11th Edition

Important Notice

● Product labels/package inserts take precedence over the formula or instructions listed in the manual.

● Generic names may be substituted for trade names in the ingredient list, providing more informationregarding animal origin, i.e. “pancreatic digest of casein” instead of Casitone.

● During 1999, catalog numbers will be changed to meet new UCC/EAN128 labeling requirements.Please visit us at www.bd.com/microbiology for more information.

Table of Contents

Foreword ................................................................................................... vii

Introduction................................................................................................. ix

Monographs .................................................................................................1

Culture Media and Ingredients, Dehydrated .............................................. 19

Culture Media, Prepared .......................................................................... 585

Stains and Indicators ................................................................................ 595

Serology and Immunology....................................................................... 607

Reference Guides ..................................................................................... 811

Indices ...................................................................................................... 843

Alphabetical Index .............................................................................845

Numerical Index .................................................................................855

vi The Difco Manual

First Edition 1927

Second Edition 1929

Third Edition 1931

Fourth Edition 1933

Fifth Edition 1935

Sixth Edition 1939

Seventh Edition 1943

Eighth Edition 1948

Ninth Edition 1953Reprinted 1953Reprinted 1956Reprinted 1958Reprinted 1960Reprinted 1962Reprinted 1963Reprinted 1964Reprinted 1965Reprinted 1966Reprinted 1967Reprinted 1969Reprinted 1971Reprinted 1972Reprinted 1974Reprinted 1977

Tenth Edition 1984Reprinted 1985Reprinted 1994Reprinted 1996

Eleventh Edition 1998

Copyright 1998 by

Difco Laboratories,Division of Becton Dickinson and Company

Sparks, Maryland 21152 USA

The Difco Manual vii

Foreword

This edition of the DIFCO MANUAL, the eleventh published since 1927, has been extensively revised and

rewritten. The purpose of the Manual is to provide information about products used in microbiology. The

Manual has never been intended to replace any official compendium or the many excellent standard text

books of scientific organizations or individual authors.

Difco is perhaps best known as the pioneer in bacteriological culture media. Numerous times one will find

the trademarks Difco® or Bacto® preceding the names of materials used by scientists in their published

papers. Because Difco products have been readily available worldwide longer than any others, Difco

products have become the common-language reagents of the microbiological community. Standardized

products readily available worldwide are essential for corroborative studies demanded by rigorous science.

Recommendation and approval have been extended to our products by the authors of many standard text

books and by the committees on methods and procedures of scientific societies throughout the world. Difco

products continue to be prepared according to applicable standards or accepted formulae. It is expected that

they will be used only by or under the supervision of microbiologists or other professionals qualified by

training and experience to handle pathogenic microorganisms. Further, it is expected that the user will be

throughly familiar with the intended uses of the formulations and will follow the test procedures outlined in

the applicable official compendia and standard text books or procedures manual of the using laboratory.

Grateful acknowledgment is made of the support we have received from microbiologists throughout the

world. It is our desire to continue and extend our services to the advancement of microbiology and related

sciences.

Difco Laboratories

Division of Becton Dickinson and Company

Foreword

The Difco Manual ix

Introduction

IntroductionMicrobiology, through the study of bacteria, emerged as a definedbranch of modern science as the result of the monumental and immortalresearch of Pasteur and Koch. In 1876, Robert Koch, for the first timein history, propagated a pathogenic bacterium in pure culture outsidethe host’s body. He not only established Bacillus anthracis as theetiological agent for anthrax in cattle, but he inaugurated a method ofinvestigating disease which ushered in the golden age of medicalbacteriology.

Early mycologists, A. de Bary and O. Brefeld, and bacteriologist,R. Koch and J. Schroeter, pioneered investigations of pure culturetechniques for the colonial isolation of fungi and bacteria on solidmedia. Koch, utilizing state-of-the-art clear liquid media which hesolidified with gelatin, developed both streak and pour plate methodsfor isolating bacteria. Gelatin was soon replaced with agar, a solidifyingagent from red algae. It was far superior to gelatin in that it wasresistant to microbial digestion and liquefaction.

The capability of Koch to isolate disease-producing bacteria on solidi-fied culture media was further advanced by manipulating the culturalenvironment using meat extracts and infusions so as to reproduce, asclosely as possible, the infected host’s tissue. The decade immediatelyfollowing Koch’s epoch-making introduction of solid culture mediafor the isolation and growth of bacteria ranks as one of the brightest inthe history of medicine because of the number, variety, and brilliance ofthe discoveries made in that period. These discoveries, which, as Kochhimself expressed it, came “as easily as ripe apples fall from a tree,”were all dependent upon and resulted from the evolution of correctmethods for the in vitro cultivation of bacteria.

The fundamental principles of pure culture isolation and propagationstill constitute the foundation of microbiological practice and research.Nevertheless, it has become more and more apparent that a successfulattack upon problems unsolved is closely related to, if not dependentupon, a thorough understanding of the subtle factors influencingbacterial metabolism. With a suitable culture medium, properlyused, advances in microbiology are more readily made than wheneither the medium or method of use is inadequate. The microbiologistof today is, therefore, largely concerned with the evolution of methodsfor the development and maintenance of microbial growth upon whichan understanding of their unique and diversified biological andbiochemical characteristics can be investigated. To this end, microbi-ologists have developed innumerable enrichment culture techniquesfor the isolation and cloning of microorganisms with specific nutri-tional requirements. These organisms and their unique characteristicshave been essential to progress in basic biological research and modernapplied microbiology.

The study of microorganisms is not easy using microscopic single cells.It is general practice to study pure cultures of a single cell type. In thelaboratory, microbiological culture media are utilized which containvarious nutrients that favor the growth of particular microorganisms inpure cultures. These media may be of simple and defined chemicalcomposition or may contain complex ingredients such as digests ofplant and animal tissue. In particular, the cultivation of bacteria is

dependent upon nutritional requirements which are known to varywidely. Autotrophic bacteria are cultivated on chemically defined orsynthetic media while heterotrophic bacteria, for optimal growth, mayrequire more complex nutrients such as peptones, meat or yeastextracts. These complex mixtures of nutrients readily supply fastidiousheterotrophic bacteria with vitamins and other growth-promotingsubstances necessary for desired cultivation. The scientific literatureabounds with descriptions of enriched, selective and differentialculture media necessary for the proper isolation, recognition andenumeration of various bacterial types.

Almost without exception whenever bacteria occur in nature, and thisis particularly true of the pathogenic forms, nitrogenous compoundsand carbohydrates are present. These are utilized in the maintenanceof growth and for the furtherance of bacterial activities. So complex isthe structure of many of these substances, however, that before theycan be utilized by bacteria they must be dissimilated into simplercompounds then assimilated into cellular material. Such metabolicalterations are affected by enzymatic processes of hydrolysis, oxidation,reduction, deamination, etc., and are the result of bacterial activities ofprimary and essential importance. These changes are ascribed to theactivity of bacterial enzymes which are both numerous and varied. Theprocesses involved, as well as their end-products, are exceedinglycomplex; those of fermentation, for example, result in the productionof such end-products as acids, alcohols, ketones, and gases includinghydrogen, carbon dioxide, methane, etc. The study of bacterialmetabolism, which defines the organized chemical activities of a cell,has led to the understanding of both catabolic or degradative activitiesand anabolic or synthetic activities. From these studies has come abetter understanding of the nutritional requirements of bacteria, and inturn, the development of culture media capable of producing rapid andluxuriant growth, both essential requisites for the isolation and studyof specific organisms.

Studies to determine the forms of carbon, hydrogen, and nitrogen whichcould most easily be utilized by bacteria for their development wereoriginally carried on by Naegeli1 between 1868 and 1880, and werepublished by him in the latter year. Naegeli’s report covered the use ofa large variety of substances including carbohydrates, alcohols, aminoacids, organic nitrogen compounds, and inorganic nitrogen salts.

The first reference to the use of peptone for the cultivation of microor-ganisms is that made by Naegeli in the report referred to above, whenin 1879, he compared peptone and ammonium tartrate. Because of itscontent amino acids and other nitrogenous compounds which arereadily utilized by bacteria, peptone soon became one of the mostimportant constituents of culture media, as it still remains. In the lightof our present knowledge, proteins are known to be complex compoundscomposed of amino acids joined together by means of the covalentpeptide bond linkage. When subjected to hydrolysis, proteins yieldpolypeptides of various molecular sizes, metapeptones, proteoses,peptones and peptides, down to the level of simple amino acids. Theintermediate products should be considered as classes of compounds,rather than individual substances, for there exists no sharp lines ofdemarcation between the various classes. One group shades byimperceptible degrees into the next. All bacteriological peptones, thus,are mixtures of various products of protein hydrolysis. Not all the

x The Difco Manual

products of protein decomposition are equally utilizable by allbacteria. In their relation to proteins, bacteria may be divided into twoclasses; those which decompose naturally occurring proteins, and thosewhich require simpler nitrogenous compounds such as peptones andamino acids.

The relation of amino acids to bacterial metabolism, and the ability ofbacteria to use these compounds, have been studied by many workers.Duval,2,3 for example, reports that cysteine and leucine are essential inthe cultivation of Mycobacterium leprae. Kendall, Walker and Day4

and Long5 reported that the growth of M. tuberculosis is dependentupon the presence of amino acids. Many other workers have studied therelation of amino acids to the growth of other organisms, as for example,Hall, Campbell, and Hiles6 to the meningococcus and Streptococcus;Cole and Lloyd7 and Cole and Onslow8 to the gonococcus; and Jacobyand Frankenthal9 to the influenza bacillus. More recently Feeley, etal.34 demonstrated that the nonsporeforming aerobe, Legionellapneumophila requires L-cysteine . HCI for growth on laboratory media.Indispensable as amino acids are to the growth of many organisms,certain of them in sufficient concentration may exert an inhibitoryeffect upon bacterial development.

From the data thus far summarized, it is apparent that the problemof bacterial metabolism is indeed complicated, and that the phaseconcerned with bacterial growth and nutrition is of the utmost practicalimportance. It is not improbable that bacteriological discoveries suchas those with Legionella pneumophila await merely the evolution ofsuitable culture media and methods of utilizing them, just as in the pastimportant discoveries were long delayed because of a lack of similarrequirements. Bacteriologists are therefore continuing to expend muchenergy on the elucidation of the variations in bacterial metabolism,and are continuing to seek methods of applying, in a practical way, theresults of their studies.

While the importance of nitrogenous substances for bacterial growthwas recognized early in the development of bacteriological technique,it was also realized, as has been indicated, that bacteria could notalways obtain their nitrogen requirements directly from protein. Itis highly desirable, in fact essential, to supply nitrogen in readilyassimilable form, or in other words to incorporate in media proteinswhich have already been partially broken down into their simpler andmore readily utilizable components. Many laboratory methods, suchas hydrolysis with alkali,10 acid,11,12,13 enzymatic digestion,8,14,15,16,17,18

and partial digestion of plasma10 have been described for the preparationof protein hydrolysates.

The use of protein hydrolysates, particularly gelatln and casein, hasled to especially important studies related to bacterial toxins byMueller, et al.20-25 on the production of diphtheria toxin; that of Tamura,et al.25 of toxin of Clostridium welchii; that of Bunney and Loerber27,28

on scarlet fever toxin, and of Favorite and Hammon29 on Staphylococcusenterotoxin. In addition, the work of Snell and Wright30 on themicrobiological assay of vitamins and amino acids was shown tobe dependent upon the type of protein hydrolysate utilized. Closelyassociated with research on this nature are such studies as those ofMueller31,32 on pimelic acid as a growth factor for Corynebacteriumdiphtheriae, and those of O’Kane33 on synthesis of riboflavin bystaphylococci. More recently, the standardization of antibiotic suscep-tibility testing has been shown to be influenced by peptones of culture

media. Bushby and Hitchings35 have shown that the antimicrobial ac-tivities of trimethoprim and sulfamethoxazole are influenced consider-ably by the thymine and thymidine found in peptones of culture media.

In this brief discussion of certain phases of bacterial nutrition, we haveattempted to indicate the complexity of the subject and to emphasizethe importance of continued study of bacterial nutrition. Difco Labo-ratories has been engaged in research closely allied to this problem inits broader aspects since 1914 when Bacto Peptone was first introduced.Difco dehydrated culture media, and ingredients of such media, havewon universal acceptance as useful and dependable laboratory adjunctsin all fields of microbiology.

References1. Sitz’ber, math-physik. Klasse Akad. Wiss. Muenchen, 10:277,

1880.2. J. Exp. Med., 12:46, 1910.3. J. Exp. Med., 13:365, 1911.4. J. Infectious Diseases, 15:455, 1914.5. Am. Rev. Tuberculosis, 3:86, 1919.6. Brit. Med. J., 2:398, 1918.7. J. Path. Bact., 21:267, 1917.8. Lancet, II:9, 1916.9. Biochem, Zelt, 122:100, 1921.10. Centr. Bakt., 1:29:617, 1901.11. Indian J. Med. Research, 5:408, 1917-18.12. Compt. rend. soc. biol., 78:261, 1915.13. J. Bact., 25:209, 1933.14. Ann. de L’Inst., Pasteur, 12:26, 1898.15. Indian J. Med. Research, 7:536, 1920.16. Sperimentale, 72:291, 1918.17. J. Med. Research, 43:61, 1922.18. Can. J. Pub. Health, 32:468, 1941.19. Centr. Bakt., 1:77:108, 1916.20. J. Bact., 29:515, 1935.21. Brit. J. Exp. Path., 27:335, 1936.22. Brit. J. Exp. Path., 27:342, 1936.23. J. Bact., 36:499, 1938.24. J. Immunol., 37:103, 1939.25. J. Immunol., 40:21, 1941.26. Proc. Soc. Expl. Biol. Med., 47:284, 1941.27. J. Immunol., 40:449, 1941.28. J. Immunol., 40:459, 1941.29. J. Bact., 41:305, 1941.30. J. Biol. Chem., 139:675, 1941.31. J. Biol. Chem., 119:121, 1937.32. J. Bact., 34:163, 1940.33. J. Bact., 41:441, 1941.34. J. Clin. Microbiol., 8:320, 1978.35. Brit. J. Pharmacol., 33:742, 1968.

Introduction

The Difco Manual 3

Section I Monographs

Original Difco LaboratoriesManufacturing facility.

Section ! Monographs

Difco Laboratories, originallyknown as Ray Chemical, wasfounded in 1895. This companyproduced high quality enzymes,dehydrated tissues and glandularproducts to aid in the digestionprocess. Ray Chemical acquiredDigestive Ferments Company,a company that specialized inproducing digestive enzymesfor use as bacterial culture mediaingredients. The experience ofprocessing animal tissues, puri-fying enzymes and performingdehydration procedures createda smooth transition to thepreparation of dehydrated

culture media. In 1913, the Digestive Ferments Company moved toDetroit, Michigan, and dropped the name, Ray Chemical.

After 1895, meat and other protein digests were developed to stimulategrowth of bacteria and fungi. The extensive research performed onthe analysis of pepsin, pancreatin and trypsin (and their digestiveprocesses) led to the development of Bacto® Peptone. Bacto Peptone,first introduced in 1914, was used in the bacteriological examinationof water and milk as a readily available nitrogen source. Bacto Peptonehas long been recognized as the standard peptone for the preparationof bacteriological culture media.

The development of Proteose Peptone, Proteose Peptone No. 2 andProteose Peptone No. 3 was the result of accumulated information thatno single peptone is the most suitable nitrogen source for growingfastidious bacteria. Proteose Peptone was developed for use in thepreparation of diphtheria toxin of high and uniform potency. BactoTryptose was originally formulated to provide the growth requirementsof Brucella. Bacto Tryptose was also the first peptone prepared thatdid not require the addition of infusions or other enrichments for theisolation and cultivation of fastidious bacteria.

The Digestive Ferments Company began the preparation of diagnosticreagents in 1923. Throughout the development of products used in thediagnosis of syphilis and other diseases, Difco worked closely withand relied on the direct involvement of expert scientists in the field.Bacto Thromboplastin, the first manufactured reagent used incoagulation studies, was developed in the early 1930s. This productwas another in a long line of many “firsts” for Difco Laboratories.

In 1934, the Digestive Ferments Company chose an acronym, “Difco,”to rename the company. The focus of Difco Laboratories was todevelop new and improved culture media formulations.

After World War II, the microbiology and health care fields expandedrapidly. Difco focused on the development of microbiological andimmunological products to meet this growing demand. In the 1940s,

Difco pursued the challenging task of producing bacterial antiseraand antigens. Lee Laboratories, a subsidiary, remains one of thelargest manufacturers of bacterial antisera. Additional “firsts” for DifcoLaboratories came in the 1950s with the development of C ReactiveProtein Antiserum, Treponemal Antigen and Antistreptolysin Reagents.

Throughout the 1950s and 1960s, Difco continued to add productsfor clinical applications. Bacto Blood Cultures Bottles were developedto aid in the diagnosis and treatment of sepsis. Difco Laboratoriespioneered in the preparation of reagents for in vitro propagation andmaintenance of tissue cells and viruses.

With the discovery of penicillin, a brand new branch of microbiologywas born. Difco initiated developmental research by preparingantibiotic disks for use in a “theorized” disk diffusion procedure.The result was Bacto Sensitivity Disks in 1946, followed by Dispens-O-Discs™ in 1965.

In the 1960s, Difco Laboratories became the largest manufacturer ofmicrobiological culture media by acquiring the ability to produce agar.Difco offers the same premier “gold standard,” Bacto Agar, today.

Bactrol™ Disks were introduced by Difco Laboratories in 1972. BactrolDisks are water-soluble disks containing viable microorganisms ofknown cultural, biochemical and serological characteristics used forquality control testing. Bactrol Disks became the first of manyproducts manufactured by Difco for use in quality control.

In 1983, Difco purchased the Paul A. Smith Company, later to be knownas Pasco®. A semi-automated instrument, the Pasco MIC/ID System, isused for bacterial identification and sensitivity testing. The Pasco DataManagement System can be used in industrial and clinical laboratories,either alone or as a back up to automated systems.

In 1992, ESP®, an automated continuous monitoring blood culturesystem, was introduced. ESP was the first blood culture system todetect both gas production and consumption by organism growth. Thetechnology continued with ESP Myco, an adaptation to the system thatallowed for growth, detection and susceptibility testing of mycobacteriaspecies. The ESP clinical system was sold to AccuMed Internationalin 1997.

In 1995, Difco Laboratories celebrated 100 years in business. In 1995,Difco was the first U.S. microbiology company to receive ISO 9001certification. The International Organization for Standardization (ISO)verifies that Difco Laboratories maintains quality standards for theworldwide microbiology industry.

In 1997, Difco Laboratories, the “industrial microbiology leader,” waspurchased by the “clinical microbiology leader,” Becton DickinsonMicrobiology Systems, to form the largest microbiology company inthe world. Together, Becton Dickinson Microbiology Systems andDifco Laboratories look forward to an even stronger future with ourcombined commitment to serving microbiologists worldwide.

History of Difco Laboratories

4 The Difco Manual

Monographs Section I

The science of microbiology evolved from a series of significantdiscoveries. The Dutch microscopist, Anton van Leeuwenhoek, wasthe first to observe bacteria while examining different water sources.This observation was published in 1676 by the Royal Society inLondon. Anton van Leeuwenhoek was also the first to describe theparasite known today as Giardia lamblia. In 1667, the discovery offilamentous fungi was described by Robert Hooke.

After microorganisms were visually observed, their growth orreproduction created a major controversy. The conflict was over thespontaneous generation theory, the idea that microorganisms will growspontaneously. This controversy continued for years until LouisPasteur’s renowned research. Pasteur realized that the theoryof spontaneous generation must be refuted for the science ofmicrobiology to advance. The controversy remained even afterPasteur’s successful experiment using heat-sterilized infusions.

Two important developments were required for the science ofmicrobiology to evolve. The first was a sophisticated microscope; thesecond was a method for culturing microorganisms. Compoundmicroscopes were developed in Germany at the end of the sixteenthcentury but it was not until the early nineteenth century thatachromatic lenses were developed, allowing the light in the microscopeto be focused.

In 1719, Leeuwenhoek was the first to attempt differentiation ofbacteria by using naturally colored agents such as beet juice. In 1877,Robert Koch used methylene blue to stain bacteria. By 1882,Robert Koch succeeded in staining the tubercle bacillus withmethylene blue. This landmark discovery was performed by usingheat to penetrate the stain into the organism. Two years later HansChristian Gram, a Danish pathologist, developed the Gram stain. TheGram stain is still widely used in the differentiation of gram-positiveand gram-negative bacteria.

In 1860, Pasteur was the first to use a culture medium for growingbacteria in the laboratory. This medium consisted of yeast ash, sugarand ammonium salts. In 1881, W. Hesse used his wife’s agar(considered an exotic food) as a solidifying agent for bacterial growth.

The study of fungi and parasites lagged behind other microorganisms.In 1839, ringworm was the first human disease found to be caused byfungi, followed closely by the recognition of Candida albicans asthe cause of thrush. It was not until 1910 that Sabouraud introduceda medium that would support the growth of pathogenic fungi. Theinterest of scientists in studying fungi was often related to cropprotection. There continues to be a close connection betweenmycology and botany today.

By 1887, a simple device called the Petri dish revolutionizedmicrobiology. With the invention of the Petri dish, the focus turned toculture media formulations. With all the research being performed,scientists began to replace gelatin with agar because it was resistant tomicrobial digestion and liquefaction.

The study of immunity began after the discovery of the tuberclebacillus by Robert Koch. With this acclaimed discovery, theinvolvement of bacteria as agents of disease became evident. The firstrational attempts to produce artificial active immunity were byPasteur in 1880 during his work with cholera.

Antibiotics had a dramatic beginning with the famous discovery ofpenicillin by Alexander Fleming in 1928. Fleming found a mold sporethat accidentally landed on a culture of staphylococci. It was not untilthe late 1930s that scientists could purify penicillin and demonstrateits antibacterial effects. Commercial production of penicillin beganas a combined wartime project between the United States andEngland. This project was the beginning of the fermentation industryand biotechnology.

Around 1930, certain growth factors, including factor X and V, wereshown to be important in bacterial nutrition. In the early 1950s, mostof the vitamins were also characterized as co-enzymes. This detailedinformation lead scientists to develop an understanding ofbiochemical pathways.

A “booming” development of microbiology began after World War II.Molecular biology, biotechnology and the study of genetics were fieldsof extraordinary growth. By 1941, the study of microbiology andgenetics came together when Neurospora crassa, a red bread mold,was used to study microbial physiology. The study of bacterialgenetics moved dramatically forward during the 1940s following thediscovery of antibiotic resistance. The birth of molecular biologybegan in 1953 after the publication by Watson and Crick of thestructure of DNA.

In 1953, viruses were defined by Luria as “submicroscopic entities,capable of being introduced into specific living cells and ofreproducing inside such cells only”. The work of John Enders onculturing viruses lead to the development of vaccines. Enders

Early years at Difco Laboratories.

History of Microbiology and Culture Media

The Difco Manual 5

Section I Monographs

Microorganism growth on culture media depends on a number ofimportant factors:

• Proper nutrients must be available.• Oxygen or other gases must be available, as required.• Moisture is necessary.• The medium must have an appropriate pH.• Proper temperature relations must prevail.• The medium must be free of interfering bioburden.• Contamination must be prevented.

A satisfactory microbiological culture medium must contain availablesources of:

• Carbon,• Nitrogen,• Inorganic phosphate and sulfur,• Trace metals,• Water,• Vitamins.

These were originally supplied in the form of meat infusion. Beef oryeast extracts frequently replace meat infusion in culture media. Theaddition of peptones, which are digests of proteins, provides readilyavailable sources of nitrogen and carbon.

The pH of the culture medium is important for microorganism growth.Temperature is another important parameter: mesophilic bacteria andfungi have optimal growth at temperatures of 25-40°C; thermophilic(“heat loving”) organisms grow only at temperatures greater than 45°C;psychrophilic (“cold loving”) organisms require temperatures below20°C. Human pathogenic organisms are generally mesophiles.

Common Media ConstituentsMedia formulations are developed on the ability of bacteria to usemedia components.

CONSTITUENTS SOURCE

Amino-Nitrogen Peptone, protein hydrolysate,infusions and extracts

Growth Factors Blood, serum, yeast extract orvitamins, NAD

Energy Sources Sugar, alcohols and carbohydrates

Buffer Salts Phosphates, acetates and citrates

Mineral Salts and Metals Phosphate, sulfate, magnesium,calcium, iron

Selective Agents Chemicals, antimicrobials and dyes

Indicator Dyes Phenol red, neutral red

Gelling agents Agar, gelatin, alginate, silica gel

Media IngredientsPeptone, protein hydrolysates, infusions and extracts are themajor sources of nitrogen and vitamins in culture media. Peptones arewater-soluble ingredients derived from proteins by hydrolysis ordigestion of the source material, e.g. meat, milk.

Carbohydrates are employed in culture media as energy sources andmay be used for differentiating genera and identifying species.

Buffers maintain the pH of culture media.

demonstrated that a virus could be grown in chick embryos and wouldlose its ability to cause disease after successive generations. Usingthis technique, Salk developed the polio vaccine.

One organism that has made a great contribution to molecularbiology is Escherichia coli. In 1973, Herbert Boyer and Stanley Cohenproduced recombinant DNA through plasmid transformation.The researchers found that the foreign gene not only survived, butcopied the genetic material. This study and similar others started abiotechnology revolution that has gained momentum over the years.

In the 1980s, instrumentation entered the microbiology laboratory.Manual procedures could be replaced by fully automated instrumentsfor bacterial identification, susceptibility testing and blood cultureprocedures. Immunoassays and probe technologies are broadening thecapabilities of the microbiologist.

With rapid advances in technologies and instrumentation, the basicculture media and ingredients listed in this Manual remain some of themost reliable and cost effective tools in microbiology today.

References1. Marti-Ibanez, F. 1962. Baroque medicine, p. 185-195. In

F. Marti-Ibanez (ed.). The epic of medicine. Clarkson N. Potter,Inc., New York, N.Y.

2. Wainwright, M., and J. Lederberg. 1992. History ofmicrobiology, p. 419-437. In J. Lederberg (ed.), Encyclopedia ofmicrobiology, vol 2. Academic Press Inc., New York, N.Y.

Microorganism Growth Requirements

6 The Difco Manual

Monographs Section I

Culture Media Ingredients – Agars

Selective Agents include Bile Salts, dyes and antimicrobial agents.Bile Salts and desoxycholate are selective for the isolation ofgram-negative microorganisms, inhibiting gram-positive cocci.

Dyes and indicators are essential in the preparation of differential andselective culture media. In these formulations, dyes act as bacteriostaticagents, inhibitors of growth or indicators of changes in acidity oralkalinity of the substrate.

Antimicrobial agents are used in media to inhibit the growth of bacteria,yeasts and fungi.

Solidifying agents, including agar, gelatin and albumin, can be addedto a liquid medium in order to change the consistency to a solidor semisolid state.

Environmental Factors in Culture MediaAtmosphereMost bacteria are capable of growth under ordinary conditions of oxygentension. Obligate aerobes require the free admission of oxygen, whileanaerobes grow only in the absence of atmospheric oxygen. Betweenthese two groups are the microaerophiles, which develop best underpartial anaerobic conditions, and the facultative anaerobes, which arecapable of growing in the presence or absence of oxygen. Anaerobicconditions for growth of microorganisms are obtained in a number of ways:

• Addition of small amounts of agar to liquid media;• Addition of fresh tissue to the medium;• Addition of a reducing substance to the medium; e.g., sodium

thioglycollate, thioglycollic acid and L-cystine;• Displacement of the air by carbon dioxide;

• Absorption of the oxygen by chemicals;• Inoculation into the deep layers of solid media or under a layer

of oil in liquid media.Many microorganisms require an environment of 5-10% CO2. Levelsgreater than 10% are often inhibitory due to a decrease in pH ascarbonic acid forms. Culture media vary in their susceptibility to formtoxic oxidation products if exposed to light and air.

Water ActivityProper moisture conditions are necessary for continued luxuriantgrowth of microorganisms. Organisms require an aqueous environmentand must have “free” water. “Free” water is not bound in complexstructure and is necessary for transfer of nutrients and toxic wasteproducts. Evaporation during incubation or storage results in loss of“free” water and reduction of colony size or total inhibition oforganism growth.

Protective Agents and Growth FactorsCalcium carbonate, soluble starch and charcoal are examples ofprotective agents used in culture media to neutralize and absorb toxicmetabolites produced by bacterial growth.

NAD (V factor) and hemin (X factor) are growth factors required bycertain bacteria; e.g., Haemophilus species, and for enhanced growthof Neisseria species.

Surfactants, including Tween® 80, lower the interfacial tension aroundbacteria suspended in the medium. This activity permits more rapidentry of desired compounds into the bacterial cell and can increasebacterial growth.

HistoryAgar was discovered in 1658 by Minora Tarazaemon in Japan.1

According to legend, this Japanese innkeeper threw surplus seaweedsoup into the winter night and noticed it later transformed into a gel bythe night’s freezing and the day’s warmth.2 In 1882, Koch was the firstto use agar in microbiology.3,4 Walter Hesse, a country doctor fromSaxony, introduced Koch to this powerful gelling agent.5 Hessehad learned about agar from his wife, Fanny Hesse, whose family hadcontact with the Dutch East Indies where agar was being used forjellies and jams.3,5,6 The term ‘agar-agar’ is a Malaysian word thatinitially referred to extracts from Eucheuma, which yields carrageenan,not agar.5

By the early 1900s, agar became the gelling agent of choice insteadof gelatin. Agar was found more suitable because it remained solid at

the temperatures required for growth of human pathogens and wasresistant to breakdown by bacterial enzymes.

Production of agar in the United States was started just before thebeginning of World War II as a strategic material.5 In the 1940s,bacteriological-grade agar manufactured by the American AgarCompany of San Diego, California, served as reference agar for theevaluation of the characteristics of other culture media components,such as peptones.5

CharacteristicsAgar is a phycocolloid, a water-soluble polysaccharide, extracted froma group of red-purple marine algae (Class Rhodophyceae) includingGelidium, Pterocladia and Gracilaria. These red-purple marine algaeare widely distributed throughout the world in temperate zones.

The Difco Manual 7

Section I Monographs

For Difco Agars, Gelidium is the preferred source of agar. The mostimportant properties of agar are:5

• Good transparency in solid and gel forms to allow identificationof colony type;

• Consistent lot-to-lot gel strength that is sufficient to withstandthe rigors of streaking but not so stiff that it affects diffusioncharacteristics;

• Consistent gelling (32-40°C) and melting (approximately85°C) temperatures, a property known as hysteresis;

• Essential freedom from metabolically useful chemicals such aspeptides, proteins and fermentable hydrocarbons;

• Low and regular content of electronegative groups that couldcause differences in diffusion of electropositive molecules(e.g., antibiotics, nutrients);

• Freedom from toxic substances (bacterial inhibitors);

• Freedom from hemolytic substances that might interfere withnormal hemolytic reactions in culture media;

• Freedom from contamination by thermophilic spores.

Agars are normally used in final concentrations of 1-2% forsolidifying culture media. Smaller quantities of agar (0.05-0.5%) areused in culture media for motility studies (0.5% w/v) and growthof anaerobes (0.1%) and microaerophiles.2

The Manufacturing ProcessDifco Laboratories selects the finest Gelidium marine algae from worldsources and requires algae harvested from water where the tempera-ture is both constant and temperate. Bacto Agar and Agar Granulatedare produced from an Ice Agar purification process. Agar is insolublein cold water but is colloidally dispersible in water above 90°C.2 Whenan agar gel is frozen, the agar skeleton contracts toward the center ofthe mass as a membrane, leaving ice as a separate phase.2

Through a variety of processes, the agar is extracted from the Gelidium,resulting in a liquid agar that is purified. The liquid agar is first gelledand then frozen, causing the soluble and suspended contaminants to be

trapped in the frozen water. The ice is then washed from the agar,eliminating the contaminants. The Ice Agar process results in greaterconsistency and freedom from interposing contaminants when usedin microbiological procedures.

Product ApplicationsBacto Agar is optimized for beneficial calcium and magnesiumcontent. Detrimental ions such as iron and copper are reduced. BactoAgar is recommended for clinical applications, auxotrophic studies,bacterial and yeast transformation studies and bacterial moleculargenetics applications.7,8

Agar Flake is recommended for use in general bacteriologicalpurposes. The quality is similar to Bacto Agar. The flakes are morewettable than the granules found in Bacto Agar.

Agar Granulated is qualified to grow recombinant strains ofEscherichia coli (HB101) and Saccharomyces cerevisiae. AgarGranulated may be used for general bacteriological purposes whereclarity is not a strict requirement. This agar was developed toaddress the special needs of the Biotechnology Industry for largescale applications.

Noble Agar is the purest form of Difco agar. It is washed extensivelyand bleached to remove extraneous material. The result is a whitepowder in dry form, clear and colorless in solution and when solidifiedin plates. This agar is suitable for immunodiffusion studies, for use insome electrophoretic applications and as a substrate for mammalianand plant tissue culture.

Agar Technical is suitable for many general bacteriologicalapplications. This agar is not as highly processed as other Difco agarsand has lower technical specifications. This agar is not recommendedfor growth of fastidious organisms.

References1. C. K. Tsend. 1946. In J. Alexander (ed.). 6:630. Colloid

Chemistry. Reinhold Publishing Corp., New York, N. Y.2. Selby, H. H., and T. A. Selby. 1959. Agar. In Whister (ed.).,

Industrial gums. Academic Press Inc., New York, NY.3. Hitchens, A. P., and M. C. Leikind. 1939. The introduction

of agar-agar into bacteriology. J. Bacteriol. 37:485-493.4. Koch, R. 1882. Die Atiologie der Turberkulose. Berl. Klin.

Wochenschr. 19:221- 230.5. Armisen, R. 1991. Agar and agarose biotechnological applications.

Hydrobiol. 221:157-166.6. Hesse, W. 1894. Uber die quantitative Bestimmung der in der

Luft enthaltenen Mikroorganismen. Mitt. a. d. Kaiserl. Gesh.Berlin 2:182-207.

7. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecularcloning, a laboratory manual, 2nd ed. Cold Spring HarborLaboratory Press, New York, N.Y.

8. Schiestl, R. H., and R. D. Geitz. 1989. High efficiencytransformation of intact yeast cells using single stranded nucleicacids as a carrier. Current Genetics 16:339-346.

Agar is derived from a group of red-purple marine algae as pictured above.

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HistoryPeptones were originally described by Naegeli in 1879.3 In this report,Naegeli compared peptone and ammonium tartrate. With the rich aminoacid and nitrogen compounds readily utilized by bacteria, peptonesoon became one of the most important constituents of culture media.The importance of peptone as a nutritive source was demonstratedby Klinger.4

Bacto Peptone was introduced commercially in 1914, and became thestandard peptone for the preparation of bacteriological culture media.The development of Bacto Proteose Peptone, Bacto Proteose PeptoneNo. 2 and Bacto Proteose Peptone No. 3 resulted from accumulatedinformation that no single peptone is the most suitable nitrogen sourcefor culturing fastidious bacteria. Extensive investigations wereundertaken at Difco Laboratories using peptic digests of animal tissueprepared under varying digestion parameters. Bacto Tryptone wasdeveloped by Difco Laboratories while investigating a peptoneparticularly suitable for the elaboration of indole by bacteria.

Other non-chemically defined ingredients, including Bacto Liver, BactoBeef Heart for Infusion and Bacto Yeast Extract can serveas nitrogen or carbon sources. Infusions of meat were first employedas nutrients in culture media. It was discovered that for many routineprocedures in the preparation of culture media, extracts have theadvantage of greater ease in preparation, uniformity and economythan infusions.

Protein BiochemistryProteins consist of amino acids joined together by means of thecovalent peptide bond linkage. When the bonds are hydrolyzed,proteins yield polypeptides of various molecular sizes, proteoses,peptones and peptides down to the level of simple amino acids.Bacteriological peptones are mixtures of various products of proteinhydrolysis, organic nitrogen bases, inorganic salts and trace elements.

Preparation of PeptonesThe composition of peptones varies with the origin and the method ofpreparation. Some common sources of peptone include:

Meat (fresh, frozen or dried)Fish (fresh, dried)CaseinGelatinKeratin (horn, hair, feathers)Ground NutsSoybean MealCotton SeedSunflower SeedsMicroorganisms (yeasts, algae, bacteria)Guar ProteinBloodCorn GlutenEgg Albumin

Demineralized water is added to these protein sources to form a thicksuspension. The digestion process follows with an acid or enzyme. Acidand alkaline hydrolyses are performed by boiling the protein withmineral acids or strong alkalis at increased pressure to raise thetemperature of the reaction. This procedure can decrease the vitamincontent of the protein and a portion of the amino acid content.Digestion with proteolytic enzymes is performed at lowertemperatures and normal atmospheric pressure. This process is oftenless harmful to the protein and amino acids. Microbial Proteoses,Papain, Pancreatin and Pepsin are used most often by DifcoLaboratories in the manufacture of peptones.

The peptone suspension is then centrifuged and filtered. Thesuspension is concentrated to approximately 67% total solids and theproduct now appears as a syrup. This peptone syrup is spray driedand packaged.

Infusions and ExtractsThe water-soluble fractions of materials such as muscle, liver, yeastcells and malt are usually low in peptides but contain valuableextractives such as vitamins, trace metals and complex carbohydrates.5

It is common practice to combine infusions and peptones to obtain thebest of both products.5 Bacto Yeast Extract, Bacto Malt Extract, BactoBeef Heart for Infusion and Bacto Beef Extract are examples ofextracts and infusions manufactured by Difco Laboratories for use inthe preparation of culture media.

Peptone PerformanceThe quality and performance of peptones, infusions and extracts arevery dependent on the freshness or preservation of the raw materials.5

Extensive quality control testing is performed on all peptones and otherculture media ingredients during the manufacturing process and on thefinal product. Certificates of Analysis supply information from themanufacturer on lot specific final testing of a product.

Typical fermentation process.

Culture Media Ingredients – Peptones and Hydrolysates

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A typical analysis was performed on Difco peptones and hydrolysatesto aid in the selection of products for research or productionneeds when specific nutritional characteristics are required. Thespecifications for the typical analysis include:

• Physical characteristics

• Nitrogen content

• Amino acids

• Inorganics

• Vitamins

• Biological testing

The quality of peptones and culture media ingredients is truly assessedby their ability to support adequate growth of various microorganismswhen incorporated into the medium.6 The nature of peptones,infusions and extracts will then play a major role in the growthperformance properties of the medium and, in turn, advance thescience of microbiology.6

Media IngredientsAutolyzed YeastAutolyzed Yeast is a desiccated product containing both the solubleand insoluble portions of autolyzed bakers’ yeast. Autolyzed Yeast isrecommended for the preparation of yeast supplements used in themicrobiological assay of riboflavin and pantothenic acid.7,8, AutolyzedYeast provides vitamins, nitrogen, amino acids and carbon inmicrobiological culture media.

BeefBeef Heart for InfusionBeef and Beef Heart for Infusion provide nitrogen, amino acids andvitamins in microbiological culture media. Beef is desiccated,powdered, fresh lean beef, prepared especially for use in beef infusionmedia. Large quantities of beef are processed at one time to secure auniform and homogenous product. Beef Heart for Infusion is preparedfrom fresh beef heart tissue and is recommended for preparing heartinfusion media. Beef Heart for Infusion is processed from largevolumes of raw material, retaining all the nutritive and growthstimulating properties of fresh tissues.

Beef ExtractBeef Extract, DesiccatedBeef Extract and Beef Extract, Desiccated are replacements forinfusion of meat. Beef Extract and Beef Extract, Desiccated providenitrogen, vitamins, amino acids and carbon in microbiological culturemedia. Beef Extract is standard in composition and reaction andgenerally used to replace infusion of meat. In culture media, BeefExtract is usually employed in concentration of 0.3%. Beef Extract,Desiccated, the dried form of Beef Extract, was developed to provide aproduct for ease of use in handling. Beef Extract is in the paste form.The products are to be used in a 1 for 1 substitution.

Bile SaltsBile Salts No. 3Bile Salts and Bile Salts No. 3 are used as selective agents for theisolation of gram-negative microorganisms, inhibiting gram-positive

cocci. Bile is derived from the liver. The liver detoxifies bile salts byconjugating them to glycine or taurine. A bile salt is the sodium salt ofa conjugated bile acid. Bile Salts and Bile Salts No. 3 contain bileextract standardized to provide inhibitory properties for selectivemedia. Bile Salts No. 3 is a modified fraction of bile acid salts,providing a refined bile salt. Bile Salts No. 3 is effective at less thanone-third concentration of Bile Salts.

Casamino AcidsCasamino Acids, TechnicalCasamino Acids, Vitamin Assay

Casamino Acids, Casamino Acids, Technical and Casamino Acids,Vitamin Assay are derived from acid hydrolyzed casein. Casein is amilk protein and a rich source of amino acid nitrogen. CasaminoAcids, Casamino Acids, Technical and Casamino Acids, VitaminAssay are added to media primarily because of their organic nitrogenand growth factor components; their inorganic components also playa vital role.9 Casamino Acids is recommended for use with microbio-logical cultures that require a completely hydrolyzed protein as anitrogen source. In Casamino Acids, hydrolysis is carried on until allthe nitrogen in the casein is converted to amino acids or other com-pounds of relative chemical simplicity. The hydrolysis of CasaminoAcids, Technical is carried out as in the preparation of CasaminoAcids, but the sodium chloride and iron content have not been decreasedto the same extent. Casamino Acids, Vitamin Assay is an acid digest ofcasein specially treated to markedly reduce or eliminate certainvitamins. It is recommended for use in microbiological assay mediaand in growth promotion studies.

Casein Digest

Casein Digest is an enzymatic digest of casein, providing a distinctsource of amino acids for molecular genetics media. Casein Digest isused as a nitrogen and amino acid source for microbiological culturemedia. Casein Digest is similar to N-Z Amine A. This product isdigested under conditions different from other enzymatic digestsof casein, including Tryptone and Casitone.

Casitone

Casitone is a pancreatic digest of casein. Casitone is recommendedfor preparing media where an enzymatic hydrolyzed casein isdesired. Casein is a rich source of amino acid nitrogen. This productis used to support the growth of fastidious microorganisms and itshigh tryptophan content makes it valuable for detecting indoleproduction.

Fish Peptone No. 1

Fish Peptone No. 1 is a non-mammalian, non-animal peptone usedas a nitrogen source in microbiological culture media. Fish PeptoneNo. 1 is a non-bovine origin peptone, to reduce Bovine SpongiformEncephalopathy (BSE) risk. This peptone was developed by DifcoLaboratories for pharmaceutical and vaccine production and canreplace any peptone, depending on the organism and productionapplication.

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Gelatin

Gelatin is a protein of uniform molecular constitution derived chieflyby the hydrolysis of collagen.10 Collagens are a class of albuminoidsfound abundantly in bones, skin, tendon, cartilage and similar tissuesof animals.10 Gelatin is used in culture media to detect gelatinliquifaction by bacteria and as a nitrogen and amino acid source.

Gelatone

Gelatone is a pancreatic digest of gelatin, deficient in carbohydrates.Gelatone is used as a media ingredient for fermentation studies and,alone, to support the growth of non-fastidious microorganisms.Gelatone is in granular form for convenience in handling and isdistinguished by a low cystine and tryptophan content.

Liver

Liver is prepared from large quantities of carefully trimmed fresh beefliver. Liver is a desiccated powder of beef liver. The nutritive factors offresh liver tissue are retained in infusion prepared from Liver.Liver is used as a source of nitrogen, amino acids and vitamins inmicrobiological culture media. The reducing substances contained inliver create an anaerobic environment, necessary to support the growthof anaerobes. One hundred thirty-five (135) grams of desiccated Liverare equivalent to 500 grams of fresh liver.

Malt Extract

Malt Extract is obtained from barley, designed for the propagation ofyeasts and molds. Malt Extract is particularly suitable for yeasts andmolds because it contains a high concentration of carbohydrates,particularly maltose. This product is generally employed inconcentrations of 1-10%. Malt Extract provides carbon, protein andnutrients for the isolation and cultivation of yeasts and molds inbacterial culture media.

Neopeptone, Difco

Neopeptone is an enzymatic digest of protein. Neopeptone containsmany peptide sizes in combination with vitamins, nucleotides,minerals and other carbon sources. Neopeptone is particularly wellsuited in supplying the growth requirements of fastidious bacteria. Thispeptone is extremely valuable in media for the cultivation ofpathogenic fungi. Growth of these microorganisms is rapid and colonyformation is uniform and typical.

Oxgall

Oxgall is manufactured from large quantities of fresh bile by rapidevaporation of the water content. Bile is composed of fatty acids, bileacids, inorganic salts, sulphates, bile pigments, cholesterol, mucin,lecithin, glycuronic acids, porphyrins and urea. The use of Oxgallensures a regular supply of bile and assures a degree of uniformityimpossible to obtain with fresh materials. It is prepared for use inselective media for differentiating groups of bile tolerant bacteria.Oxgall is used as a selective agent for the isolation of gram-negativemicroorganisms, inhibiting gram-positive bacteria. The majorcomponents of Oxgall are taurocholic and glycocholic acids.

Peptamin

Peptamin, referred to as Peptic Digest of Animal Tissue, complieswith the US Pharmacopeia XXIII (USP).11 Peptamin providesnitrogen, amino acids, vitamins and carbon in microbiological culturemedia. Diluting and rinsing solutions, Fluid A and Fluid D, contain0.1% Peptamin.

Peptone, BactoPeptone Bacteriological, Technical

Bacto Peptone and Peptone Bacteriological, Technical are enzymaticdigests of protein and rich nitrogen sources. Bacto Peptone wasintroduced in 1914 and became the standard peptone for thepreparation of culture media. Peptone Bacteriological, Technical canbe used as the nitrogen source in microbiological culture media whena standardized peptone is not essential. Both peptones have a highpeptone and amino acid content and only a negligible quantity ofproteoses and more complex nitrogenous constituents.

Proteose PeptoneProteose Peptone No. 2Proteose Peptone No. 3

The development of Proteose Peptone, Proteose Peptone No. 2 andProteose Peptone No. 3 is the result of accumulated informationdemonstrating that no single peptone is the most suitable nitrogensource for culturing fastidious bacteria. Proteose Peptone is anenzymatic digest of protein high in proteoses. Many factors accountfor the suitability of Proteose Peptone for the culture of fastidiouspathogens, including the nitrogen components, buffering range and thehigh content of proteoses. Proteose Peptone No. 2 and ProteosePeptone No. 3 are enzymatic digests of protein. Proteose PeptoneNo. 2 is used for producing bacterial toxins and is suitable for mediaof nutritionally less-demanding bacteria. Proteose Peptone No. 3 isa modification of Proteose Peptone, adapted for use in the preparationof chocolate agar for propagation of Neisseria species and chocolatetellurite agar for Corynebacterium diphtheriae.

Sodium DeoxycholateSodium Taurocholate

Sodium Desoxycholate is the sodium salt of desoxycholic acid. SinceSodium Desoxycholate is a salt of a highly purified bile acid, it is usedin culture media in lower concentrations than in naturally occurringbile. Sodium Taurocholate is the sodium salt of a conjugated bile acid.Sodium Taurocholate contains about 75% sodium taurocholate inaddition to other naturally occurring salts of bile acids. SodiumDesoxycholate and Sodium Taurocholate, like other bile salts, are usedas selective agents in microbiological culture media. They are usedto aid in the isolation of gram- negative microorganisms, inhibitinggram-positive organisms and spore forming bacteria.

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Soytone

Soytone is an enzymatic digest of soybean meal. The nitrogen sourcein Soytone contains the naturally occurring high concentrations of vi-tamins and carbohydrates of soybean.

TC Lactalbumin HydrolysateTC Yeastolate

TC Lactalbumin Hydrolysate is an enzymatic digest of lactalbumin foruse as an enrichment in tissue culture media. Lactalbumin is a proteinderived after removal of casein from milk. TC Yeastolate is adesiccated, clarified, water soluble portion of autolyzed fresh yeastprepared and certified for use in tissue culture procedures.TC Yeastolate is a source of vitamin B complex.

Tryptone Peptone

Tryptone Peptone is a pancreatic digest of casein used as a nitrogensource in culture media. Casein is the main protein of milk and is a richsource of amino acid nitrogen. Tryptone Peptone is rich in tryptophan,making it valuable for use in detecting indole production.12 The ab-sence of detectable levels of carbohydrates in Tryptone Peptone makesit a suitable peptone in differentiating bacteria on the basis of theirability to ferment various carbohydrates.

Tryptose

Tryptose is a mixed enzymatic hydrolysate with distinctive nutritionalproperties. The digestive process of Tryptose results in assortedpeptides, including those of higher molecular weight. Tryptose wasoriginally developed as a peptone particularly adapted to the growthrequirements of Brucella.

Yeast ExtractYeast Extract, Technical

Yeast Extract and Yeast Extract, Technical are water soluble portionsof autolyzed yeast containing vitamin B complex. Yeast Extract isan excellent stimulator of bacterial growth and used in culture media.The autolysis is carefully controlled to preserve the naturallyoccurring B-complex vitamins. Yeast Extract is generally employedin the concentration of 0.3-0.5%, with improved filterability at20%. Yeast Extract, Technical is used in bacterial culture media whena standardized yeast extract is not essential. Yeast Extract, Technicalwas developed to demonstrate acceptable clarity and growthpromoting characteristics. Yeast Extract and Yeast Extract, Technicalalso provide vitamins, nitrogen, amino acids and carbon inmicrobiological culture media.

References1. Nash, P., and M. M. Krenz. 1991. Culture Media, p. 1226-1288.

In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg,and H. J. Shadomy (ed.), Manual of clinical microbiology, 5th ed.American Society for Microbiology, Washington, D.C.

2. De Feo, J. 1986. Properties and applications of hydrolyzedproteins. ABL. July/August, 44-47.

3. Naegeli. 1880. Sitz’ber, math-physik. Klasse Akad. Wiss.Muenchen. 10:277.

4. Klinger, I. J. 1917. The effect of hydrogen ion concentration onthe production of precipitates in a solution of peptone and itsrelation to the nutritive value of media. J. Bacteriol. 2:351-353.

5. Bridson, E. Y. 1990. Media in microbiology. Rev. Med. Microbiol.1:1-9.

6. Alvarez, R. J., and M. Nichols. 1982. Formulating microbio-logical culture media-a careful balance between science and art.Dairy Food Sanitation 2:356- 359.

7. J. Ind. Eng. Chem., Anal. Ed. 1941. 13:567.

8. J. Ind. Eng. Chem., Anal. Ed. 1942. 14:909.

9. Nolan, R. A., and W. G. Nolan. 1972. Elemental analysis ofvitamin-free casamino acids. Appl. Microbiol. 24:290-291.

10. Gershenfeld, L., and L. F. Tice. 1941. Gelatin for bacteriologicaluse. J. Bacteriol. 41:645-652.

11. United States Pharmacopeial Convention. 1995. The UnitedStates pharmacopeia, 23rd ed. The United States PharmacopeialConvention. Rockville, MD.

12. J. Bacteriol. 1933. 25:623.

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The preparation of culture media from dehydrated media requiresaccuracy and attention to preparation. The following points areincluded to aid the user in successful and reproducible preparationof culture media.

Dehydrated Media and Ingredients

• Store in a cool (15-30°C), dark and dry area unless otherwisespecified.

• Note date opened.

• Check expiry (applied to intact container).

• Verify that the physical characteristics of the powder are typical.

Glassware / Plasticware

• Use high quality, low alkali borosilicate glass.

• Avoid detergent residue.

• Check for alkali or acid residue with a few drops of brom thymolblue pH indicator (yellow is acidic; blue is alkaline).

• Use vessels at least 2-3 times the volume of medium.

• Discard (recycle) etched or chipped glassware.

• Do not used etched glassware.

Equipment

• Use measuring devices, scales, pH meters, autoclaves and otherequipment that are frequently and accurately calibrated.

Water

• Use distilled or deionized water.

• pH 5.5-7.5.

Dissolving the Medium

• Accurately weigh the appropriate amount of dehydrated medium.

• Dissolve the medium completely.

• Agitate the medium while dissolving.

• Take care to not overheat. Note media that are very sensitive tooverheating. Overheated media will frequently appear darker. Donot heat in a microwave.

Sterilization

• The autoclave set-temperature should be 121°C.

• Routine autoclave maintenance is important. Ask manufacturerto check for “hot” and “cold” spots.

• The recommended 15 minute sterilization assumes a volumeof 1 liter or less. Larger volumes may require longercycles. Check with your autoclave manufacturer forrecommended load configurations.

• Quantities of media in excess of two liters may require anextended autoclave time to achieve sterilization. Longersterilization cycles can cause nutrient concentration changesand generation of inhibitory substances.

Adding Enrichments and Supplements

• Enrichments and supplements tend to be heat sensitive.

• Cool medium to 45-55°C in a waterbath prior to addingenrichments or supplements.

• Ensure adequate mixing of the basal medium with enrichmentsor supplements by swirling to mix thoroughly.

• Sterile broths may be cooled to room temperature before addingenrichment.

pH

• Commercial dehydrated media are designed to fall within thespecified pH range after steam sterilization. The pH tends to fallapproximately 0.2 units during steam sterilization.

• For filter sterilization, adjust the pH, if necessary, prior to filtering.

• Avoid excessive pH adjustments.

Dispensing Media

• Ensure gentle mixing during dispensing.

• Cool the medium to 50-55°C prior to dispensing to reducewater evaporation.

• Dispense quickly.

• If using an automatic plate dispenser, dispense general purposemedia before dispensing selective media.

• Immediately recover or recap tubes to reduce the chance ofcontamination. Leave Petri dish covers slightly open for 1-2 hoursto obtain a dry surface.

Storage and Expiry

• In general, store steam-sterilized plated media inverted in aplastic bag or other container in a dark refrigerator for up to1-2 weeks.

Quality Control

• For media prepared in-house, each lot of every medium mustbe tested.

• Maintain Quality Control Organisms appropriately.

• Maintain appropriate records.

• Report deficiencies to the manufacturer.

The following table is a troubleshooting guide to assist in thepreparation of reliable culture media.

Media Preparation

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PROBLEM A B C D E F G H OTHER CAUSES

Abnormal color of medium • • •

Incorrect pH • • • • • • • Storage at high temperatureHydrolysis of ingredientspH determined at wrong temperature

Nontypical precipitate • • • • • •

Incomplete solubility • Inadequate heatingInadequate convection in a too small flask

Darkening or carmelization • • • •

Toxicity • • Burning or scorching

Tract substances (Vitamins) • Airborne or environmental sourcesof vitamins

Loss of gelation property • • • • Hydrolysis of agar due to pH shiftNot boiling medium

Loss of nutritive value or • • • • • • • Burning or scorchingselective or differential Presence of strong electrolytes, sugarproperties solutions, detergents, antiseptics, metallic

poisons, protein materials or othersubstances that may inhibit the inoculum

Contamination Improper sterilizationPoor technique in adding enrichments andpouring platesNot boiling agar containing medium

Media Sterilization

Sterilization is any process or procedure designed to entirely eliminateviable microorganisms from a material or medium. Sterilization shouldnot be confused with disinfection, sanitization, pasteurization orantisepsis which are intended to inactivate microorganisms, but maynot kill all microorganisms present. Sterilization can be accomplishedby the use of heat, chemicals, radiation or filtration.1

Sterilization with Heat1The principal methods of thermal sterilization include 1) moist heat(saturated steam) and 2) dry heat (hot air) sterilization. Heat killsmicroorganisms by protein denaturation and coagulation. Moist heathas the advantage of being more rapid and requiring lower temperaturesthan dry heat. Moist heat is the most popular method of culture mediasterilization. When used correctly, it is the most economical, safe andreliable sterilization method.

Moist Heat SterilizationWater boils at 100°C, but a higher temperature is required to killresistant bacterial spores in a reasonable length of time. A temperaturerange of 121-124°C for 15 minutes is an accepted standard conditionfor sterilizing up to one liter of culture medium. The definition of“autoclave at 121°C for 15 minutes” refers to the temperature of thecontents of the container being held at 121°C for 15 minutes, not to thetemperature and time at which the autoclave has been set.2 The steampressure of 15 pounds per square inch at this temperature aids in thepenetration of the heat into the material being sterilized. If a largervolume is to be sterilized in one container, a longer period should beemployed. Many factors can affect sterility assurance, including sizeand contents of the load and the drying and cooling time. Certainproducts may decompose at higher temperature and longer cycles. Forthis reason, it is important that all loads be properly validated.

The basic principles for validation and certification of a sterilizingprocess are enumerated as follows:3

1. Establish that the processing equipment has the capability ofoperating within the required parameters.

KeyA Deteriorated Dehydrated Medium D Incorrect Weighing G Repeated Remelting

B Improperly Washed Glassware E Incomplete Mixing H Dilution by a Too Large Inoculum

C Impure Water F Overheating

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2. Demonstrate that the critical control equipment andinstrumentation are capable of operating within the prescribedparameters for the process equipment.

3. Perform replicate cycles representing the required operationalrange of the equipment and employing actual or simulatedproduct. Demonstrate that the processes have been carried outwithin the prescribed protocol limits and, finally, that theprobability of microbial survival in the replicate processescompleted is not greater than the prescribed limits.

4. Monitor the validated process during routine operation.Periodically as needed, requalify and recertify the equipment.

5. Complete the protocols and document steps 1-4, above.

For a complete discussion of process validation, refer to appropriatereferences.

Ensuring that the temperature is recorded correctly is vital. Thetemperature must reach all parts of the load and be maintained for thedesired length of time. Recording thermometers are employed for thechamber and thermocouples may be buried inside the load.

For best results when sterilizing culture media, plug tubes or flasksof liquids with nonabsorbent cotton or cap loosely. Tubes should beplaced in racks or packed loosely in baskets. Flasks should never bemore than two-thirds full. It is important to not overload the autoclavechamber and to place contents so that there is a free flow of steamaround the contents. After sterilizing liquids, the chamber pressure mustbe reduced slowly to atmospheric pressure. This allows the liquid tocool below the boiling point at atmospheric pressure before openingthe door to prevent the solution from boiling over.

In autoclave operation, all of the air in the chamber must be expelledand replaced by steam; otherwise, “hot spots” and “cold spots” willoccur. Pressure-temperature relations of a properly operated autoclaveare shown in the table below.

Over-sterilization or prolonged heating will change the composition ofthe medium. For example, carbohydrates are known to break down incomposition upon overheating. Over-sterilizing media can cause anumber of problems, including:

• Incorrect pH;

• A decrease in the gelling properties of agar;

• The development of a nontypical precipitate;

• Carmelization or darkening of the medium;

• Loss of nutritive value;

• Loss of selective or differential properties.

There are certain media (e.g., Hektoen Enteric Agar and Violet RedBile Agar) that should not be autoclaved. To dissolve these mediaformulation, heat to boiling to dissolve completely. It is importantto follow all label directions for each medium. Media supplementsshould be sterile and added aseptically to the sterilized medium,usually at 45-55°C.

Dry Heat Sterilization1Dry heat is employed for materials such as metal instruments that couldbe corroded by moist heat, powders, ointments and dense materialsthat are not readily penetrated by steam. Because dry heat is effectiveonly at considerably higher temperatures and longer times than moistheat, dry heat sterilization is restricted to those items that willwithstand higher temperatures. The dry heat time for sterilization is120 minutes at 160°C.

Chemical Sterilization1Chemical sterilization employs gaseous and liquid sterilants forcertain medical and industrial instruments. The gases include ethyleneoxide, formaldehyde and beta-propiolactone. The liquid sterilantsinclude glutaraldehyde, hydrogen peroxide, peracetic acid, chlorinedioxide and formaldehyde. Chemical sterilization is not employed inthe preparation of culture media. For a complete discussion of thistopic, consult appropriate references.

Radiation Sterilization1Radiation sterilization is an optional treatment for heat-sensitivematerials. This includes ultraviolet light and ionizing radiation.

Ultraviolet light is chemically active and causes excitation of atomswithin the microbial cell, particularly the nucleic acids, producinglethal mutations. This action stops the organism from reproducing. Therange of the ultraviolet spectrum that is microbiocidal is 240-280 nm.There is a great difference in the susceptibility of organisms toultraviolet radiation; Aspergillus niger spores are 10 times moreresistant than Bacillus subtilis spores, 50 times more resistant thanStaphylococcus aureus and Escherichia coli, and 150 times moreresistant than influenza virus.

Because most materials strongly absorb ultraviolet light, it lackspenetrating power and its applications are limited to surface treatments.Much higher energy, 100 to millions of times greater, is generated byionizing radiations. These include gamma-rays, high energy X-rays andhigh energy electrons.

Ionizing radiation, unlike ultraviolet rays, penetrates deeply intoatoms, causing ionization of the electrons. Ionizing radiation maydirectly target the DNA in cells or produce active ions and free radicalsthat react indirectly with DNA.

Gamma radiation is used more often than x-rays or high-energyelectrons for purposes of sterilization. Gamma rays are generated by

PRESSURE IN POUNDS TEMPERATURE (°C) TEMPERATURE (°F)

5 109 228

10 115 240

15 121 250

20 126 259

25 130 267

30 135 275

Pressure-Temperature Relations in Autoclave4

(Figures based on complete replacement of air by steam)

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radioactive isotopes, cobalt-60 being the usual source. Gammaradiation requires many hours of exposure for sterilization. Validationof a gamma irradiation procedure includes:4

• Establishment of article materials compatibility;

• Establishment of product loading pattern and completion of dosemapping in the sterilization container;

• Establishment of timer setting;

• Demonstration of the delivery of the required sterilization dose.

The advantages of sterilization by irradiation include low chemicalreactivity, low measurable residues, and few variables to control.3

Gamma irradiation is used for treating many heat-sensitive productsthat can also be treated by gaseous sterilization, including medicalmaterials and equipment, pharmaceuticals, biologicals, certainprepared media and laboratory equipment.

Sterilization by Filtration1,3Filtration is a useful method for sterilizing liquids and gases. Filtrationexcludes microorganisms rather than destroying them. Two major typesof filters may be used, depth filters and membrane filters.

The membrane filter screens out particles, while the depth filterentraps them. Membrane filters depend largely on the size of the poresto determine their screening effectiveness. Electrostatic forces are alsoimportant. A membrane filter with an average pore size of 0.8 µm willretain particulate matter as small as 0.05 µm. For removing bacteria, apore size of 0.2 µm is commonly used. For retention of viruses andmycoplasmas, pore sizes of 0.01-0.1 µm are recommended. Cocci andbacilli range in size from about 0.3 to 1 µm in diameter. Most virusesare 0.02-0.1 µm, with some as large as 0.25 µm.

Rating the pore size of filter membranes is by a nominal rating thatreflects the capability of the filter membrane to retain microorganismsof size represented by specified strains. Sterilizing filter membranesare membranes capable of retaining 100% of a culture of 107

microorganisms of a strain of Pseudomonas diminuta (ATCC® 19146)per square centimeter of membrane surface under a pressure ofnot less than 30 psi. These filter membranes are nominally rated 0.22µm or 0.2 µm. Bacterial filter membranes (also known as analyticalfilter membranes), which are capable of retaining only largermicroorganisms, are labeled with a nominal rating of 0.45 µm.

Membrane filters are used for the commercial production of a numberof pharmaceutical solutions and heat-sensitive injectables. Serumfor use in bacterial and viral culture media are often sterilized byfiltration, as well as some sugars that are unstable when heated.Membrane filtration is useful in testing pharmaceutical and medicalproducts for sterility.

Sterility Assurance1Sterility Assurance is the calculated probability that a microorganismwill survive sterilization. It is measured as the SAL, SterilityAssurance Level, or “degree of sterility”. For sterility assurance,Bacillus stearothermophilus which contains steam heat-resistant sporesis employed with steam sterilization at 121°C.

Testing Sterilizing Agents1,5Sterilization by moist heat (steam), dry heat, ethylene oxide and ioniz-ing radiation is validated using biological indicators. The methods ofsterilization and their corresponding indicators are listed below:

For moist heat sterilization, paper strips treated with chemicals thatchange color at the required temperature may be used.

The heat-resistant spores of B. stearothermophilus are dried on papertreated with nutrient medium and chemicals. After sterilization, thestrips are incubated for germination and growth, and a color changeindicates whether they have or have not been activated. Spore stripsshould be used in every sterilization cycle.

Glossary1,6

Bioburden is the initial population of living microorganisms in theproduct or system being considered.

Biocide is a chemical or physical agent intended to produce the deathof microorganisms.

Calibration is the demonstration that a measuring device producesresults within specified limits of those produced by a referencestandard device over an appropriate range of measurements.

Death rate is the rate at which a biocidal agent reduces the numberof cells in a microbial population that are capable of reproduction.This is determined by sampling the population initially, duringand following the treatment, followed by plate counts of the survivingmicroorganisms on growth media.

D value stands for decimal reduction time and is the time required inminutes at a specified temperature to produce a 90% reduction in thenumber of organisms.

Microbial death is the inability of microbial cells to metabolize andreproduce when given favorable conditions for reproduction.

Process validation is establishing documented evidence that aprocess does what it purports to do.

Sterility Assurance Level is generally accepted when materialsare processed in the autoclave and attain a 10-6 microbial survivorprobability; i.e., assurance of less than one chance in one million thatviable microorganisms are present in the sterilized article.3

Sterilization process is a treatment process from which the probabilityof microorganism survival is less than 10-6, or one in a million.

STERILIZATION METHOD BIOLOGICAL INDICATOR

Steam Bacillus stearothermophilus

Dry heat Bacillus subtilis var. niger

Ethylene oxide Bacillus subtilis var. globigii

Filtration Pseudomonas diminuta

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Monographs Section I

Thermal Death Time and Thermal-Chemical Death Time are termsreferring to the time required to kill a specified microbial populationupon exposure to a thermal or thermal-chemical sterilizing agentunder specified conditions. A typical thermal death time value withhighly resistant spores is 15 minutes at 121°C for steam sterilization.

References1. Block, S. 1992. Sterilization, p. 87-103. Encyclopedia of

microbiology, vol. 4. Academic Press, Inc., San Diego, CA.

2. Cote, R. J., and R. L. Gherna. 1994. Nutrition and media,p. 155-178. In P. Gerhardt, R. G. E. Murray, W. A. Wood, and N. R.Krieg (ed.), Methods for general and molecular bacteriology.American Society for Microbiology, Washington, D.C.

3. The United States Pharmacopeia (USP XXIII) and TheNational Formulary (NF 18). 1995. Sterilization andsterility assurance of compendial articles, p. 1976-1980.United States Pharmacopeial Convention Inc., Rockville, MD.

4. Perkins, J. J. 1969. Principles and methods of sterilizationin health sciences, 2nd ed. Charles C. Thomas, Springfield, IL.

5. Leahy, T. J. 1986. Microbiology of sterilization processes. In F. J.Carleton and J. P. Agalloco (ed.), Validation of asepticpharmaceutical processes. Marcel Dekker, Inc. New York, N.Y.

6. Simko, R. J. 1986. Organizing for validation. In F. J. Carleton andJ. P. Agalloco (ed.), Validation of aseptic pharmaceutical processes.Marcel Dekker, Inc., New York, N.Y.

Quality Control Organisms

Bacteria Control Strain SourceAn integral part of quality control testing includes quality controlorganisms. Microorganisms should be obtained from reputable sources,for example, the American Type Culture Collection (ATCC®) orother commercial sources.

Maintenance / Frozen Stock CulturesIf using commercial stock cultures, follow the manufacturer’srecommendations for growth and maintenance.

To prepare frozen stock cultures of Staphylococcus species,Streptococcus species, Enterobacteriaceae and Pseudomonasaeruginosa:

1. Reconstitute the stock culture, if necessary.

2. Inoculate multiple plates of a general purpose medium (e.g., TSAor blood agar).

3. Incubate plates for 18-24 hours in an appropriate atmosphere andat the recommended temperature.

4. Check for purity and correct colony morphology.

5. If necessary, verify biochemical tests.

6. Remove sufficient growth from a confluent area to prepare a 0.5McFarland standard (1-2 x 108 CFU/ml). For fastidious organisms,adjust to a 1 McFarland.

7. Suspend the growth in 50-100 ml of cryoprotective medium, e.g.,Tryptic Soy Broth with 10-15% Glycerol, Skim Milk or steriledefibrinated sheep blood.

8. Dispense 0.5-1.0 ml into sterile glass or plastic freezing vials.Prepare enough vials for one year of storage. Assume onlyone freeze/thaw cycle per vial. Assume at least one fresh cultureevery four weeks.

9. Store vials at or below -50°C (freezer) for one year. Organismswill keep longer (indefinitely) if stored in an ultra low temperaturefreezer or in a liquid nitrogen tank.

To use a frozen culture:1. Thaw the vial quickly.

2. Use the culture directly or subculture.

3. Discard any unused cell suspension.

Working CulturesPrepare no more than three serial subcultures from a frozen stockculture.

1. Inoculate an agar slant or plate with the frozen stock culture andincubate overnight.

2. Store the working culture at 2-8°C or at room temperature for upto four weeks.

3. Check for purity and appropriate colony morphology.

OR

1. Use the frozen stock culture directly as a working culture.

Maintain anaerobic cultures in Cooked Meat Medium or another suit-able anaerobic medium. Alternatively, use frozen anaerobic cultures.

Test Procedure1. Inoculate an agar plate from the “working culture”.

2. Incubate overnight.

3. Suspend 3-5 isolated colonies with typical appearance in a smallvolume (0.5-1.0 ml) of TSB. Incubate 4-5 hours in an appropriateatmosphere and temperature.

4. Adjust the turbidity to 0.5 McFarland and 0.08-0.1 absorbance unitsat 625 nm.

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Section I Monographs

OR

1. Adjust an overnight culture to a 0.5 McFarland.

2. Plate 0.01 ml of the specimen to confirm a colony count of1-2 x 108 CFU/ml. If using a frozen culture, confirm the appropriatedensity.

To Test Cultural ResponseNon-Selective MediaDilute the cell suspension 1:100 in normal saline or purified water.Inoculate each plate with 0.01 ml to give 1-2 x 104 CFU/plate. Reducethe inoculum ten fold, if necessary, to obtain isolated colonies.

Selective Media and Tubed MediaDilute the cell suspension 1:10 in normal saline or purified water. Streakeach plate with 10.01 ml of the suspension to provide 1-2 x 105 CFU/plate. Reduce the inoculum ten fold, if necessary, to avoidoverwhelming some selective media.

ResultsFor general-purpose media, sufficient, characteristic growth andtypical colony morphology should be obtained with all test strains.For selective media, growth of designated organisms is inhibited andadequate growth of desired organisms is obtained. Color and hemolyticreaction criteria must be met.

ReferenceNational Committee for Clinical Laboratory Standards. 1996.Quality assurance for commercially prepared microbiological culturemedia, 2nd ed. Approved standard. M22-A2, vol. 16, no. 16. NationalCommittee for Clinical Laboratory Standards, Wayne, PA.

Typical Analysis

“Typical” chemical compositions have been determined on mediaingredients. The typical analysis is used to select products for researchor production needs when specific nutritional characteristics arerequired. The specifications for the typical analysis include:

• Physical characteristics,

• Nitrogen content,

• Amino acids,

• Inorganics,

• Vitamins, and

• Biological testing.

All values are presented as weight/weight; % = g/100 g.

GlossaryAshThe higher the ash content, the lower the clarity of the preparedingredient. The ash content includes sodium chloride, sulfate, phos-phates, silicates and metal oxides. Acid-insoluble ash is typicallyfrom silicates found in animal fodder.

MoistureLower moisture levels (

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Monographs Section I

maximal at low concentrations, 0.1- 0.2 mg/l, and inhibited at highconcentrations). Chelating agents (e.g., citrate) may be added toculture media to sequester trace metals and clarify the media.

Antigenic Schema for SalmonellaUpdate of the Kauffmann-White Schema1The Centers for Disease Control has modified the Kauffmann-Whiteantigenic schema originally proposed by Ewing.1-3 The updated schemaare used with Difco Salmonella Antisera as an aid in the serologicalidentification of Salmonella.

All of the Salmonella serovars belong to two species, S. bongoricontaining 18 serovars and S. enterica containing the remaining2300-plus serovars which are divided among six subspecies.1 The sixsubspecies of S. enterica are:

S. enterica subsp. enterica (I or 1)S. enterica subsp. salamae (II or 2)S. enterica subsp. arizonae (IIIa or 3a)S. enterica subsp. diarizonae (IIIb or 3b)S. enterica subsp. houtenae (IV or 4)S. enterica subsp. indica (VI or 6)

The legitimate species name for the above strains is S. choleraesuis.However, this name may be confused with the serotype named“choleraesuis.” At the International Congress for Microbiology in 1986,the International Subcommittee for Enterobacteriaceae agreed to adoptthe species name “S. enterica.”4 LeMinor and Popoff5 published arequest to the Judicial Commission to use S. enterica as the officialspecies name. The Judicial Commission ruled that S. choleraesuis isthe legitimate name.6,7 S. enterica is used in many countries and isfavorably accepted as the species name.3,8 The Centers for DiseaseControl has adopted this designation until the problem of naming thisspecies is resolved.1

Nomenclature and classification of these bacteria are ever changing.9

Salmonella and the former Arizona should be considered a singlegenus, Salmonella.10 All serovars in subspecies enterica are named.Serovars in other subspecies (except some in subspecies salamae andhoutenae) are not named. It is recommended that laboratories reportnamed Salmonella serovars by name and unnamed serovars byantigenic formula and subspecies. For the most recent information onnomenclature, consult appropriate references.1,3,9,10,12

Serotypes of Salmonella are defined based on the antigenic structureof both somatic or cell wall (O) antigens and flagellar (H) antigens.The antigenic formula gives the O antigen(s) first followed by theH antigen(s). The major antigens are separated by colons and thecomponents of the antigens separated by commas. For example, theantigenic formula for Salmonella typhimurium is Salmonella1,4,5,12:i:1,2. This means that the strain has O antigen factors 1,4,5and 12, the flagella phase 1 antigen I, and flagella phase 2 antigens1 and 2.

Complete identification of Salmonella requires cultural isolation,biochemical characterization and serotyping. Any serological resultsobtained before biochemical identification must be considered as

presumptive identification only. Consult Reference 1 and other appro-priate references for complete identification of Salmonella.1,3,9,11-14

References1. McWhorter-Murlin, A. C., and F. W. Hickman-Brenner. 1994.

Identification and serotyping of Salmonella and an update of theKauffmann-White Scheme. Centers for Disease Control andPrevention, Atlanta, GA.

2. Kauffmann, F. 1969. Enterobacteriaceae, 2nd ed. Munksgaard,Copenhagen.

3. Ewing, W. H. 1986. Edwards and Ewing’s identification ofEnterobacteriaceae, 4th ed. Elsevier Science Publishing Co. Inc.,New York, NY.

4. Penner, J. L. 1988. International committee on systematicbacteriology taxonomic subcommittee on Enterobacteriaceae. Int.J. Syst. Bacteriol. 38:223-224.

5. LeMinor, L., and M. Y. Popoff. 1987. Request for an opinion.Designation of Salmonella enterica sp. nov., nom. rev., as the typeand only species of the genus Salmonella. Int. J. Syst. Bacteriol.37:465-468.

6. Wayne, L. G. 1991. Judicial Commission of the InternationalCommittee on Systematic Bacteriology. Int. J. Syst. Bacteriol.41:185-187.

7. Wayne, L. G. 1994. Actions of the Judicial Commission of theInternational Committee on Systematic Bacteriology on requestsfor opinions published between January 1985 and July 1993. Int.J. Syst. Bacteriol. 44:177.

8. Old, D. C. 1992. Nomenclature of Salmonella. J. Med. Microbiol.37:361-363.

9. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, andR. H. Yolken. 1995. Manual of clinical microbiology, 6th ed.American Society for Microb