular masses of very small Gram-negative cocci, cultured from various parts of therespiratory and alimentary tracts and genitalia.They do not appear to cause disease.

Growth may be improved by adding 1% sodiumpyruvate and 0.1% potassium nitrate to the basalmedium.

Taxonomists now recognize seven species:Veillonella atypica, V. dispar and V. parvula arefound in humans; and V. caviae, V. criceti, V. rattiand V. rodentium in rodents. In clinical laborato-ries it is not necessary to identify species.

Fluorescence under UV light is generally weak,fades rapidly and is medium dependent. It worksonly on brain infusion agars, except for V. cricetiwhich fluoresces on a range of media (Brazier andRiley, 1988). Phenotypic differentiation is based oncatalase, fermentation of fructose and the origin of

the strain. For further information, see Mays et al.(1982).

REFERENCES

Bennett, K. W. and Duerden, B. I. (1985) Identificationof fusobacteria in a routine diagnostic laboratory.Journal of Applied Bacteriology 59: 171–181.

Brazier, J. S. (1986) Yellow fluorescence offusobacteria. Letters in Applied Microbiology 2:125–126.

Brazier, J. S. and Riley, T. V. (1988) UV redfluorescence of Veillonella spp. Journal of Clinical

Microbiology 26: 383–384.Brazier, J., Citros, D. M. and Goldstein, E. J. C. (1991)

A selective medium for Fusobacterium spp. Journal

of Applied Bacteriology 71: 343–346.Holdeman, L. V., Cato, E. P. and Moore, W. E. C.

(eds) (1977) Anaerobic Laboratory Manual, 4thedn. Blacksburg, VA: Virginia State University.

Mays, T. D., Holdeman, L. V., Moore, W. E. C. andJohnson, J. L. (1982) Taxonomy of the genusVeillonella Prevot. International Journal of

Systematic Bacteriology 32: 28–36.Phillips, K. D. (1976) A simple and sensitive technique

for determining the fermentation reactions ofnonsporing anaerobes. Journal of Applied

Bacteriology 41: 325–328.

References 369

44

Clostridium

This is a large genus of Gram-positive (but seebelow), spore-bearing anaerobes (a few are aerotol-erant) that are catalase negative. They are normallypresent in soil; some are responsible for human andanimal disease, whereas others are associated withfood spoilage.

Clostridia are classified according to the shapeand position of the spores (Table 44.1) and bytheir physiological characteristics. Most speciesare mesophiles; there are a few important ther-mophiles and some psychrophiles and psy-chrotrophs. They may be either predominantly

saccharolytic or proteolytic in their energy-yield-ing activities. Saccharolytic species decomposesugars to form butyric and acetic acids, and alco-hols. The meat in Robertson’s medium is red-dened and gas is produced. Proteolytic speciesattack amino acids. Meat in Robertson’s mediumis blackened and decomposed, giving the culture afoul odour.

The clostridia are conveniently considered underfour headings: food poisoning, tetanus and gas gan-grene, pseudomembranous colitis and antibioticrelated diarrhoea, and food spoilage.

Table 44.1 Morphology and colonial appearance of some species of Clostridium

Species Spores Bacilli Haemolysis Colony appearance on blood agar

C. botulinum OC or S normal + large, fimbriate, transparentC. perfringens a large thick + flat, circular, regularC. tetani RT normal + small, grey, fimbriate, translucentC. novyi OS large + flat, spreading, transparentC. septicum OS normal + irregular, transparentC. fallax OS thick − large, irregular, opaqueC. sordellii OC or S large thick + small, crenatedC. bifermentans OC or S large thick + small, circular, transparentC. histolyticum OS normal − small, regular, transparentC. sporogenes OS thin + medusa head, fimbriate, opaqueC. tertium OT long thin − small, regular, transparentC. cochlearium thin − circular, transparentC. butyricum OC normal − white, circular, irregularC. nigrificans normal blackC. thermosaccharolyticum normal granular, feather edgesC. difficile OS normal − grey, translucent, irregular

Spores: O, oval; R, round; S, subterminal; C, central; T, terminala not usually seen

FOOD POISONING

Botulism

This is the least common but most often a fatalkind of food poisoning (see Chapter 13); it iscaused by Clostridium botulinum. This is a strictanaerobe which requires a neutral pH. It is difficultorganism to isolate in pure culture because it com-petes poorly with other micro-organisms that maybe present in food (e.g. canned, underprocessed).

Emulsify the suspected material in 0.1% peptonewater and inoculate several tubes of Robertson’scooked meat medium, which have been heated todrive off air and then cooled to room temperature.Heat some of these tubes at 75–80°C for 30 minand cool. Incubate both heated and unheated tubesat 35°C for 3–5 days. Plate them on blood agarthat has been pre-reduced in an anaerobic jar orcabinet and on egg yolk agar, and incubate understrict anaerobic conditions at 35°C for 3–5 days.

If the material is heavily contaminated, heat someof the emulsion as described above, make severalserial dilutions of heated and unheated materialand add 1-ml amounts to 15–20 ml glucose nutri-ent agar melted and at 50°C in 152 × 16 mm tubes(Burri tubes, see p. 377). Mix, cool and incubate.Cut the tube near the sites of suspected colonies,aspirate these with a Pasteur pipette and plate onblood agar or on egg yolk agar containing (per mil-lilitre): cycloserine, 250 μg; sulphamethoxazole,76 μg; trimethoprim, 4 μg (Dezfullian et al., 1981).

Clostridium botulinum gives a positive egg yolkand Nagler (half-antitoxin plate) reaction (seebelow). Types A, B and F liquefy gelatin, blackenand digest cooked meat medium, and produce H2S.Types C, D and E do none of these. All strains pro-duce acid from glucose, fructose and maltose andare indole negative (Table 44.2, and see p. 374 andTable 44.1).

It is best to use fluorescent antibody methods andto send suspected cultures to a reference labora-tory.

Type A C. botulinum is usually associated withmeat. Type E is found in fish and fish products andestuarine mud, and is psychrotrophic.

Clostridium perfringens food poisoning

Clostridium perfringens is a common cause of foodpoisoning (see Chapter 13). However, not allstrains are capable of causing symptoms. About5% of strains carry the gene responsible for theproduction of C. perfringens enterotoxin (CPE)that causes abdominal pain and diarrhoea.

FaecesIt is pointless to examine stools of food handlersand ‘contacts’ in outbreaks of C. perfringens foodpoisoning because the disease is not spread byhuman agency.

C. perfringens food poisoning may be diagnosedby demonstrating CPE in the faeces using anenzyme immuno-assay kit (C. perfringensEnterotoxin WIA, Bioconnections Ltd., Leeds) or alatex kit (Berry et al. 1987).

The ethanol shock method may be useful.Emulsify the faeces in ethanol (industrial grade) togive a 1:1 suspension. Mix well and stand for 1h.Inoculate culture media, incubate anaerobicallyand proceed as under Identification (see p. 373).

FoodOutbreaks involving both heat-resistant and heat-sensitive strains have been reported (see Chapter13).

Use a Stomacher to prepare a 10% suspension ofthe food in peptone water diluent. Make dilutionsof 10−1 to 10−3 and plate both suspensions and dilu-tions on Willis and Hobbs medium, tryptose sul-phite cycloserine agar and neomycin blood agar.Place a metronidazole disc on the inoculum.Incubate anaerobically at 37°C overnight.

To count the clostridia do pour plates with thedilutions in oleandomycin polymyxin sulphadi-azine perfringens (OPSP) agar. Alternatively, usethe Miles and Misra method (see p. 148) withneomycin blood agar. Incubate duplicate platesaerobically and anaerobically to distinguishbetween clostridia and other organisms.

Add some of the suspension to two tubes ofRobertson’s cooked meat medium. Heat one tube

Food poisoning 371

Table 44.2 Cultural and biochemical properties of some species of clostridia

Species RCM Litmus Acid from Indole Gelatin Lecithinase

Colour Digestion Odour Gasmilk

Glucose Sucrose Lactose Salicinpro- liquefactionduction

C. botulinum black + − + D + v − − − + −C. perfringens black + + + CD + + + v − + +C. tetani black + + − C − − − − + + −C. novyi red − − + GC + − − − v + vC. septicum red − − + AC + − + + − + −C. fallax red − − + AC + + + + − − −C. sordellii a black + + + CD + − − − + + +C. bifermentans a black + + + CD + − − v + + +C. histolyticum black + + − D − − − − − + −C. sporogenes black + + + D + − − v − + −C. tertium − − + AC + + + + − − −C. cochlearium red − − + − − − − − − −C. butyricum − − + ACG + + + + − − −C. nigrificans − − − − − − − − − −C. thermosaccharolyticum − − + + + + + − − −

RCM: Robertson’s cooked meat mediumLitmus milk: A, acid; C, clot; D, digestion; G, gasa C. sordellii also splits urea; C. bifermentans does not

for 10 min at 80°C. This helps to select the heat-resistant strains that are most likely to be associ-ated with food poisoning. Incubate overnight andplate out as indicated above.

Commercial kits are available for the detection ofenterotoxins (see Berry et al., 1987).

Identification

It is usually sufficient to identify C. perfringens bythe Nagler half-antitoxin test (see below) but otherproperties are given in Tables 44.1 and 44.2. Nogreat reliance should be placed on the ‘stormyfermentation’ of purple milk. General culturalmethods are given on p. 371.

Nagler half-antitoxin plate testSpread good quality antitoxin to C. perfringensalpha toxin on one half of a plate of nutrient agarcontaining 10% egg yolk. Dry the plates and inoc-ulate the organism across both halves of the plate.Incubate anaerobically overnight at 37°C.

The lecithinases of C. perfringens and C. novyiproduce white haloes round colonies on theuntreated half of the plate. This activity is inhibitedon the half treated with antitoxin. The lecithinasesof C. bifermentans and C. sordellii are partiallyinhibited by C. perfringens antitoxin, but they maybe distinguished by a positive indole reaction andthe presence of spores, rarely seen in vitro with C.perfringens.

On egg yolk medium, some clostridia show a‘pearly layer’ resulting from lipolysis. This is a use-ful differential criterion for species of Clostridium.

TETANUS AND GAS GANGRENE

Tetanus is caused by toxigenic strains of C. tetani.Clostridia associated with gas gangrene include C.novyi, C. perfringens, C. septicum and C. sordellii.C. histolyticum is a possible pathogen and someother species, such as C. sporogenes, C. tertium

and C. bifermentans, may also be found in woundsbut are not known to be pathogenic.

Examine Gram- and fluorescent antibody-stainedfilms of wound exudates. Bacilli with swollen ter-minal spores (‘drumstick’) suggest C. tetani but arenot diagnostic. Thick, rectangular, box-like bacillisuggest C. perfringens. Very large bacilli might beC. novyi.

Inoculate two plates of blood agar, one for anae-robic and the other for aerobic culture. Inoculatetwo tubes of Robertson’s cooked meat medium.Heat one at 80°C for 30 min and cool. Incubateblood agar and all the other media anaerobically at37°C for 24 h. Plate out the Robertson’s mediumon blood agar and other clostridial media, andincubate anaerobically with 10% carbon dioxide.

Identification

Commercially available kits for the identificationof anaerobes, including clostridia, are commonlyused but strains giving equivocal results should besent to a reference laboratory.

PSEUDOMEMBRANOUS COLITISAND ANTIBIOTIC-ASSOCIATEDDIARRHOEA

Pseudomembranous colitis is almost exclusivelycaused by C. difficile but antibiotic-associated diarr-hoea may also be caused by CPE-producing strainsof C. perfringens.

Treat faeces by the ethanol shock method (seep. 371). Plate on selective agar containing cefoxitin8 μg/ml and cycloserine 250 μg/ml (CCY agar,Bioconnections Ltd., UK) and inoculate cookedmeat medium. Alternatively plate on Columbia agarcontaining those antibiotics and 5% egg yolk so thatlecithinase-producing colonies may be ignored (C.difficile is lecithinase negative). Incubate anaerobi-cally for 48 h. Enterotoxigenic C. perfringens maybe identified by ELISA as described above.

Colonies of C. difficile are 2–5 mm in diameter,

Pseudomembranous colitis and antibiotic-associated diarrhoea 373

irregular and opaque. Confirm by latex agglutina-tion (Mercia Diagnostics). There may be cross-reaction with C. glycolicum.

Examine blood agar cultures under long-waveultraviolet (UV) light. C. difficile colonies (and thoseof some other organisms) fluoresce yellow–green.

In most diagnostic laboratories, faecal toxindetection is used to identify C. difficile disease. It isrecommended that either tissue culture methods ordiagnostic kits that detect both A and B toxins(Bioconnections Ltd., UK and Premier MeridianDiagnostics, USA) be used, as toxin A-negative, B-positive strains have been reported.

There are diagnostic kits for the toxin (Porton,Cambridge, UK) and Premier C. difficile Toxin A(Meridian Diagnostics, USA).

SPECIES OF CLOSTRIDIUM OFMEDICAL IMPORTANCE

Clostridium botulinum

This is a strict anaerobe that requires a neutral pHand absence of competition to grow in food (e.g.canned, underprocessed). Free spores may be pres-ent. Cultural characteristics are variable within andbetween strains. There are six antigenic types (A–F).Types A, B and F are proteolytic, causing clearingon Willis and Hobbs medium; types C, D and E arenot. It is the causative organism of botulism, one ofthe least common but the most fatal kind of foodpoisoning. Type A is usually associated with meat;type E (a psychrotroph) is associated with fish prod-ucts and is found in soil and marine mud.

Clostridium perfringens

This highly aerotolerant anaerobe may grow inbroth, e.g. MacConkey broth inoculated withwater. Spores are rarely seen in culture (a diagnos-tic feature) but can be obtained on Ellner’s mediumand medium with added bile, bicarbonate and

quinoline (Phillips, 1986). There are six antigenictypes (A–F). Type A is associated with gas gangreneand with food poisoning and antibiotic-associateddiarrhoea. It causes lamb dysentery, sheep ‘struck’and pulpy kidney in lambs. It is found in soil, waterand animal intestines.

Clostridium tetani

A strict anaerobe, this dies readily on exposure toair. It swarms over the medium, but this may bedifficult to see. It is, however, an important diag-nostic characteristic. It is found in soil, especiallyanimal manured, and in the animal intestine. It isthe causative organism of tetanus, rarely seen inwestern countries where the disease is largely pre-vented by immunization and prophylaxis. It may,however, present as jaw stiffness in the elderly witha history of recent, often minor, trauma.

Clostridium novyi (C. oedematiens)

This strict anaerobe dies rapidly in air. There arefour antigenic types (A–D). Types A and B causegas gangrene, and type D bacillary haemoglobin-uria in cattle and ‘black disease’ of sheep.

In 2000, C. novyi type A was implicated in anoutbreak of serious illness, with deaths, amonginjecting drug users in the UK. A contaminatedbatch of heroin was believed to be the source. Theidentification of clostridia from such sources is dis-cussed by Brazier et al. (2002).

Clostridium septicum and C. chauvoei

Both are strict anaerobes; once considered to be asingle species, they are now known to be antigenicallydistinct. C. septicum causes gas gangrene in humans,and braxy and blackleg in sheep. C. septicum is oftenisolated from blood cultures in patients with malig-nant disease of the colon. C. chauvoei is not patho-

374 Clostridium

genic for humans but causes quarter evil, blackleg,and ‘symptomatic anthrax’ of cattle and sheep. Bothcan be identified by fluorescent antibody methods.

Clostridium bifermentans and C. sordellii

These are separate species, distinguished biochemi-cally, but they are conveniently considered togetherbecause both have a toxin (lecithinase) similar tothat of C. perfringens and give the same reaction asthat organism on half-antitoxin plates with C. per-fringens type A antiserum. C. sordellii is ureasepositive, C. bifermentans urease negative, C. bifer-mentans not pathogenic, but C. sordellii is associ-ated with wound infections and gas gangrene. Bothare found in soil.

Clostridium fallax

This is a strict anaerobe associated with gas gan-grene. Spores are not very resistant to heat and theorganism may be killed in differential heatingmethods. It is found in soil.

Clostridium difficile

This may cause a wide spectrum of predominantlynosocomial diarrhoeal disease ranging from mildantibiotic-associated diarrhoea to full-blownpseudomembranous colitis that may be fatal.

SPECIES OF CLOSTRIDIUM OFDOUBTFUL OR NOSIGNIFICANCE IN HUMANPATHOLOGICAL MATERIAL

Clostridium histolyticum

This is not a strict anaerobe. Filamentous formsmay grow as surface colonies on blood agar

aerobically. Associated with gas gangrene, butusually in mixed infection with other clostridia.It is found in soil, in bone products such asgelatin and in the intestines of humans andanimals.

Clostridium sporogenes

This is not pathogenic but is a common contami-nant of wounds. It is widely distributed, andfound in soil and animal intestines. It is pheno-typically identical with proteolytic strains of C.botulinum. Differentiation is by the demonstra-tion (in a reference laboratory) of toxin produc-tion.

Clostridium tertium

This is aerotolerant and non-pathogenic.

Clostridium cochlearium

The only common clostridium that is biochemicallyinert. It is not pathogenic.

FOOD-SPOILAGE CLOSTRIDIA

Clostridium species are known to be responsible forspoilage of a wide variety of foods, including milk,meat products, fresh water fish and vegetables. Italso causes spoilage in silage.

Prepare 10% emulsions of the product in 0.1%peptone water using a Stomacher or blender. Heatsome of this emulsion at 75–80°C for 30 min (pas-teurized sample). Inoculate several tubes ofRobertson’s cooked meat medium and/or liquidreinforced clostridium medium with pasteurizedand unpasteurized emulsion and incubate pairs atvarious temperatures, e.g. 5–7°C for psy-chrophiles, 22 and 37°C for mesophiles, and 55°C

Food-spoilage clostridia 375

for thermophiles. Inoculate melted reinforcedclostridial agar (RCM) in deep tubes with dilu-tions of the emulsions. Allow to set. Incubate atthe desired temperature and look for blackcolonies, or blackening of the medium, which sug-gests clostridia. Inoculate several tubes oflitmus/purple milk medium each with 2 ml suspen-sion and incubate at the required temperatures.This gives a useful guide to the identity of theclostridias.

Appearance in litmus milk medium

• C. putrifaciens, C. sporogenes, C.oedematiens and C. histolyticum are indicatedby slightly alkaline reaction, gas, soft curdsubsequently digested leaving clear brownliquid, a black sediment and a foul smell.

• C. sphenoides gives a slightly acid reaction,soft curd, whey and some gas.

• C. butyricum produces acid, firm clot and gas.• C. perfringens usually gives a stormy clot.• C. tertium usually gives a stormy clot.

Note that aerobic spore bearers may give similarreactions, so plate out and incubate both aerobi-cally and anaerobically.

Thermophiles associated with cannedfood spoilage

Two kinds of clostridial spoilage occur as a resultof underprocessing: ‘hard swell’ and ‘sulphurstinkers’.

Examine Gram-stained films for Gram-positivespore bearers. Inoculate reinforced clostridial mediumor glucose tryptone agar and iron sulphite medium indeep tube cultures by adding about 1-ml amounts ofdilutions of a 10% emulsion of the food material inpeptone water diluent to 152 × 16 mm tubes con-taining 15–20 ml medium melted and at 50°C. Eithersolid or semisolid media may be used. Incubate dupli-cate sets of tubes for up to 3 days at 60 and 25°C.

Clostridium thermosaccharolyticumThis produces white lenticular colonies in all threemedia and changes the colour of glucose tryptoneagar from purple to yellow. This organism isresponsible for ‘hard swell’.

Clostridium nigrificansThis produces black colonies, particularly in mediathat contain iron. It causes ‘sulphur stinkers’.

Neither organism grows appreciably at 25°C(Table 44.3).

376 Clostridium

Table 44.3 Food-spoilage species

Species Comments

ThermophilesC. nigrificans Sulphur stinkers

C. thermosaccharolyticum Hard swell

Mesophiles

C. butyricum Cheese disorders; butter and milk products

C. sporogenes

C. sphenoides Meat and dairy productsC. novyi �C. perfringens

Psychophiles

C. putrefaciens Bone taint, off-odours

COUNTING CLOSTRIDIA INFOODS

Emulsify 10 g of the food in 90 ml 0.1% peptonewater in a homogenizer. Divide into two portionsand heat one at 75°C for 30 min.

Do MPN tests with 10-, 1- and 0.1-ml amountsand using the five- or three-tube method (seeChapter 10), on each portion, in liquid differentialreinforced clostridial medium (DRCM). Clostridiaturn this medium black (‘black tube method’). Theunheated portion gives the total count, the heatedportion the spore count.

For the pour-plate method add 0.1-ml amounts ofserial dilutions of the emulsion to melted OPSPagar. Incubate anaerobically for 24 h and count thelarge black colonies.

It may be necessary to dilute the inoculum 1 : 10or 1 : 100 if many clostridia are present. If the loadof other organisms is heavy, add 75 units/ml ofpolymyxin to the medium used for the unheatedcount.

To recover individual colonies, Burri tubes maybe used. These are open at both ends and are closedwith rubber bungs. Use solid DRCM and, afterinoculation, cover the medium at both ends with alayer of melted paraffin wax about 2 cm deep.These tubes need not be incubated anaerobically.

After incubation and counting, remove both stop-pers and extrude the agar cylinder. Cut it with asterile knife and aspirate colonies with a Pasteurpipette for subculture. Membrane filters may beused for liquid samples. Roll up the filters and placein test tubes; cover with melted medium.

DRCM is suitable for most counts but forC. nigrificans iron sulphite medium is good. Selectincubation temperatures according to the species tobe counted.

General identification procedure

Subculture each kind of colony of anaerobic Gram-positive bacillus in the following media:Robertson’s cooked medium; purple milk; peptone

water for indole production; gelatin medium; andglucose, lactose, sucrose and salicin peptonewaters. These media should be in cottonwool-plugged test tubes, not in screw- capped bottles.Omit the indicator from these because it may bedecolourized during anaerobiosis. Test for acidproduction after 24–48 h with bromocresol purple.It may be necessary to enrich some media withFildes’ extract. An iron nail in each tube assistsanaerobiosis. Gas production in sugar media is notvery helpful and Durham’s tubes can be omitted.The API 20A system for anaerobes is very helpful.

Do half-antitoxin (Nagler) plates as above usingC. perfringens type A and C. novyi antitoxin (seeTables 44.1 and 44.2).

‘Gram-negative clostridia’Some Clostridium spp. appear to be Gram nega-tive. Clostridias, however, are sensitive to a 5-μgvancomycin disc whereas true Gram-negativebacteria are resistant (Engelkirk et al., 1992). Forfurther information on clostridia see Willis (1977),Holdeman et al. (1977) and Phillips et al.(1985).

REFERENCES

Berry, P. R., Weinecke, A. A., Rodhouse, J. C. et al.

(1987) Use of commercial tests for the detection ofClostridium perfringens and Staphylococcus aureus

enterotoxins. In: Grange, J. M., Fox, A. andMorgan, N. L. (eds), Immunological Techniques in

Microbiology. Society for Applied BacteriologyTechnical Series No. 24. London: Blackwells, pp.245–250.

Brazier, J. S., Duerden, B. I., Hall, V. et al. (2002)Isolation and identification of Clostridium spp.from infections associated with the injections ofdrugs; experiences of a microbiologicalinvestigation team. Journal of Medical

Microbiology 51, 985–989.Dezfullian, M., McCroskey, C. L., Hatheway, C. L.

and Dowell, V. R. (1981) Isolation of Clostridium

botulinum from human faeces. Journal of Clinical


References 377

Engelkirk, P. G ., Duen-Engelkirk, J. and Dowell, V. R.(1992) Principles and Practice of Anaerobic

Bacteriology. Belmont, NY: Star.Holdeman, L. V., Cato, E. P. and Moore, W. E. C.

(eds) (1977) Anaerobic Laboratory Manual, 4thedn. Blacksburg, VA: Virginia State University.

Phillips, K. D. (1986) A sporulation medium forClostridium perfringens. Letters in Applied


Phillips, K. D., Brazier, J. S., Levett, P. N. et al. (1985)Clostridia. In: Collins, C.H and Grange, J.M. (eds),Isolation and Identification of Micro-organisms of

Medical and Veterinary Importance. Society forApplied Microbiology Technical Series No. 21.London: Academic Press, pp. 215–236.

Willis, A. T. (1977) Anaerobic Bacteriology, 3rd edn.London: Butterworths.

378 Clostridium

The mycobacteria are acid fast: if they arestained with a strong phenolic solution of anarylmethane dye, e.g. carbol–fuchsin, they retainthe stain when washed with dilute acid. Otherorganisms are decolourized. There are nogrounds for the commonly held belief that somemycobacteria are acid and alcohol fast, butothers are only acid fast. These properties varywith the technique and the organism’s physiolog-ical state.

The most important members of this genus areobligate parasites. They include the Myco-bacterium tuberculosis complex, containing vari-ous species as described below; and the leprosy(Hansen’s) bacillus M. leprae. In addition, around100 named species of mycobacteria, and anunknown number of unnamed ones, live freely inthe environment and some of these have been iden-tified as opportunist pathogens of humans and ani-mals. These species are often therefore termed‘environmental mycobacteria’ (EM) and are alsotermed ‘non-tuberculous’, ‘opportunistic’ and‘MOTT’ (‘mycobacteria other than typical tuber-cle’) bacilli.

The M. tuberculosis complex, and in somecountries certain other species of mycobacteria(see national lists), are Risk Hazard Group 3pathogens and infectious by the airborne route.All manipulations that might produce aerosolsshould be done in microbiological safetycabinets in Biosafety Containment Level 3laboratories.

THE M. TUBERCULOSIS COMPLEX(TUBERCLE BACILLI)

Direct microscopic examination

SputumThis is the most common material suspected ofcontaining tubercle bacilli. It is often viscous anddifficult to manipulate. To make direct films usedisposable 10-μl plastic loops. These avoid the useof Bunsen burners in safety cabinets. Discard theminto disinfectant. Ordinary bacteriological loopsare unsatisfactory and unsafe. Spread a small por-tion of the most purulent part of the material care-fully on a slide, avoiding hard rubbing, whichreleases infected airborne particles. Allow the slidesto dry in the safety cabinet then remove them forfixing and staining. This is quite safe; aerosols arereleased during the spreading, not from the driedfilm, although these should not be left unstained forany length of time. Fixing may not kill the organ-isms and dried material is easily detached.

For direct microscopy of centrifuged depositsafter homogenizing sputum for culture spread a 10-μl loopful over an area of about 1 cm. Dryslowly and handle with care. The material tends tofloat off the slide while it is being stained.

Cerebrospinal fluidMake two parallel marks 2–3 mm apart and10 mm long in the middle of a microscope slide.

45

Mycobacterium

Place a loopful of the centrifuged deposit betweenthe marks. Allow to dry and superimpose anotherloopful. Do this several times before drying, fixingand staining. Examine the whole area.

UrineDirect microscopy of the centrifuged deposit isunreliable because urine frequently contains envi-ronmental acid-fast bacilli, which have entered thespecimen from the skin or the inanimate environ-ment.

Aspirated fluids, pus Centrifuge if possible and make films from thedeposit. Otherwise prepare thin films. Thick filmsfloat off during straining. Material containingmuch blood is difficult to examine and may givefalse positives.

Gastric lavage Direct microscopy is unreliable because environ-mental mycobacteria are frequently present in foodand hence in the stomach contents.

Laryngeal swabs Direct microscopy is unrewarding.

Blood, faeces and milk Direct microscopy is unreliable because acid-fastartefacts and environmental mycobacteria may bepresent. Microscopic examination of faeces andcentrifugates of lysed blood may be useful in theprovisional diagnosis of disseminated mycobacter-ial disease, usually caused members of theM. avium complex (MAC) in patients withacquired immune deficiency syndrome (AIDS;Kiehn et al., 1985).

TissueCut the tissue into small pieces. Work in a safetycabinet. Remove caseous material with a scalpeland scrape the area between caseation and soft tis-sue. Spread this on a slide. It is more likely to con-tain the bacilli than other material.

StainingStain direct films of sputum or other material bythe Ziehl–Neelsen (ZN) method. The bacilli maybe difficult to find and it is often necessary to spendseveral minutes examining each film. Fluorescencemicroscopy, using the auramine phenol (AP)method, is popular in some clinical laboratories. Alower-power objective may be used and morematerial examined in less time. False positives arenot uncommon with this method and the presenceof acid-fast bacilli should be confirmed by over-staining the film with ZN stain. These stainingmethods are described in Chapter 7.

ReportingReport ‘acid-fast bacilli seen/not seen’. If they arepresent report the number per high power field.Table 45.1 gives a reporting system in common usein Europe for sputum films.

‘False’ positives Single acid-fast bacilli should be regarded with cau-tion. They may be environmental contaminants,e.g. from water. They may be transferred from oneslide to another during staining or if the same pieceof blotting paper is used for more than one slide.

Isolation from, or detection in,pathological material

Traditionally, tubercle bacilli are isolated by cou-ture on solid egg-based media but several weeks

380 Mycobacterium

Table 45.1 Scale for reporting acid-fast bacilli (AFB) in sputumsmears examined microscopically

No. of bacilli observed Report

0 per 300 fields Negative for AFB 1–2 per 300 fields (±) Repeat test 1–10 per 100 fields +1–10 per 10 fields + +1–10 per field + + +≥ 10 per field + + + +

Note that this is a logarithmic scale; this facilitates plotting.

elapse between setting up the culture and theappearance of colonies visible to the naked eye.Other techniques have therefore been developedand the most widely used are automated radiomet-ric and non-radiometric systems, and those basedon nucleic acid-based technology such as the poly-merase chain reaction (PCR). Other techniquesused in certain specialized reference centres includethe detection of specific mycobacterial lipids bymass spectrometry (French et al., 1987) andmycobacterial antigen in clean specimens such ascerebrospinal fluid (CSF) by immunologicalmethods (Drobnieski et al., 2001).

In most tuberculosis laboratories world wide, thetraditional culture methods are still in use soemphasis will be given to these in this chapter.

Preparation of pathological materialfor culture

Mycobacteria are often scanty in pathologicalmaterial and not uniformly distributed. Manyother organisms may be present and the platingmethods used in other bacteriological examina-tions are useless. Acid-fast bacilli usually grow at avery much slower rate than other bacteria and areovergrown on plate cultures. Some specimens arefrom normally sterile sites (blood, bone marrow,CSF, certain tissues) and may be inoculated directlyon culture media. Others are likely to contain manybacteria and fungi and must first be treated with areagent that is less lethal for mycobacteria than forother organisms, and that reduces the viscosity ofthe preparation so that it may be centrifuged. Noideal reagent is known; there is a choice between‘hard’ and ‘soft’ reagents.

Hard reagents are recommended for specimensthat are heavily contaminated with other organisms.Exposure times are critical or the mycobacteria arealso killed. Soft reagents are to be preferred whenthere are fewer other organisms, e.g. in freshlycollected sputum. The reagent can be left in contactwith the specimen for several hours without signi-ficantly reducing the numbers of mycobacteria. Soft

reagents may permit the recovery of mycobacteriaother than tubercle bacilli, which are often killed byhard reagents. Alternatively, specimens may beinoculated on media containing a ‘cocktail’ ofantimicrobial agents that will kill virtually all micro-organisms other than mycobacteria.

The methods described below are intended toreduce the number of manipulations, particularlyin neutralization and centrifugation, so that thehazards to the operator are minimized.

Culture of the centrifuged deposit after treatmentgives optimum results, but is avoided by someworkers because of the dangers of centrifugingtuberculous liquids. Scaled centrifuge buckets(safety cups) overcome this problem (see Chapter1). These considerably reduce the hazards ofaerosol formation should a bottle leak or break inthe centrifuge. They should be opened in a safetycabinet.

If material is not centrifuged, more tubes of cul-ture media should be inoculated and a liquidmedium should be included. This should containan antibiotic mixture (see p. 383).

Sputum culture Encourage the sputum from the specimen containerinto the preparation bottle with a pipette madefrom a piece of sterile glass tubing (200 × 5 mm)with one end left rough to cut through sputumstrands and the other smoothed in a flame to accepta rubber teat.

Hard method Add about 1 ml sputum to 2 ml 4% NaOH solu-tion or a mixture of 2% NaOH and 1% N-acetyl-L-cysteine (a mucolytic agent) in a 25-mlscrew-capped bottle. Stopper securely, place in aself-sealing plastic bag to prevent accidental disper-sal of aerosols and shake mechanically, but not vig-orously, for not less than 15 min but not more than30 min. Thin specimens require the shorter time.Incubation does not help. Ten per cent of myco-bacteria survive.

Remove the bottle from the plastic bag and add3 ml 14% (approximately 1 mol/l) monopotassiumhydrogen orthophosphate (KH2PO4) solution

The M. tuberculosis complex (tubercle bacilli) 381

containing enough phenol red to give a yellowcolour. This neutralizing fluid should be dispensedready for use and sterilized in small screw-cappedbottles. It should not be pipetted from a stockbottle.

The colour change of the phenol red from yellowto orange pink indicates correct neutralization. Re-stopper, mix gently and centrifuge with the bottlesin sealed centrifuge buckets (‘safety cups’) for15 min at 3000 rev./min. Pour off the supernatantfluid carefully into disinfectant. Wipe the neck ofthe bottle with a piece of filter paper, which is thendiscarded into disinfectant. Culture the deposit.

Soft method To 2–4 ml sputum in a screw-capped bottle, add anequal volume of 23% trisodium orrthophosphatesolution. Mix gently and stand at room tempera-ture or in a refrigerator for 18 h. Add the contentsof a 10-ml bottle of sterile distilled water to reducethe viscosity, centrifuge, decant and culture thedeposit using the methods and precautionsdescribed above.

Laryngeal and other swabs Place the swab in a tube containing 1 mol/l sodiumhydroxide solution for 5 min. Remove, drain andplace in another tube containing 14% potassiumdihydrogen orthophosphate (KH2PO4) solution for5 min. Drain and inoculate culture media.

Urine culture Use fresh, early morning mid-stream specimens. Donot bulk several specimens because this often leadsto contamination of cultures. Centrifuge 25–50 mlurine at 3000 rev./min and assess the number oforganisms in the deposit by examining a Gram-stained film. Suspend the deposit in 2 ml 4% sul-phuric acid for 15–40 min depending on the load ofother organisms. Neutralize with 15 ml distilledwater, centrifuge and culture the deposit.

Alternatively plate some of the urine on blood agar,incubate overnight and, if this is sterile, filter50–100 ml of the urine through a membrane filter.Cut the filter into strips and place each strip on thesurface of the culture media in screw-capped bottles.

Blood and bone marrow Since the advent of AIDS it is often necessary todetect disseminated mycobacterial disease byexamination of blood and bone marrow. Collect8.5 ml blood aseptically and add to a tube contain-ing 1.5 ml 0.35% sodium polyethanolium sulphate(SPS; this is the least toxic of the anticoagulants formycobacteria and lyses white cells). Mix to avoidcoagulation and inoculate 10–20 volumes of liquidmedia, e.g. Middlebrook 7H9 or 13A liquidmedium containing 0.025% SPS and, if necessary,an antibiotic cocktail (see p. 383). The Isolator-10lysis centrifugation system (Du Pont Co.,Wilmington, DE, USA) is also suitable (Kiehn et al.,1985). Or collect 10 ml blood into a tube that con-tains an anticoagulant and saponin which lyses allblood cells. Centrifuge at 3000 × g for 30 min andinoculate deposit on Middlebrook 7H11 agar.

Alternatively, add blood or bone marrow directlyto the medium. Check broth weekly for bacterialgrowth. Confirm acid fastness by ZN smear andinoculate solid media.

Automated techniques are ideal for blood cultureand are used according to the manufacturer’sinstructions (see Chapter 8).

FaecesCulture of faeces of AIDS patients with suspectedintestinal disease caused by the M. avium complexmay be required (Kiehn et al., 1985). Suspend 1 gfaeces in 5 ml Middlebrook 7H9 broth, decontam-inate as for sputum by the ‘hard’ method (seep. 000) and inoculate on Mitchison’s antibioticmedium (see p. 383).

Culture of CSF, pleural fluids, pus, etc. Plate original specimen or centrifuged deposit onblood agar and incubate overnight. Keep theremainder of specimen in a deep-freeze. If theblood agar culture is sterile, inoculate media formycobacteria without further treatment. Use asmuch material and inoculate as many tubes as pos-sible. If the blood agar culture shows the presenceof other organisms, proceed as for sputum and/orculture directly into media containing antibiotics(see below). Half-fill the original container with

382 Mycobacterium

Kirchner medium and incubate. Some tuberclebacilli may adhere to the glass or plastic.

Culture of tissue Grind very small pieces of tissue, e.g. endometrialcurettings, or emulsify them in sterile water withglass beads on a Vortex mixer. Homogenize largerspecimens with a Stomacher or blender. Cheek thesterility and proceed as for CSF, etc. Keep some ofthe material in a deep-freeze in case the cultures arecontaminated and the tests need repeating.

Isolation from the environment

This may be done to trace the sources of environ-mental mycobacteria that contaminate clinicalmaterial and laboratory reagents, and also to studythe distribution of these mycobacteria in nature.

WaterPass up to 2 litres water from cold and hot tapsthrough membrane filters (as for water bacteriology,see p. 254). Drain the membranes and place themin 3% sulphuric or oxalic acid for 3 min and thenin sterile water for 5 min. Cut them into strips andplace each strip on the surface of the culture mediumin screw-capped bottles. Use Lowenstein–Jensenmedium and Middlebrook 7H11 medium contain-ing antibiotics (see below).

Swab the insides of cold and hot water taps andtreat them as laryngeal swabs.

MilkCentrifuge at least 100 ml from each animal.Examination of bulk milk is useless. Treat creamand deposit separately by the NaOH method andculture deposit on several tubes of medium.

Dust and soil Place 2–5 g samples in 1 litre sterile distilled watercontaining 0.5% Tween 80. Mix gently to avoidtoo much froth. Pass through a coarse filter toremove large particles and allow to settle in a

refrigerator for 24 h. Decant the supernatant andpass it through membrane filters as described abovefor water. Centrifuge the sediment and treat it withNaOH as for sputum. Neutralize, centrifuge andinoculate several tubes of medium.

Culture media

Egg media are most commonly used and there seemslittle to choose between any of them. In the UK,Lowenstein–Jensen medium is most popular but, inthe USA, ATS or Piezer medium is often used. Bothglycerol medium (which encourages the growth ofthe human tubercle bacillus) and pyruvate medium(which encourages the bovine tubercle bacillus andM. malmoense) should be used. Pyruvate mediumshould not be used alone because some opportunistmycobacteria grow very poorly on it.

Some workers prefer agar-based media, e.g.Middlebrook 7H10 or 7H11. Kirchner liquidmedium is also useful for fluid specimens that can-not be centrifuged or when it is desirable to culturea large amount of material. Aspirated pleural, peri-cardial and peritoneal fluids may be added directlyto an equal volume of double-strength Kirchnermedium at the bedside.

The antibiotic media of Mitchison et al. (1987)are useful for contaminated specimens, even aftertreatment with NaOH or other decontaminatingagents, etc. They offer a safety net for non-repeat-able specimens such as biopsies. These mediashould be used as well as, not in place of, egg mediafor tissues, fluids and urines. They are prepared asfollows. Add the following to complete Kirchner orMiddlebrook 7H11 media: polymyxin 200 units/ml, carbenicillin 100 mg/1, trimethoprim 10 mg/1,and amphotericin 10 mg/1. Dispense fluid mediumin 10-ml amounts and make slopes of solidmedium.

An alternative antibiotic mixture (PANTA) isused, particularly in automated systems. This con-tains (final concentrations) polymyxin 50 units/ml,amphotericin 5 mg/l, nalidixic acid 20 mg/1,trimethoprim 5 mg/1 and azlocillin 10 mg/l.


Inoculating culture media

Use an inoculum of at least 0.2 ml (not a loopful) foreach tube of medium. Inoculate two tubes each ofegg medium, one containing glycerol and the otherpyruvic acid and other media as indicated above.

Incubation

Incubate all cultures at 35–37°C and cultures fromsuperficial lesions also at 30–33°C for at least 8weeks. Prolonging the incubation for a further 2–4weeks may result in a small increment of positives,especially from tissues and for some species.Examine weekly.

Automated culture

Commercial instruments (e.g. BACTEC, BectonDickinson) have been developed for detecting theearly growth of mycobacteria by a radiometric orinfrared method (see Chapter 8).

Sputum or other homogenates, decontaminated ifnecessary, are added to Middlebrook brothmedium containing antibiotics, to discourage thegrowth of other organisms, and 14C-labelledpalmitic acid. The medium is prepared commer-cially (Becton Dickinson) in rubber-sealed bottlesand is inoculated with a syringe and hypodermicneedle. If growth occurs, 14CO2 is evolved. The airspace above the medium in each bottle is sampledautomatically at fixed intervals and the amount ofradioactive gas is estimated and recorded. Growthof mycobacteria may be detected in 2–12 days, butpositive results require further tests to distinguishbetween tubercle bacilli and other mycobacteria. Inthe BACTEC system, p-nitro-α-acetylamino-β-pro-piophenone (NAP) is used and this takes another2–5 days. Mycobacteria that grow in media con-taining this substance are subcultured and identi-fied by traditional methods (see Heifets, 1986, andbelow) or by the use of DNA probes (see below).

Disposal of radioactive waste is a major problem

with the radiometric system and in many centres ithas been replaced by non-radiometric systemsincluding the Mycobacterial Growth IndicatorTube (MGIT Bactec 960, Becton Dickinson,Franklin Lakes, USA) and the MB BacT Alert(Organon Technika, Boxtel, The Netherlands) sys-tems. These permit detection of bacterial growthwithin 12–18 days and are less demanding on stafftime than the radiometric system. They are basedon a change in the colour of a dye on release of CO2

(MB BacT Alert) or unquenching of a fluorescentsubstance on consumption of oxygen during bacil-lary growth (MGIT). The systems are used accord-ing to the manufacturers’ instructions.

Ideally, these automated systems should be usedin conjunction with solid media-based culture(Drobniewski et al., 2001). Automated systemsbased on nucleic acid amplification are consideredin Chapter 8.

Identification of tubercle bacilli

In most parts of the world, the majority ofmycobacteria cultured from pathological materialwill be tubercle bacilli. No growth will be evidenton egg media until 10–14 days. Colonies of thehuman tubercle bacillus, M. tuberculosis, arecream coloured, dry and look like bread crumbs orcauliflowers. Those of the bovine tubercle bacillus,M. bovis, are smaller, whiter and flat. Growth ofthis organism may be very poor; it grows much bet-ter on pyruvate medium than on glycerol medium.(A smooth tubercle bacillus, M. canetti, has beendescribed but is exceedingly rare.)

On Middlebrook agar media, colonies of bothM. tuberculosis and M. bovis are flat and grey. InKirchner fluid media, colonies are round and gran-ular, and may adhere to one another in strings ormasses. They grow at the bottom of the tube andsettle rapidly after the fluid has been shaken.

Make ZN films to check for acid fastness. Someyeasts and coryneform organisms grow on egg mediaand their colonies may resemble those of tuberclebacilli. Make the films in a drop of saturated mercuricchloride as a safety measure against dispersing live

384 Mycobacterium

bacilli in aerosols, and spread the films gently. Notewhether the organisms are difficult or easy to emul-sify. Check the morphology microscopically. Tuberclebacilli may be arranged in serpentine cords, usuallyuniformly stained and usually 3–4 μm in length.

Make suspensions of the organisms as follows.Prepare small, glass, screw-capped bottles contain-ing two wire nails shorter than the diameter of thebase of the bottles, a few glass beads 2–3 mm indiameter and 2 ml phosphate buffer pH 7.2–7.4.(The nails will rust if water is used; wash both nailsand beads in dilute HCl and then in water beforeuse.) Sterilize by autoclaving. Place several coloniesof the organisms in a bottle and mix on a magneticstirrer. Allow large particles to settle and use thesupernatant.

Inoculate two tubes of egg medium and one tubeof the same medium containing 500 μg/mlp-nitrobenzoic acid (PNBA medium). To make thep-nitrobenzoic acid stock solution, dissolve 0.5 g ofthe compound in 50 ml water to which a smallvolume of 1 mol/l NaOH solution has been added. Carefully neutralize with HCl and make upto 100 ml with water. This solution keeps forseveral months in a refrigerator. Incubate one eggslope at 25 ± 0.5°C, the other in an incubatorwith an internal light at 35–37°C. Incubate the p-nitrobenzoic acid egg slope at 35–37°C. If aninternally illuminated incubator is not available,grow the organisms for 14 days and then expose tothe light of a 25-W lamp at a distance of 1–1.5 mfor several hours and re-incubate for 1 week.

Tubercle bacilli have the following characteristics:

• Not easily emulsified in water (orconcentrated HgCl2 which is safer).

• Regular morphology, 3–4 μm in length andusually showing serpentine cords.

• Relatively slow growing, taking 10 days ormore to show visible growth.

• Produce no yellow, orange or red pigment. • Fail to grow in the presence of 500 μg/ml

p-nitrobenzoic acid (the bovine organism mayshow a trace of growth).

• Fail to grow at 25°C (the bovine organismmay show a trace of growth).

Acid-fast organisms which grow in 1 week or less,or yield yellow or red colonies, or emulsify easily,or are morphologically small, coccoid, or long, thinand beaded or irregularly stained, are unlikely to betubercle bacilli.

Variants of tubercle bacilli and BCG

Although the identification of ‘species’ within theM. tuberculosis complex is not essential for themanagement of the patient, it may be of value forepidemiological work, such as determining the roleof cattle as a source of human tuberculosis in someregions. It is also useful to identify BCG, particu-larly if it may have been responsible for dissemi-nated disease as a result of immunization ofneonates, HIV infection or the treatment of malig-nancy. Although nucleic acid-based methods areavailable for such typing, a simple system based oncultural characteristics and advocated by theWorld Health Organization (Grange et al., 1996)differentiates between the major variants withinthe M. tuberculosis complex.

Use suspensions prepared as described above andinoculate media for the following tests and incu-bate at 37°C.

TCH susceptibility Use Lowenstein–Jensen medium containing5 μg/ml thiophen-2-carboxylic acid hydrazide.Read at 18 days.

Pyruvate preference Compare the growth on Lowenstein–Jensenmedium containing glycerol with that containingpyruvate after 18 days’ incubation.

Nitrate reduction test Use Middlebrook 7H10 broth and test after 18days by the second method described under‘Nitrate reductase test’ in Chapter 7 (see p. 98).

Oxygen preference Use Kirchner medium made semisolid with 0.2%agar. Inoculate with about 0.02 ml suspension.


Mix gently to avoid air bubbles and incubateundisturbed for 18 days. Aerobic growth occurs ator near the surface, and microaerophilic growth asa band 1–3 cm below the surface, sometimesextending upwards.

Pyrazinamide susceptibility and pyrazinamidaseactivityUse the method described on p. 397 for pyrazi-namide susceptibility testing. As an alternative tothe rather complex susceptibility test, pyrazinami-dase activity (which correlates very closely withsusceptibility to pyrazinamide) may be detected.Inoculate butts of Wayne’s medium (Middlebrook7H9 broth, 1000 ml; pyrazinamide, 100 mg;sodium pyruvate, 2 g; agar, 15 g. Dissolve bysteaming, dispense on 5-ml amounts in screw-capbottles and autoclave at 115°C for 15 min. Allowto cool in the upright position to form butts ratherthan slopes). Incubate at 37°C for 7 days, add 1 mlfreshly prepared 1% w/v ferrous ammonium sul-phate in distilled water and refrigerate at 4°C for4 h. A distinct pink band in the upper part of thebutt indicates pyrazinamidase activity. Faint pinkbands, seen with some strains of M. bovis, shouldbe ignored. Include controls of M. tuberculosis(positive) and M. bovis (negative) (Table 45.2).Identification of a strain as ‘Asian’ or ‘African’ doesnot indicate origin of strain or ethnic group of thepatient. The variants are widely distributed.

The niacin test It was originally claimed that this test distinguishedbetween human tubercle bacilli, which synthesizeniacin, and bovine tubercle bacilli, which do not.Niacin test strips are commercially available.Unfortunately some human strains give negativeniacin results and some environmental mycobac-teria do synthesize niacin. It is an unreliable testand has little to commend it.

Cycloserine resistance This test is useful for identifying BCG because it ismore resistant to cycloserine than other membersof the M. tuberculosis complex. Use Lowenstein–Jensen medium containing 20 μg/ml cycloserine.Read after 18 days.

Species within the M. tuberculosiscomplex

Mycobacterium tuberculosis This, the human tubercle bacillus, species growswell (eugonic) on egg media containing glycerol orpyruvate. Colonies resemble breadcrumbs and arecream coloured. Films show clumping and cordformation – the bacilli are orientated in ropes orcords, especially on moist medium. There is nogrowth at 25 or 42°C or on PNBA medium. It is

386 Mycobacterium

Table 45.2 Variants within the M. tuberculosis complex

Species TCH Nitrate reductase Oxygen preference Pyrazinamide resistancea

M. bovis S − M R M. africanum type I S − M S M. africanum type II S + M S M. tuberculosis Asian S + A S M. tuberculosis Classical R + A S BCGb S − A R

S, sensitive; R, resistant; M, microaerophilic; A, aerobic; TCH, thiophen-2-carboxylic acidaPyrazinamidase activity correlates with pyrazinamide susceptibilitybDiffers from the others in being resistant to cycloserine

usually resistant to TCH (the Asian variants aresensitive), is nitrate reduction positive and aerobic,and usually susceptible to pyrazinamide. It causestuberculosis in humans and may infect domesticand wild animals (usually directly or indirectlyfrom humans). Simians are particularly likely tobecome infected.

Mycobacterium bovis This, the bovine tubercle bacillus, grows poorly(dysgonic) on egg medium containing glycerol butgrowth is usually enhanced on pyruvate medium.Colonies are flat and grey or white; growth may beeffuse. Cords are present in films. There is no orvery poor growth at 25°C and on PNBA mediumcompared with a control slope at 37°C. It issensitive to TCH, nitrate reduction negative,microaerophilic and resistant to pyrazinamide. Itwas common in dairy cows before eradicationschemes were introduced in the industrializedcountries, but it is still present in many developingcountries. It causes human tuberculosis, both pul-monary and extrapulmonary, the latter usuallyresulting from the consumption of infected milk. Inthe industrialized countries, the few cases encoun-tered usually represent reactivation of infectionsacquired much earlier.

Mycobacterium africanum This name is given to the African group of strainswith properties intermediate between those of thehuman and bovine tubercle bacillus (see Collins etal., 1982). Growth is usually dysgonic, enhancedby pyruvate. It is susceptible to TCH andmicroaerophilic, and susceptible to pyrazinamide.The nitrate reduction test may be negative (mostWest African strains) or positive (most East Africanstrains). It causes tuberculosis, clinically indistin-guishable from that caused by the other tuberclebacilli.

BCGThis is the bacillus of Calmette and Guérin, anorganism of attenuated virulence used in immu-

nization against tuberculosis. It is eugonic; growthis not enhanced by pyruvate. It is susceptible toTCH, nitrate reduction negative, aerobic, resistantto pyrazinamide and (unlike other strains in thisgroup) resistant to cycloserine.

Other variants within the M. tuberculosis com-plex include: M. microti, a cause of disease in thevole and other small mammals; ‘M. canetti’, a veryrarely encountered variant of M. tuberculosis pro-ducing smooth colonies on solid media; the caprine(goat) variant of M. bovis which has caused diseasein a few veterinary surgeons; and some unnamedstrains with unusual properties isolated from sealsand the rock hyrax.

ENVIRONMENTALMYCOBACTERIA

Many species of mycobacteria inhabit the naturalenvironment, and are especially associated withwatery environments: marshes, streams, rivers andindustrial and domestic water supplies. Around100 species have been named and new ones areregularly being described. Some species have oftenbeen incriminated as opportunist pathogens ofhumans and animals, others rarely or never. Thereis, however, no clear distinction between potentialpathogens and non-pathogens, and it is probablethat any mycobacterial species, given a suitableopportunity, could cause disease. Environmentalmycobacteria may contaminate specimens or beintroduced during collection and/or laboratoryprocessing. It is also difficult to assess the signifi-cance of opportunists. Determining whether anisolate is the cause of disease or a contaminant isnot easy, especially in the case of isolates fromsputum.

Identification of some of the more unusual envi-ronmental mycobacteria requires the use ofnucleic acid-based technology, such as sequencingof the 16-S ribosomal RNA. Most of the speciesisolated in clinical laboratories may be identifiedwith the aid of the following tests and Table45.3.

Environmental mycobacteria 387

Table 45.3 Usual properties of some opportunist mycobacteria and others that may be encountered in clinical material

Species Pigment TZ Nitrate Tween Growth at Arylsulphatasea Catalaseb Telluritec Growth on Rapidreductase hydrolysis 20 25 33 42 44 3 days 21 days reduced N medium growth

(°C)

M. kansasii P S + + – + + v – – + +++ – –M. marinum P R – + (late) + + + – – – ++ ++ v +M. xenopi – /Sc R – – – – – + + – +++ – – – –M. avium-intracellulare – /Sc R – – – + + + v – v – + – –M. scrofulaceum Sc v – – v + + – – – v +++ – – –M. malmoense – R + + (late) – + + – – – – – – – –M. simiae P R – – – + + – – – + ++ – – –M. szulgai Sc/Pd R + + – + + – – – +++ + – – –M. fortuitum – R + – + + + v – +++ +++ v + + +M. chelonei – R – – + + + – – +++ +++ v + + +M. gordonae Sc R – + + + + – – – + ++ – –M. flavescens Sc R + + + + + – – – + + + +M. gastri – R – + + + + – – – + + – – –M. terrae – R + + v + + – – – + +++ – – –M. triviale – R – + – + + – – – + + – – –M. nonchromogenicum – R – + v + + – – – + +++ – – –M. smegmatis – R + + + + + + + – + ++ + + +M. phlei Sc R + + + + + + + – + ++ + + +M. ulcerans – /Sc R – – – – + – – – – – – – –

TZ, thiacetazone; P, photochromogen; Sc, scotochromogen; S, sensitive; R, resistant; + , usually positive; – , usually negative or none; v, variablea Sulphatase: +++ , deep pink; ++ , pink; + , pale pinkb Catalase: Amount of foam, +++ , > 20 mm; ++ , 10–20 mm; + , 5–10 mm; – , < 5 mmc Test not done on pigmented strainsd M. szulgai: At 25°C = photochromogen; at 37°C = scotochromogen

Inoculation

Unless otherwise stated, inoculate the media withone loopful or a 10-μl drop of a suspension pre-pared as described on p. 385.

Pigment production

Inoculate two egg medium slopes. Incubate both at35–37°C, one exposed to light and the other in a light-proof box. Examine at 14 days. Photochromogensshow a yellow pigment only when exposed to light.Scotochromogens are pigmented in both light anddark, but the culture exposed to light is usually deeperin colour. False photochromogenicity may arise if theinoculum is too heavy and pigment precursor is car-ried over. Continue to incubate the slope exposed tolight and look for orange-coloured crystals ofcarotene pigment that may form in the growth.

Temperature tests

Inoculate egg media slopes with a single streakdown the centre and incubate at the following(exact) temperatures: 20, 25, 42 and 44°C.Examine for growth at 3 and 7 days and thereafterweekly for 3 weeks.

Thiacetazone susceptibility

Inoculate egg medium slopes containing 20 μg/mlthiacetazone (TZ). Make a stock 0.1% solution ofthis agent in formdimethylamide (caution). Thissolution keeps well in a refrigerator. Incubate slopesfor 18–21 days and observe the growth in compar-ison with that on an egg slope without the drug.

Nitrate reduction test

Use the second method on p. 98 of Chapter 7(under ‘Nitrate reductase test’). Strains that reducenitrite may not accumulate enough from nitrate

(nitrate reductase present) to score as nitrate reduc-tase positive.

Sulphatase test

Use the method described on p. 100 of Chapter 7.If the organism is a rapid grower, add the ammoniaafter 3 days.

Catalase test

Prepare the egg medium in butts in screw-cappedtubes (20 × 150 mm). Inoculate and incubate at35–37°C for 14 days with the cap loosened and thenadd 1 ml of a mixture of equal parts 30% hydrogenperoxide (caution) and 10% Tween 80. Allow tostand for a few minutes and measure the height inmillimetres of the column of bubbles. More than40 mm of froth is a strong positive result (+++).

Tween hydrolysis

Add 0.5 ml Tween 80 and 2.0 ml of a 0.1% aque-ous solution of neutral red to 100 ml M/15 phos-phate buffer. Dispense this in 2-ml lots andautoclave at 115°C for 10 min. This solution keepsfor about 2 weeks in the dark and should be a pale-straw or amber colour. Add a large loopful of cul-ture from solid medium and incubate at 35–37°C.Examine at 5 and 10 days for a colour change fromamber to deep pink.

Tellurite reduction

Grow the organisms in Middlebrook 7H10medium for 14 days or until there is a heavygrowth. Add four drops of sterile 0.02% aqueoussolution of potassium tellurite (caution) and incu-bate for a further 7 days. If tellurite is reduced therewill be a black deposit of metallic tellurium. Ignoregrey precipitates. This test is of no value for highlypigmented mycobacteria.


Growth in N medium

Inoculate the medium with a straight wire andincubate. Rapid growers give a turbidity with orwithout a pellicle in 3 days. ‘Frosting’, i.e. adher-ence of a film of growth to the walls of the tubeabove the surface of the medium, is often seen.Ignore a very faint turbidity.

Resistance to antibacterial agents

Methods of doing these tests are described below.Some mycobacteria exhibit consistent patterns,which may be useful for identification.

Aerial hyphae

Some Nocardia spp. are partially acid fast andresemble rapidly growing mycobacteria. Do a slideculture (see p. 133) and look for aerial hyphae. Invery young cultures of mycobacteria, a myceliummay be seen, but it fragments early into bacilli.Aerial hyphae are not formed except by M. xenopi.

Emulsifiability and morphology

When making films for microscopic examination,note whether the organisms emulsify easily. Notethe morphology, whether very short, coccobacilli,long, poorly stained filaments or beaded forms.

Other more specialized techniques for identifyingenvironmental mycobacteria include lipid chro-matography (Jenkins, 1981) and pyrolysis massspectrometry (Sisson et al., 1991). DNA probes forsome of the more commonly encountered speciesare available commercially (see p. 398).

Species of opportunist and othermycobacteria

Mycobacterium kansasii This is a photochromogen (but see below).Optimum pigment production is observed if the

inoculum is not too heavy, if there is a plentifulsupply of air (loosened cap) and if exposure to lightis continuous. Young cultures grown in the darkand then exposed to light for 1 h will develop pig-ment, but old cultures that have reached the sta-tionary phase may not show any pigment afterexposure. Continuous incubation in the lightresults in the formation of orange-coloured crystalsof carotene. The morphology is distinctive; thebacilli are long (5–6 μm) and beaded. M. kansasii isnitrate reduction positive, sensitive to 20 μg/mlthiacetazone, hydrolyses Tween 80 rapidly, andgrows at 25°C but not at 20°C; some strains growpoorly at 42°C but not at 44°C. The arylsulphatasetest is weakly positive and the catalase stronglypositive (more than 40 mm by the methoddescribed above). It is resistant to streptomycin,isoniazid and p-aminosalicylic acid (PAS), but sus-ceptible to ethionamide, ethambutol andrifampicin. Some strains are susceptible toamikacin and erythromycin by the disc technique.

Occasional nonchromogenic or scotochro-mogenic strains have been reported but these maybe recognized by their biochemical reactions andmorphology.

This organism is an opportunist pathogen, associ-ated with pulmonary infection. It is rarely signifi-cant when isolated from other sites. It has beenfound in water supplies.

The Mycobacterium avium complexThe species, M. avium and M. intracellulare, areusually grouped together as the M. avium complex(MAC). The two closely related species may be dis-tinguished by DNA probes or agglutination serol-ogy in some reference laboratories. The complexalso contains M. avium ssp. paratuberculosis (pre-viously M. paratuberculosis), the cause of hyper-trophic enteritis or Johne’s disease in cattle andother ruminants, and M. lepraemurium, the causeof skin lesions in rats and other small rodents andcats.

MAC bacilli are small, almost coccoid. Althoughusually non-chromogenic, a feeble yellow pigmentis produced by some isolates. Some strains are sus-ceptible to thiacetazone. All are nitrate reduction

390 Mycobacterium

negative, catalase negative or weakly positive,reduce tellurite and do not hydrolyse Tween 80.The sulphatase reaction varies from strongly posi-tive to negative. There is growth on egg medium at25°C; growth at 20°C and 42°C is variable.Resistance to antituberculosis drugs is usual, butsome strains are susceptible to ethionamide.

MAC bacilli are opportunist pathogens ofhumans, associated with cervical adenitis (scrof-ula), especially in young children. Pulmonary dis-ease, usually but not always with predisposing lungconditions, occurs in adults. They frequently causeopportunist disease in patients with the AIDS. Suchdisease is often disseminated and the organismsmay be isolated from many sites including blood,bone marrow and faeces (Kiehn et al., 1985). Theyare also opportunist pathogens of pigs and birdsand have been found in soil and water. There havebeen claims that M. avium ssp. paratuberculosis isa cause of Crohn’s disease in humans but theseclaims require substantiation.

Strains of M. avium ssp. paratuberculosis andsome strains of M. avium, notably those isolatedfrom wood pigeons, require the addition ofmycobactin, an iron-binding lipid extracted frommycobacterial cell walls, to media for their growth.

Mycobacterium xenopi Pigment, if any, is more obvious on cultures incu-bated at 42–44°C, which become pale yellow.Morphologically it is easily recognized; it stainspoorly and the bacilli are long (5–6 μm) and fila-mentous. It is nitrate reduction negative, resistantto thiacetazone and does not hydrolyse Tween 80.It is a thermophile, growing well at 44°C, slowly at35°C and not at 25°C. (Almost all other environ-mental mycobacteria grow at 25°C.)

The arylsulphatase test is strongly positive, andthe catalase test negative. It does not reduce tellu-rite. It is more susceptible to isoniazid than otheropportunist mycobacteria, usually giving a resist-ance ratio of 4. It is susceptible to ethionamide, butits susceptibility to other antituberculosis drugsvaries between strains. Some strains are susceptibleto amikacin and erythromycin by the disc method.

It is an opportunist pathogen in human lung

disease and is rarely significant in other sites. It is afrequent contaminant of pathological material,especially urine, and has been found in hospital hotwater supplies. ‘Outbreaks’ of laboratory contami-nation are not uncommon. This organism is verycommon in south-east England and north-westFrance.

Mycobacterium celatumStrains of this species were originally isolated inFinland and Somalia, principally from respiratorytract specimens. Strains grow at 25°C and 45°C,they do not hydrolyse Tween 80 and some developa light-yellow pigment in the dark. They resembleM. xenopi in being strongly arylsulphatase posi-tive, but differ from this species in being susceptibleto ethambutol and ethionamide. Another species,M. branderi, is phenetically similar but differs in itsdrug susceptibility pattern.

Mycobacterium genavenseThis species is an occasional cause of disseminateddisease in AIDS patients and has been isolated frompet and zoo birds. It is cultivable only with greatdifficulty: slow and feeble growth occurs in Bactec13A medium used for the radiometric detection ofmycobacterial growth. It is identifiable by uniquebase sequences in its ribosomal 16-S RNA.

Mycobacterium scrofulaceum This, the ‘scrofula scotochromogen’, is pheneticallysimilar to the MAC bacilli but is scotochromogenicand fails to reduce tellurite to tellurium. It may alsobe differentiated from MAC by agglutinationserology.

Mycobacterium marinum This is an opportunist pathogen responsible forsuperficial skin infections, known as swimmingpool granuloma, fish tank granuloma or fish-fanciers’ finger. It is found in sea-bathing pools andin tanks where tropical fish are kept, and is apathogen of some fish (see the review by Collins etal., 1985). It is missed in clinical laboratories thatdo not culture material from superficial lesions at30–33°C. Primary cultures do not grow at


35–37°C. It is a photochromogen. Beading orbanding similar to that of M. kansasii may be evi-dent. It is nitrate reduction negative and resistant tothiacetazone, and hydrolyses Tween 80. After lab-oratory subculture its temperature range is modi-fied so that it grows at 25°C and 37°C but not at44°C. The catalase and arylsulphatase tests areweakly positive and growth may occur in Nmedium. Resistance to streptomycin and isoniazidis usual; resistance to other drugs is variable. Somestrains are susceptible to co-trimoxazole, erythro-mycin and amikacin by the disc method.

Mycobacterium gordonae This is also known as the tap-water scotochromogen,although other scotochromogens are found in watersupplies. Organisms in this group grow slowly, pro-duce a deep-orange pigment in light and usually ayellow pigment in the dark. No crystals of caroteneare formed. If such crystals are seen, the organismmay be a scotochromogenic strain of M. kansasii.Morphology is not distinctive. It is nitrate reductionnegative, resistant to thiosemicarbazone and hydrol-yses Tween 80. Growth occurs at 20°C but not at44°C. Some strains are psychrophilic. The arylsul-phatase reaction is weak and the catalase test isusually strongly positive. Growth does not occur inN medium. They are usually resistant to isoniazidand PAS but susceptible to streptomycin and otherantituberculosis drugs.

The organisms in this group are rarely associatedwith human disease. They are not infrequent con-taminants of pathological material but usuallyappear as single colonies on egg medium. They maybe found in tap water, dust and soil.

Mycobacterium szulgai This scotochromogen differs from M. gordonae inbeing nitrate reductase positive and strongly aryl-sulphatase positive, and giving a weak catalasereaction. It hydrolyses Tween 80. It is a rare humanopportunist pathogen.

Rapidly growing scotochromogens There are many species in this group, e.g.M. flavescens, M. aurum, M. gilvum, M. duvalii

and M. vaccae. With very rare exceptions they arenot known to cause human disease but occur in theenvironment and sometimes contaminate patho-logical material.

Mycobacterium fortuitum and M. chelonaeThese two non-pigmented rapid growers are conve-niently considered together. There have been taxo-nomic problems: M. fortuitum has subdivided intoM. fortuitum and M. peregrinum whereas M. che-lonae has been subdivided into M. chelonae andM. abscessus. On subculture, growth occurs within3 days on most media, but on primary isolationfrom clinical or environmental specimens growthmay, paradoxically, not be apparent for severalweeks or months. The bacilli tend to be rather fatand solidly stained. The nitrate reduction test ispositive (M. fortuitum and M. peregrinum) ornegative (M. chelonae and M. abscessus). Tween 80is not hydrolysed. There is growth at 20°C and insome strains at 42°C. Psychrophilic strains, whichfail to grow at 37°C, are not uncommon, particu-larly on primary isolation. The arylsulphatase testis positive at 3 days and the catalase reaction isvariable. Tellurite is reduced rapidly. There isgrowth in N medium in 3 days. There is a generalresistance to all antituberculosis drugs, except thatM. fortuitum and M. peregrinum are usuallysusceptible to ethionamide and the quinolones, butM. chelonei and M. abscessus are resistant.

These are opportunist pathogens usually occurringin superficial infections (e.g. injection abscesses) andoccasionally as secondary agents in pulmonarydisease. They are common in the environment andfrequently appear as laboratory contaminants.

Mycobacterium smegmatis and M. phlei These two rapidly growing saprophytes are morecommon in textbooks than in clinical laboratories.Growth occurs in 3 days. They are nitrate reduc-tion positive and hydrolyse Tween 80 in 10 days.They grow at 20°C and 44°C; M. phlei grows at52°C. The 3-day sulphatase test is negative, butlonger incubation gives positive reactions. Thecatalase test is strongly positive. Tellurite isreduced. There is growth in N medium.

392 Mycobacterium

They are not associated with human or animal dis-ease. They have contributed to the myth of acid-fastversus acid- and alcohol-fast bacilli (see p. 379).

Slowly growing nonchromogens There are at least four species: M. terrae,M. nonchromogenicum, M. triviale and M. gastri.All grow at 25°C but poorly or not at all at 42°C.All hydrolyse Tween 80 and none reduces tellurite,which permits differentiation from MAC bacilli.M. terrae and M. triviale give a positive nitratereduction test but M. nonchromogenicum andM. gastri are negative. M. gastri is occasionallyphotochromogenic and is usually susceptible to thi-acetazone and may therefore be confused withM. kansasii to which, although non-pathogenic, itis genetically closely related. Susceptibility to anti-tuberculosis drugs is variable.

Mycobacterium ulcerans This causes Buruli ulcer, a serious skin infection incertain regions within the tropics and in Australia.It is difficult to cultivate because it has a very nar-row temperature range of growth, 31–34°C, andgrowth is very slow, taking 10–12 weeks to givesmall colonies resembling those of M. bovis. It isbiochemically inert and undistinguished.

Mycobacterium simiae Originally isolated from monkeys, this is a pho-tochromogen and is nitrate reduction negative, anddoes not hydrolyse Tween 80. Pulmonary and dis-seminated human disease has been reported.

Mycobacterium malmoense This resembles the MAC organisms, grows veryslowly and is sometimes confused with M. bovis byinexperienced workers. This suggests that it is morecommon than might be expected. It is difficult toidentify except by lipid chromatography. It causespulmonary disease in adults and cervical adenopa-thy in children.

Mycobacterium haemophilum This is another rare species, possibly missedbecause it grows poorly or not at all on media that

do not contain iron or haemin. It grows at 30°C onLJ medium containing 2% ferric ammonium citrateand on Middlebrook 7H11 medium containing60 μg/ml haemin. All the usual biochemical testsare negative. It has been isolated from the skin andsubcutaneous tissues of immunologically compro-mised patients and from cases of cervical lym-phadenopathy in otherwise healthy children.

DRUG SUSCEPTIBILITY TESTS

Although most cases of tuberculosis yield tuberclebacilli that are sensitive to the commonly used anti-tuberculosis drugs, drug and multidrug resistance isan increasing problem, particularly in certain‘hotspot’ regions. Where facilities are available,drug susceptibility tests are of value, particularly ifthe patient’s condition does not improve as a resultof therapy. Susceptibility tests for environmentalmycobacteria causing disease are of questionablevalue because there is a poor correlation between invitro results and response to therapy.

Treatment of tuberculosis with several drugs isessential to prevent the emergence of drug resist-ance. The WHO has made definitive recommenda-tions on the therapy of tuberculosis. Most patientsreceive four drugs – rifampicin, isoniazid, ethamb-utol and pyrazinamide – for 2 months (the inten-sive phase) and two of these – rifampicin andisoniazid – for a further 4 months (the continuationphase). Streptomycin, being an injectable drug, isnow usually only used in regimens for the re-treat-ment of cases showing bacteriological relapse.Patients with multidrug resistance (by definition,resistance to rifampicin and isoniazid with or with-out resistance to other drugs) require treatmentwith second-line drugs including fluoroquinolonesand ethionamide.

Susceptibility tests with mycobacteria are com-plex and are usually done in specialist and refer-ence laboratories. There are four principalmethods:

1. The absolute concentration method which ispopular in Europe

Drug susceptibility tests 393

2. The proportion method, also used in Europeand popular in America

3. The resistance ratio method used in the UK4. Radiometric and non-radiometric automated

methods.

Special techniques are used for pyrazinamide sus-ceptibility tests and nucleic acid-based methods areavailable for the rapid determination of resistanceto certain agents.

The absolute concentration method

Carefully measured amounts of standardized inoc-ula are placed on control media and media con-taining varying amounts of drugs, and the lowestconcentration that will inhibit all, or nearly all, ofthe growth is reported. It is very difficult, however,to standardize the active concentration of drugs inthe media and the method gives different results indifferent laboratories. It is not possible to use amedium that must be heated after the addition ofthe drug (e.g. egg medium), because some drugs arepartially heat labile and heating times are rarelyconstant even in the same laboratory. Middlebrook7H10 or 7H11 media are used but this method hasnot found much favour in the UK.

The proportion method

Several dilutions of the inoculum are made andmedia containing no drug and standard concentra-tion of drugs are inoculated. The number ofcolonies growing on the control from suitable dilu-tions of the inoculum are counted, and also thenumber growing on drug-containing mediumreceiving the same inoculum. Comparison of thetwo shows the proportion of organisms that areresistant. This is usually expressed as a percentage.

This method is popular in the USA and Europe,but it is technically very difficult and, as it is usuallydone in Petri dishes, we regard it as highly haz-ardous. There are also more risks attached to stan-dardizing the inocula than with the resistance ratiomethod.

The resistance ratio method

The minimal inhibitory concentrations of the drugthat inhibit test strains are divided by those thatinhibit control strains to give the resistance ratios.Ratios of 1 and 2 are considered susceptible, andthose of ≥ 4 resistant (see below). These resultsusually correlate with the clinical findings. Thismethod is used extensively in the UK and isdescribed below.

Each of the above methods may be used for‘direct’ tests on sputum homogenates that containenough tubercle bacilli to give positive direct filmsas well as for ‘indirect’ tests on cultures. The latterare more reliable and reproducible.

For background information about these tests, seeCanetti et al. (1969) and Vestal (1975). For a dis-cussion on the design of resistance ratio tests, seeCollins et al. (1997).

Technique of resistance ratio method

Dilutions of drugs are incorporated inLowenstein–Jensen medium, which is then inspis-sated (i.e. heat coagulated). As some of these drugsare affected by heat, it is essential that the inspissa-tion procedure be standardized. The inspissatormust have a large circulating fan so that all tubesare raised to the same temperature in the sametime. The load (i.e. number of tubes) and the timeof exposure must be constant. To ensure that thedrug-containing medium is not overheated, themachine should be raised to its correct temperaturebefore it is loaded and racks can be devised thatallow loading in a few seconds. A period of 45 minat 80°C is usually sufficient to coagulate thismedium and no further heat is necessary if it is pre-pared with a reasonably aseptic technique.

Control strains The resistance ratio method compares the mini-mum inhibitory concentration (MIC) of theunknown strain with that of control strains on thesame batch of medium.

394 Mycobacterium

Some workers use the H37Rv strain of M. tuber-culosis as the control strain, but the susceptibilityof this to some drugs does not parallel that of ‘wild’tubercle bacilli and may give misleading ratios. It isbetter to use the modal resistance method of Leatand Marks (1970), in which the unknown strainsare compared with the modal resistance (i.e. thatwhich occurs most often) of a number of knownsusceptible strains of recent origin.

Drug concentrations Each laboratory must determine its own ranges,because these will vary slightly according to localconditions. Initially use those suggested in Table45.4 and inoculate at least 12 sets with known sus-ceptible organisms to arrive at a baseline for futurework. A drug-free control slope must be included.

Stock solutions of the drugs are conveniently pre-pared as 1% solutions in water, except forrifampicin, which should be dissolved in formdi-methylamide (caution). All stock solutions keepwell at −4°C.

Bacterial suspensions Smooth suspensions must be used. Large clumps orrafts of bacilli give irregular results and make read-ings difficult.

Sterilize 7-ml (bijou) screw-capped bottles con-taining a wire nail (shorter than the diameter of thebottle), a few glass beads and 2 ml phosphatebuffer (13.3 g anhydrous Na2HPO4 and 3.5 gKH2PO4 in 2 litres water, pH 7.4). Into each bottleplace a scrape of growth equal to about three orfour large colonies and place the bottle on a mag-

netic stirrer for 3–4 min. Allow to stand for a fur-ther 5 min for any lumps to settle and use thesupernatant to inoculate culture media.

Methods of inoculation Place the bottle containing the suspension on ablock of Plasticine at a suitable angle. Inoculateeach tube with a 3-mm loopful of the suspension,withdrawn edgewise. Plastic disposable 10-μl loopsare best.

A better and quicker method uses an automaticmicropipette (the Jencon Micro-Repette is ideal)fitted with a plastic pipette tip. Deposit 10 μl of thesuspension near to the top of each slope ofmedium. As it runs down it spreads out to give asuitable inoculum. The plastic tips are sterilizedindividually in small, capped test tubes and afteruse are deposited in a jar containing glutaralde-hyde, which is subsequently autoclaved. The tipsmay be re-used.

IncubationIncubate at 37°C for 18–21 days.

Reading results Examine tubes with a hand lens and record as fol-lows: confluent growth, CG; innumerable discretecolonies, IC; between 20 and 100 colonies, +; < 20colonies, 0.

The drug-free control slope must give growthequal to CG or IC. Less growth invalidates the test.The modal resistance, i.e. the MIC occurring mostfrequently in the susceptible control strains, shouldgive readings of CG or IC on the first two tubes

Drug susceptibility tests 395

Table 45.4 Susceptibility testing by the resistance ratio method: suggested concentrations of drugs in Lowenstein–Jensen medium

Drug Final concentration (μg/ml)

Isoniazid 0.007 0.015 0.03 0.06 0.125 0.25 0.5 Ethambutol 0.07 0.15 0.31 0.62 1.25 2.5 5 Rifampicin 0.53 1.06 3.12 6.25 12.5 25 50 Streptomycin 0.53 1.06 3.12 6.25 12.5 25 50

These are for the preliminary titration. Choose six that give confluent or near confluent growth in the two lowest and no growth inthe next four

(Table 45.5). The range must therefore be adjustedto give this result during the preliminary exercises.Once the range is set it is seldom necessary to varyit by more than one dilution.

InterpretationThe resistance ratio is found by dividing the MICof the test strain by the modal MIC of the controlstrains. When the readings are all CG or IC, this iseasy but, when there are tubes showing +, care andexperience may be required in interpretation. Ingeneral, a resistance ratio of ≤ 2 can be reported assusceptible, 4 as resistant and 8 as highly resistant.A ratio of 3 is borderline except for ethambutol,

when it probably indicates resistance. Mixed sus-ceptible and resistant strains occur. Examples ofthese findings are shown in Table 45.6.

Automated methods

The radiometric and non-radiometric systems (seep. 384) may be used to obtain rapid results, e.g.within 7 days. Appropriate amounts of each drugare added to the vials followed by the inoculum.For more information see the manufacturers’brochures.

396 Mycobacterium

Table 45.5 ‘Modal resistance’

Strain Drug concentrations in tube no.

1 2 3 4 5 6

Strain A CG CG 0 0 0 0 Strain B CG IC 0 0 0 0 Strain C CG CG + 0 0 0 Strain D IC IC 0 0 0 0 Strain E CG CG + 0 0 0 Mode CG CG 0 0 0 0

Table 45.6 Interpretation of results of drug susceptibility by the resistance ratio method based on ‘modal resistance’

Strain Drug concentrations in tube no. Resistance ratio Interpretation

1 2 3 4 5 6

Mode CG IC 0 0 0 0 Strain A CG 0 0 0 0 0 0.5 Susceptible Strain B CG GC 0 0 0 0 1 Susceptible Strain C CG IC + 0 0 0 1 Susceptible Strain D CG CG CG 0 0 0 2 Susceptible Strain E CG CG CG + 0 0 3 Borderlinea

Strain F CG CG CG IC 0 0 4 Resistant Strain G CG CG CG CG IC 0 8 Highly resistant Strain H CG CG CG CG CG CG 16 Highly resistant Strain I CG CG CG + + + − Mixed susceptible

and resistant

a Probably resistant with ethambutolCG, confluent growth; IC, innumerable discrete colonies

Pyrazinamide sensitivity tests

Pyrazinamide acts upon tubercle bacilli in the lyso-somes, which have a pH of about 5.2. For reliablesusceptibility tests the medium should therefore beat this pH. Tubercle bacilli do not grow well on eggmedium at pH 5.2 and agar medium (e.g.Middlebrook 7H11) is therefore frequently used.We have had good and consistent results withYates’ (1984) modification of Marks (1964)stepped pH method. This is a Kirchner semisolidmedium layered on to butts of Lowenstein–Jensenmedium.

Pyrazinamide stock solution Dissolve 0.22 g dry powder in 100 ml distilledwater; sterilize by filtration or steaming.

Solid medium Use Lowenstein–Jensen medium containing onlyhalf the usual concentration of malachite green (atan acid pH, less dye is bound to the egg and thehigher concentration of ‘free’ dye is inhibitory totubercle bacilli). Adjust 600 ml medium to pH 5.2with 1 mmol/l HCl. To 300 ml add 9 ml water(control); to the other 300 ml add 9 ml of the stockpyrazinamide solution (test). Tube each batch in 1-ml amounts and inspissate upright, to makebutts, at 87°C for 1 h.

Semisolid medium Add 1 g agar and 3 g sodium pyruvate to 1 litreKirchner medium. Adjust pH to 5.2 with 5 mol/lHCl. To 500 ml add 15 ml water (control); to theother 500 ml add 15 ml of the stock pyrazinamidesolution. Steam both bottles to dissolve the agar.Cool to 40°C and add to each bottle 40 ml OADCsupplement.

Final medium Layer 2 ml of the control semisolid medium onbutts of the control Lowenstein–Jensen medium.Do the same with the test media. Store in a refrig-erator and use within 3 weeks. (It may keep longer;we have never tried it.)

Bacterial suspensions Two inocula are required. Use one as describedabove for other susceptibility tests and also a 1 : 10dilution of it in sterile water.

InoculationWith a suitable pipette (e.g. the JenconsMicroRepette and plastic tip) add 20 μl (approxi-mately) of (1) the undiluted and (2) the diluted sus-pension to pairs of test and control media.

InterpretationTwo concentrations of inoculum are used because:

1. Some strains require a heavy inoculum to givegrowth at an acid pH, even in the controlmedium.

2. A heavy inoculum of some other strains mayovercome the inhibitory action ofpyrazinamide.

Colonies of tubercle bacilli should be distributedthroughout the semisolid medium in one or bothcontrols. If there is no growth in the test bottlefrom either the heavy or light inoculum, report thestrain as susceptible. If there is growth in the testbottle that received the heavy inoculum but not inthat which received the diluted suspension, reportthe strain as susceptible. If there is growth in bothtest bottles, comparable with that in their respec-tive controls, report the strain as resistant.

Note that resistance to pyrazinamide in strains ofM. tuberculosis from previously untreated patientsis uncommon but strains of M. bovis are naturallyresistant. Other methods for pyrazinamide suscep-tibility testing, including a radiometric (BACTEC)technique, have been compared by Cutler et al.(1997).

APPLICATION OF NUCLEIC ACID-BASED TECHNOLOGY TO DIAGNOSTICMYCOBACTERIOLOGY

Nucleic acid-based technology has been used in thetuberculosis laboratory for the rapid detection of

Application of nucleic acid-based technology to diagnostic mycobacteriology 397

members of the M. tuberculosis complex and othermycobacteria in clinical specimens, the identifica-tion of mycobacteria cultivated in various mediaand systems, the typing or ‘fingerprinting’ of iso-lates of M. tuberculosis for epidemiological pur-poses, and the rapid detection of drug (principallyrifampicin) resistance.

Detection of mycobacteria in clinicalspecimens

This is based on DNA or RNA amplification sys-tems, principally the PCR. Various amplificationtechniques that have been evaluated are reviewedby Drobniewski et al. (2001). Some methods arecommercially available and a widely used. One isthe Cobas Amplicor PCR system (RocheMolecular Systems, USA), which may be auto-mated. The latter system was found to be highlyspecific (99.7%) in a multicentre evaluation(Bogard et al., 2001) but, although its sensitivitywas high (96.1%) when applied to microscopi-cally positive respiratory specimens, it was lesssensitive (71.7%) when applied to microscopicallynegative specimens.

Identification of isolated mycobacteria

DNA probes are commercially available for theidentification of commonly encountered mycobac-teria in clinical material. Such probes may be usedto identify mycobacteria isolated in automated cul-ture systems as well as conventional culture.Currently available systems such as the INNO-LiPA Mycobacteria (Innogenetics, Ghent,Belgium), a reverse-hybridization-based line probeassay, and the AccuProbe assay (Gen-Probe Inc.,San Diego, CA, USA) detect species-specific varia-tions in the variable regions of the 16-S rRNA geneor the 16-S to 23-S rRNA spacer region (Scarparoet al., 2001).

Typing or ‘fingerprinting’ of M. tuberculosis

Fingerprinting of M. tuberculosis is usually per-formed by restriction fragment length polymor-phism (RFLP) analysis, which detects certainrepetitive DNA sequences inserted in the bacterialgenome. Up to 25 copies of the insertion sequenceIS6110 is found in most isolates of M. tuberculosisand is usually found in 5–25 copies throughout thechromosome. After digestion of the mycobacterialDNA by restriction endonucleases, the DNA frag-ments are separated by electrophoresis and thenumber and position of the insertion sequences arerevealed by DNA hybridization. Standardizedmethods and criteria for establishing identity ofisolates have been developed (Fletcher, 2001; vanSoolingen, 2001).

An alternative typing method, useful for strainscontaining few insertion sequences, is known asspacer oligonucleotide typing (spoligotyping) anddetects short DNA sequences situated near the sitesof the insertion elements mentioned above. UnlikeRFLP, spoligotyping can be performed on DNAamplified by PCR directly from the clinical speci-men.

Applications of DNA fingerprinting to epidemio-logical studies of tuberculosis are discussed indetail by Godfrey-Faussett (1998).

Rapid detection of rifampicinresistance

Rifampicin resistance is associated with variousmutations in the RpoB gene, which are detectableby use of a commercially available line probe assaysystem. In a study of 411 strains of M. tuberculosisfrom several countries, this system correctly identi-fied all 145 rifampicin-sensitive and 262 (98.5%)of 266 resistant strains (Traore et al., 2000). Asrifampicin resistance is usually accompanied byisoniazid resistance, this method is a good surro-gate indicator of multidrug resistance.

398 Mycobacterium

The sequencing of the genome of M. tuberculosisis facilitating the development of rapid moleculartests for other forms of drug resistance (Caws andDrobniewski, 2001) and suitable kits may soon beavailable.

REFERENCES

Bogard, M., Vincelette, J., Antinozzi, R. et al. (2001)Multicenter study of a commercial, automatedpolymerase chain reaction system for the rapiddetection of Mycobacterium tuberculosis inrespiratory specimens in routine clinical practice.European Journal of Clinical Microbiology and

Infectious Disease 20: 724–731.Canetti, G., Fox, W., Khomenko, P. et al. (1969)

Advances in techniques of testing mycobacterialdrug sensitivity and the use of sensitivity tests intuberculosis control programmes. Bulletin of the

World Health Organization 41: 21–43.Caws, M. and Drobniewski, F. A. (2001) Molecular

techniques in the diagnosis of Mycobacterium

tuberculosis and the detection of drug resistance.Annals of the New York Academy of Science 953:138–145.

Collins, C. H., Yates, M. D. and Grange, J. M. (1982)Subdivision of Mycobacterium tuberculosis intofive variants for epidemiological purposes: methodsand nomenclature. Journal of Hygiene (Cambridge)

89: 235–242.Collins, C. H., Grange, J. M. and Yates, M. D. (1985)

Mycobacterium marinum infections in man.Journal of Hygiene (Cambridge) 94: 135–149.

Collins, C. H., Grange, J. M. and Yates, M. D. (1997)Tuberculosis Bacteriology. Organization and

Practice, 2nd edn. London: Butterworths.Cutler, R. R., Wilson, P., Villarroel J. et al. (1997)

Evaluating current methods for determination ofthe susceptibility of myobacteria to pyrazinamide,conventional, radiometric BACTEC and twomethods of pyrazinamidase testing. Letters in

Applied Microbiology 24: 127–132.Drobniewski, F. A., Caws, M., Gibson, A. et al. (2001)

Modern laboratory diagnosis of tuberculosis.Lancet Infectious Diseases 3: 141–147.

Fletcher, H. (2001) Molecular epidemiology oftuberculosis: recent development and applications.Current Opinions on Pulmonary Medicine 7:154–159.

French, G. L., Chan, C. Y., Cheung, S. W. and Oo, K.T. (1987) Diagnosis of pulmonary tuberculosis bydetection of tuberculostearic acid in sputum byusing gas chromatography-mass spectrometry withselected ion monitoring. Journal of Infectious

Diseases 156: 356–362.Godfrey-Faussett, P. (1998) The use of DNA

fingerprinting in the epidemiology of tuberculosis.In: Davies, P. D. O. (ed.), Clinical Tuberculosius,

2nd edn. London: Chapman & Hall, pp 53–65.Grange, J. M., Yates, M. D. and de Kantor, I. N.

(1996) Guidelines for speciation within theMycobacterium tuberculosis complex.WHO/EMC/ZOO/96.4. Geneva: World HealthOrganization.

Heifets, L. (1986) Rapid automated methods (BACTECsystem) in clinical mycobacteriology. Seminars in

Respiratory Infections 1: 242–249.Jenkins, P. A. (1981) Lipid analysis for the

identification of mycobacteria: an appraisal.Reviews of Infectious Diseases 3: 862–866.

Kiehn, T. E., Edwards, F. F., Brannon, P. et al. (1985)Infections caused by Mycobacterium avium

complex in immunocompromised patients:diagnosis by blood culture and fecal examination,antimicrobial susceptibility tests, andmorphological and scroagglutinationcharacteristics. Journal of Clinical Microbiology

21: 168–173.Leat, J. L. and Marks, J. (1970) Improvements in drug

sensitivity tests on tubercle bacilli. Tubercle 51:68–73.

Marks, J. (1964) A ‘stepped pH’ technique for theestimation of pyrazinamide sensitivity. Tubercle

45: 47–50.Mitchison, D. A., Allen, B. J. and Manickayasagar, D.

(1987) Selective Kirchner medium for the cultureof specimens other than sputum for mycobacteria.Journal of Clinical Pathology 36: 1357–1361.

Scarparo, C., Piccoli, P., Rigon, A., Ruggiero, G.,Nista, D. and Piersimoni, C. (2001) Directidentification of mycobacteria from MB/BacT alert

References 399

3D bottles: comparative evaluation of twocommercial probe assays. Journal of Clinical

Microbiology 39: 3222–3227.Sisson, P. R., Freeman, R., Magee, J. G. and Lightfoot,

N. F. (1991) Differentiation between mycobacteriaof the Mycobacterium tuberculosis complex bypyrolysis mass spectrometry. Tubercle 72:206–209.

Traore, H., Fissette, K., Bastian, I., Devleeschouwer,M. and Portaels, F. (2000) Detection of rifampicinresistance in Mycobacterium tuberculosis isolatesfrom diverse countries by a commercial line probeassay as an initial indicator of multidrug resistance.

International Journal of Tuberculosis and Lung

Disease 4: 481–484.van Soolingen, D. (2001) Molecular epidemiology of

tuberculosis and other mycobacterial infections:main methodologies and achievements. Journal of

Internal Medicine 249: 1–26.Vestal, A. L. (1975) Procedures for the Isolation and

Identification of Mycobacteria, DHEW (CDC) 75-8230. Washington DC: Government Printing Office.

Yates, M. D. (1984) The differentiation andepidemiology of the tubercle bacilli and a studyinto the identification of other mycobacteria.Master of Philosophy thesis, University of London.

400 Mycobacterium

This group contains Gram-positive aerobes thatmay form filaments, branches and aerial mycelium.Some are partly acid fast.

ISOLATION

Culture pus directly or wash some of it to recovergranules as described on p. 404. Treat sputum bythe ‘soft’ method used for culturing mycobacteria(see p. 382). Culture on blood agar and on dupli-cate Lowenstein–Jensen (LJ) and Middlebrookmedium slopes. Incubate at 37°C for several days.Incubate one of the slopes at 45°C at which tem-perature nocardias may grow whereas other organ-isms are discouraged.

IDENTIFICATION

Nocardias grow on most standard bacteriologicalmedia. Media suitable for primary isolation include

brain–heart infusion agar, trypticase–soy agarenriched with blood Lowenstein–Jensen medium,and Sabouraud’s dextrose agar containing chlo-ramphenicol as a selective agent.

Subculture aerobic growth on blood agar andincubate at 37°C for 3–10 days. Colonies vary insize and may be flat or wrinkled, or sometimes ‘starshaped’. They may be pigmented (pink, red, cream,orange, brown or yellow) and are often coveredwith a whitish, downy or chalky aerial mycelium.Colonies of nocardias are usually on the surfacealthough they adhere firmly to the medium,whereas those of streptomyces may be embedded inthe medium.

Examine Gram- and Ziehl–Neelsen (ZN)-stainedfilms (minimum decolourization with the latter).Do slide culture (see below) and inoculate lysozymebroth: dissolve 20 mg lysozyme in 2 ml 50%ethanol in water and add 50 μl to 2 ml nutrientbroth at pH 6.8. Test for the hydrolysis of casein,xanthine, hypoxanthine and tyrosine (Table 46.1).The API ZYM system may be used to distinguish

46

Nocardia, Actinomadura, Streptomycesand Rhodococcus

Table 46.1 Nocardia, Rhodococcus, Actinomadura and Streptomyces spp.

Species Acid fast Aerial mycelium Lysozyme Hydrolysis of

Casein Xanthine Tyrosine

N. asteroides +a + R − − −N. brasiliensis +a + R + − +N. otitidis caviarum +a + R − + −Actinomadura − + S + v +Streptomyces − + S v v vRhodococcus − − S − − −

R, resistant; S, sensitive; v, variablea Rarely complete. Acid-fast elements may be few in number or absent

between nocardias, streptomyces and relatedorganisms.

Slide cultures

Melt a tube of malt agar, dilute with an equal vol-ume of distilled water and cool to 45°C. Drawabout 1 ml of this mixture into a Pasteur pipette,followed by a drop of a thin suspension of theorganisms. Run this over the surface of a slide in amoist chamber. No cover glass is needed. Examinedaily with a low-power microscope until amycelium is seen. Spores may be observed as pow-dery spots on the surface of the medium.

Partially acid-fast, Gram-positive mycelia that donot branch and that fragment early in slide culturesmay be tentatively identified as nocardias. Non-acid-fast, Gram-positive mycelia that brancheswith aerial mycelia and conidiophores abstricted inchains suggest streptomyces.

Nocardia species

The species of nocardia that are of medical impor-tance are Nocardia asteroides, N. brasiliensis andN. otitidis caviarum. Less common pathogensinclude N. nova and N. transvalensis. SomeNocardia-like organisms that are very rare causesof human disease are included in the generaGordona, Oerskovia, Rothia and Tsukamurella.

Acid-fast elements may be rare or occur in cul-tures at different times and are best observed onMiddlebrook media. Nocardias are resistant tolysozyme and may be differentiated by hydrolysisof casein, xanthine and tyrosine (Table 46. 1).Identification of other species and members ofrelated genera is not easy and is usually undertakenin reference laboratories by sequence analysis ofthe 16S ribosomal RNA (rRNA).

Nocardia asteroides This is the most common; it causes severe pul-monary infections, brain abscesses and occasion-ally cutaneous infections.

Nocardia brasiliensis This is mostly confined to North America and thesouthern hemisphere, and usually causes cutaneousinfections although it may be associated withsystemic disease.

Nocardia otitidis caviarum (N. caviae)This is a rare cause of human infections.

Actinomadura and Streptomyces species

Actinomadura madurae causes madura foot, andA. pelleteri and S. somaliensis cause mycetomas.They are difficult to distinguish bacteriologically,although A. madurae is said to hydrolyse aesculinwhereas S. somalensis does not. Unlike the sapro-phytic streptomyces, they do not produce acid fromlactose and xylose. Cell wall analysis or 16S rRNAsequence is necessary to sort out these and associ-ated species.

Rhodococcus species

Several species are found in plant material but noneis pathogenic for humans, although one, R. equi,formerly known as Corynebacterium equi, is apathogen of horses. Rhodococci are important onlyin that they may be confused with nocardias, actin-omaduras and S. somaliensis (see also p. 354).

For more information on the organisms in thischapter, see Collins et al. (1988), Gyles (1995),Hay (1995) and Grange (2002), and on these andother aerobic Gram-positive bacilli, see Schaal(1998), and Bortolussi and Kennedy (1999).

REFERENCES

Bortolussi, R. and Kennedy, W. (1999) Aerobic Gram-positive bacilli. In: Armstrong, D. and Cohen, J.(eds), Infectious Diseases, Vol. 2. London: Mosby,Chapter 8, Sections 15, pp. 1–20.

Collins, C. H., Uttley, A. H. C. and Yates, M. D.(1988) Presumptive identification of nocardias in a

402 Nocardia, Actinomadura, Streptomyces and Rhodococcus

clinical laboratory. Journal of Applied Bacteriology

65: 55–59.Grange, J. M. (2002). Actinomyces and nocardia. In:

Greenwood, D., Slack, R. and Peutherer, J. (eds),Medical Microbiology, 16th edn. Edinburgh:Churchill Livingstone, pp. 221–224.

Gyles, C. L. (1995) Nocardia; actinomyces;dermatophilus. In: Gyles, C. L. and Thoen, C. O.(eds), Pathogenesis of Bacterial Infections in

Animals. Ames: Iowa State University Press, pp.124–132.

Hay, R. J. (1995) Nocardiosis. In: Weatherall, D. J.,Ledingham, J. G. G. and Warrell, D. A. (eds),Oxford Textbook of Medicine, 3rd edn, Oxford:Oxford University Press, pp. 686–687.

Schaal, K. P. (1998) Actinomycosis, actinobacillosisand related diseases. In: Collier, L., Balows, A. andSussman, M. (eds) Topley and Wilson’s

Microbiology and Microbial Infections, 9th edn.Vol. 3, Bacterial Infections. London: Arnold, pp.777–798.

References 403

This group contains Gram-positive, aerobic, anaer-obic or microaerophilic organisms that tend tobranch. Actinomyces spp. require CO2 for growthand are catalase negative; Propionibacterium sp.does not require CO2 and is catalase positive;Bifidobacterium sp. does not require CO2 and iscatalase negative. Species of medical and veterinaryimportance occur in pus and discharges as coloniesor granules.

A recently described actinomycete, Tropherymawhippelii, is the cause of Whipple’s disease, a mul-tisystem disease presenting with diarrhoea andmalabsorption, often with arthralgia and fever andsometimes with central nervous system (CNS)involvement.

ACTINOMYCES

Isolation

Wash pus gently with saline in a sterile bottle orPetri dish. Look for ‘sulphur granules’, about thesize of a pinhead or smaller. Aspirate granules witha Pasteur pipette and crush one between two slidesand stain by the Gram method. Look for Gram-positive mycelia in an amorphous matrix sur-rounded by radiating large Gram-negative,club-like structures. The club forms are acid fast onZiehl–Neelsen staining, modified by use of 1% sul-phuric acid for decolourization. Sulphur granulesand mycelia in tissue sections are also identifiableby use of fluorescein-conjugated specific antisera.

Crush granules with a sterile glass rod in a smallsterile tube containing a drop of broth. Inoculate

blood agar plates, brain heart infusion agar andenriched thioglycollate medium. These may bemade selective for Gram-positive non spore-bearersby adding 30 μg/ml nalidixic acid and 10 μg/mlmetronidazole. Do cultures in triplicate and incu-bate:

• anaerobically plus 5% carbon dioxide• in 5% carbon dioxide in air• aerobically

doing all at 37°C for 2–7 days.Subculture any growth in the broth media to solid

media and incubate under the same conditions. Lookfor colonies resembling ‘spiders’ or ‘molar teeth’.

Identification

Do catalase tests on colonies from anaerobic ormicroaerophilic cultures that show colonies ofGram-positive coryneform or filamentous organ-isms.

Actinomyces are catalase negative but corynebac-teria and bifidobacteria are usually catalase positive.It is often difficult to distinguish actinomyces frombifidobacteria and propionibacteria by simple tests.Analysis of the metabolic products of carbohydratefermentation by gas–liquid chromatography (GLC)may be necessary. The spot test for indole pro-duction applied to colonies is useful; Actinomycesspp. are negative, unlike Propionibacterium spp.

Subculture for nitrate reduction test and test foracid production from mannitol, xylose andraffinose, and aesculin hydrolysis (Table 47.1). TheAPI ZYM system may be useful in distinguishingbetween actinomyces and related genera.

47

Actinomyces, Propionibacterium, Bifidobacteriumand Tropheryma

Actinomyces 405

Species of Actinomyces

Actinomyces bovis Colonies at 48 h on blood agar are pinpoint sizedand smooth, later becoming white and shining withan entire edge. Spider and molar tooth colonies arerare. In thioglycollate broth, growth is usually dif-fuse but occasionally there are colonies that resem-ble breadcrumbs. Microscopically the organismsare coryneform, and rarely branching.

Nitrates are not reduced, and acid is not producedfrom mannitol, xylose or raffinose. Starch ishydrolysed; aesculin is not hydrolysed.

This is a pathogen of animals, usually of bovines,causing lumpy jaw.

Actinomyces israelii Colonies at 48 h on blood agar are microscopic,with a spider appearance, later becoming white andlobulated with the appearance of molar teeth. Inthioglycollate broth, there are distinct colonieswith a diffuse surface growth and a clear medium.The colonies do not break when the medium isshaken. Microscopically, the organisms are coryne-form, with branching and filamentous forms.

Nitrates may be reduced, and acid is producedfrom xylose and raffinose; aesculin is hydrolysedbut not starch. Its identity may be confirmed bystaining with specific fluorescent antisera

This species causes human actinomycosis andmay be responsible for pelvic infections in womenfitted with intrauterine contraceptive devices.

Actinomyces naeslundii Colonies at 48 h on blood agar are similar to thoseof A. israelii; spider forms are common, but notmolar tooth forms. In thioglycollate broth, growthis diffuse and the medium is turbid.Microscopically the organisms are irregular andbranched, with mycelial and diphtheroid forms.

Nitrates are reduced, and acid is produced fromraffinose; aesculin is hydrolysed; starch hydrolysisis variable. This is a facultative aerobe.

It is not known to be a human pathogen but hasbeen found in human material.

Actinomyces odontolyticus Colonies at 48 h on blood agar are 1–2 mm in diam-eter and grey, but they may develop a deep-reddishcolour after further incubation. CO2 is required forgrowth. The organisms are coryneform althoughbranching is rare. Nitrate is reduced; acid is rarelyproduced (except occasionally from xylose); starchand aesculin hydrolysis is variable.

The normal habitat is the human mouth but theorganisms have been isolated from the tear ducts.

Actinomyces meyeri Colonies at 48 h on blood agar are pinpoint sized,greyish white and rough. CO2 is required for bothaerobic and anaerobic growth. The organisms areshort and coryneform but branching is rare.Nitrate is not usually reduced, and acid is producedfrom xylose and raffinose; starch and aesculin arenot hydrolysed.

Table 47.1 Actinomyces and Bifidobacterium species

Species Nitrate reduction Acid from Hydrolysis of

Mannitol Xylose Raffinose Starch Aesculin

A. bovis − − − − + −A. israelii v v + + − +A. naeslundii + − v + v +A. odontolyticus + − v − v vA. meyeri − − + + − −A. pyogenes v − − − v −B. eriksonii − + + + − v

The normal habitat is the human mouth but theorganism has been isolated from abscesses.

Actinomyces pyogenes This is involved in suppurative lesions and mastitisin domestic and wild animals. On blood agar it pro-duces pinpoint α-haemolytic colonies at 36–48 h.For more information about actinomyces, see Gyles(1995); Schaal and Lee (1995); and Schaal (1999).

PROPIONIBACTERIUM

These are pleomorphic coryneforms varying fromcoccoid to branched forms. They are aerobic butmay be aerotolerant, non-motile and grow poorlyin the absence of carbohydrates. They are catalasepositive and some strains may liquefy gelatin. Theoptimum temperature is 30°C. There are severalspecies of interest to food microbiologists. Theyoccur naturally in the bovine stomach and hence inrennet, and are responsible for flavours and ‘eyes’in Swiss cheese. A method for presumptive identifi-cation is given on p. 241. It is difficult to identifyspecies.

There is one species of medical interest: P. acnes.

Propionibacterium acnes

The bacilli are small, almost coccoid, or about2.0 × 0.5 μm, and may show unstained bands. Thebest growth is obtained anaerobically. The colonieson blood agar are either small, flat, grey–white andbuttery, or larger, heaped up and more granular.Both colony forms are β-haemolytic. Acid is notusually produced. The spot test for indole pro-duction is positive. It is nitrate reduction negative,catalase positive and liquefies gelatin. Slide aggluti-nation is useful.

It is a commensal on human skin, in hair folliclesand in sweat glands. It appears to be involved in thepathogenesis of acne. Oral and systemic infectionshave been suspected, and it is an occasional causeof delayed-onset postoperative endophthalmitis.

This genus is described by Cummings andJohnson (1986).

BIFIDOBACTERIUM

Bifidobacteria are small Gram-positive rods resem-bling coryneforms, which predominate in thefaeces of breast-fed infants, but they are alsocommensals in the adult bowels, mouth andvagina. They are anaerobic and may need to bedifferentiated from anaerobic corynebacteria.Bifidobacteria are catalase negative and nitratereduction negative, and do not produce gas fromglucose. CO2 is not required but it does improvegrowth.

There is one pathogen, B. eriksonii, formerlyActinomyces eriksonii, associated with mixedinfections in the upper respiratory tract.

For information about bifidobacteria, seeSeardovi (1986).

TROPHERYMA WHIPPELII

Gene amplification techniques have shown that aGram-positive bacillus detectable microscopicallyin intestinal tissue from patients with Whipple’sdisease is an actinomycete with a unique 16S rRNAsequence. This organism, Tropheryma whippelii,does not grow on conventional culture media, butreplicates within cells of the human fibroblast(HEL) line. Confirmation of the diagnosis ofWhipple’s disease may be made by polymerasechain reaction (PCR) examination of intestinalbiopsies and specimens from other tissues involvedin the disease process.

For further details, see Veitch and Farthing(1999) and Puechal (2002).

REFERENCES

Cummings, C. S. and Johnson, J. L. (1986) The genusPropionibacterium. In: Starr, M.P., Stolp, H. andTroper, H.C (eds), The Prokaryotes: A handbook

406 Actinomyces, Propionibacterium, Bifidobacterium and Tropheryma

References 407

on habitats, isolation and identification of bacteria,

Vol. 2. New York: Springer, p. 1864 .Gyles, C. L. (1995) Nocardia; actinomyces;

dermatophilus. In: Gyles, C.L. and Thoen, C.O.(eds), Pathogenesis of Bacterial Infections in

Animals. Ames, Iowa: Iowa State University Press,pp 124–132

Puechal, X. (2002) Whipple’s disease. Joint, Bone,

Spine 69: 133–140.Seardovi, V. (1986) The genus Bifidobacterium. In:

Sneath, P.H.A., Mair, N.S. and Holt, J.G. (eds),Bergey’s Manual of Systematic Bacteriology, Vol.2. Baltimore: Williams & Wilkins, p. 1418.

Schaal, K. P. (1999) Actinomycosis, actinobacillosisand related diseases. In: Collier, L., Balows, A. andSussman, M. (eds), Topley and Wilson’s

Microbiology and Microbial Infections, 9th edn.Vol. 3, Bacterial Infections, CD-ROM. London:Arnold, Chapter 39.

Schaal, K. P. and Lee, H-J. (1995) Actinomyceteinfections in humans – a review. Gene 115:201–211.

Veitch, A. M. and Farthing, M. J. G. (1999) Whipple’sDisease. In: Armstrong, D. and Cohen, J. (eds),Infectious Diseases. London: Mosby, pp.2.36.1–2.36.2.4.

These two organisms are considered together herealthough they are not related. Bartonella hasaroused interest recently as a result of taxonomicchanges. The significance of Mobiluncus in thevagina is not well documented.

BARTONELLA

This genus contains at least three species of small,Gram-negative bacilli that are oxidase negative andfastidious in their growth requirements.

Isolation

Perform blood cultures using one of the highlyenriched media (automated systems are usuallysatisfactory, but the organisms may not produceenough CO2 for detection by some of them).Incubate duplicate cultures at 25–30°C and35–37°C for at least 4 weeks.

Subculture to lysed blood agar (5–10% rabbitblood). Incubate cultures for at least 14 days in ahigh humidity and under 5–10% CO2. Coloniesmay be dry, of ‘cauliflower’ appearance, or small,circular and entire with pitting of the medium.

Identification is difficult and it is best to sendspecimens or cultures to a reference laboratory.

Bartonella bacilliformisThis causes Oroya fever (Carrion fever; bartonel-losis) and is transmitted by the sandfly(Phlebotomus spp.).

It is best observed in Giemsa-stained blood films

and the bacilli are seen in and on red blood cells.They are pleomorphic, coccobacilli, often in chainsor arranged like Chinese letters.

Bartonella henseliaeFormerly in the genus Rochalimaea this is nowrecognized as the agent of cat-scratch fever (seeWindsor, 2001). It has also been isolated fromimmunocompromised and other patients withbacteraemia and fever.

Bartonella quintanaThis is the agent of trench fever, suffered bymilitary personnel. It is spread by the human bodylouse.

For more information about Bartonella, seeWelch and Slater (1995).

MOBILUNCUS

This old species, revived in 1980, is now of interest.It is found in vaginal secretions along with otheragents of vaginitis but its significance is not fullydocumented.

Isolation

Culture vaginal fluids on enriched media contain-ing horse or rabbit blood and incubate at 35–37°Cfor at least 5 days. Colonies are entire, convex,smooth and translucent. It grows at pH 12.0(Pålson et al., 1986).

48

Bartonella and Mobiluncus

References 409

REFERENCES

Pålson, C., Hallén, A. and Forsum, U. (1986) Improvedyield of Mobiluncus species from clinical specimensafter alkaline treatment. Acta Pathologia,

Microbiologia et Immunologia Section B 94:113–116.

Welch, D. F. and Slater, L. N. (1995) Bartonella. In:

Murray, P. R., Barron E. J., Pfaller, F. A. et al.(eds), Manual of Clinical Microbiology, 6th edn.Washington DC: American Society forMicrobiology.

Windsor, J. J. (2001) Cat scratch fever: epidemiology,aetiology and treatment. British Journal of

Biomedical Science 58: 101–110.

Three genera that are of medical importance areconsidered briefly here: Borrelia, Treponema andLeptospira.

BORRELIA

There are at least 19 species, classified by theirarthropod vectors. Only three are consideredbriefly here.

Borrelia duttonii and B. recurrentis cause relaps-ing fever, transmitted by lice and ticks. B. burgdor-feri is responsible for Lyme disease (inflammatoryarthropathy), first described in Old Lyme,Connecticut, USA and now known in Europe(Burgdorfer, 1984, 1985). It is a tick-borne zoono-sis and small woodland rodents and wild deer arereservoirs. Infection is also transmitted to domesti-cated and farm animals

Identification

Take blood during a febrile period. For the relaps-ing fever species examine wet preparations by darkfield using a high-power dry objective. Stain thickand thin films with Giemsa stain.

Fix smears in 10% methyl alcohol for 30 s. Stainwith 1 part Giemsa stock stain and 49 partsSorensen’s buffer at pH 7 in a Coplin jar for45 min. Wash off with Sorensen’s buffer. Dry in airand examine. Borrelias appear as shallow, coarse,irregular, highly motile coils 0.25–0.5 × 8–16 μm.

In cases of suspected Lyme disease diagnosis maybe made by fluorescence antibody methods

(Technicon kit), enzyme-linked immunosorbentassay (ELISA) or silver staining of biopsies.

It is not usual practice to attempt culture of bor-relias from ticks and host animals, but methods aregiven by Johnson (1999).

TREPONEMA

There are at least 14 species. In medicine the mostimportant are T. pallidum, T. pertenue, T. cara-teum and T. vincentii.

Identification

In suspected syphilisExamine exudate (free from blood and antiseptics)from lesion by dark-field or fluorescencemicroscopy. For detailed methods, see Sequira(1987).

Treponema pallidumThis is the causative organism of syphilis. It formstightly wound slender coils, 0.1–0.2 × 6–20 μm,with pointed ends, each of which has three axialfibrils. They are sluggishly motile with driftingflexuous movements.

In suspected Vincent’s anginaStain smears with dilute carbol–fuchsin. Largenumbers of spirochaetes are seen together withfusobacteria.

49

Spirochaetes

Treponema vincentiiThis is found in cases of Vincent’s angina (ulcera-tive lesions of the mouth or genitals) and pul-monary infections. It is a loosely wound singlecontoured spirochaete 0.2–0.6 × 7–18 μm.

Other treponemes

Treponema pertenueThis organism causes yaws and is principally trans-mitted by direct contact. It is morphologicallyindistinguishable from T. pallidum. Laboratorytests are unhelpful.

Treponema carateumThis causes pinta. The mode of spread is, in com-mon with yaws, by direct contact. It is morpholog-ically indistinguishable from T. pallidum.Diagnosis is by silver impregnation of tissue. Forfurther information, see Penn (1999).

LEPTOSPIRA

Identification is important only for epidemiologicalpurposes. Leptospira interrogans (23 serogroupsand about 200 serotypes) is primarily an animalparasite, but in humans causes an acute febrile ill-ness with or without jaundice, conjunctivitis andmeningitis. The more severe manifestations areknown as Weil’s disease. Animal hosts includerodents, dogs, horses, cattle and pigs. L. biflexa isnon-pathogenic.


Examine blood during the first week of illness andurine thereafter. Add 9 parts blood to 1 part 1%sodium oxalate in phosphate buffer at pH 8.1 andcentrifuge 15 min at 1500 rev./min. Examine clearplasma under dark-field microscopy using low-power magnification. If this is negative, centrifuge

the remainder of the plasma at 10 000 rev./min for20 min and examine the sediment. Direct filmsrarely show leptospiras either in dark-field orGiemsa-stained preparations. Centrifuge urineand examine deposit by dark field within15 min.

MorphologyLeptospiras are short, fine, closely wound spirals,0.25 × 6–20 μm, resembling a string of beads. Theends are bent at right angles to the main body toform hooks.

CultureThe methods given here are adapted from those ofWaitkins (1985). Add two drops of fresh blood,cerebrospinal fluid (CSF) or urine (at pH 8) to 5 mlEMJ/5FU medium (see p. 75) and make five serialdilutions in the same medium to dilute out anti-body. Incubate at 30°C. Examine daily for 1 week,then weekly for several weeks. Use dark-field,phase contrast or fluorescence microscopy.

IdentificationIf leptospiras are seen. subculture to EMJHmedium (se p. 75) and incubate at 30 and 13°C.Subculture also to EMJ medium containing 225 mgazoguanine/l.

Leptospira interrogans grows at 30°C, not at13°C and not in azoguanine medium. L. biflexagrows at 13 and 30°C and also in azoguaninemedium.

Serological diagnosis

Macroslide agglutinations may be done withpatients’ sera and (commercial) genus-specific anti-gen, but further agglutination tests are best done inreference laboratories.

There is a rapid test kit (Leptese: BradsureBiologicals). For more information about lep-tospires, culture, serology and epidemiology, seeWaitkins (1985) and Faine (1999).

Leptospira 411

412 Spirochaetes

REFERENCES

Burgdorfer, W. (1984) Discovery of the Lymespirochaete and its relationship to tick vectors.Yale Journal of Biology and Medicine 57:165–168.

Burgdorfer, W. (1985) Borrelia. In: Lennette, E. H.,Balows, A., Hauser, W. J. et al. (eds), Manual of

Clinical Microbiology, 4th edn. Washington DC:Association of American Microbiologists, pp.154–175.

Faine, S. (1999) Leptospira. In: Collier, L., Balows, A.and Sussman, M. (eds), Topley and Wilson’s


Johnson, R. C. (1999). Borrelia. In: Collier, L., Balows,A. and Sussman, M. (eds), Topley and Wilson’s


Penn, C. W. (1999). Treponema. In: Collier, L.,Balows, A. and Sussman, M. (eds), Topley and

Wilson’s Microbiology and Microbial Infections,9th edn. Vol. 2, Systematic Bacteriology, CD-ROM. London: Arnold, Chapter 55.

Sequira, P. J. L. (1987) Syphilis. In: Jephcott, A.E. (ed.),Sexually Transmitted Diseases. London: PublicHealth Laboratory Service, pp. 6–22.

Waitkins, S. A. (1985) Leptospiras and leptospirosis.In: Collins, C.H. and Grange, J.M. (eds), Isolation

and Identification of Micro-organisms of Medical

and Veterinary Importance. Society for AppliedBacteriology Technical Series No. 21, London:Academic Press, pp. 251–296.

Yeasts are fungi, the main growth form of which isunicellular; they usually replicate by budding.Many can also grow in the hyphal form and thedistinction between a yeast and a mould is one ofconvention only. Some yeast-like fungi such asAcremonium and Geotrichum spp. and the ‘blackyeasts’ Exophiala, Aureobasidium and Phialo-phora spp. are traditionally excluded from texts onyeasts.

The yeasts are not a natural group. Many areAscomycotina, a few are Basidiomycotina and

others are asexual forms of these groups (Deutero-mycotina or Fungi Imperfecti).

IDENTIFICATION

The methods given in Chapter 9 yield informationabout morphology, sugar fermentation and assimi-lation of carbon and nitrogen sources. The mor-phological key (Table 50.1) allows most yeastsisolated in clinical and food laboratories to be

50

Yeasts

Table 50.1 Key to identification of genera of yeasts

Cells reproducing by fission, not budding Schizosaccharomyces

Budding cells only (corn meal and coverslip preparation); no ascospores1. Cells small (2–3 μm), bottle shaped, with broad base to daughter bud Malassezia2. Cells oval with prominent lateral ‘spur’; colony pinkish, depositing mirror image of colony Sporobolomyces

on lid of inverted Petri dish3. Cells not as in 1 or 2:

(a) urease negative Asexual forms of Candida Saccharomycesetc.

3. (b) urease positive; colonies pink/red; cells large; encapsulated Rhodotorula(c) urease positive; colonies white or cream; cells large; encapsulated Cryptococcus

Budding cells, some with ascospores1. Ascospores liberated from parent cells, kidney-shaped or elongate Kluyveromyces2. Ascospores liberated, round with or without disc-like flange; nitrate assimilated Pichia3. Ascospores as in 2; nitrate not assimilated Pichia4. Ascospores retained in parent cells, round, smooth Saccharomyces5. Ascospores retained, round, warty and ridged Debaromyces

Budding cells borne on pseudomycelium; no ascospores Candida (most species)

Budding cells born on pseudomycelium which grows out to true mycelium after 3–4 days1. Chlamydospores present C. albicans/

C. dubliniensis2. No chlamydospores C. tropicalis

Extensive true mycelium, fragmenting to form arthrospores1. Budding on short side branches Trichosporon or

Blastoschizomyces2. No budding Geotrichum

identified to genus level. Specific identification maythen be made by reference to Table 50.2 and Figure50.1 (see also Campbell et al., 1996).

Candida

Candida species are commensals in the human gut.The principal species of medical importance isCandida albicans, which can cause superficial,mucosal and systemic infections, particularly inimmunocompromised patients.

Candida parapsilosis is a normal skin commensaland may infect nails. Systemic infections are oftenrelated to the use of parenteral nutrition and havebeen particularly noted in paediatric intensive careunits. It is also a cause of candida endocarditis.

The other species most commonly implicated inhuman disease include C. glabrata, C. tropicalis,C. krusei, C. famata and C. lusitaniae. The increas-ing use of azole antifungal therapy in both prophyl-axis and treatment may have contributed to theirincreasing incidence. C. krusei is innately resistantto fluconazole and C. glabrata is less susceptible tofluconazole and other azole drugs than C. albicans.Identification to species level is therefore an impor-

tant consideration in the choice of treatment ofthese infections.

All Candida spp. produce creamy, white, smoothcolonies on malt or peptone agar after 2 days at30–37°C, except C. krusei, which has a ground-glass appearance (see Table 50.2).

Cryptococcus

Cryptococcus neoformans causes meningitis (whichmay be fulminant or slowly progressive) and sub-cutaneous or deep granulomata. It is mainly foundin HIV-infected patients. Two varieties are found:C. neoformans var. neoformans, which has a globaldistribution, and C. neoformans var. gattii, foundmainly in Australia and central Africa. As with otherencapsulated yeasts (Rhodotorula, Trichosporonspp.), these are the asexual forms of Basidiomycetes,although the sexual forms are not normally encoun-tered.

Colonies on Sabouraud’s agar incubated 24–48 hat 37°C are large, white and butyrous, and may‘flow’ down a vertical slope.

The most sensitive method of diagnosis of C. neo-formans infection is the detection of the capsular

414 Yeasts

Table 50.2 Biochemical differentiation of some common yeasts

Species Fermentation of Assimilation ofNitrate

glu ma su la glu ma su la mn ra ce er reductase

Candida albicans + + − − + + + − + − − − −C. tropicalis + + + − + + + − + − + − −C. kefyr + − + + + − + + + + + − −C. parapsilosis + − − − + + + − + − − − −C. guillermondii + − + − + + + − + + + − −C. krusei + − − − + − − − − − − − −C. glabrata + − − − + − − − − − − − −Cryptococcus neoformans − − − − + + + − + +w +w ± −C. albidus − − − − + + + ± + +w + − +C. laurentii − − − − + + + + − − − − −Trichosporon beigelii − − − − + + + + ± ± ± ± −Blastoschizomyces capitus − − − − + − − − − − − − −Saccharomyces cerevisiae + + + − + + + − ± ± − − −

ce, cellobiose; er, erythritol; glu, glucose; la, lactose; ma, maltose; mn, mannitol; NO3, nitrate; ra, raffinose; su, sucrose; w, weak; + , gas produced or growth occurs; − , no gas produced or no growth occurs

polysaccharide antigen by latex agglutination usingserum or cerebrospinal fluid. Commercial kits areavailable for this purpose and may also be used tomonitor the course of infection following treatmentby titration of the samples.

In advanced infections agglutination tests for anti-body production may be negative because the cap-sular material is poorly metabolized by the body,and the accumulation of it causes a high-doseimmunological tolerance.

Other species of Cryptococcus such as C. albidusand C. laurentii are sometimes found contaminat-ing solutions but are rarely pathogenic as they donot grow well above 30°C.

Rhodotorula

Members of this genus are frequent skin contami-nants. They form red or orange colonies on malt or

Identification 415

Figure 50.1 Yeasts

Budding yeasts

Mycelium withchlamydospores andblastospores

Chlamydospores

Myceliumwitharthrospores

Fission yeasts

Pseudomycelium withblastospores

Asci withascospores

Diversity ofascospore shape

Saccharomyces

Zygosaccharomyces

Myceliumwith asci andbudding cells

Sterigma

Ballistospore formation

glucose peptone agar after 2 days’ incubation at30°C. Morphologically they resemble cryptococciand may be capsulated.

Sporobolomyces

These yeasts live on leaf surfaces and reproduce byballistospores, which are ejected into the air. Thesalmon-pink colonies of S. roseus produce mirrorimages on the lids of Petri dishes if left undisturbed.They are of no clinical significance.

Trichosporon

Trichosporon beigelli is the one most frequentlyisolated in clinical work. It is a skin contaminantbut occasionally causes deep infections in immuno-compromised patients. It is also the cause of ‘whitepiedra’, in which masses of yeasts grow on theouter surfaces of axillary and other hair that is keptpermanently moist. T. beigelli actually encom-passes a group of species including T. asahii.T. cutaneum, T. inkin, T. mucoides and T. ovoides.

Blastoschizomyces

Blastoschizomyce capitatus colonies are whitish,wrinkled, tough, and often slightly ‘hairy’ on thesurface, as a result of the presence of aerial hyphae.It sometimes causes systemic infection in immuno-compromised patients.

Pneumocystis

Pneumocystis carinii was originally thought to be aprotozoan but was later identified as a fungus onthe basis of molecular studies, which place itbetween the Basidiomycota and Ascomycota. Morerecently it has been attributed to the Archiasco-mycetes together with Schizosaccharomyces and is

described here in the yeast chapter, although it hasnot been classified as a yeast.

Pneumocystis carinii has emerged as a commoncause of pulmonary infection in AIDS patients. Theorganism cannot be cultivated on routine labora-tory media. Diagnosis is therefore based on poly-merase chain reaction (PCR) detection orimmunofluorescence staining of the thick-walledcysts in samples of broncheoalveolar lavage.

Malassezia species

Malassezia spp. are lipophilic yeasts characterizedby monopolar budding from a broad base. Theyare common skin commensals but may cause dis-seminated infection in patients, particularlyneonates, receiving lipid infusions. Overgrowth ofthe fungus may cause seborrhoeic dermatitis.

Seven species have been described: M. furfur,M. globosa, M. pachydermatis, M. obtusa,M. restricta, M. slooffiae and M. sympodialis. OnlyM. pachydermatis is capable of growth on labora-tory culture media without the addition of lipids.

Pityriasis versicolor is a mild skin infection of thechest or back characterized by depigmentation orhyperpigmentation. M. furfur is the name usuallygiven to the organism in this condition, althoughsome of the other species listed may also beinvolved. Diagnosis of pityriasis versicolor is bymicroscopic examination of infected skin scrap-ings, which show clumps of yeast cells interspersedby short hyphal fragments.

YEASTS OF IMPORTANCE INFOOD PROCESSING ANDSPOILAGE

Saccharomyces and similar yeasts

This group contains the brewing (‘pitching’) andbaking yeasts. S. cerevisiae is the ‘top yeast’, used inmaking beer, and S. carlsbergensis the ‘bottomyeast’, used in making lager. There are many

416 Yeasts

special strains and variants used for particularpurposes.

Saccharomyces pastorianusThis is a ‘bottom yeast’ with long sausage-shaped cells, which produces unpleasant flavours inbeer.

Saccharomyces cerevisiaeThis is sometimes isolated as a non-significantorganism from faecal and oral samples but can be acause of genital or systemic infection.

ZygosaccharomycesZygosaccharomyces rouxii and Z. mellis are‘osmophilic yeasts’ which will grow in concen-trated sugar solution (compare with aspergilli ofthe glaucus group) and spoil honey and jam.

Zygosaccharomyces baillii can grow at low pH inthe presence of some preservatives and has becomea major problem in some areas of the food andbeverage industry.

Pichia spp.These occur as contaminants in alcoholic liquors.They can use alcohol as a source of carbon, and area nuisance in the fermentation industry. They growas a dry pellicle on the surface of the substrate.Some species occur as the ‘flor’ in sherry and in cer-

tain French wines, to which they give a distinctiveflavour. They also cause pickle spoilage, particu-larly in low-salt (10–15%) brines.

Debaromyces spp.These are also ‘pellicle-forming’ yeasts. They arefound in animal products such as cheese, glue andrennet, and are also responsible for spoilage inhigh-salt (20–25%) pickle brines.

Apiculate yeastsThese lemon-shaped yeasts are common contami-nants in food. Perfect forms are placed in the genusHanseniaspora and imperfect forms in the genusKloeckera.

Yeasts are considered in detail by Kurtzman andFell (1998).

REFERENCES

Campbell, C. K., Johnson, E. M., Philpot, C. M. andWarnock, D. W. (1996) Identification of

Pathogenic Fungi. London: Public HealthLaboratory Service

Kurtzman, C. P. and Fell, J. W. (1998) The Yeasts, a

Taxonomic Study. Amsterdam: Elsevier Science.

References 417

Many common moulds may occur as air-borne con-taminants on ordinary culture media if care is nottaken. Most of them are saprophytes, causingspoilage of food and other commodities; others areplant pathogens. Some, e.g. Aspergillus,are describedin this chapter although they are also recognizedcauses of human disease, especially in immunosup-pressed patients, or are of public health importance

because they produce mycotoxins (e.g. Penicillium).Each strain of a mould isolated from human mate-rial must therefore be assessed individually and somereliance must be placed on the observation of hyphaeby direct microscopy. The preliminary identificationguide, based on colony colour and texture (Table51.1), includes genera in which some of the pathogensdescribed in Chapter 52 occur.

ALTERNARIA AND ULOCLADIUM

These are two large genera of plant pathogens andsaprophytes. Without knowledge of the host plantspeciation is very difficult (see Ellis, 1971). Rarecases of subcutaneous granulomata in humans andcats have been reported. Alternaria species havecaused cutaneous infection on the faces of cats.

Colonies on Saboraud’s medium after incubationat 20–25°C for 3–4 days are high, densely fluffymats, white at first, becoming dark-grey, deepest incolour in the centre. On some media the wholecolony is black, with less aerial growth. Spores arelarge, brown pigmented, club shaped and multicel-lular, with some septa longitudinal or oblique. InAlternaria spp. (Figure 51.1) the spores are inchains mostly with an apical beak; in Ulocladiumspp. they are single or only occasionally joinedtogether. A tape impression mount will demon-strate this difference. Alternaria species are some-times slow to produce spores.

Incubation at room temperature in daylight canencourage sporulation. Ulocladium chartarum maybe responsible for discoloration of paintwork andwall paper on damp walls. Some species of Alternariamay produce mycotoxins in food plant products.

51

Common moulds

Table 51.1 Colour guide to mould cultures

Black or dark brown mycelium, spores or bothAlternaria Cladosporiuma

Arthrinium Exophialaa

Aureobasidium Fonsecaeaa

Botrytis Madurellaa

Chaetomium Phialophoraa

Aspergillus (niger) Sporothrixa

Stachybotrys

Some shade of green predominantTrichoderma Aspergillus

(flavus/parasiticus)Chaetomium Penicillium

Some shade of red predominantMonascus Trichophytona

Paecilomyces (lilacinus) AcremoniumAureobasidium Fusarium (some strains)a

Colonies white or creamChrysosporium Coccidioidesa

Geomyces Blastomycesa

Dermatophytesa Mucoraceous speciesHistoplasmaa Fusariuma

Colonies sandy-brown to khakiPaecilomyces (variotii) Epidermophyton floccosuma

ScopulariopsisAspergillus terreus

a Described in Chapter 52

Alternaria and Ulocladium 419

Figure 51.1 Common moulds (bar = 10 μm)

ARTHRINIUM

These are common contaminants of no medicalimportance. Most species are from plant material,on which they look quite unlike the in vitrogrowth.

The growth on laboratory media is dense, vigor-ous and at first pure white in colour. The centres ofolder cultures become grey because of the abun-dance of spores, which are unicellular, roundedand black, often with a conspicuous germ slit (Ellis,1971).

CHAETOMIUM

These are saprophytes on plant material. Some aretroublesome pests of paper and cellulosic materialscausing discoloration and degradation They haveno medical implications, although some species areknown to produce toxic metabolites.

Colonies on ordinary laboratory media are flatwith low, often sparse aerial growth, with black orgreenish discrete bodies standing on the agar sur-face. Low power examination shows that thesebodies are covered with long black spines (Figure51.1). Under the high power these are seen to bestraight, spiral or irregularly branched according tospecies. Large numbers of globose asci are pro-duced inside the perithecium, each containing darkbrown, ovoid ascospores. In Chaetonium the ascuswalls rapidly break down, releasing the spores andgiving the impression that the perithecium itself isfull of loose spores.

CLADOSPORIUM

There are many species, most of which are of noclinical significance. Long chains of conidia areproduced by acropetal budding and may branchwhere a conidium develops two buds (Figure 51.1).They differ from the pathogenic Cladophialophoraspp., which have longer, rarely branching chains ofsmooth, slender and thin-walled conidia (see Figure51.1).

AUREOBASIDIUM

This mould occurs on damp cellulosic materialsand grows on painted surfaces. Colonies on ordi-nary laboratory media are at first yeast like, oftenmucoid, with pink, white and black areas. In timethe black areas increase in size and the colonybecomes drier and rough surfaced. There may beaerial mycelia. They are often referred to as ‘blackyeasts’.

Microscopy shows a bizarre mixture of oval tolong budding yeasts and pigmented, irregularlyshaped, hyphal cells. Under low power the edge ofthe colony shows yeasts clustered in round massesalong the submerged hyphae. This is best seen in aslide culture or needle mount (see Figure 51.1).A. pullulans is the most common of the saprophyticspecies in the group.

BOTRYTIS

This is a group of plant pathogens, of which thecommon B. cinerea has a very wide host range andis also a successful saprophyte. It is well known asthe grey mould of fruit such as strawberries.

Growth on ordinary laboratory media is vigor-ous, producing a high, grey turf which developsdiscrete black bodies (sclerotia) at the agar surface,especially at the edge of the culture vessel. Thelarge, colourless unicellular spores are produced ingrape-like clusters on long, robust black conidio-phores (see Figure 51.1).

PHOMA

This is a representative of a large number of pycni-dial genera, all plant parasites, any of which maybe encountered as contaminants. It is included herebecause it is probably one of the most common andmany others were once classified with it.

Some species produce low, flat colonies on labo-ratory media with areas of black mixed with pink(spore masses). Others may give dense, fluffygrowths with black or grey shading. Microscopically

420 Common moulds

Scopulariopsis 421

they all show pycnidia – spherical structures con-taining large numbers of small conidia, oftenreleased in a wet, worm-like mass through a roundostiole. These are best seen by examining the grow-ing colony in situ under low power (see Figure 51.1).

TRICHODERMA

Growth is white, rapidly spreading and cobweblike, with irregular patches of dark-green spores.These are abundant, small, ovoid, arising in ball-like groups on short right-angled branches (seeFigure 51.1). This fungus may be mistaken for aPenicillium but the rapidly spreading growth form,often covering the agar surface and even the sidesof the Petri dish, would be very unusual forPenicillium.

MONASCUS

The colonies of this saprophyte are flat, rathergranular with a deep-red pigment. The characteris-tic ascospores are produced inside spherical asco-carps. These are unusual in that each develops froma single (or a very few) hypha and they have a thinwall (see Figure 51.1). Chains of conidia super-ficially resembling Scopulariopsis may also bepresent.

PAECILOMYCES

Usually seen as a spoilage fungus, there is onespecies, P. variotti, which has been found in rare,deep infections in immunosuppressed patients.

Growth resembles that of Penicillium, producingeither pale purple or yellowish olive coloniesdepending on species. This feature, the distinctlyelongated spores, and the long, tapering tips, whichare often curved, to the spore-producing cells of the‘penicillus’ distinguish it from Penicillium (seeFigure 51.1).

CHRYSOSPORIUM

These are soil dwellers, many of which specialize inusing keratinaceous substrates; some may be capa-ble of causing infection of the skin and nails.

This genus is close to the dermatophytes and, likethem, is unaffected by cyclohexamide in themedium. Colonies of most species are white orcream coloured and flat with felt-like surfaces.Spores are similar to those of dermatophyte micro-conidia but mostly longer than 5 μm (see Figure51.1).

GEOMYCES

One species, G. pannorus, a soil organism, is oftenmistaken for a dermatophyte and it has beenreported on the surfaces of meat carcasses in coldstorage at temperatures as low as –6°C (usuallyunder the name Sporotrichum carnis).

Colonies are restricted, often heaped up, with avery thin white crust of aerial spores. These resem-ble dermatophyte microconidia but are smallerthan most (< 1.5 μm long) and produced onminute, acutely branched, Christmas tree-likestructures (see Figure 51.1).

SCOPULARIOPSIS

One species, S. brevicaulis, is a saprophyte, livingin soil although it does seem to be adapted to usekeratin and is one of the more common invaders ofhuman nail tissue. It is also notable for releasinggaseous arsines from the arsenical pigment Parisgreen.

Colonies are often folded and heaped up, but theymay be flat. When sporing they are cinnamon–brown in colour and have a powdery surface. Thespores are large, rounded, with a flat detachmentscar and are produced in long chains from a peni-cillium-like branching structure. In most strains thespore surfaces are distinctly roughened (see Figure51.1).

STACHYBOTRYS

One species, S. chartarum (S. atra) may producevery toxic macrocyclic trichothecenes such as thesatratoxins. This species is cellulolytic and mayoccur as black patches on the walls of damp houseswhere it has been implicated in some forms ofdamp building illness. It forms effuse, black orblackish green colonies, developing black glisteningheads of unicellular phialospores with verrucosewalls.

ASPERGILLUS AND PENICILLIUM

These genera are closely related but, although thereare several species that appear intermediate in mor-phology, there is generally no difficulty in placingisolates in one genus or the other. Both producelong, dry chains of conidia by repeated budding

through the end of a bottle-shaped cell, thephialide.

In Penicillium the phialides are clustered at the tipsof tree-like conidiophores and develop unevenly.

In Aspergillus they are clustered on a club-shapedor spherical vesicle and develop synchronously(Figure 51.2).

In the subgenus Aspergilloides of Penicillium, thephialides are attached directly to the tip of the coni-diophore (monoverticillate), which may be dis-tinctly swollen, giving the impression of a verysimple aspergillus fruiting body. However, theconidiophore of Penicillium is septate, and verysimilar in appearance to the mycelium from whichit has branched, whereas that of Aspergillus is amore specialized structure, usually non-septate anddeveloping from a distinct foot cell differentiatedclearly from the supporting mycelium.

In both groups the mycelium is usually colourless,but may be coloured by the production of pigments

422 Common moulds

Conidiospore

Phialide

Metula

Ramus orbranch

Conidiophore

Phialide

Metula

Conidiophore

Foot cell

(d) BiseriateAspergillus

(c) UniseriateAspergillus

(b) TerverticillatePenicillium

(a) MonoverticillatePenicillium

Figure 51.2 (a, b) Penicillium; (c, d) Aspergillus

Aspergillus and penicillium 423

retained in the cytoplasm or secreted into themedium; any colour on the obverse is the result ofthe coloured spores and on the reverse is the resultof secreted pigments. Penicillium spores are usuallysome shade of green or blue. Aspergillus sporesmay be green, ochre-yellow, brown or black. Inboth genera there are species with white spores.

Some Penicillium spp. are plant parasites but themajority of both genera occur on decaying vegetation.As a result of their wide range of secondary metabo-lites several are of importance in industrial fermenta-tion and synthetic processes. They are also importantin that they may produce mycotoxins in foods.

A single species, P. marneffii, is implicated ininfections of people who lived in or visited someregions of south-east Asia in which the species isendemic (see Chapter 52).

Aspergillus glaucus group

Members of this group flourish in conditions ofphysiological dryness (low water activity), e.g. onthe surface of jam, on textiles and on tobacco. Theyare very distinctive in appearance, with bluish-green or grey–green conidia and large yellow cleis-tothecia of the ascomycete genus Eurotium.

Aspergillus restrictus group

Like A. glaucus, these will grow on dry materials,such as slightly dry textiles. As their name suggests,they are slow-growing organisms.

Aspergillus fumigatus

This is so called because of its smoky-green colour. Itis a common mould in compost, and can grow at tem-peratures above 40°C; hence it can survive in the tis-sues of birds, for which it is a pathogen. It spores morereadily at 37°C than at 26°C. It is now well-recog-nized as a human pathogen, underlying pulmonaryeosinophilia. In this condition, the fungus may befound in the mucus plugs that are expectorated. Italso invades old tuberculous cavities and gives rise toaspergilloma of the lung. When isolated from suchcases, the organism often does not produce spores;incubation at 42°C may encourage it to do so. Patientswho harbour this organism develop precipitatingantibody, which is rather weak in pulmonaryeosinophilia but strong in cases of aspergilloma. Formethods, see p. 135. If A. fumigatus is examined witha lens, the conidiophores are seen to be columnar. Itlooks like a test-tube brush (see Figure 51.2).

Aspergillus niger

In contrast to A. fumigatus, this organism hasround heads that are large enough to appear dis-crete to the naked eye. This, together with theirblack colour, makes them easy to recognize. It is acommon cause of ear infections.

The flavus–oryzae group

Members of this group are notorious for theproduction of aflatoxin in groundnuts, maize, and

Table 51.2 Morphology of Aspergillus species

Aspergillus

fumigatus flavus niger terreus versicolor nidulans glaucus

Finely roughened stalks − + − − − − −Stalks pale brown − − − − − + −Green colony + + − − + + +Black colony − − + − − − −Sand-brown colony − − − + − − −Metulae present − + / − + + + + −

several other foods derived from tropical and sub-tropical plants, when badly stored (see Moss et al.,1989). They are also of value commercially, as asource of amylase and protease.

The specific name, which means yellow, is anerror; the colony colour in this series is green. Theheads are round, but smaller than those of A. niger.

Table 51.2 shows the morphological properties ofsome Aspergillus species/groups.

ZYGOMYCOTINA

These are fungi with non-septate, wide hyphae inwhich sexual fusion results in a thick-walled restingzygospore. They are seldom seen, however, and

distinction of genera and species is largely based onthe asexual structures. The group contains twomain orders. Many of them grow rapidly and pro-duce aerial mycelium resembling sheep wool.

Mucorales

These reproduce by many-spored sporangia. Themajority belong to three genera: Rhizopus, Absidiaand Mucor.

RhizopusThe columella enlarges greatly after rupture of thesporangium and collapses to form a characteristicmushroom shape. Spores are angular and delicately

424 Common moulds

Figure 51.3 Zygomycetes

References 425

striated. Root-like rhizoids may occur at the baseof the sporangium stalk (Figure 51.3).

AbsidiaA trumpet-shaped columella and sporangium stalkare more or less continuous (see Figure 51.3).

MucorThe absence of features characteristic of the lattertwo genera suggests Mucor spp. The commonpathogenic species, all causing mucormycosis, areRhizomucor pusillus, Absidia corymbifera andRhizopus arrizhus. They grow at 37°C, which isuseful in identification (see Figure 51.3).

Non-sporing moulds

Some moulds do not readily produce spores bywhich they may be identified. If they are consideredsignificant on the basis of direct microscopic evi-dence, sporulation may be encouraged by sub-culturing on a variety of different media, such aspotato sucrose agar or dilute strength cornmeal

agar, and on exposure to daylight at room temper-ature.

For detailed information about these moulds, seeSutton (1980), Pitt and Hocking (1997) and deHoog et al. (2000).

REFERENCES

de Hoog, G. S., Guarro, J., Gené, J. and Figueras, M. J.(2000) Atlas of Clinical Fungi, 2nd edn. Utrecht:Centraalbureau voor Schimmelcultures/UniversitarRovira I Virgili.

Ellis, M. B. (1971) Dematiaceous Hyphomycetes.Farnham: Farnham Royal, CommonwealthAgricultural Bureau.

Moss, M. O., Jarvis, B. and Skinner, F. A. (1989)Filamentous fungi in foods and feeds. Journal of

Applied Bacteriology (Symposium Suppl. No. 18)67: 1S–144S.

Pitt, J. I. and Hocking, A. D. (1997) Fungi and Food

Spoilage, 2nd edn. London: Blackie Academic.Sutton, B. C. (1980) The Coelomycetes. Farnham:

Farnham Royal, Commonwealth AgriculturalBureau.

The following fungi are considered in this chapter:dermatophytes, causing infections of epidermal tis-sues; some opportunistic deep-tissue pathogens(but see Chapter 51), agents of subcutaneous infec-tions, and the dimorphic systemic pathogens inHazard Group 3.

DERMATOPHYTES

There are three genera of common dermatophytes,classified according to the types of conidia theyproduce in culture:

• Microsporum: macroconidia spindle shaped,roughened, and microconidia clavate

• Trichophyton: macroconidia cylindrical andsmooth, and microconidia clavate or round

• Epidermophyton: macroconidia clavate,

smooth or slightly roughened, nomicroconidia.

The nature of the disease assists in identificationas many of these fungi have characteristic infectionpatterns (Table 52.1). This is particularly helpful inidentifying strains with abnormal morphology.

Identification to the species level depends uponcolonial morphology, pigment production and themicroscopic study of these conidia. Strains withmany microconidia and no macroconidia usuallybelong to Trichophyton species.

Appearance in clinical material

In nail and skin cleared as described in Chapter 9,fungal hyphae may be seen, some of them breaking

52

Pathogenic moulds

Table 52.1 Characteristic infection patterns of dermatophytes

Patient Body site Geographical origin Organism

Adult Feet, hands, groin Any T. rubrumT. interdigitaleE. floccosum

Adult or child Scalp, face, arms, chest, legs Any M. canisM. gypseumT. verrucosumT. mentagrophytes

Adult or child Scalp, face, arms, chest, legs N Africa, E and SE Asia T. violaceumChild Scalp Any T. tonsuransChild Scalp W and Central Africa, Caribbean M. audouiniiChild Scalp N and E Africa T. soudanaenseChild Scalp N and E Africa, Middle East T. schoenleinii

Dermatophytes 427

into arthrospores, although specific identificationcannot be made from their arrangement.

Some dermatophyte-infected hairs fluoresce undera Wood’s lamp (examine in a darkened room).Table 52.2 indicates the appearance of infectedhairs and Figure 52.1 shows various types ofinfected hairs, skin and nails.

Table 52.2 Appearance of infected hairs

Identification

Identification to species level depends on colonialand microscopic appearances, both of which areinfluenced by the growth medium.

Use Sabouraud’s dextrose agar which has beenshown to give adequate pigmentation and sporeproduction.

Microsporum audouinii

This is a scalp fungus with no animal or soil reser-voir. It spreads from child to child in schools, andinfection usually resolves at puberty. The organismis of medium growth rate, with a thinly developed,white-to-pale buff, aerial mycelium. The reverse ofthe colony is typically a pale apricot colour.Macroconidia and microconidia are rare (Figure52.2).

Spore arrangement Fluorescence Organism

Small spore ectothrix + M. canis

+ M. audouinii

− T. mentagrophytes

Large spore ectothrix − M. gypseum

− T. verrucosum

Endothrix − T. soudanense

− T. violaceum

‘Favic’ hairs weak T. schoenleinii

Hair

Endothrix

Linear small sporeMosaic small spore Large spore

Ectothrix

Large spore

Airspaces

Favic Mycelium

Arthrospores

Skin and nail

Figure 52.1 Infected hair, skin and nail

428 Pathogenic moulds

Microsporum canis

As its name suggests, this is a common parasite ofdogs and cats, where the infection is often difficultto detect. Children are commonly infected frompets. This organism, which is fast growing, usually

produces a vivid yellow pigment and fairly abun-dant macroconidia. Occasionally, strains are non-pigmented and slow growing. To differentiate themfrom M. audouinii, grow on rice (5 g rice and 20 mlwater in a 100-ml flask, autoclaved at 115°C for 20min and cooled). Under these conditions M. canis

Figure 52.2 Microscopic appearances of some dermatophytes (bar = 10 μm)

Dermatophytes 429

produces aerial mycelium; M. audouinii does not(see Figure 52.2).

Microsporum gypseumThis occurs as a saprophyte in soil and has a low vir-ulence for humans. Infections are therefore uncom-mon and typically present with a solitary lesion,related to contact with the soil. The fungus growsfast, and is soon covered with a buff-coloured gran-ular coating of spores. These tend to be arranged inradial strands on the surface, rather like a spider’sweb. Macroconidia are abundant (see Figure 52.2).

Trichophyton rubrumThis is the most common dermatophyte seen indermatology clinics in developed countries, but theless common T. interdigitale is probably morewidespread in the general population.

There are many varieties of T. rubrum; the oneseen most often gives a downy, white colony with ared–brown pigment and a narrow white edge onreverse. Microscopy shows only club-shapedmicroconidia (see Figure 52.2).

Other varieties are ‘melanoid’ in which a dark-brown pigment diffuses into the medium, ‘yellow’,which is a non-sporing pale yellow form, and‘granular’, which has the usual dark-red reverse butpowdery aerial growth with patches of pink. Thisvariety has larger microconidia and usually pro-duces macroconidia (see Figure 52.2).

Non-pigmented strains occur which resemble thedowny variety of T. interdigitale (see Figure 52.2).To distinguish these, subculture on urea agar.T. rubrum does not usually produce the colourchange by 7 days but most other dermatophytes do.

Trichophyton mentagrophytes complex

The most common form in this group is the anthro-pophilic T. interdigitale, which causes athlete’sfoot. Colonies resemble those of the zoophilicT. mentagrophytes (see below), with a uniformlypowdery, cream-coloured surface made up of anabundance of almost spherical microconidia. Spiralhyphae and macroconidia may be present (seeFigure 52.2). The downy variety forms a pure

white cottony colony and has elongated microconi-dia resembling those of T. rubrum.

The zoophilic (usually from rodents) T. menta-grophytes has coarsely granular colonies with areasof agar surface showing between radiating zones ofwhite or cream sporing growth. Reverse pigmenta-tion often shows dark brown ‘veins’.

Trichophyton tonsuransThis is a cause of scalp ringworm. Its colonies areslow growing with a dark red–brown edge, andgranular to velvety centre, often with folding. Moststrains show a mixture of microconidia andswollen hyphal cells (chlamydospores).Microconidia are large, oval and arranged alongwide, often empty hyphae (see Figure 52.3).

Trichophyton verrucosumThis is the causative organism of cattle ringworm.It also causes scalp, beard or nail infections in farmworkers, and other people such as slaughtermenand veterinary surgeons who come into contactwith cattle. In culture it grows very poorly; sus-pected cultures should be examined carefully witha lens after incubation, for 14 days, becausecolonies are often submerged and minute. This,together with the large, thin-walled, balloon-likechlamydospores at the end of a straight hypha andchains of thick-walled cells (see Figure 52.3), is suf-ficient to identify the organism.

Trichophyton schoenleiniiThis is the cause of favus. The colony is slowgrowing, hard and leathery, with a surface likewhite suede. The only microscopic features arethick, knobbly hyphae, like arthritic fingers – theso-called ‘favic chandeliers’ – and chlamy-dospores.

Trichophyton violaceumThis is another cause of scalp ringworm, micro-scopically similar to T. verrucosum, although thehyphae are usually thinner. The characteristic fea-ture is a deep red–purple pigment, which appearsas a spot in the centre of a young colony, and grad-ually spreads to the edge.

EpidermophytonThere is only one species, E. floccosum which isone cause of tinea cruris. It also occasionally infectsfeet. Its growth on malt agar is khaki in colour witha powdery surface caused by masses of macronidiawhich have a very characteristic shape (see Figure52.3)

ENTOMOPHTHORALES

These insect pathogens are seen rarely in clinicalmaterial in tropical countries. The asexual sporeform is a single, relatively large ‘conidium’ which isforcefully discharged.

Colonies are wrinkled, cream coloured and waxy.A haze of discharged spores forms a mirror imageof the colony on the lid of the Petri dish.

BasidiobolusThis is a cause of tropical subcutaneous zygo-mycosis.

Conidiobolus coronatus (Entomophthora coronata)This causes tropical rhinoentomophthoromycosisof the nasal mucosa.

MISCELLANEOUS PATHOGENICMOULDS

MadurellaMadurella mycetomatis and M. grisea cause tropi-cal black grain mycetoma. The former producesflat or wrinkled, yellowish colonies, often releasingmelanoid pigments into the medium. The lattergrows as domed, densely fluffy, mouse-grey toblack colonies. Neither forms conidia.

ExophialaColonies are dark grey to black, intensely fluffy orwet and yeast-like depending on degree of conidialbudding, strain and growth conditions. All produceconidia from small, tapering nipples which grow inlength as more conidia are cut off (Figure 52.4).


Figure 52.3 Microscopic appearances of some dermatophytes (bar = 10 μm)

Miscellaneous pathogenic moulds 431

Exophiala jeanselmei is a cause of mycetoma, E.werneckii of tinea nigra, E. dermatitidis of chromo-mycosis and E. spinifera of subcutaneous cysts.

PhialophoraMembers of this genus are black moulds in whichthe spore apex has a definite collarette or funnel-shaped cup in which the conidia are formed (seeFigure 52.4). P. verrucosa is one cause of chromo-mycosis; P. parasitica is occasionally found in sub-cutaneous cysts.

FonsecaeaMembers of this genus are black moulds in whichthe conidia are formed in short chains by apical

budding of the cell below. They differ fromCladosporium and Cladophialophora spp. only inthat the chains consist of no more than four spores(see Figure 52.4). F. pedrosi is the common cause ofchromoblastomycosis; F. compacta is less com-mon.

CladophialophoraThe most frequent Cladosporium-like fungi caus-ing human including cerebral phaeohyphomycosishave been moved from Cladosporium toCladophialophora. The common species are C. car-rionii (subcutaneous chromoblastomycosis) andC. bantiana (a pathogen of the brain, now rated inthe UK as in Hazard Category 3). They differ from

Figure 52.4 Microscopic appearances of some pathogenic fungi (bar = 10 μm)

Cladosporium (see Figure 51.1) in having verylong, rarely branching chains of conidia (see Figure52.4). The conidia are slender, smooth and thinwalled in comparison with Cladosporium. Somestrains also produce phialides of the phialophoratype (see Chapter 51).

ScedosporiumVariously named Allescheria, Petriellidium orMonosporium spp. It causes pale grain mycetomaand miscellaneous other infections (otitis externa inthe UK). Two species cause a variety of clinicalinfections, S. apiospermum (the asexual form ofPseudallescheria boydii) and S. prolificans.S. apiospermum colonies are fluffy, white to lightgrey, producing oval, slightly yellowish spores insuccession at the tips of long, straight hyphae. Eachspore has a basal scar (see Figure 52.4). Occasionalstrains may produce black ascomata beneath theagar.

Scedosporium prolificans colonies are flatter andslower growing, often with areas of black wet sur-face. Under the microscope they have short, lateralconidia bearing hyphae, which are swollen at thebasal half and taper to a narrow filament-like tiparea. A number of fatal cases of deep opportunisticinfections by this species have been described.S. prolificans is particularly resistant to modernsystemic antifungal drugs.

AcremoniumThis is a very large group of white or pink mouldswith wet heads of conidia produced one by onefrom the tips of straight hyphae or lateral nipples(see Figure 52.4). In some, the conidia bud second-ary spores and the colony becomes yeast like. A.kilense is a cause of pale grain mycetoma; otherspecies invade nail tissue.

FusariumColonies are often loose and fluffy with a pink orpurple pigment. They are characterized by curved,multiseptate macroconidia, although these may beabsent. Many strains produce microconidia similarto those of Acremonium spp. but usually somewhatcurved (see Figure 52.4).

Fusarium solani and F. oxysporum cause a varietyof infections including mycetomas and keratitis.They are plant pathogens.

SporothrixAt low temperatures (25–30°C) colonies ofSporothrix are grey to black, dry, membranous andwrinkled. Hyaline or pigmented spores are pro-duced initially as rosettes on minute, teeth-likepoints on expanded apical knobs (see Figure 52.4).Culture at 37°C when they convert to yeast formfacilitates differentiation from non-pathogenicspecies. Causes sporotrichosis in which it exists asbudding, spindle-shaped yeast.

DIMORPHIC SYSTEMICPATHOGENS

These cause systemic or generalized infectionsresulting from the inhalation of airborne conidia.Heavily sporing mould cultures are more haz-ardous than those in the yeast-like phase. Theseorganisms are classified as Hazard Group 3pathogens and they must be handled in microbio-logical safety cabinets in Containment Level 3laboratories.

A cultural feature of these organisms is that theyare not inhibited by cycloheximide at 0.5 mg/ml.

Blastomyces dermatitidisThis causes North American blastomycosis.Infection is almost always by inhalation of spores.Skin lesions are usually secondary to a pulmonaryinfection, even a mild one. The mycelial form, at26°C, is a moist colony at first, developing a fluffyaerial mycelium with microconidia. At 37°C or intissue, large oval yeasts are seen (Figure 52.5).

Histoplasma capsulatumThis causes the pulmonary disease histoplasmosis,which is very common in the Ohio River valley inthe southern half of the USA, although it is alsofound in other countries. Infection is associatedwith exposure to dust containing faecal material inbarns and sheds. Most people recover rapidly: only


Dimorphic systemic pathogens 433

an occasional patient develops progressive disease.The route of infection is inhalation, and the pathol-ogy similar to that of tuberculosis (see Figure 52.5).(See caution above.)

At 26°C, the organism grows with a white aerialmycelium, which becomes light brown. The sporesare striking and characteristic. At 37°C, small, ovalbudding yeasts are found. In tissue, these yeasts areintracellular, except in necrotic material.

Paracoccidioides brasiliensisThis causes South American blastomycosis. Theprimary lesion is almost always in the oral mucosa.The spread is through the lymphatics. The organ-ism is very slow growing, white and featureless at

26°C. At 37°C on rich media, characteristic yeastcells are formed (see Figure 52.5).

Coccidioides immitisThis causes another lung infection, coccidioidomy-cosis. This disease is limited to a few special areasin the south west of the USA and in Mexico andVenezuela. It is inhaled in desert dust. Again, mostpeople suffer only a mild, transient, ‘flu-like’ ill-ness. Occasionally, cases progress to generalizeddestructive lesions. (See caution above.)

At 26°C, the colony is at first moist, then developsan aerial mycelium, becoming brown with age andbreaking up into arthrospores. There is no secondform in artificial culture. In the clinical lesions,

Figure 52.5 Microscopic appearances of Blastomyces dermatitidis; Histoplasma capsulatum; Coccidioides immitis; Paracoccidioides brasiliensis:(i) mycelial form; (ii) tissue form (bar = 10 μm)

however, large, thick-walled cells are seen. Whenmature, these are full of spores (see Figure 52.5).

Penicillium marneffeiPenicillium marneffei has become one of the mostfrequent causes of opportunist infection in AIDSpatients who have resided in or visited south-eastAsia. At lower temperatures (25–35°C) this dimor-phic fungus is a hyphal mould with classic penicil-lium conidial heads (see Chapter 51). The conidiaare pale green in mass and contribute this colour tothe colony surface. A deep-red diffusing pigment isproduced on most media. Microscopically the coni-dial heads are more spread out and irregular thanthose of the saprophytic P. purpurogenum, whichis sometimes encountered as a contaminant andalso has a deep-red diffusing pigment.

At blood temperature on nitrogen-rich media(blood agar, brain–heart agar) the fungus convertsto a yeast-like colony, which is composed of cylin-drical cells reproducing by fission.

In view of the difficulty of identification ofPenicillium spp., this Hazard Group Category 3

pathogen should be sent to a reference laboratoryfor confirmation.

For more details about pathogenic fungi, pleasesee Campbell and Stewart (1980), Campbell et al.(1996), Midgeley et al. (1997) and de Hoog et al.(2000).

REFERENCES

Campbell, M. C. and Stewart, J. C. (1980) The Medical

Mycology Handbook. New York and Chichester,Wiley.

Campbell, C. K., Johnson, E. M., Philpot, C. M. et al.

(1996) Identification of Pathogenic Fungi. London:Public Health Laboratory Service.

de Hoog, G. S., Guarro, J., Gené, J. et al. (2000) Atlas

of Clinical Fungi., 2nd edn. Utrecht: Reus,Centraalbureau voor Schimmelcultures/UniversitatRovira I Virgili.

Midgley, G., Clayton, Y. and Hay, R. J. (1997)Diagnosis in Color. Medical mycology. Chicago:Mosby-Wolfe.


All employers whose work involves the deliberateexposure of their employees to biological agentsneed to comply with the law. The informationgiven here provides the reader with the frameworkof the primary legislation in the UK that applies inthe microbiology laboratory, thus providing a basisupon which a good health and safety policy can beproduced.

HEALTH AND SAFETY AT WORK

The Health and Safety at Work, etc. Act (the Act)1974 (HSE, 1990) provides the foundations for thehealth and safety system as we know it today.Before the Act, health and safety legislation wasprescriptive, out of date, complex and inflexible,and would not have been able to meet the demandsof managing the type of risks encountered in amodern microbiology laboratory. The implementa-tion of the Act established the Health and SafetyCommission (HSC) and the Health and SafetyExecutive (HSE). The Act places on theCommission the responsibility of undertaking revi-sion and replacement of legislation, to recommendnew legislation based on developments in technol-ogy and to comply with European UnionDirectives. The Commission’s main instrument inoperating these responsibilities is the HSE.

The Act is described as goal-setting, leavingemployers the freedom to decide how to control therisks that they identify. There are therefore generalduties, which employers have towards employeesand members of the public, and which employeeshave to themselves. These duties are qualified bythe principle ‘so far as is reasonably practicable’.

This means that the degree of risk in a particularjob, e.g. the culture of Hazard Group 3 biologicalagents, needs to be balanced against the time, trou-ble, cost and physical difficulty of taking measuresto avoid or reduce the risk.

REGULATIONS, CODES OFPRACTICE AND GUIDANCE

The Act states that legislation passed before 1974should be ‘progressively replaced by a system ofregulations and approved codes of practice’. Infact, the general principle has been that regulations,like the Act itself, should, so far as is possible,express general duties, principles and goals, andthat subordinate detail should be set out inGuidance and Approved Codes of Practice (ACoP).

Regulations, e.g. the Control of SubstancesHazardous to Health (COSHH) Regulations, arelaw, approved by Parliament. An ACoP givesadvice, but employers are free to take other equallyeffective measures provided that they do what isreasonably practicable. Some risks are so great, orthe proper control measures so costly, that it wouldnot be appropriate to leave employers the discre-tion to decide what to do about them. Regulationsidentify these risks and set out specific action thatmust be taken. These requirements may be absoluteand must be complied with, without the qualifica-tion of reasonable practicability, e.g. a Contain-ment Level 3 Laboratory must be sealable to permitdisinfection.

An ACoP offers practical examples of good prac-tice. Advice is provided on how to comply with thelaw, by providing a guide to what is ‘reasonably

Appendix

Regulatory safety issues that affect work withmicro-organisms in the UK

practicable’, e.g. regulation 6 of the COSHHRegulations requires an ‘assessment of health riskscreated by work involving substances hazardous tohealth’. This assessment should be ‘suitable andsufficient’. The general ACoP accompanying theCOSHH Regulations informs that a suitable andsufficient assessment should include:

• an assessment of the risks to health• consideration of the practicability of

preventing exposure to hazardous substances

• the steps that need to be taken to achieveadequate control of exposure whereprevention is not reasonably practicable

• identification of other action necessary tocomply with the remaining COSHHregulations.

For biological agents, as distinct from other sub-stances hazardous to health, the assessment shouldreflect the ability they may have to replicate, betransmitted and infect. Additional clarification forbiological agents is provided in the main body of theACoP of the COSHH Regulations. ApprovedCodes of Practice have special legal status: ifemployers are prosecuted for a breach of health andsafety law, and it is proved that they have not fol-lowed the relevant provisions of the ACoP, a courtcan find them at fault unless they can show that theyhave complied with the law in some other way.

The HSE publishes guidance on a range of sub-jects, which may be specific to the health and safetyproblems of an industry or of a particular processused in a number of industries, e.g. the manage-ment, design and operation of microbiological con-tainment laboratories (Advisory Committee onDangerous Pathogens, ACDP, 2001) and successfulhealth and safety management (HSE, 1997). Themain purposes of guidance are:

• to interpret – helping people to understandwhat the law says

• to help people comply with the law• to give technical advice.

Following guidance is not compulsory andemployers are free to take other action; however, if

they do follow guidance they will normally bedoing enough to comply with the law.

MANAGEMENT RESPONSIBILITIES

Successful control of exposure to biological agentsdepends on having effective arrangements for man-aging health and safety. An overview of the rele-vant health and safety legislation and guidance thatshould be consulted when working with biologicalagents in microbiological containment laboratoriesis shown in Figure A.1. Employers need to considera number of key areas related to work in microbi-ology laboratories, including:

• risk assessment• information, instruction and training• buildings and accommodation• safe working practices• health surveillance and immunization• checking what has been done.

Detailed information on local arrangements andprocedures is best provided in standard operatingprocedures. These should be based on a risk assess-ment and are a good place to record the findings ofassessments. Standard operating procedures needto be written down and kept up to date. All staffneed to be aware of the local procedures that applyto them.

HEALTH AND SAFETY LAW: KEYREGULATIONS

The following section provides some information onthe Key Regulations that apply to the prevention ofinfection in the laboratory. The information pro-vided is not intended to be comprehensive and man-agers must ensure that all other risks are identifiedand all other legal obligations fulfilled. Examples ofadditional regulations that will apply in the labora-tory include the Manual Handling OperationsRegulations 1992 (HSE, 1992), the Display ScreenEquipment Regulations 1992 (HSE, 1994a) and theNoise at Work Regulations 1989 (HSE, 1998a).

436 Regulatory safety issues that affect work with micro-organisms in the UK

Health and safety law: key regulations 437

The Health and Safety at Work, etc.Act

As previously stated, the Act places general dutieson employers, employees and others.

Key dutiesEmployers must:

• protect the health and safety of theiremployees

• protect the health and safety of others who

might be affected by the way they go abouttheir work (e.g. cleaners, visitors orcontractors working in the laboratory)

• prepare a statement of safety policy and theorganization and arrangements for carrying itout (if five or more people are employed, thisstatement must be written down).

Employees must:

• take care of their own health and safety, andthat of others

• cooperate with their employer.

Primary legislation Health and Safety at Work etc Act 1974

Control of SubstancesHazardous to HealthRegulations – general andbiological agents provisions

SecondaryRegulationsand ApprovedCodes ofPractice

Generalguidance

Guidance oncontainmentand controlmeasures

Guidanceon workwith specificagents

Categorisation of biological agentsaccording to hazard and categories ofcontainment – Approved List ofBiological Agents

Infection risksto new andexpectantmothers in theworkplace

Management of Heathand Safety at WorkRegulations

Containmentlaboratories

Blood-borneviruses

ACDP guidance

TransmissibleSpongiformEncephalopathies

Viralhaemorrhagicfevers

Researchanimals,includingsimians

Largescale

Clinicallaboratoriesa

ACGMCompendiumof Guidance

A guideto theregulations

GMContainedUseRegulations

Biotechnology in educationb

Disposal ofclinical wastea

Figure A.1 Health and safety legislation and guidance relevant to work with biological agents in microbiological containmentlaboratories. a Health Service Advisory Committee guidance. b Education Services Advisory Committee guidance

The Management of Health and Safetyat Work Regulations 1999

The general duties in the Act are developed in theManagement of Health and Safety at WorkRegulations 1999 (the Management Regulations)and other more specific pieces of law. The ACoP onthe Management of Health and Safety at WorkRegulations provides further guidance (HSE,2000).


• assess risks to staff and others, includingyoung people, and new and expectantmothers (HSE, 1994b; ACDP, 1997)

• make appropriate health and safetyarrangements, which must be recorded if fiveor more people are employed

• appoint competent people to help themcomply with health and safety law

• establish procedures to deal with imminentdanger

• cooperate and coordinate with otheremployers and self-employed people whoshare the workplace.

Employees must:

• work in accordance with training andinstruction given by their employer

• report situations, which they believe to beunsafe.

The Control of Substances Hazardousto Health Regulations

The COSHH Regulations 2002 apply to substancesthat have been classified as being very toxic, toxic,harmful, corrosive, sensitizing or irritant (HSE,1999a). Biological agents are also treated by theRegulations as substances hazardous to health.Additional requirements of the Regulations, per-mitting work with biological agents, are describedin Schedule 3 of the Regulations (see Table 1.3,

p. 4) which lists some of the containment measuresfor laboratories that are necessary to ensure com-pliance with the Regulations. Further practicalguidance can be found in Schedule 3 and Appendix2, ‘Additional provisions relating to work with bio-logical agents’, of COSHH (HSE, 2002).


• carry out risk assessments • select and use appropriate control measures

where there is a likelihood of exposure tobiological agents

• maintain, examine and test control measures• inform, instruct and train employees• keep records of employees exposed to Group

3 biological agents• carry out health surveillance of employees.

Reporting of Injuries, Diseases andDangerous Occurrences Regulations1995

The Reporting of Injuries, Diseases and DangerousOccurrences Regulations (RIDDOR) (HSE, 1999b)require employers and others to report accidentsand specified diseases, which arise out of or in con-nection with work, to the HSE.


• report any acute illness that requires medicaltreatment, where there is reason to believethat this resulted from exposure to abiological agent or its toxins or infectedmaterial

• report any accident or incident that resulted,or could have resulted, in the release or escapeof a Hazard Group 3 or 4 biological agent

• report specified infectious diseases, such ashepatitis and tuberculosis

• report any infection reliably attributable towork in the laboratory.


RISK ASSESSMENT

Risk assessment is not simply a paper exercise. Itspurpose is to ensure that there are appropriate pre-cautions in place to prevent and control risk. Theprocess can be broken down into the following fivesteps (HSE, 1998b):

1. Identify the hazards.2. Ascertain how the hazards cause harm.3. Assess the risks – determine what preventive

measures or controls are required.4. Record and implement the findings of the

assessment.5. Monitor, review and revise the assessment.

Step 1: Identify the hazards

The first step in any risk assessment is to look forthe hazards. A hazard is the potential for somethingto cause harm. Employers will need to consider:

• which biological agents may be present• the virulence of these agents• the route(s) of infection• their transmissibility.

Step 2: Ascertain how the hazardscause harm

Employers must acknowledge the fact that theirstaff may all potentially be exposed to infectiousagents. Those employees who may be affected,along with the routes of transmission, need to beconsidered when assessing risks and decidingwhich precautions are needed.

Step 3: Assess the risks – determinewhat preventive measures or controlsare required

A risk is the likelihood of harm occurring.Employers need to evaluate likely risks arising from

any exposure to hazardous agents, taking intoaccount:

• the severity of the diseases caused• prevalence of particular infections in the

community• the nature of any likely contact with a patient

specimen• the likelihood of an accident involving

exposure to biological agents• the likelihood of infection from biological

agents present• the individual susceptibility of staff.

Employers need to devise and implement a mech-anism for reporting and responding rapidly andeffectively to accidents and incidents of ill-health.An official local record should be made of all inci-dents and occurrences with infectious or poten-tially infectious material involving the exposure ofindividuals. This record should include near misses.It must be remembered that, in addition to localreporting of accidents and exposures, in some casesthe HSE must be notified under the RIDDORRegulations.

When deciding whether existing preventivemeasures are adequate or whether further con-trols are required, employers need to consider thelaboratory facilities and the activities therein ofthe staff. Where assessments identify the need forimprovements, employers need to plan how to dothis and make sure that their plans are imple-mented.

Step 4: Record and implement thefindings of the assessment

The main findings of the risk assessment must bewritten down in all but the simplest of cases.Employers often incorporate the findings in thestandard operating procedures. These proceduresare then used as working documents for managers,employees and safety representatives.

Risk assessment 439

Step 5: Monitor, review and revise theassessment

Employers should review the assessment(s) regu-larly to check that they are still valid. This is bestdone as part of the day-to-day management of thelaboratory. It may include verifying that:

• standard operating procedures are relevant tocurrent practices

• staff have received appropriate informationand training about their work

• the system for reporting and responding toaccidents, incidents and ill-health is in placeand being followed

• equipment, such as microbiological safetycabinets, is working appropriately.

If the review identifies deficiencies in the assess-ment, employers should revise it and implementany necessary further precautions. Employers alsoneed to make practical checks to ensure that staffare following the standard operating proceduresappropriately.

INFORMATION, INSTRUCTIONAND TRAINING

All staff need sufficient information, instruction andpractical training about the risks associated withtheir work and on the precautions required to con-trol/prevent the risks. Until staff have received ade-quate instructions about hygiene and safety at workthey should not work with biological agents.Employers need to ensure that staff, contractors andvisitors know the relevant safety standards applica-ble to them while working in or attending the labo-ratory. Employers are required to identify particularstaff needs, including any gaps in knowledge and/orexperience, and to provide the necessary informa-tion, instruction and training to fill these gaps.

To be useful, instructions and information should:

• take account of the level of training,knowledge and experience that staff andothers have

• be easily understood, e.g. by those withlanguage or communication difficulties.

INCIDENT REPORTING

Management needs to have clear procedures in placefor dealing with incidents and accidents that couldoccur to staff or visitors. In addition, employees andother workers should be encouraged to report inci-dents of ill-health. Standard operating proceduresshould cover the arrangements for:

• immediate action in the event of an accident,fire or other emergency, especially where thereis a risk of infection

• the action to take in the event of a significantspillage, e.g. dropping of culture plates

• reporting, recording and investigatingaccidents, incidents and ill-health includingthose with the potential to cause injury or ill-health

• notifying employees and their representativesof the causes of the incident and any remedialmeasures.

As previously described, the RIDDO Regulationsrequire employers to report and keep records ofspecified types of accident, incident and diseases. Foreffective monitoring of health and safety arrange-ments, the internal reporting system needs to takeaccount of all incidents and accidents that may occurin the laboratory, not just the more serious ones.Following up all such occurrences will help man-agers, safety representatives, safety managers andothers monitor the adequacy of their precautions,check performance, learn from mistakes, identifyjobs or activities that cause the greatest number ofproblems and identify deficiencies in staff trainingneeds. This information is also very useful when car-rying out the risk assessment process.

HEALTH SURVEILLANCE

Employers need to advise on the health surveillancerequirements and immunization arrangements for


their staff. Advice should be obtained from a qual-ified occupational health practitioner. Moredetailed information is provided by the HSE(1999c), Health Services Advisory Commission(HSAC, 1984) and Association of National HealthService Occupational Physicians (ANHSOP, 1986).

Pre-employment screening

Routine medical examination before employmentis not considered necessary; instead, candidates cancomplete a questionnaire. One purpose of the ques-tionnaire would be to determine the immune statusof employees and offer the appropriate immuniza-tion or to identify those who may be more suscep-tible to infection because of a pre-existing medicalcondition. Only those whose replies suggest that aproblem may exist need to seek advice from anoccupational health professional.

Immunization

Employers need to ensure that occupational healtharrangements include agreed immunization proce-dures for all staff. They should base these require-ments on guidance from the Joint Committee onVaccination and Immunisation (Department ofHealth, 1996). Generally, microbiologists will needto be immunized. Selected immunization proce-dures should be implemented as soon as possibleafter the appointment and ideally before startingwork. The need for immunization will be deter-mined as part of the risk assessment; invariablyprotection against hepatitis B, tetanus and tubercu-losis will be included. Immunization should, how-ever, be seen only as a useful supplement,reinforcing procedural controls and the use of pro-tective equipment. The COSHH Regulationsrequire employers to make effective vaccines avail-able to employees exposed to biological agents. Asthis is a specific requirement under the Regulations,employers cannot charge their employees for suchvaccines. In providing vaccines, employers should

ensure that employees are made aware of theadvantages and disadvantages of immunizationand its limitations. Adequate records should bekept of any vaccines given and checks made toensure that protection has been provided.

Monitoring

This involves checking employees’ health to detectworkplace illness by, for example, following upsickness absence or explaining symptoms of infec-tion to employees so that they can monitor theirown health. In some cases, it may be useful to pro-vide medical contact cards to alert medical practi-tioners about the nature of the work in the event ofsudden unexplained illness.

Record keeping

Accurate health records are needed for all staff.Records that are more detailed should be kept forthose who may be exposed regularly to a high riskof infection. It is important that, where hazardsexist, employers monitor the health of their staffand note and act upon occurrences of work-relatedillness, e.g. sickness absence. Active health surveil-lance will be required for needlestick injuries andfollowing exposure to significant pathogens, e.g.Mycobacterium tuberculosis. The health record isdifferent from a clinical record and may be keptwith other confidential personnel records. Recordsthat include medical information arising from clin-ical examination should be held in confidence bythe occupational health doctor and can be releasedonly with the written consent of the individual.

Employers are required to keep the followingdetails about employees exposed to a Group 3 bio-logical agent:

• the type of work that the employee does• the biological agent(s) to which he or she has

been exposed (where this is known)• records of exposures, accidents and incidents.

Health surveillance 441

Health records will need to be maintained in mostcases for a period of 40 years after the last knownexposure.

CHECKING WHAT HAS BEENDONE

Measuring performance and review

Employers need to measure how effectively theyare implementing their standard operating proce-dures and assess how effectively they are control-ling risks in the laboratory. Actively monitoringsystems before something goes wrong is a key partof a successful monitoring regimen and part of amanager’s responsibility. Information that is moredetailed can be found in HSE guidance – SuccessfulHealth and Safety Management (HSE, 1997).

Bio-security

Historically, security measures in laboratoriesmaintaining or researching pathogenic cultureshave focused on the containment of the pathogenand the safety of workers. The supply of pathogensto such laboratories requires those laboratories tohave suitable facilities for the containment of thoseorganisms and, in order to be supplied withpathogens, to have notified the HSE about theirintent. It is recognized that the choice of contain-ment measure is largely determined by the catego-rization of the biological agent (ACDP, 1995).

Recent events, e.g. 11 September 2001, havefocused attention on the threat posed by the terror-ist use of biological agents as ‘weapons of massdestruction’ (WMD). Consequently, there is anincreasing requirement for greater scrutiny of dis-tribution of biological agents and making it moredifficult to access areas where such agents arestored and used. A full discussion on the implica-tions of bioterrorism and subsequent impact on themicrobiology laboratory is beyond the scope of thisbook, and the reader is referred to other publica-

tions (McGinness et al., 2001; Beeching et al.,2002) (see also p. 10).

REFERENCES

Advisory Committee on Dangerous Pathogens (ACDP)(1995) Second supplement to: Categorisation of

Biological Agents According to Hazard and

Categories of Containment, 4th edn. Sudbury: HSEBooks.

ACDP (1997) Infection Risks to New and Expectant

Mothers in the Workplace: A guide for employers.Sudbury: HSE Books.

ACDP (2001) The Management, Design and Operation

of Microbiological Containment Laboratories.Sudbury: HSE Books.

Association of National Health Service OccupationalPhysicians (ANHSOP) (1986) Health Assessment

for Employment in the NHS: Physician’s guidance

Note 9. Bath: ANHSOP, Royal United Hospital.Beeching, N. J., Dance, D. A. B., Miller, A. R. O. and

Spencer, R. C. (2002) Biological warfare andbioterrorism. British Medical Journal 324:336–339.

Department of Health (1996) Immunisation Against

Infectious Disease. Department of Health JointCommittee on Vaccination and Immunisation.London: HMSO.

Health Services Advisory Committee (HSAC) (1984)Guidelines on Occupational Health Services in the

Health Service. Sudbury: HSE Books.Health and Safety Executive (HSE) (1990) A Guide to

the Health and Safety at Work etc Act 1974 (L1).Sudbury: HSE Books.

HSE (1992) Manual Handling. Manual Handling

Operations Regulations 1992 Guidance on

Regulations (L23). Sudbury: HSE Books.HSE (1994a) VDUs: An easy guide to the regulations

(HSG90). Sudbury: HSE Books.HSE (1994b) New and Expectant Mothers at Work: A

guide for employers (HSG 122). Sudbury: HSEBooks.

HSE (1997) Successful Health and Safety Management

(HSG65). Sudbury: HSE Books.


References 443

HSE (1998a) Reducing Noise at Work: Guidance on

The Noise at Work Regulations 1989 (L108).Sudbury: HSE Books.

HSE (1998b) Five Steps to Risk Assessment

(INDG163). Sudbury: HSE Books.HSE (1999a) Approved Guide to the Classification and

Labelling of Substances Dangerous for Supply:

Chemicals (hazard information and packaging for

supply) regulations 1994: guidance on regulations

(L100). Sudbury: HSE Books.HSE (1999b) A Guide to the Reporting of Injuries,

Diseases and Dangerous Occurrences Regulations

1995: Guidance on regulations (L73). Sudbury:HSE Books.

HSE (1999c) Health Surveillance at Work (HSG 61).Sudbury: HSE Books.

HSE (2000) Management of Health and Safety at

Work, Management of Health and Safety at Work

Regulations 1999 Approved Code of Practice and

guidance (L21). Sudbury: HSE Books.HSE (2002) Control of Substances Hazardous to

Health: Approved Codes of Practice (L5), 4th edn.Sudbury: HSE Books.

McGinness, S., Sleator, A and Youngs, T. (2001) Theanti-terrorism, crime and security bill Parts VI &VII: pathogens, toxins and weapons of massdestruction. Parliamentary Library Research Paper01/94. www.parliament.uk

www.parliament.uk

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Abiotrophia sppnutritional variant ‘streptococci’

345properties 334

abortion, contagious 305Absidia 424–5accreditation, quality assessment 16–18acetic acid bacteria 272Acetobacter spp 272Achromobacter spp 270acid-fast bacilli (AFB) 380

fluorescent stain 91acid-fast stain 90Acinetobacter spp 359

breakpoints 175isolation and identification 269properties 260, 271species 269

acne 406Acremonium spp 432Actinobacillus spp 303

A. actinomycetemcomitans 311, 318A. equuli 317A. lignieresii 317A. suis 318isolation and identification 317species 317–18

Actinomadura madurae 401–2Actinomadura pelleteri 401–2Actinomyces spp

A. bovis 405A. israelii 318, 405A. meyeri 405A. naeslundii 405A. odontolyticus 405A. pyogenes 354, 406isolation and identification 404water microbiology 257Ziehl–Neelsen staining 404

Actinomyces (Bifidobacterium) eriksonii406

adonitol fermentation 285, 286Advisory Committee on Dangerous

Pathogens (ACDP, 2001) 436Aerococcus spp 342–3

isolation and culture 342properties 334

Aeromonas spphydrophila group 280isolation 280properties 260, 279–80salmonicida group 280

aesculin hydrolysis 93, 317–18aflatoxins 217

Aspergillus flavus-oryzae 423–4agar 63

see also culture mediaagar dilution methods

breakpoints 181MIC 178–9

agglutination tests 101–2, 139, 336air sampling 246–7

Aspergillus 132moulds 132

airmailing, specimen transport, safetyprecautions 9–11

Albert’s stain 350Laybourn’s modification 91

albumin, in media 169Alcaligenes spp

butter 240isolation and identification 269–70milk 235, 236properties 260species 270

Alcaligenes faecalis 270alcohols, as disinfectants 53aldehyde disinfectants, properties 51,

52Alicyclobacillus spp 223alkaline peptone water (APW) 274, 281

formula 75alkaline phosphatase (ALP) test, milk

234–5Allescheria see ScedosporiumAltenaria spp 418, 419ammonia test 93ammonium salt ‘sugars’ 73anaerobes

antibiogram patterns 368BSAC tests 174carbohydrate fermentation test 368in cheese 241cocci 368CO[U]2[u] culture 84–5counting methods 150culture methods 83–5, 161fastidious anaerobe (FA) agar 367Gram-negative bacilli and cocci

367–9identification 368incubation 85low-acid canned foods 221‘mixed’: reporting 368

phosphomycin test 368potassium hydroxide test 368redox indicators 84UV fluorescence 368Wilkins–Chaldren agar 174, 368

anaerobic cabinets 84anaerobic ‘sugars’ 73Anaerococcus 368anthrax 362–3antibiogram patterns, anaerobes 368antibiotic selective agents 66–7antibody detection, mycological

methods 136antifungal drugs 139–42

assays 141–2disc diffusion methods 140E test methods 140–1M27-A2 and M38-A 139susceptibility testing 141–2

antigen detection, commercial kits 138–9antimicrobial susceptibility tests

168–86beta-lactamase test 183breakpoint methods 169, 181–2comparative methods 177–80direct susceptibility tests 183disc diffusion methods 168, 171–6disc storage 170E-test 182–3incubation 170–1inoculum size 169–70media 169MIC/MBC 168–9purity plates 181quality assurance 183–5time–kill curves 180tolerance 181

API 2ONE kit, pseudomonads 262API 2YM system 270API 20E kit, bacilli 365API 32 ATB kit 368API 50 CHB kit 363, 365API CORYNE kit 354Approved Codes of Practice (ACoPs)

435–6arginine broth 72–3arginine dihydrolase test 277, 280, 285arginine hydrolysis 93arginine test 264, 275Arthrinium spp 420arylsulphatase test 391

phenolphthalein sulphate agar 75

Index

Page numbers in bold type refer to tables.

Aschaffenberg–Mullen test, milk 235ascospores 415Aspergillus spp 422–4

A. flavus-oryzae group 423–4A. fumigatus 423A. glaucus group 423A. niger 423A. restrictus group 423air sampling 132commercial antibody detection

methods 138–9morphology 422

ATP measurement, automated methods122–3

Aureobasdium pullulans 420autoclaves 35, 46–50

British Standards 43gravity displacement 47–9plastics 40testing 48–9

automated methods 110–28ATP measurement 122–3blood culture instrumentation

116–20direct epifluorescence techniques

(DEFT) 123, 150flow cytometry 123identification and antimicrobial

susceptibility 110–16impedance instrumentation 121–2molecular microbiology 124–7PCR technology 125–6pyrolysis mass spectrometry 124urine examination 120–1

autopsy material, specimens 163auxanograms 86

yeasts 135

baby foods 225Bacillus spp 362–6

B. anthracis 362–3B. cereus 364, 365

dehydrated vegetables 215food poisoning 188, 191, 363–4in milk 243plating media 203

B. circulans 364, 366B. coagulans 364, 366B. licheniformis 364, 365

‘ropey’ bread 217B. macerans 364, 366B. megaterium 364, 365B. mesentericus see Bacillus subtilisB. polymyxa 364, 366B. pumilis 364, 365B. sphaericus 364, 366B. stearothermophilus 364, 365

flat-sour spoilage 222, 365see also thermophilic bacteria

B. subtilis 364, 365‘ropey’ bread 217

food poisoning/spoilage 363–5isolation 363rapid staining 363–4

bacitracin disc, streptococci 337bacon and cured meat 210–11

canning 223organisms 200

BacT/Alert blood culture automatedmethod 118–19

BACTECblood culture automated methods

116–19, 384pyrazinamide susceptibility tests

386, 397Bacteroides spp 367

culture 161–2Bacteroides urealyticus 367Bactofoss, ATP measurement 122–3Bactometer, impedance system 122Bactoscan, direct epifluorescence

technique 123Baird–Parker carbohydrate medium

203, 217, 237, 329–30formulation 73modification 74

ballistospores 415Bartonella bacilliformis 408Bartonella henseliae 408Bartonella quintana 408Basic (risk) laboratory 3Basidiobolus 430baskets 42bathing beaches 258

swimming pools 257BCG (bacillus of Calmette and Guérin)

387BCIG agar 231beta-lactamase test 183

extended spectrum beta-lactamases(ESBLs) 183

Bifidobacterium eriksonii 406Biolog automated method 113–15biological indicators, autoclaves 49bioluminescence, ATP automated

measurement 122–3biotechnology plant 249bioterrorism and biosecurity 10, 442Blastomyces dermatiditis 432, 433Blastoschizomyces spp 416

differentiation 414bleaches (hypochlorite disinfectants)

51, 52blenders 36blood culture, M. tuberculosis complex

382blood culture automated methods

116–20, 157BACTEC 116–19ESP 119–20

blood cultures, likely pathogens 157–8blood-borne infections, safety

precautions 7blue-green algae 258Boas–Oppler bacillus 347bone marrow culture, M. tuberculosis

complex 382bone meal, anthrax 362–3Bordetella spp

B. bronchiseptica 307B. parapertussis 307B. pertussis 307

isolation and identification 306–7properties 260, 303, 306

Borrelia sppB. burgdorferi 410B. duttonii 410B. recurrentis 410

Botrytis cinerea 419, 420bottles/containers 248–50Bowie–Dick autoclave tape 49brain–heart agar 362Branhamella see Moraxellabread, ropey 217breakpoints, antimicrobial susceptibility

169, 181–2BSAC 176quality assessment 182quality assurance 184–5reading and interpretation 169,

181–2Brevundimonas diminuta 263, 267Brevundimonas vesicularis 263, 267brilliant green agar (BGA) 242, 255,

295brilliant green bile lactose broth (BGBB)

231, 234, 284British Society for Antimicrobial

Chemotherapy (BSAC)anaerobes 174breakpoints 176controls 172, 173disc diffusion method 171–4inoculum 172, 173

British Standards, laboratory equipment43–4

Brochothrix campestris 358Brochothrix thermosphacta 358

on meat 207–9plating media 202

Brucella spp 303–6detection in milk 236, 305whey agglutination test 306

Brucella abortus, undulant fever 236,305

Brucella melitensis, brucellosis 305Brucella suis, contagious abortion 305brucellosis, humans 305bubonic plague 318buffer salts 63buffered charcoal yeast extract (BCYE)

agar 256buffered peptone water 76buffered yeast extract agar 174Bunsen burners 37Burkholderia spp 265–6

B. cepacia 266B. mallei 267B. pseudomallei 266–7B. thailandensis 267reactions in biochemical tests 263

burns, specimens 165Buruli ulcer 393butter 239–40

see also dairy productsbutyric blowing, cheese 241Byssochlamys fulva 219, 223Byssochlamys nivea 219

446 Index

CAC, food for babies and children 225cake mixes 216calcium ion, in media 169calgon Ringer’s 76CAMP test 93–4, 357Campylobacter spp 303, 314–15

C. coli 315C. fetus 315C. hyointestinalis 315C. jejuni 315C. lari 315C. sputorum 315CLO-test 316food poisoning 188, 190, 212plating media 203in water 255, 256

Candida spp, differentiation 414Candida albicans

ATCC 140, 142commercial antibody detection

methods 138–9germ tube test 134

Candida dubliniensis 134Candida kefyr (NCPF) 140, 414Candida parapsilosis 414canned foods 219–23

‘blown’ cans 220–1leaker spoilage 222, 223low-acid 220, 221

capacitance, impedance instrumentation121

Capnocytophaga 367capsule stains 91carbohydrate fermentation test 73, 94,

276, 368carbohydrate oxidation 94carcass sampling 205–7Cardiobacterium spp 303caries 348Carnobacterium spp 348carrion fever 408casein hydrolysis 94Castenada system 157, 304cat-scratch fever 408catalase test 94, 389cefoxime tellurite sorbitol MacConkey

(CT-SMAC) agar 203, 256centrifuges 28–9

British Standards 43cephaloridine fucidin cetrimide (CFC)

medium 203cereals 216–17cerebral phaeohyphomycosis 431cerebrospinal fluid (CSF) 158–9,

379–80culture 382–3

cervical adenitis 391Chaetomium spp 419, 420charcoal agar 306charcoal yeast extract (BCYE) agar 256cheeses 240–1

anaerobes 241butyric blowing 241organisms 200–1, 240–1propionibacteria 241starters 240

chemical disinfection 50–4chemical selective agents 66Chemunex, flow cytometry 123Chlamydia trachomatis, eye discharges

159chlamydospores 415chloral hydrate agar 301chocolate 216cholera 160Christie, Atkins and Munch-Petersen

(CAMP) test 357Chromobacterium spp, properties 260Chromobacterium violaceum 271chromogenic agars 135Chrysosporium spp 419, 421citrate agar 284citrate utilization test 95Citrobacter spp

C. freundii 288C. koseri 288general properties 283, 285see also coliforms

Cladophialophora spp 419, 420, 431C. bantiana 431C. carrionii 431

Cladosporium spp 206, 419, 420, 431classifications

hazardslevels of containment 3risk basis 1–2

clean air work stations 37clean rooms, pharmaceuticals 250clinical material see specimensCLO-test 316Clostridium spp 370–8

C. botulinumcanned foods 219food poisoning 191

C. butyricum, cheeses 241C. estertheticum, meat 207C. perfringens

cake mixes 216dehydrated vegetables 215food poisoning 160, 188, 191

C. thermosaccharolyticum, hardswell 223

reinforced clostridial medium (RCM)249

sulphite-reducing 255cloths/towels 249clue cells 312clumping factor test, S. aureus 330coagulase test 95COBAS Amplicor PCR technology 126Coccidioides immitis 433–4coconut, desiccated 218cold storage, psychrotrophs 207, 225,

241coliforms 283, 284–8

bathing beaches 258defined 282in icecream 243isolation and identification

284–7short set method 286

media 284

in milk 234, 236swimming pools 257in water 251–3see also Citrobacter; E. coli;

Enterobacteriaceae; Klebsiella;most probable number (MPN)methods

Colilert-18 medium 254Comamonas see Delftia acidovoranscombustible waste

incineration 59plastic bags 57–8

commercial kits see kitsconductance, impedance

instrumentation 121Conidiobolus 430contagious abortion 305Containment (risk) laboratory 3–5Control of Substances Hazardous to

Health Regulations (2002,COSHH) 436, 438

conventional tests 93–101cook–chill catering 224Corynebacterium spp 350–4

C. bovis 354C. diphtheriae 350, 352–3

gravis/intermedius/mitis types 353C. jeikeium 353C. kutscheri (C. murium) 354C. pseudodiphtheriticum 353C. pseudotuberculosis (C. ovis) 354C. renale 354C. ulcerans 353C. xerosis 353coryneform bacilli 354diphtheroids 350–1isolation and identification 353–4species from animal sources 353–4species from human sources 352–3see also diphtheria group;

Rhodococcus spp.COSSH (Control of Substances

Hazardous to Health Regulations2002) 436, 438

CO[U]2[u] cultureanaerobes 84–5blood cultures 157

CO[U]2[u] incubators 27–8counterimmunoelectrophoresis 137counting methods 144–55

counting chamber 144–5dip slides 150direct vs viable counts 144–5drop count method 148errors 144membrane filter counts 149most probable number (MPN)

methods 150–4plate counts 146–7roll-tube count 148spiral plate method 149spread-drop plate count 149surface count method 147–8

Craigie tubes 362–3motility 98sloppy agar 76

Index 447

creamimitation 237pasteurized/sterilized/UHT 236samples

coliform count 236–7pathogens 237total viable count 234

see also dairy productsCrohn’s disease 391Cryptococcus spp

C. albidus 415C. laurentii 415C. neoformans 414–15differentiation 414

Cryptosporidium spp 255, 258culture media/reagents 61–78

common components 62–3complex polypeptides in 67–8defined vs undefined 63–4future of 71–2history 61–2identification media 78laboratory-prepared media 72–8management 15performance testing 70–1plating technique 77–8protection of prepared plates 69–70quality assurance 15–16quality standards 71selective and elective media 66–7storage and protection 68–9stressed organisms and resuscitation

64–6culture methods 81–8

anaerobes 83–5, 161auxanograms 86culture recognition 85–6general bacterial 81–3preservation of cultures 87–8serial subculture 87stock cultures 86–7

culture procedures 92–3cyanobacteria 258cycloserine cefotoxin egg yolk fructose

agar 160cystine lactose electrolyte-deficient

(CLED) medium 276, 277, 284,301

cystine selenite broths 242, 295cytochrome oxidase test 99Czapek–Dox agar 132

dairy equipment 236dairy products 233–44

organisms 200–1plating media 202–3

Debaromyces spp 417decarboxylase tests 95, 275–7decontamination

and disposal of waste 55–9laboratory equipment 24safety cabinets 34–5

dehydrated fruit and vegetables 215Delftia acidovorans 263, 267dental disease 348dental specimens 159

deoxycholate agar (DCA) 318depuration, shellfish 231dermatophytes 426–30

appearance 426–7identification 427

de(s)oxycholate citrate agar (DSA) 160Hynes 295

desserts, instant 216Desulfotomaculum nigrificans 223DG18 agar 216diagnostic mycobacteriology, nucleic

acid-based technology 397–9diagnostic specimens, safety precautions

7–102,4-diamino-6,7-diisopropyl pteridine

(0/129) 274, 276dichloran glycerol medium (DG18)

131, 203dichloran Rose Bengal chloramphenicol

agar (DRBC) 203Diene’s stain, mycoplasmas 92diluents 146dilutions 146dinoflagellates 232dip slides 150diphtheria group 350–3

isolation and identification 350–2plate toxigenicity test 351–2species from human sources 350–3see also Corynebacterium spp

direct epifluorescence techniques(DEFT), automated methods 123,150

direct susceptibility, antimicrobials 183disc diffusion methods

antifungals 140antimicrobial susceptibility 168,

171–6disc storage 170quality assurance 183–4Stokes’ method 177–8

discard bags/bins/jars 56–7, 58–9disinfectants 50–4

properties compared 51testing 53–4

disposal of waste 55–9DNA amplification-based technology

125–6DNA fingerprinting see typingDNA probe-based technology 124DNase test 95Doderlein’s bacillus 347Donovan’s medium, formulation 74Dorset’s egg medium 74double diffusion test 136–7drains 295dried fruit and vegetables 215dried infant feeds 225drop count method 148dry discard jars 57Ducrey’s bacillus 311Durham tubes 237, 254

E testantifungal drugs 140–1antimicrobial susceptibility 182–3

ear/eye discharges, specimens 159–60EC Directives

pasteurizationeggs 242milk 233

in vitro diagnostic devices (IVDs)20–2, 26–7

water 251education see staff trainingEdward’s medium, streptococci 335Edwardsiella spp, general properties

283egg medium, Dorset’s 74egg yolk salt broth/agar 74eggs 241–2Ehrlich’s method, indole formation 97elective media 67Elek’s plate toxigenicity test, diphtheria

351–2ELISA tests 138, 139EMJ medium 75

Leptospira 75Endo medium 284Enterobacter spp 283, 285, 288–9

E. aerogenes 283, 285, 289E. cloacae 283, 285, 288–9E. faecalis 172, 173E. liquefaciens 235E. sakazaki 225

infant foods 225see also coliforms; Enterobacteriaceae

Enterobacteriaceae 282–3, 284–9antigens 287bile salts in VRB/VRBG agar 224breakpoints 175BSAC disc diffusion (E. faecalis) 173in fresh flour 217gluconate broth 74as indicator organisms 195in infant feeds 225on meat 206plating media 202–3on poultry 212properties 260, 283see also coliforms

Enterococcus spp 340–2breakpoints 175E. avium 341E. casseliflavus 342E. durans 341E. faecalis 341E. faecium 341E. gallinarum 342E. malodoratus 342E. mundtii 342isolation and culture 341plating media 202properties 334

Entomophthora coronata 430Entomophthorales 430environmental microbiology 245–50

air sampling 246–7bottles/containers 248–50surface sampling 245–6

Epidermophyton floccosum 430epifluorescence techniques, direct

448 Index

(DEFT), automated methods 123,150

equipmentbottles/containers 248–50British Standards 43–4

Erwinia spp, general properties 283Erysipelothrix spp 348–9

E. rhusiopathiae 348–9E. tonsillarum 348–9isolation and identification 348

Escherichia coli 284–8BSAC disc diffusion method 173cake mixes 216control strain 172dehydrated vegetables 215food poisoning 188, 192general properties 283, 285, 287isolation and identification 284–7MIC breakpoints 176MPN method, shellfish 231pathotypes 287plating media 202serological testing 287–8see also butter; cheese; cream; eggs;

milkEscherichia coli O157 288

meat 207in water 256

ESP blood culture automated methods119–20

estimation see counting methodsEuropean Commission, directives see

EC DirectivesExophiala spp 430–1EXPERT automated method 111–12

faecal streptococci 255faeces

culture 382specimens 160

fastidious anaerobe (FA) agar 367feedstuffs, anthrax 362–3Fildes’ extract 311filtration units 50Finegoldia 368fingerprinting see typingfish 229–30

scombroid fish poisoning 232shellfish 231–2

fish tank granuloma 391fission yeasts 415flagella stain 91flat-sour spoilage 222, 365Flavobacterium spp 235, 241, 270

F. meningosepticum 270, 271properties 260

flour 216–17flow cytometry 123fluconazole 140flucytosine (5-fluorocytosine) 140

bioassay 141–2fluorescence microscopy 129fluorescence technology 318

automated methods 112–13fluorescent antibody techniques 102–3Fonsecaea spp 431

food microbiology 194–204investigations 195methods 197–204organisms 200–1plating media 202–3pre-testing 197quality assurance systems 196sampling/plans 196–7standards 195–6

food poisoning 187–93agents 190–3examination of material 193infectivity 187–90public health issues 190

formaldehyde 34as disinfectant 51, 52

fowl typhoid 302Francisella spp 303Francisella tularensis 320–1freezers 30frozen foods

fruit and vegetables 215other 223–5

fruit juices 201, 226fruit and vegetables

dried 215fresh 214–15frozen 215, 223–5

fuchsin stain 90Fuller’s precipitin tube method 336–7fungi

microfungi 257see also antifungals; mycology

Fusarium spp 432Fusobacterium spp 367, 368

Gaffkya (Micrococcus) 332Gaffkya (Peptostreptococcus) tetradius

368beta-galactosidase 253, 276, 285Gardnerella spp 303, 312Gardner’s STAA medium 203, 356,

357gastric lavage 380GC agar 174gelatin, for ice-cream 218gelatin (dry) 217–18gelatin liquefaction test 95–6gelling agents (agar) 63Gemella haemolysans 360Geomyces spp 419, 421Giardia spp 255glassware 39–41

bottles/containers 248–50British Standards 43maintenance 36–7

gluconate broth 74gluconate oxidation 96Gluconobacter spp 272glucose peptone (Sabouraud’s) agar

131, 132glucose tryptone agar 215, 304, 363beta-glucuronidase test 253, 285glutaraldehyde 34

as disinfectants 51, 52glycerol medium 383

gonococcal arthritis 162Gram stain 90Gram-negative anaerobic bacilli 367–8Gram-negative anaerobic cocci 368–9Gram-negative bacilli see coliformsGram-negative rods 260–73Griffiths’tubes 362–3Grocott’s stain 130

H and O antigen suspensions, testingunknown/known sera 101–2

haemadsorption test, mycoplasmas325–6

haemolysis test, mycoplasmas 326haemolytic streptococci, breakpoints

175haemolytic–uraemic syndrome 160Haemophilus spp 303, 310–11

H. aphrophilus 311, 318H. ducreyi 311H. haemoglobinophilus 311H. haemolyticus 311H. influenzae 310–11

breakpoints 176BSAC disc diffusion method 173control strain 172

H. parahaemolyticus 311H. parainfluenzae 311isolation and identification 310media 172

haemorrhagic colitis 160Hafnia spp, general properties 283Hansen’s bacillus see Mycobacterium

lepraeHansen’s paraffin cheese wax 240Haverhill fever 312Hazard Analysis Critical Control Point

(HACCP) system 219hazards

biohazard sign 5group 3 pathogens 131, 431, 432,

441–2incident reporting 440levels of containment 2–5risk assessment, steps 1–5 (HSE)

439–40risk classifications 1–2

Health and Safety at Work Act (1974)435, 437–40

Guidance and Approved Codes ofPractice (ACoPs) 435–6

health surveillance 440–2Helber counting chamber 144–5Helicobacter pylori 303, 315–16hides, anthrax 362–3hippurate hydrolysis test 96, 312histamine, scombroid fish poisoning

232Histoplasma capsulatum 432–3Hoffman’s bacillus 353Hugh and Leifson medium 262, 264,

303, 312, 318formulation 74test 96

human milk 243–4humidifier fever 250

Index 449

hydrogen sulphide 284, 368test 96–7

hydrotherapy pools 257hypochlorite disinfectants, properties

51, 52

ice-creamcoliform count 218total viable count 218, 242–3

identification methods 89–109agglutination tests 101–2conventional tests 93–101culture procedures 92–3fluorescent antibody techniques

102–3media 78microscopy 89–92typing 103–7

identification systems 110–16assessment 116ATCC and NCTC control tests 92

immunization for staff 441immunodiffusion

agar base 136mycological methods 136

immunoelectrophoresis 137impedance instrumentation

automated methods 121–2ompared 122

in vitro diagnostic devices (IVDs) 20–2,26–7

point-of-care-testing (POCT) 21incident reporting 440incubation, antimicrobial susceptibility

170–1incubators/rooms 27–8indicator organisms 195indole production 97, 270, 276, 404infant foods 225

E. sakazaki 225infections

laboratory-associated 5–7primary, secondary, tertiary

barriers 5–7routes 2

inoculating loops/wires 37–8multipoint inoculators 87–8

inoculum size, antimicrobialsusceptibility 169–70

inositol fermentation 285, 286iodophor disinfectants, properties 51,

53iron agar 230, 284Islam’s medium, group B streptococci

334Iso-Sensitest agar 169, 172, 173Iso-Sensitest agar with horse blood

(ISTHBA) 172, 173Isolator-10 lysis centrifugation 382itraconazole

bioassay 142extraction 141

jacuzzi pools 257Jamieson’s plate toxigenicity test,

diphtheria 351–2

Janthinobacterium spp 271properties 260

JK diphtheroids (Corynebacteriumjeikeium) 353

Johne’s disease 390

Kanagawa haemolysis test 278kanamycin aesculin azide medium

(KAA) 203Kaufmann–White system, Salmonella

spp 292ketchups 216Kingella kingii 308King’s B medium 264, 265Kinyoun stain 90–1Kirchner medium 383, 384kits 92–3, 354, 363, 365, 368

antibiogram patterns 368antigen detection 138–9bacilli 365corynebacteria 354pseudomonads 262yeasts 135

Klebsiella spp 283, 285K. aerogenes 235, 283, 285, 289K. oxytoca 285, 288K. ozaenae 285, 288K. pneumoniae 285, 288K. rhinoscleromatis 285, 288media 284

Donovan’s medium 74see also coliforms

Kligler medium 284Kovac’s method, indole formation 97Kovac’s reagent 231, 253, 276

laboratory designBasic/Containment (of risk) 3–5incubator rooms 27–8quality assurance issues 12–13safety cabinets 30–5

laboratory equipment 20–44bench/glass/plastics 37–42bottles/containers 248–50British Standards 43–4capital 27–37capital/consumable, lists 20decontamination 24management 22–6

maintenance/repair 14, 24–5preventive monitoring 14records 23–4, 441–2selection 22–3staff training 24

in vitro diagnostic devices (IVDs)20–2, 26–7

see also named equipmentlaboratory waste, decontamination and

disposal 55–9laboratory-associated infections,

primary, secondary, tertiarybarriers 5–7

laboratory-prepared media 72–8beta-lactamase test 183lactic acid bacteria

in butter 240

on meat 207media 215on pastry 217

lactobacillus 346–8counting lactobacilli 348identification 347isolation 347L. acidophilus 347L. bulgaricus (delbrueckii) 239, 347L. casei 347L. leichmannii 348L. plantarum 347L. thermophilus 235MRS broth 73other species 348species 347–8in yoghurt and milk 235, 239

lactobacillus fermentation medium 73Lactococcus spp 342

isolation and culture 342properties 334

lactophenol–cotton blue 129–30lactose peptone water 253laminar flow (clean air) work stations

37Lancefield’s precipitin tube method,

streptococci 336–7latex agglutination tests 101–2, 139,

336lauryl tryptose broth 284Laybourn’s modification, Albert’s stain

91lecithinase, egg yolk salt broth/agar 74Legionella spp 174, 322–4

L. bozemanii 323L. dumoffii 323L. gormanii 323L. jordanii 323L. longbeachii 323L. micdadei 323L. pneumophila 323serology 174, 323–4in water 256

Leptospira biflexa 411EMJ medium 75

Leptospira interrogans 411Leuconostoc spp 343–4

isolation and culture 343L. cremoris 343–4L. dextranicum 343–4L. lactis 343–4L. mesenteroides 343–4L. paramesenteroides 343–4properties 334

Levan production 97lipolytic activity 97Listeria spp 356–7

L. grayi 357L. innocua 357L. ivanovii 357L. monocytogenes 356–7

cheeses 240–1culture

identification 356–7plating media 203, 356

cured meats 211, 212

450 Index

food poisoning 188, 192vegetables 214see also butter; cream; milk

L. murrayi 357L. seeligeri 357L. welshimeri 357

Listeria selective medium 203, 356Loeffler medium 307, 351–2looping-out culture method 81–2Lowenstein–Jensen (LJ) medium 383,

385, 393pyrazinamide susceptibility tests

386, 397resistance ratio method 394, 395

luciferin, ATP automated measurement122–3

Lyme disease 410lysine decarboxylase medium 297lysine decarboxylase test 277, 285

M27-A2 and M38-A, antifungal drugs139

MacConkey agarcoliforms 284CT-SMAC 203, 256sorbitol 288

MacConkey broth, contraindications 284Madurella spp 430magnesium ion, in media 169mailing, specimen transport, safety

precautions 8–11Malassezia spp 416malonate test 97Malthus 2000, impedance system 122Management of Health and Safety at

Work Regulations (1999) 438de Mann, Rogosa and Sharp agar

(MRS) 203, 215, 249, 347mannitol media

egg-yolk polymixin agar (MEYP)203

lysine crystal violet (MLCV) agar242

selenite broths 242, 295yeast extract agar 271

manuals, standard operating procedure(SOP) 14–15

MAST system 286mastitis 236Maxted’s precipitin tube method 336–7Mayer’s stain 130mayonnaise, in salads 219meat 205–13

British fresh sausages 208–9carcass sampling 205–7comminuted meat 208cured meat 210–11organisms 200–1pies 209–10plating media 202–3vacuum-packed 211–12

mediaantimicrobial susceptibility tests 169

BSAC 172NCCLS 174

plating media 202–3

Medical Devices Agency (MDA) 22Mediterranean fever 305melioidosis 266membrane campylobacter-selective

medium (mCCD) 203membrane enterococcus agar (MEA)

255membrane enterococcus medium (m-E)

203membrane filter count method 149,

248–9faecal streptococci 255sulphite-reducing clostridia 255water 254–5

membrane lauryl sulphate broth (MLSB)254

meningitis, CSF specimens 158meningococcal arthritis 162mercurial compounds, as disinfectants

53metal block heaters 30methicillin-resistant Staphylococcus

aureus (MRSA) 332methylene blue stain 89–904-methylumbelliferyl-beta-D-glucuronide

(MUG) agar 231, 237, 286M’Fadyean’s reaction 362microarrays, molecular microbiology

126–7Microbacterium lacticum 354Micrococcus spp 332–3

see also Aerococcus; PediococcusMicrococcus lactis, milk 235microscope slides 38microscopes 27microscopy 89–92

direct examination of moulds andyeasts 129–31

prepared films 89–92Microsporum audouinii 427, 428Microsporum canis 427, 428Microsporum gypseum 427–9Middlebrook 7H9 medium 382, 383,

384nitrate reduction test 385

Midi identification system 116Miles and Misra count, plating

technique 77–8milk 233–9

buttermilk 239dried 238–9fermented products 239human milk 243–4milk drinks 239M. tuberculosis complex 383pasteurized/sterilized/UHT 233, 235processed 238–9raw 233, 234‘ropey’ 236samples

coliform count 234collection/transport 234microbial content 235–6pathogens 236phosphatase test 234–5turbidity test 235

total viable count 234, 238m, M, n and c 234

milk ring test 305–6milk salt agar (MSA) 329minimum bactericidal concentration

(MBC) 168–9, 180minimum inhibitory concentration

(MIC) 168–9agar dilution methods 178–9broth dilution methods 179–80E test 182–3quality assurance 184see also breakpoint

Mitchison’s antibiotic medium 382, 383Mobiluncus 408modified alkaline peptone water

(MAPW) 274, 281modified lipovitellin-salt-mannitol agar

(M-5LSMA) 257modified minerals glutamate medium

(MMG) 231modified tryptone soya broth (MTSB)

256Moeller’s medium 275molecular microbiology 124–7

DNA fingerprinting 124–5microarrays 126–7moulds 134, 139PCR technology 125–6probe-based technology 124–7

molluscs 231–2Monascus 419, 421Monosporium see ScedosporiumMoraxella spp 303, 307, 359

M. bovis 308M. catarrhalis 308

breakpoints 176M. kingii 308M. lacunata 308M. liquefaciens 308M. nonliquefaciens 308properties 260, 307see also Psychrobacter spp

Morganella morganii 283, 302most probable number (MPN) methods

150–4cream 237dried milk 238shellfish 231Vibrio spp 275water 251–3

motility study 97–8moulds 418–34

common 418–25exoantigen methods 133identification and culture 418pathogenic 425–34

dermatophytes 426–30dimorphic systemic 432–4

plating media 132, 202seed-borne 135slide culture 133

Mucor spp 206, 424–5Mucorales 424–5Mueller–Hinton agar 140, 169, 172,

173, 311

Index 451

MUG (4-methylumbelliferyl-beta-D-glucuronide) agar 231, 237, 286

Muller–Chermack stain 90Muller–Kaufmann tetrathionate broth

(MK-TB) 298multilocus enzyme electrophoresis

(MLEE) 105Mycobacterium spp 379–400

diagnostic nucleic acid-basedtechnology 397–9

drug susceptibility tests 393–7environmental (EM)/opportunistic

387–93growth in N medium 390identification tests 389–90pigment production 389usual properties 388

N medium 75see also tuberculosis/tubercle bacilli

mycobactin 391M. africanum 387M. aurum 392M. avium complex (MAC) 380, 390–1M. bovis 387

identification 384pyrazinamide susceptibility 386

M. branderi 391M. canetti 387M. celatum 391M. chelonae 392M. duvalii 392M. flavescens 392M. fortuitum 392M. gastri 393M. genavense 391M. gilvum 392M. gordonae 392M. haemophilum 393M. intracellulare see M. avium complex

(MAC)M. kansasii 390, 392M. leprae, nasal septum 161M. lepraemurium 390M. malmoense 393M. marinum 391M. nonchromogenicum 393M. phlei 392–3M. scrofulaceum 391M. simiae 393M. smegmatis 392–3M. szulgai 392M. terrae 393M. triviale 393M. tuberculosis complex 386–7

automated culture 384characteristics 385CSF 158, 379–80culture of pathological material

381cycloserine resistance 386diagnostic nucleic acid-based

technology 397–9drug susceptibility tests 393–7ESP blood automated culture

119–20milk 236

Mycobacterial Growth IndicatorTube (MGIT) 384

niacin test 386pyrazinamide susceptibility 386sputum 379, 380, 381–2typing (fingerprinting) 398urine 380, 382variants 385–6Ziehl–Neelsen method 380, 384see also tuberculosis

M. ulcerans 393M. vaccae 392M. xenopi 391mycological methods 129–43

antifungal drugs 139–42direct examination 129–31isolation of moulds and yeasts

131–2molecular methods 139serology 135–9yeasts 134–5

mycoplasmas 324–8diagnostic tests 328Diene’s stain 92haemadsorption test 325–6haemolysis test 326isolation and culture 324–5M. genitalium 325–7M. hominis 325–7M. pneumoniae 325–7

mycotoxins 135food poisoning 192

NAD (V factor) 310Nagler test 98National Committee for Clinical

Laboratory Standards (NCCLS),disc diffusion method 174–6

needle-stick injuries 441Neisseria flavescens 360Neisseria gonorrhoeae 165–6, 359–61

breakpoints 176control strain 172CO[U]2[u] incubation 174media 172

Neisseria lactamica 360Neisseria meningitidis 360

breakpoints 176Neisser’s stain 350niacin test 386nitrate reductase test 98nitrate reduction test 385, 404p-nitro-alpha-acetylamino-beta-

propiophenone (NAP) 384p-nitrobenzoic acid (PNBA) 385, 387o-nitrophenyl-beta-D-galactopyranoside

253nivalenol 217Nocardia spp 401–2

differentiation from Mycobacterium390

N. asteroides 401–2N. brasiliensis 401–2N. nova 401–2N. otitidis caviarum 401–2N. transvalensis 401–2

non-puerperal infections, specimens165–6

nucleic acid-based technologyamplification 125–6diagnostic mycobacteriology 397–9

O antigen suspensions, testingunknown/known sera 101–2

O/129 (2,4-diamino-6,7-diisopropylpteridine) 274

odours 86Omnilog ID automated method 113–15onion powder 218ONPG test 98, 276, 277, 283, 285optochin test 99ornithine decarboxylase test 277, 285Oroya fever 408Oxfam–Del Agua, water testing 258Oxford listeria medium 203oxidase test 99

p-nitrobenzoic acid (PNBA) 385, 387Paecilomyces spp 419, 421Pandoraea see Burkholderia sppPANTA antibiotic medium 383Paracoccidioides brasiliensis 433Paracolobacterium spp 241paraformaldehyde 34PAS stain 130pasta 217Pasteur pipettes 38Pasteurella spp 303

P. gallinarum 320P. haemolytica 320P. multocida 319–20P. pneumotropica 320properties 260

pasteurizationeggs 242milk 233

pastry 217pathogens

hazard 3 grouping 131, 431, 432levels of containment 2–5

PCR technology 125–6, 106–7microarrays 126–7real-time assays 126

Pediococcus spp 344–5isolation and culture 344–5P. acidi-lacti 344P. damnosus 344P. pentosaceus 344P. urinae-equi 344–5

Penicillium spp 206, 422–3cheese 240P. marneffei 434P. purpurogenum 434

peptides vs amino acids, culture media 68Peptococcus niger 368Peptoniphilus 368Peptostreptococcus tetradius 368Petri dishes 40Petriellidium see ScedosporiumpH

adjustment 77media 169

452 Index

pH meters, British Standards 44phage typing 105pharmaceuticals, clean rooms 250phenolic disinfectants, properties 51, 52phenolphthalein phosphate polymixin

agar (PPPA) 329phenolphthalein sulphate agar 75phenylalanine, PPA test 99, 301Phialophora spp 431Phlebotomus, vector of Bartonella

bacilliformis 408Phoenix automated method 115Phoma spp 419, 420phosphatase test 99

milk 234–5S. aureus 331

phosphate-buffered saline 76phosphomycin test, anaerobes 368photochromogens 389phytoplankton 231Pichia spp 417pickles 215pies, meat 209–10pinta 411pipette jars 57pipettes

aids 38–9graduated 38Jencon Micro-Repette 395making dilutions 146, 147Pasteur 38re-usable 59

pipettors 39plague 318plant pathogens 7plastic bags, combustible waste 57–8plastics ware

autoclavable/non-autoclavable 40British Standards 44consumable 39–41

plate counts 146–7plate toxigenicity test, diphtheria group

351–2plating media, food microbiology

202–3plating technique

Miles and Misra count 77–8protection of prepared plates 77–8

Plesiomonas spp, properties 260Plesiomonas shigelloides 279PLET (polymixin lysozyme EDTA agar)

362pleural fluid, culture 382–3pneumococci, differentiation from

streptococci 340Pneumocystis carinii see Pneumocystis

jirovecipoint-of-care-testing (POCT) 21polymerase chain reaction amplification

106–7, 125–6arbitrary primed PCR 107

polymixin mediamannitol egg-yolk polymixin agar

(MEYP) 203phenolphthalein phosphate polymixin

agar (PPPA) 329

polymixin egg-yolk mannitolbromothymol blue agar(PEMBA) 203

polymixin lysozyme EDTA (PLET)brain–heart agar 362

salt polymixin broth (SPB) 274sodium dodecylsulphate polymixin B

sucrose (SPS) agar 75, 278, 312tellurite polymixin egg-yolk agar

(TPEY) 329–30polypeptide mixtures, and ribozymes

68polypeptides

as redox agents 67–8transport 68

Porphyromonas spp 367postmortem material, specimens 163potassium hydroxide test 368potato glucose agar 240poultry 213pour plate culture method 81precipitin tube methods, Streptococcus

spp 336–7Preisz–Nocard bacillus 354prepacked foods 219–23prepared plates, protection 69–70preservation of cultures 87–8

drying 87freeze-drying 87–8freezing 87

Prevotella spp 367primary barriers (to infections) 5–6probe-based technology 124Propionibacterium spp, cheeses 241Propionibacterium acnes 406proportion method, drug susceptibility,

tuberculosis 394proteolysis 100Proteus spp 301–2

general properties 283vs Salmonella 295

Proteus mirabilis 302MIC breakpoints 176

Providencia spp 302general properties 283P. alcalifaciens 302P. rettgeri 302P. stuartii 302

Pseudomonas spp 262–5ammonium salt ‘sugars’ 73isolation and identification 262–4properties 260, 271P. aeruginosa 264–5

antibiotics 265breakpoints 175BSAC disc diffusion method 173characteristics 264–5control strain 172plating media 202on poultry 212serotypes 265swimming pools 257

P. alcaligenes 265P. fluorescens 235, 265P. pseudoalcaligenes 265P. putida 265

P. stutzeri 265reactions in biochemical tests 263

psychrotrophson cheese 241on meat 207sous vide meals 225

puerperal infections, specimens 165pulsed-field gel electrophoresis (PFGE)

105–6purity plates, antimicrobial susceptibility

181pus

culture 382–3specimens 161, 367

pyocyanin 256, 264pyomelanin 262pyorubrin 262pyrazinamide susceptibility tests 386,

397pyrolysis mass spectrometry 124, 390pyruvate medium 383

QACs, disinfectants 51, 53quality assessment 16–18

accreditation 16–18quality assurance 12–16

antimicrobial susceptibility 183–5food microbiology 196

RABIT, impedance system 122racks and baskets 42Ralstonia pickettii 266Rambach agar 203RAMUS automated system, urine 164Rappaport–Vassiliades soya broth

(RVSB) 242, 255, 296recombinant organisms, safety

precautions 7record keeping, safety 23–4, 441–2rectal swabs 160red heat 45redox indicators, anaerobiosis 84refrigerators 30

psychrotrophs 207, 225, 241regulatory safety issues 435–43reinforced clostridial medium (RCM)

249relaying, shellfish 231repetitive extragenic palindromic (REP)

sequence 106–7Reporting of Injuries, Diseases and

Dangerous OccurrencesRegulations (1995, RIDDOR) 438

resistance ratio method, drugsusceptibility, tuberculosis 394,395

restriction fragment lengthpolymorphism 106

resuscitation, of stressed organisms64–6

Rhizopus 424–5Rhodococcus spp 401–2Rhodococcus equi 354, 401–2Rhodotorula spp 415–16

cheese 241Rickettsia burnetti, in milk 236

Index 453

RIDDOR (Reporting of Injuries,Diseases and DangerousOccurrences Regulations (1995)438

rifampicin resistance, rapid detection398

Ringer’s solution 76risk assessment

steps 1–5 (HSE) 439–40see also safety

Robinson’s serum water sugars 73–4Robinson’s sugar medium 350Rochalimaea see Bartonella henseliae

408Rogosa agar 203, 215, 217roll-tube count method 148, 249Rose Bengal chloramphenicol agar

(RBC) 203, 257rotary plating 82Rpo gene, rifampicin resistance 398rubber teats for pipettes 39

Sabouraud’s agar 131, 132, 414Saccharomyces carlsbergensis 416–17Saccharomyces cerevisiae 416–17

differentiation 414Saccharomyces pastorianus 417safety 1–11, 435–43

bioterrorism and biosecurity 10, 442blood-borne infections 7Control of Substances Hazardous to

Health Regulations (2002,COSHH) 436, 438

hazardsgroup 3 pathogens 131, 431, 432,

434, 441–2levels of containment 2–5risk classifications 1–2summary 4

Health and Safety at Work Act(1974), Guidance and ApprovedCodes of Practice (ACoPs)435–6

health surveillance 440–2immunization for staff 441Management of Health and Safety at

Work Regulations (1999) 438needle-stick injuries 441odour sniffing 86prevention of laboratory-associated

infections 5–7primary, secondary, tertiary

barriers 5–7radioactive waste 384records 23–4, 441–2regulatory safety issues 435–43risk assessment, steps 1–5 (HSE)

439–40specimen transport 7–11

safety cabinets 30–5British Standards 43–4classes I, II and III 30–1decontamination 24, 34–5maintenance 35siting 32testing airflows 32–3

salad cream 218salads 214–15

mayonnaise 219saline 76Salmonella serotypes 292–300

agglutination tests 296–7antigenic formulae 297antigens 292–4, 293biochemical reactions 298chocolate products 216classification 294food poisoning 188, 190, 212general properties 283identification 296–9isolation 295–6plating media 203poultry 213Vi antigen 297in water 255see also butter; cheese; cream; eggs;

milksalt polymixin broth (SPB) 274salt tolerance 276sample containers 41Sarcina ventricula 368–9

see also Micrococcussatellitism 310sauces 216sauerkraut 347sausages 208–9Scedosporium spp 432scombroid fish poisoning 232Scopulariopsis spp 419, 421scotochromogens 389–92scrofula 391secondary barriers (to infections) 6selective and elective media 66–7selenite broths 242, 295Sensititre automated method 112–13serial subculture methods 87serology

Escherichia coli 287–8Legionella spp 174, 323–4mycological methods 135–9

antibody detection 136immunodiffusion 136

Streptococcus spp 336typing (fingerprinting) 104–5

serotyping 104–5serous fluids, specimens 162Serratia spp, general properties 283sewage 258sewers 295shake tube cultures 83shakers 36shellfish and crustaceans 231–2Shewanella putrefaciens 207, 241

plating media 202Shigella spp 292–300

agglutination test 299antigens 292–3biochemical reactions 298classification 294food poisoning 192general properties 283identification 296–9

isolation 295–6reference laboratories 298–9S. boydii 300S. dysenteriae (shiga) 299S. flexneri 299S. sonnei 299–300species and serotypes 294–5,

298–300sick building syndrome 250Simmons’ citrate agar 284sloppy agar 76sodium dichlorisocyanate 52sodium dodecylsulphate polymixin B

sucrose (SPS) agar 278, 312sodium ion, in media 169sodium polyethanolium sulphate (SPS)

anticoagulant 382soft drinks 225–6soil, M. tuberculosis complex 383sorbitol MacConkey agar 288Sorensen’s buffer 410‘sous vide’ foods 224–5South American blastomycosis 433specimens 156–67

collection 13–14containers 41dental 159quality assessment 17safety precautions 156

transport 7–11Sphingomonas paucimobilis 263, 267spices 218spiral plating 82

counting 149spirochaetes 410–12Sporobolomyces 416Sporothrix spp 432Sporotrichum spp 206spot test

indole formation 97indole production 404

spread-drop plate count 149spreader culture method 81spreaders 38SPS (sodium dodecylsulphate polymixin

B sucrose) agar 75, 278, 312sputum

culture 381–2M. tuberculosis complex 379, 380specimens 162–3

stab culture methods 83Stachybotrys chartarum 422staff training

immunization for staff 441management of laboratory equipment

24safety cabinets 35safety issues 10

standard operating procedure (SOP)manual 14–15

standard strains, stock cultures 86–7Staphylococcus spp 329–32

breakpoints 175BSAC disc diffusion method 173isolation 329methicillin-resistant (MRSA) 332

454 Index

plating media 203S. aureus 331–2

cake mixes 216CAMP test 357clumping factor test 330control strain 172cooked meats 211food poisoning 188, 191, 330–1phosphatase test 331poultry processing 212swimming pools 257tube coagulase test 330–1see also specific

substances/locationsS. chromogenes 332S. cohnii 332S. epidermidis 332S. hyicus 332S. intermedius 332S. saprophyticus 332S. warneri 332

starch agar 76starch ampicillin agar 281starch hydrolysis 100Stenotrophomonas maltophilia 263,

267sterilization 45–50

autoclave 46–50dry heat 45holding times 46hot air 46red heat 45steam 49–50

stock cultures 86–7Stokes’ method

direct tests 183disc diffusion 177–8

storage bottles 41Streptobacillus spp, S. moniliformis

303, 312Streptococcus spp 334–40

antigens 335cheese starters 240differentiation from pneumococci

340faecal 255groups 337–40

anginosus group 339mitis group 340pyogenic group 337–8

haemolytic species 337isolation and culture 334–5nutritional variants 345precipitin tube methods 336–7properties 334serology 336S. agalactiae 337–40S. bovis 339–40S. canis 338–9S. dysgalactiae 337–40S. equi 338–9S. equinus 339–40S. inae 338–9S. lactis, milk 235S. mutans 339–40S. parasanguis 338–9

S. pneumoniae 338–9breakpoints 175BSAC disc diffusion method 173CO[U]2[u] incubation 174media 172

S. porcinus 338–9S. pyogenes 337–40

milk 236S. salivarius 338–9S. sanguis 338–9S. suis 339–40S. thermophilus 338–9, 342

yoghurt 239S. uberis 338–9S. zooepidemicus 338–9

Streptomyces somaliensis 402stressed organisms, and resuscitation

64–6sugar and confectionery 216sulphatase test 100sulphite polymixin sulphadiazine agar

75sulphite-reducing clostridia 255surface count method 147–8surface sampling, environmental

microbiology 245–6swimming pool granuloma 391swimming pools 257

teats for pipettes 39tellurite media 350

polymixin egg-yolk agar (TPEY)329–30

tellurite reduction 100, 389tertiary barriers (to infections) 6–7test tubes and stoppers 40Thamnidium spp 206thermophilic bacteria 216, 217, 222

spore growth 222thiacetazone susceptibility 389thiosulphate citrate bile salt-sucrose

agar (TCBSA) 256, 274thiosulphate Ringer’s 76Thornley’s arginine broth 262, 264throat swabs 161, 163thymidine, in media 169time–kill curves, antimicrobial

susceptibility 180tinned food see canned foodstissue grinders/homogenizers 39tolerance, antimicrobial susceptibility

181Torulopsis spp, cheese 241total viable count

cheese 234, 240–1ice-cream 242–3m, M, n and c defined 234milk 234, 238

training see staff trainingtransport media 312

transport of specimens 7–11Treponema carateum 411Treponema pallidum 410Treponema pertenue 411tributyrin agar 218Trichoderma 419, 421

Trichomonas vaginalis 165Trichophyton mentagrophytes complex

428, 429Trichophyton rubrum 428, 429Trichophyton schoenleinii 429Trichophyton tonsurans 429, 430Trichophyton verrucosum 429, 430Trichophyton violaceum 429Trichosporon beigelii 416

differentiation 414trichothecenes 217triple sugar iron (TSI) agar 284, 297,

314Tropheryma whippelii 404, 406tryptone soya broth 248, 281

modified 256tryptone water 231, 253tryptose sulphite cycloserine agar

(TSCA) 255tube coagulase test, S. aureus 330–1tuberculosis/tubercle bacilli

bacterial suspensions 395diagnostic nucleic acid-based

technology 397–9identification of isolated

mycobacteria 398mycobacteria in clinical specimens

398rifampicin resistance 398typing (fingerprinting) 398

drug susceptibility tests 393–7absolute concentration method

394automated methods 396–7control strains 394–5drug concentrations 395modal resistance 396proportion method 394resistance ratio method 394

therapy (WHO) 393see also M. tuberculosis complex;

Mycobacterium spptularaemia 321turbidity test, milk 235Tween hydrolysis 100, 389tyndallization, steam sterilization

49–50typing (fingerprinting) 103–7

multilocus enzyme electrophoresis(MLEE) 105

phage typing 105polymerase chain reaction

amplification 106–7pulsed-field gel electrophoresis

(PFGE) 105–6reasons for typing 103requirements of targets 103–4restriction fragment length

polymorphism 106serotyping 104–5tuberculosis 398typing targets 103

tyrosine agar 75tyrosine decomposition 100

Ulocladium spp 418, 419

Index 455

undulant fever 305urea breath test 316Ureaplasma urealyticum 325, 326, 327urease test 100urethral discharges, specimens 163–4urine culture, M. tuberculosis complex

380, 382urine examination, automated methods

120–1, 164–5urines, disposal 59utensils/containers 248–50UV fluorescence, anaerobes 368

V factor (NAD) 310vacuum-packed meat 211–12vaginal discharges, specimens 165, 312vancomycin-resistant enterococci (VRE)

343vats and hoppers 249VBNC organisms 65vegetables see fruit and vegetablesVeillonella spp 369vending machines 226veronal buffer 137viable non-culturable (VBNC)

organisms 65Vibrio spp 274–9

alkaline peptone water (APW) 75enumeration 275growth on thiosulphate citrate bile

salt agar 275O/129 sensitivity 277properties 260, 276–8V. alginolyticus, properties 278–9V. anguillarum 278V. cholerae

O:139 (Bengal) 278properties 276–8serovar O:1 276, 278water 256

V. damsela 278V. fluvialis 278V. furnissi 278V. hollisae 278V. metschnikovii 278V. mimicus 278V. parahaemolyticus

food poisoning 192, 232isolation 274properties 278

V. vulnificus, properties 276–8Vincent’s angina 367, 410–11violet red bile lactose agar (VRBL) 203,

234VITEK automated method 110–12Voges–Proskauer reaction (VP) 100–1,

276

water microbiology 251–9coliforms, MPN 251–4colony counts 251EC Directives 251membrane filter method 254–5microfungi and actinomycetes 257M. tuberculosis complex 383pathogens 255–7standards, various sources 251–2testing in remote areas 258see also coliforms

waterbaths 29–30Wayne’s medium 386Weil’s disease 411whey agglutination test 306Whipple’s disease 404, 406whooping cough 307Wilkins–Chaldren agar, anaerobes 174,

368woolsorter’s disease 362wounds/ulcers, specimens 166

X factor 310xanthine agar 75xanthine decomposition 101xylose–lysine desoxycholate citrate agar

(XLD) 160, 242, 255, 295Ryan’s 281

yaws 411yeast extract agar 248, 251yeast morphology agar 140yeasts 134–5, 413–17

apiculate 417auxanograms 135fermentation tests 134germ tube test 134identification key 413morphology 134, 415plating media 202

Yersinia spp 318–19isolation and identification 318–19properties 260, 303Y. enterocolitica 318–19

food poisoning 192in milk 236

Y. fredericksenii 319Y. intermedia 319Y. kristensenii 319Y. pestis 318Y. pseudotuberculosis 319

Yersinia selective agar 160yoghurt 239

Ziehl–Neelsen method 380, 384, 404Ziehl–Neelsen stain 90Zygomycotina 424–5Zygosaccharomyces baillii 216, 417Zygosaccharomyces mellis 417

456 Index

Documents

Microbiological Methods