Microbiological Criteria

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Development and use ofmicrobiological criteriafor foodsGuidanceforthoseinvolvedin using and interpreting microbiologicalcriteriaforfoods PrefaceWith the increased use ofHazard Analysis and Risk Assessment in the management of food safetyand, to some extent, quality, it is reasonable to ask whether microbiological testing and criteria arestill necessary, other than when required by legislation. Certainly, there is considerable debate onwhether criteria should be published at all. In fact, information on the numbers ofparticularmicrobial populations and their safe limits is still needed in many instances.Microbiological criteria are necessary to assist in setting critical limits in HACCP systems,verification ofHACCP plans, and in shelf life studies where storage trials and challenge tests areneeded. In microbiological risk assessments, knowledge of microbial population distribution andnumbers in food forms an essential part of the information required, in conjunction with populationexposure, infective dose and pathogenicity of organisms. When mathematical models are used topredict microbial growth, survival or decline, it is also necessary to appreciate the numbers that areinevitable and those that cause concern or signal the end of shelf life.For larger companies, such information is generated in-house and is readily available on theirdatabases, but this information is likely to remain confidential and will not be published for reasonsof competitive advantage. Small-to-medium sized companies in supply, manufacture, catering andretailing have little or no such information readily available to them. The information, where itexists, can only e found in a wide diversity of literature, including ICMSF publications, industrycodes ofpractice, legislation and peer-reviewed technical publications. Much of this information isnow elderly and therefore incomplete with respect to current microbiological issues, because recentlyemerged pathogens and new products and processes are inevitably excluded.In order to help redress the balance, this discussion of the development and use ofcriteria isoffered for guidance. It includes a list ofproduct-group related microbiological limits, compiled fromthe experience, knowledge and publications ofmany experts in food microbiology. It acknowledgesthat all tests included in each group are not always needed and that extra tests are sometimes applicable, but within that framework it gives an extensive array of information on what can be achieved yGMP and what represents the limit of shelf-life and safety.This paper is intended for practical use Debate and comment are encouraged and welcomed.The second edition will be published soon as an I ST Monograph. you wish your comments tobe considered, please write to Dr CBell, c/oIFST,I Cambridge Court, 210Shepherd sBush Road, London W6 7NJ by the end of November 1997.

Dr Cathy Stannard, ChairmanI ST Professional Food Microbiology Group

AcknowledgementsWorkingGroupMembers:ChrisBell(Convenor),MelodyGreenwood,Jane Hooker,AlecKyriakides,RebeccaMills.Gratefulthanks are dueto manycolleagueswhogavegenerouslyoftheir timein contributingtoand commentingonvariousdraftsof thisdocument,alsoto MrsMargaretBatten forher patienceand fortitudein typing,retyping etcnumerousdraftsofthisdocumentpriorto its finalappearan::ain the outsideworld.

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Page NoCONTENTS1 INTRODUCTION.1 Aims and scope 1391.2 Background 1401.3 Definition and practical application of microbiological criteria 1401.4 Components of microbiological criteria 1411.5 Limitat ions of microbiological criteria 1412 FACTORS AFFECTING THE MICROBIOLOGY OF FOODS2.1 General 1422.2 Factory structure, environment and personnel 1422.3 Raw materials 1422.4 Process technology 1432.4.1 General 1432.4.2 Temperature 1432.4.3 Water activity 1432.4.4 pH and acidity 1432.4.5 Atmosphere 1432.4.6 Combination of factors 1432.5 Shelf life 144

2.6 Application of microbiology 144 .3 SAMPLING FOR MICROBIOLOGICAL TESTING3.1 Introduction 1443.2 Terms 1443.3 Selection of sampling plan 1453.3.1 Sampling plan structure 1453.3.3 Enumeration (counts) of microorganisms 146.3.2 Detection (presence/absence) tests 1453.3.4 Censored values 146 G

4 CONSIDERATIONS IN CHOOSING MICROBIOLOGICAL METHODS4.1 General 1464.2 Choice of test 1464.3 Accuracy of the result 1474.4 Limit of detection of the method 1474.5 Recovery of target organisms 1474.6 Comparability of results between different methods 1474.7 Inhibition of growth of microorganisms by the constituents of the food tested 1484.8 Competitive growth 1484.9 Test methods 1485. FOOD CATEGORIES5.1 Introduction 148 -5.2 Frozen foods 148

5.3 Dried foods, low water activity foods and other ambient stable foods 1485.4 Fresh & chilled foods 1495.5 Heat-treated foods 1495.6 Fermented foods 1506 MICROBIOLOGICAL LIMITS6.1 Introduction 1506.2 Use of Tables 1516.3 Index for Foods 1526.4 Abbreviations 1536.5 Tables 153

A: Raw Poultry 154B: Raw Meat 154C: Raw Fish & Shellfish 155D: Doughs, Pasta & Batters 156E: Fruit and Fruit Juices 156

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"F: Raw and Prepared Vegetables (including Salad Vegetables) 157G: Milk, Cream & Dairy Products 158H: UHT Milk, Cream & Dairy Products 159I: Part Cooked foods 159J: Processed Foods 160K: Dried Foods, to be cooked . 161L: Dried Raw Foods, ready-to-eat 161M: Dried Heat Processed Foods 162N: Dried Heat Processed Foods, ready-to-eat after rehydration 1620: Dried Baby Foods 163P: Canned, Pouched or Bottled Foods (F0>3 process) 163Q: Preserved Foods - Heat Treated (Fo


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from the practical experience of the authors as wellas existing guidelines and standards. Such adocument can never be exhaustive and the foodcategories used encompass a wide range of products.Where microbiological testing and criteria areconsidered useful by a food business theinformation and figures given in this document maybe used as a basis for determining relevant criteriafor individual products.

I t is hoped that this document will stimulate aproductive discussion on this important subject.1.2 BackgroundFoodborne disease and microbial spoilage of foodresult from the failure or inability to controlmicroorganisms at one or more stages of the foodchain, from raw material production to consumptionof the final product. The implications of situationsthat result in food poisoning outbreaks or foodspoilage can be severe for food producers, retailers,consumers and regulatory authorities.

Traditionally, control of microorganisms in a foodhas been demonstrated by microbiological testing ofsamples at various stages of production and ofthe final product. Criteria have been developedto give some degree of assurance that food issafe and of suitable quality, and that it will remainso to the end of its shelf life provided it is handledappropriately. Conformance to microbiologicalcriteria is determined by testing. I t is recognisedthat microbiological testing can never give anabsolute assurance of product safety or quality,particularly because of the problems involved inproduct sampling and the distribution ofmicroorganisms in food Used judiciously however,microbiological testing is a valuable tool in the foodindustry.

In the food industry today, microbiological testingcan be effective when used alongside physical andchemical process control systems as part of hazardanalysis approaches such as HACCP ICMSF, 1988)and other HACCP-based systems. The HACCPsystem is a structured approach to identifyinghazards and defining and implementing systems ofcontrol. Microbiological examination can be used tohelp verify that these critical control points remainunder control.1.3 ~ i n i t i o n and practical application ofmicrobiological criteriaMicrobiological criteria are essentially of threetypes:

Standard Guideline SpecificationThese terms as defined by the Codex AIimentariusCommission Codex, 1981), were principally for usein Codex standards and codes of practice.140

Microbiological standards guidelines andspecifications have been redefined by CodexCodex, 1993), the International Commission onMicrobiological Specifications for Foods ICMSF,1986) and the United States Subcommittee onMicrobiological Criteria NRC, 1985) and forsimplicity the following working definitions aregenerally recognised:tandard - This is a microbiological criterioncontained in a law or a regulation where complianceis mandatory. These are introduced by governmentsor regulatory authorit ies. Typical examples include

most criteria in European CommunityEC)lEuropean Union EU) Directives and StatutoryInstruments of England and Wales. The foodindustry must ensure full compliance with thesestandards which are monitored by enforcementagencies. s well as being an offence, products not / -complying with standards will be rejected as unfitfor the intended use. Results not conforming tostandards may be used by enforcement agencies asthe basis for prosecution or for refusing importationof foods. Current EU microbiological standards aresummarised in Section 7Guideline - A microbiological guideline is a criterionapplied at any stage of food processing and retailingwhich indicates the microbiological condition of the ,ample and aids in identifying situations requiringattent ion for food safety or quality reasons. Results 5btained from testing against a microbiologicalguideline also assist in trend analysis. Results thatdeviate significantly from the trend may indicate atendency towards a situation which is out of controland highlight the need for attention before control islost. Guidelines are usually self imposed by the foodindustry but may occasionally be included inlegislation. Products not complying with guidelinesshould result in investigative action to identify andrectify the cause. Guidelines on the levels and typesof microorganism relevant in specified foodsproduced under good manufacturing practice may -also be provided by industrial associations for theirmembers. In some EU Directives there areguidelines for indicator organisms e.g. Directive92/46IEEC on milk and milk products. In the UK,the Public Health Laboratory Service has publishedmicrobiological guidelines PHLS, 1996) to assistfood examiners and enforcement officers in thedetermination of the microbiological quality of foodsand to indicate levels of bacterial contaminationconsidered to be a potential health risk in readyto-eat foods at point of sale.Specification - This is a microbiological criterionapplied to raw materials, ingredients or the endproduct which is used in a purchase agreement.Criteria may include pathogens, toxins, indicatormicroorganisms or spoilage microorganisms wherenon-compliance may affect product safety and/orquality during shelf life. End product specifications

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are usually more stringent than microbiologicalstandards n order to provide a margin of safety.

Products not complying with specificationsshould be investigated to determine the cause.Rejection of products may occur even i f they are nothazardous or unwholesome at the time of testing.

It is important to ensure that the components ofmicrobiological specifications are relevant andrealistic and fully understood by both parties in theagreement.1.4 Components of microbiological criteriaA microbiological criterion consists of a statement ofat least the following:

the microorganism or microbial toxin of concern the foo concerned and sample type the sampling plan (see 3.3.1) the microbiologicallimit(s)onformance to microbiological criteriashould be monitored using specified, relevantmethods which have been validated for the

microorganism or toxin o f concern in the foodbeing examined see also 4.9 and 6.1).Microorganism or microbial toxin of concernThe contaminants detailed in the criterion may befoodborne pathogens, food spoilage organisms,indicator organisms or microbial toxins (ICMSF,1978). Specified microorganisms must be relevant tothe foo and/or food process (see Appendix: Specificorganisms and the principles of test methods).Pathogen testing is applied for reasons of publichealth where evidence exists ofa potential hazard tohealth from a specific food/organism. Testing forindicator and spoilage microorganisms is used forthe following reasons:Cost effectiveness: More analyses can be conductedfor microbial indicators of unhygienic practice andspoilage microorganisms than for specific pathogensbecause testing for pathogens is usually more expensive.Simplicity: Test for indicator microorganisms arefrequently simpler to conduct than tests for specificpathogens.Rapidity: Tests for indicator organisms usually provide results more rapidly than those for pathogens,thus allowing for faster remedial action.Trend analysis: Indicator and spoilage microorganisms are usually present in higher numbers thanpathogens and indicate unsatisfactory conditionswhen levels increase significantly. Since levels canbe monitored, trends can be established so thattrend analysis can identify situations before theybecome out of control. Tests for pathogens or toxinsusually result in numerous negative results; any

positive results indicate situations which oftenrequire immediate remedial action.Food concerned and sample typeMicrobiological criteria must indicate the food andstage of processing to which they relate. Ideally, theyshould give a description of the food, indicating keyprocessing features and conditions under which thematerial should be stored and used. Such factorssignificantly influence the content and validity of amicrobiological criterion. For example, a specification for frozen foo may allow higher aerobic platecounts at the point of production compared with thesame chilled foo since bacteria will not grow underfrozen conditions but may grow during chilledstorage. However, bacterial growth during or afterdefrosting should be taken into account for frozenproducts.Also, the microbial levels in a perishable sampletested at the beginning of its shelf life are likely todiffer from those at the end of shelf life. Suchdifferences must be taken into account whenspecifying a microbiological criterion and it shouldbe made clear at what point in the product shelf lifethe relevant criterion is to be applied. It isalso important to indicate the nature of the samplei f this is likely to influence the end result andits subsequent interpretation. Microbiologicallysensitive components of multi-component foods e.g.sandwiches may need to be tested separately. In thefood industry such details are usually contained inproduct specifications.Microbiological limitThe microbiological limit defined in a criterionrepresents the level above which action is required.Levels must be realistic and should be determinedfrom a knowledge of the microbiology of rawmaterials and the effects of processing, producthandling, storage and end use on the microbiologyof the final product. Limits may be derived bydetermining microbial levels in products whereconditions of processing, storage and productcharacteristics are known. They should take intoaccount the levels that indicate inadequate controland may represent a hazard to health.

t is not possible to prove the absence of anorganism in a product and negative results fromdetection (presence/absence) tests should bedeclared as not detected (ND) e.g. not detected in25g or not detected in 1ml.

For quantitative tests in which no growth occurs,it is normal to record the result as less than thelimit of detection of the test e.g.


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number of factors which should always be clearlyunderstood when using them:a) Methods

No microbiological method is capable of detect ing allrepresentatives of the target microorganism beingsought; even the best are probably capable of recovering no more than 90% of representatives. Thepresence of sub-lethally injured cells or cells thatmay be viable but non-eulturable, contaminantspresent in the food but at a level below the sensitivity of the method and variants not capable of growthunder the conditions of the test will all result in lessthan total recovery of the target organism.Confidence limits for results from enumerative techniques are also relatively wide (BS 5763: Part :1996).b) Sampling

Microorganisms are rarely distributed homogeneously in a sample or a product batch andpathogens, if present, are usually at low levels. Anysingle test for detect ion or enumeration only reflectsthe situation in that particular sub-sample of thebatch. The effectiveness ofthe test is also dependenton sample size and type and sampl ing techniques;aseptic sampling procedures are of particularimportance for preventing extraneous contamination.c) Laboratory competence

Laboratory staff competence and the operation ofquality systems in laboratories are very importantfor achieving valid microbiological results.Independent auditing, use of extemal quality assur-ance schemes and accreditation of laboratories helpto provide assurance of laboratory competence.d) Cost effectiveness

The only way microbiological testing and associatedcriteria can be totally reliable is by adoption of 100%inspection with methods of 100% reliability andaccuracy. Since current methods are effectivelydestruct ive, 100% testing would leave no food to consume and whilst this would undoubtedly reducefoodbome illness it is not a feasible option

In the absence of 100% inspection but by theapplication ofstatistical techniques to sampling, thedegree of assurance derived from a result will bedependent on the number and size of samples taken.Detection of foodbome pathogens, present at lowlevels and distributed heterogeneously in a food,requires an exceptionally high number of samples toe analysed to achieve a reasonable degree of assure (ICMSF, 1986).Despite the limitations described (which are well

use of microbiotesting may play an important part in

hazard analysis process. When used regularly,

non-statistical sampling and testing schemes arecost effective in that they are a simple form ofmonitoring (trend analysis) that can allow trends tobe identified and add to the assurance that theprocess is under control. This constructive use of rmicrobiology alongside other process controlsystems helps to ensure that the food supply is safeand of good quality.

2. Factors affecting the microbiologyof foods2.1 GeneralThe food industry uses a wide variety oftechnologiesto produce an enormous range of foodstuffs, fromchilled foods with a short shelf life to shelfstable intermediate moisture foods and long lifepackaged foods. Each technology involves a complexseries of interlinked processes that combine toproduce safe and wholesome products. t is essentialto understand the interrelationship ofthese processes so that relevant criteria can be developed andapplied. Monitoring procedures should be derivedfrom HACCP-based assessments of all the processesinvolved.

Factory structure, manufacturing equipment,raw materials, process flows, temperature/timecontrols and hygiene systems (personnel andenvironment) all affect the microbiological nature ofthe product and each requires careful evaluation.2.2 Factory structure, environment andpersonnelIn establishing criteria, consideration must be givento the potential for contamination of food from theenvironment. For example, a cooked, sliced meatwill be more susceptible to environmental contamination than the same type of meat cooked in its finalcontainer; this should be reflected in the associatedcriteria. The IFST booklet Food and Drink - Good -Manufacturing Practice: A Guide to its ResponsibleManagement (1991) contains useful advice concerning premises, equipment and approaches to goodmanufacturing practice (GMP).2.3 aw materialsIt is essential to understand the nature of potentialhazards that may be presented by raw materials.Specifications for raw materials should containrelevant microbiological, toxicological and physicochemical parameters. Microbiological criteria forfinished product should take account of the level andtypes of microorganism present in a raw materialand the processing conditions which will affect thelevels of organisms throughout the process. Evenwhere a manufacturing process may be expected toeliminate a particular potential microbial hazardfrom the raw material, the possible production by

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the microorganism of heat-stable toxins which maycause illness or enzymes proteases and lipases)which may cause spoilage must be taken intoaccount in microbiological criteria.2.4 Process technology2.4.1 GeneralA clear understanding of the effect of food processtechnologies on microorganisms and their survivaland growth characteristics in foods is necessarybefore relevant crit eria can be assigned ICMSF,1996).2.4.2 1er.nperatureFoods may be chilled, frozen, mildly heat treatedthermised, pasteurised, sous vide) or exposed tohigh heat treatments Ultra High TemperatureURT) or those used in some drying or canningprocesses).

Freezing to temperatures below approximatelyminus lOoC will stop microbiological growth butenzyme activity can still occur and may causeorganoleptic changes. The microbial population atthe point of freezing generally remains stablethroughout the frozen shelf life althoughsome organisms are more susceptible to freezingtemperatures and may die.Chill temperatures prevent the growth of manymicroorganisms, but some pathogens and a widevariety of food spoilage organisms may grow, albeitrelatively slowly. Criteria applied at the point ofmanufacture must take account of the potential forsubsequent growth of psychrotrophic microorganisms during shelf life.Mild heat treatments such as pasteurisationreduce the levels of vegetative microorganisms infoods but thermotolerant organisms and spores maysurvive. Criteria must take account of these organisms together with post-process contaminants.High temperature processes such as UHT andsome canning processes are used to produce shelfstable products. Vegetative microorganisms shouldbe completely destroyed and only a few types ofspore forming bacteria are able to survive suchprocesses although pathogenic strains are killed.2.4.3 Water activityWater activity Ow) is a measure of the wateravailable for microbial growth; reducing the a w in afood can restrict the growth of many microorganisms. Water activity may be reduced from themaximum 1.0 for pure water) to low levels 0.75by direct removal of water e.g. drying, or by theaddition of solutes such as salt or sugar. Themicroorganisms capable ofgrowth or survival at theaw of a particular food should be considered whensetting criteria. For example, most bacteria cannotgrow below aw 0.9 but Staphylococcus aureus

S. aureus) can grow down to approximately aw 0.85and may be important n criteria for some driedproducts whilst some yeasts and moulds can grow atlevels as low as Ow 0.6.2.4.4 p and acidityThe pH/acidity of a product affects the range oforganisms that can survive and grow. Manyproducts have a low pH Le. 3.0 see section 5.5) oracidify the product to ensure its safety. The possibility of outgrowth of organisms that may survive anyheat treatment and raise the product pH to a levelat which pathogens may grow should also be takeninto account.2.4.5 Atr.nosphereThe atmosphere within product packaging is oftenaltered to help improve product keeping quality andincrease shelf life. Air may be removed altogether tocreate a vacuum as with some meat joints, or airmay be replaced by other gases e.g. nitrogen and/orcarbon dioxide in various proportions as with manysliced cooked meats , fresh salad mixes and fish products. Most of these latter types are tenned modifiedatmosphere packs MAP) and the atmosphere within these packs changes over time because of thedynamic processes which occur between the foodand the atmosphere.Strictly controlled atmosphere packs CAP) arethose in which these dynamic processes arebalanced throughout the storage life of the product,the atmosphere therefore remaining constant. Somenovel packaging systems incorporate a chemicalscavenging system to keep oxygen levels low. In eachparticular case, the packaging material is carefullyselected to ensure its permeability characteristicsare appropriate for the required atmosphericconditions to be maintained. Such packagingsystems are often used for chilled foods andalthough the changed atmospheric conditionscontribute to the inhibition of some microbiologicalgrowth, lactic acid bacteria and yeasts in particularmay cause spoilage. Consideration must be given tothe potential for some modified atmospheres toprovide conditions which select for the growth ofclostridia including C. botulinur.n ACMSF, 1992).2.4.6 Cor.nbination offactorsThe combined effect of non-optimal conditions oftemperature, aw pH and packaging atmosphere onthe growth and survival of microorganisms usually

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has agreater effectthan an individualfactorusedatthe same level (ICMSF, 1996). Preservatives canalso beused in combination with these factors toenhance microbial suppression. The ICMSF bookFactors Affecting Life and Death of Microorganisms(1980a)givesextensiveinformationoneachoftheabove factors in addition to listing some 'limitsfor growth'ofsome microorganismsfor aw and pHvalues.2.5 Shelf lifeThepotentialforgrowthand/ortoxinproductionofresidualmicrobialpopulationsin finishedproductsdependsonthe typesoforganismspresent and theirability to grow to a level of concern under thestorageconditionsappliedduringthe productshelflife.Microbiological criteria should take account ofany organisms likely to be present. The levels oftoleranceappliedat the pointofmanufactureshouldbesuchthat allowingforpredictablegrowthoftheseorganisms, the productwillremain safeand wholesome to the end of shelf life provided it is storedunder the correctconditions.It isindustrypracticeto bui ld in a safety margin for chilled productswhichallowsformildtemperature abuseofproductduring storage.2.6 Application of microbiologyMicrobiological testing, evenus ing rapid methods,takes time(sometimesmany days)and costsmoney.Thereisatendencytoincludetoomanyorganismsand toapplyover-stringentlimits in microbiologicalcriteriain amisguidedattempt toachieveproductsafety.Inappropriatecriteriaare costlyin timeandresources(humanand financial)whichcouldbe puttobetter usein the practicalapplication of HACCPand GMP systems. t is therefore important toensure that any such testing isbothrelevant andmeaningfulwithinthe contextof the foo productionoperation. Answering the following questions willassistin obtainingthe mostsensible,practicalandcosteffectiveuseof microbiology: what informationisneeded? whyisthis informationnecessary? what isthe best meansof obtainingtheinformation? i f amicrobiologicaltest istobeused,whereisitbest applied? what criteriaare appropriateand practicable? what response is appropriate to 'out of limit'results?Forexample,tensofthousands offmishedproductexaminationsforS.aureus are carriedout eachyearin industry on extensively handled foods and theorganismisrarelydetected.In order tominimisethelevelof S.aureus in the finishedproduct, it may bemore effective to apply a structured personnel

hand-swabbingand training programme for thoseinvolvedin directhandlingofthe productand/oritscomponents.Thisapplicationofthe test couldyieldmore positive benefits in heightened personnelawarenessand improvedpersonalhygienepracticesand handling procedures than would be accruedfromsomany tests onproducts.Product safety and due diligence obligationswill be supported more cost effectively by theapplication of directly relevant micro-biological testing regimes and criteria basedon sound product and process knowledge inassociation with H CCPbased systems and theoperation ofGMP

3.Sampling for microbiological testing3.1IntroductionNoamount of testingwillevermake afoodsafe.Thesheer volume and variety of food produced, thedistributionand state ofmicroorganismsin the food,and industry resourcelimitationsmake it essentialto ensure that food production systems areinherently safe. Microbiological testing should beregarded as a helpful tool in support of thesesystems,notas ameans wherebya'satisfactory'or'unsatisfactory'result froman end productisusedtodemonstratethe safetyofthe food.Thereliabilityofa result from the analysisofafoo isdependentuponthe accuracyand precisionofthe method, the quality of the aseptic samplingproceduresusedand the extent ofsampling.

Inappropriate sampling can lead to invalidconclusions being reached. The purpose andlimitationsof samplingand the relevanceof statisti-caltechniquestosamplingneedtobeunderstoodifthe results from microbiological testing are tobecorrectlyinterpreted.3.2TermsTheterm 'sample'referstothe material removedforexaminationfrom an identifiedbatch oroperation.This sample may consist of one or more sampleunits.Theindividualsampleunit may be split intoone or more test portions,or sampleunits may becombinedfortesting.Thetest portionwillusually beonly part of the sampleunit and it is essential tounderstand that the microorganisms in a sampleunit are unlikelytobedistributedevenly. For thefollowingdiscussion,unlessotherwiseindicated,nodistinctionismadebetweensample,sampleunit ortest portion.Statistical significance is used to describe theconfidence in any decision made concerning thesafety or quality of the food. A high level ofconfidence (significance) is assumed to begreaterthan 95%certaintyof beingright.Mostmicrobiologicalsamplingplans in commonuseworkwith less

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confidence than this. This does not make these plans, invalid, but the user should be aware of a plan'sperformance when using it. A full discussion ofstatistical sampling and methods is given in theICMSF book ampling for Microbiological Analysis:Principles and Specific Applications 1986).The criteria given by the ICMSF are generallyrecommended for port-of-entry sampling andexamination of foods, circumstances in which verylittle may be known concerning the food productionprocesses (see 2.1). Food manufacturers however,should have full knowledge of raw materials,processing technology and other factors contributingto the product's microbiological characteristics.With this knowledge, product sampling and microbiological testing in food businesses can be appliedin a more practical way (see 1.2).The size, number, composition and method oftaking samples will affect the outcome of a test andshould be specified. Attention to consistency withrespect to these points will maximise repeatability,but uneven microbial distribution may still causeresults to vary significantly between tests.Sampling should also take account of the natureof the hazard and the likelihood of it occurring giventhe raw materials, the process and process controls.Hazard categories and associated sampling planshave been detailed (ICMSF, 1986 and 1994).Historical data demonstrat ing the absence of a hazard are often used in determining a sampling plan,but in the absence of a hazard analysis approachassumptions based on such data are unsound.3.3 Selection of sampling plan3.3.1 Sampling plan structureThe sampling plan should describe the samplingstrategy used in assessing the microbial target andindicate the action to be taken in response to testresults. The principles of 2-class and 3-classattribute plans are described by ICMSF (1986) andthese are commonly applied in industry.Sampling plan definitions may be summarised asfollows:Two-class sampling plann = number of samples analysedm = maximum acceptable level of targetorganism(s) or toxin(s)c = the number of samples allowed to exceed mIn industry, 2-elass sampling plans are appliedmainly when testing for specific pathogens usingdetection (presence/absence) tests and c=O.Example: In a two-class sampling plan forSalmonella in a ready meal, where 5 samples eachof 25g are taken and tested and the presence of anypositives is unacceptable, the following applies:

n=5, c=O, m=not detected in 25g

This is a simple test protocol. A specified samplesize and number are examined. f a not detected'result in these samples i s achieved, the test requirements are satisfied. At a given level of contamination for organisms distributed homogeneously, tbelarger the sample examined e.g. 100g, the greaterthe chance of detecting the target organism.If organisms are unevenly distributed, a greaterconfidence is achieved by specifying larger numbersof samples for examination. Table 1 illustrates thisapproach.Table 1: Probability of accepting a batch containingspecific percentages of defective product

%Probability of acceptanceNumber of samplesexamined Actual % of defective samples

10% 20% 30% 40%3 73 51 34 225 59 33 17 810 35 3


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number of samples are the two main considerationswhen determining the probability of detecting atarget organism with a given frequency of contamination. This type of test is not used to quantify bacteria in a food f a high degree of confidence isrequired to detect very low numbers of positives(defects) in a batch, many samples must be taken(lCMSF, 1986). For practical and economic reasons,it is common practice to combine a number ofsamples to reduce the number of tests required.Continuous sampling devices are useful forremoving large numbers of small samples,particularly for powders and liquids, which are thenmixed thoroughly before removal of test portions forexamination.3.3.3 Enumeration (counts) ofmicroorganismsEnumeration procedures are normally carried outusing a minimum test portion of 109. A homogenateand further dilutions are prepared from the testportion and used to enumerate the level of organisms in the portion; counts are typically expressed inresults as colony forming units per gram (cfulg).Estimates based on countslg assume that perfectmixing has occurred at all stages of sampling beforeany aliquot is removed for examination.ICMSF three-class sampling plans are often usedto describe specifications for microbiological counts.For example, where five samples ofa ready meal aretaken and it is acceptable for the aerobic plate countto exceed 108 cfu/g on 2 occasions but not to exceed10' cfu/g at all, the following applies:

n =5, m =103 cfu/g, M = cfu/g, c =2In many manufacturing situations the sample numbers do not readily lend themselves to sampleattributes plans; this may occur for instance, when amicrobiological specification relates to one sampleonly (n = 1). In such cases, practical strategies aresometimes employed in industry in which samplesfrom different batches are examined and resultsused for trend analysis.3.3.4 Censored valuesA censored value is one that is outside a measurab lerange. These are common in food microbiology. Thecommonest examples are 'less than' estimatese.g.10 3/g)do occur.

For statistical analyses of results, it is importantto avoid censored values; this can be done by extending the dilution ranges examined in 'count'tests.4. Considerations in choosing microbio-logical methods4.1 GeneralFactors to be considered when choosing amicrobiological.method include:

1) Proven capability of recovering the targetorganism(s) from the product being examined(validation).2) How soon the result is required. r3 Ability to achieve the sensitivity and specificityrequired.4 Cost.The microorganisms being detected or enumeratedare effectively defined by the method used. Forexample, an aerobic plate count (APC; also referredto as the standard plate count or total viable count)will only determine the number of organismscapable of forming visible colonies after a specifiedtime on the growth medium used and under thespecific temperature and atmosphere used for incubation. Thus an APe at 300C for 48 hours may givedifferent information from an APC at 220 C or 370 Cfor 48 hours. \4.2 Choice of testInformation about relevant target organisms (and the basis for their use in microbiological specifications can be found in the Appendix: Specific organisms and the principles of test methods.a) Indicator and spoilage organisms

Examination of a product for indicator or spoilageorganisms can provide simple, reliable and rapidinformation about processing failure, postprocessing contamination, contamination from theenvironment and the general level of hygiene.Methods normally involve estimation of numbers oforganisms in the food. These methods cannotreplace examination for specific pathogens wheresuitable methods exist and where such testing isappropriate, but usually provide information in ashorter time than that required for isolation andidentification of specific organisms or pathogens.(b) PathogensMost methods of examination for pathogens aim todetect very low numbers e.g. a single viable cell ofthe target organism in a defined amount of sample.Although detection tests are also referred to aspresence/absence tests, absence of a pathogen froma food canno t be proven; microorganisms can only bedemonstrated to be 'not detected' by the test methodused. Classical microbiological methods are oftenslow and labour intensive requiring resusc itation ofsub-lethally injured organisms, selective enrichment and isolation of the target organism usingmedia which encourage the growth of thetarget organism whilst suppressing growth of thebackground flora. This is then followed by confirmatory tests on suspect isolates. Because detectiontests generally incorporate a resuscitation stagethey allow better recovery of stressed organisms

.than moat direct plating methods.Food Science and Technology Ibday 11 (3) 199746


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4.3 Accuracy of the resultAccuracy depends on the methods used, thetechnical competence of the laboratory staff, and thenumber, physiological state and distribution of thetarget organisms present. ISO 7218, MicrobiologyGeneral guidance for microbiological examinations(1985, revised 1994), tabulates the statisticalvariability of counts arising from plating methodsand most probable number (MPN) procedures. Anunderstanding of the capability of the method andthe error margins of the results is essential for thesensible application of microbiological criteria andinterpretation of results obtained.A count of 10,000 cfu/g appears very exact butcould have been calculated from only ten colonies ona medium inoculated with 1ml from a 1 in 1000dilution of the test portion. There are clearly definedrules relating to the number of colonies acceptablefor counting under such circumstances, but thesecannot always be followed. The lower accuracy ofestimates made outside this acceptable rangee.g. low numbers of colonies (typically lower than20) needs to be recognised. The confidence limits ofcounts ranging from 3 - 320 are illustrated inTable 2.Most probable number estimates are often usedby food microbiologists but are not precise estimatesof numbers of organisms. Nevertheless MPNtechniques are widely used (Mossel et aI, 1995) forsamples in which levels of target organisms areexpected to be low i.e.


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4.7 Inhibition of growth of microorganisms by 5. Food categoriesthe constituents of the food tested 5.1 IntroductionGrowth of microorganisms is often affected by thechemical composition of the food; if insufficientlydiluted or neutralised, these factors may preventgrowth of the target microorganisms during the testprocedure. Salt or sugar content, concentrations ofchemical preservatives and the acidity of theproduct are examples of factors that may influencethe test results. Chocolate, many herbs and spices,garlic and onion contain inhibitory compounds andmany flavourings may contain high levels of saltand acids. These inhibitory compounds may affectthe results of tests. Examination of samplesinoculated with known levels of representativestrains of the target organisms can help toverify both media and method efficacy (Mossel et al1995).4.8 Competitive growthThe microflora of foods can vary widely, for examplebetween the fresh product and the same product atthe end of shelf life. Growth of the target organismmay be suppressed or the organisms may beoutgrown by background flora. Validation studiesmay be useful to demonstrate that the method useddetects the target organism in the presence of thecompetitive flora in the products to be examined.4.9 Test methodst is important to ensure that the method useddetects the organism of concern in the food

being examined. t may be necessary to initially validate the method and in routine usequality assurance systems must be employed toverify test efficacy.Reference and industry test methods areavailable from several sources e.g. BritishStandards Institution, International StandardsOrganisation, International Dairy Federation,International Commission on MicrobiologicalSpecifications for Foods, Public Health LaboratoryService (UK), Association of Official AnalyticalChemists (USA), Campden and Chorleywood FoodResearch Association (UK). Many microbiologicalstandards in legislation also prescribe methods. Theappendix describes the principles of classical testmethods and discusses the relevance and limitationsfor some of the more commonly specified targetorganisms. New rapid test methods are becomingavailable for many bacteria and their toxins, andtheir performance is generally measured by comparison with results obtained by classical methods. Forsome microorganisms e.g. viruses and protozoa,there are currently no methods appropriate forrout ine applicat ion to foods.

In establishing microbiological criteria it isimportant to consider the technologies applied in thepreparation of the food. The following sectionsdiscuss some aspects of the technologies and theireffect on microorganisms.5.2 Frozen foodsFrozen foods are microbiologically stable Le. thereis no growth of micro,organisms providing thetemperature is below about minus lOoC. Mostbacteria do not grow below DoC, but somepsychrotrophic organisms, particularly some fungi,will grow slowly at a few degrees below freezingpoint. The shelf life of frozen foods is determinedby physical and chemical/enzymic changes e.g.dehydration and oxidation, and not by microbiological growth. Some bacteria and fungi die in a frozenfood and others become sublethally damaged.However, most microorganisms are preserved byfreezing; Salmonella for example has been recoveredfrom frozen foods after years ofstorage. Therefore, itmust not be assumed that freezing can improve themicrobiological safety or quality of a food. Freezingdoes not inactivate the toxins of microorganisms butcan reduce the infectivity of some pathogenic protozoa and nematodes e.g. in raw meat and fish(ICMSF,1980a).The application of microbiological criteria tofrozen foods is dependent on the intended use of thefood e.g. stringent criteria may be applied to frozenfoods which are ready-to-eat after being thawed.Food should be handled and stored hygienicallybefore freezing so that its quality does notdeteriorate. Microbiological testing is occasionallyused to monitor the efficacy of such handlingprocesses. The rate of freezing and thawing cansignificantly affect the microbiology of a food andfactors affecting the rate of freezing such as packsize and temperature gradients must be considered.Frozen foods are often examined after controlleddefrosting of the sample and microbiological testsused should incorporate a resuscitation procedure tomaximise the recovery of any damaged cells.5.3 Dried foods, low water activity foods andother ambient stable foodsDried foods with an llw of


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Drying without heat preserves many bacteria sothe process cannot be relied upon to improvemicrobiological quality; in fact the net effect can beto increase the concentration of microorganisms.Numbers of organisms generally decline slowly indried foods (over a period of months or years)although Salmonella has been shown to survive foryears in chocolate (lCMSF, 1980b). However somefoodbome pathogens, notably Campylobacter do notsurvive well in a dry environment.Microbiological criteria for dried foods are variedand take account of the intended use of the food(ready-to-eat or not), the intended consumer (babyfood or not) and the type of food (heat processed,fermented, dried).

The inhibitory factors in many products (seeSection 4.7) must be diluted out or neutralised priorto microbiological examination. Pathogens presentin dried foods may be stressed and a resuscitationstage must be incorporated into detectionprocedures.5.4 Fresh chilled foodsMost fresh foods are not microbiologically stable andsome commodities are highly perishable. Theassociated microflora and consequent shelf life willbe affected by the quality and hygienic conditions ofhusbandry, slaughtering, harvesting, storage andprocessing. The application of rigorous hygienemeasures at all stages will help to prevent productcontamination and achieve a realistic shelf life.Extension of shelf life of many fresh foods can beachieved by chilling, and the type of packaging usedmay also have an effect on shelf life. During chilledstorage the microbial profile will change from predominantly mesophilic to psychrotrophic, andspoilage will be due to the nature and activity ofthese psychrotrophic microorganisms. In contrast,the microbiological quality of foods such as nuts andcitrus fruits that have a natural protective outercasing depends on maintaining the integrity of thatouter barrier and the control of moisture duringstorage. The contents are comparative ly microbiologically stable unless penetration of the outercasing occurs.

The occasional presence of pathogens can beexpected in most raw foods. When selecting relevantmicrobiological criteria, the type of food, therequired shelf life, storage conditions, intended useand destination of the commodities must all betaken into account. An aerobic plate count may beused to indicate the quality and the probable shelflife of many raw fresh foods; whilst normally this isperformed at 300C to measure the mesophilicpopulation, a temperature of 20-250C may bepreferable for chilled foods in order to estimate thelevels of psychrotrophic organisms present andhence the potential spoilage organisms. Yeast ormould counts are more relevant indicators of shelflife than bacteria for commodities with a low pHsuch as fruits and fruit juices. Indicator organisms

may be used to monitor the hygiene of processing,and may also give information about the temperature conditions of storage. In addition, E. coli levelsmay be used to assess the quality ofirrigation waterused for fruit and vegetables, to monitor slaughterhouSe hygiene and the conditions for production andmarketing of live bivalve molluscs (Directive9V4921EEC .5.5 Heat-treated foodsHeat-treated food may be ready to eat either hot orcold, may require reheating or further cookingbefore consumption, or may undergo further cookingas an ingredient in a composite food product. Thedegree of heat treatment it has received will affectits microbiological profile and stability. Some formsof heat treatment such as blanching or scalding arenot designed to cook the food, but to inactivateenzymes or assist in skin or shell removal. This typeof mild heat treatment and others such as pasteurisation and sous-vide processes will kill most vegetative bacteria, leaving only heat resistant strains andspores. Pasteurisation or an equivalent process alsokills the majority of common foodbome yeasts andmoulds but does not destroy mycotoxins.

Growth of any surviving organisms in many heatprocessed foods is minimised by chilled storage,acidity, chemical preservation, or some othercontrolling factor.A more severe heat treatment such as the UltraHigh Temperature (URT) process used for certainliquids results in a shelf stable product at roomtemperature provided that aseptic filling occursafter heat treatment or that the liquid is heated inthe final packaging. Oven baking of biscuits andcertain cakes may also result in stable products atambient temperature as long as contact withmoisture and surface mould contamination can beprevented. Cooking of meats and meat products mayallow the survival of bacterial spores, and so chilledstorage will be necessary after cooking.Long-term microbiological stability without theuse of refrigeration for non-dried foods is obtainedby prolonged heating in hermetically sealed containers such as cans; the heat process will need to bemore severe for low acid foods (Hersom Hulland,1980). Low acid foods (pH greater than 4.5) mustreceive a minimal thermal process ofFo 3 (121.1oCfor 3 minutes) known as a botulinum cook' or anequivalent heat process. 'Ib minimise spoilage due tothermophilic spore forming bacteria in canned foodsdestined for or manufactured in countries with hotclimates, it may be necessary to apply a very muchhigher thermal process e.g. F0>10.

The bactericidal effect of heat will depend on thenature of the food, for example the fat or sugarcontent, moisture content or acidity, as well as thetemperature and exposure time employed. Theparameters of the heat treatment must take accountof the characteristics of the food. Measuring thetemperature and duration of heat treatment in all

Food Science nd Techrwlogy Today (3) 1997 149


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processes is of prime importance; the integritythe containers is also essential to prevent ingress

after heat treatment. The maintesting of heat processed

after heat processing may affectmicrobiological status of the product and hencecriteria that are applicable.The nature and extent of contamination

and equipment after heatwill affect the resulting shelf life andthe food. These criteria should reflect theuse of the commodity; for example the

of a compositeas pizza which receives a further

than if noheat process is to be applied. The type of

and expected shelf life should also beinto consideration, for example Listeria mono-should be absent from products cooked incontainers and/or products

its growth and have long refrigeratedlives. However, a low level of Listeria might bein a chilled food likely to be eaten within a

Fermented foodsterm fermentation describes the process of

in foods catalysed byFermented foods may be

with the use of starter microorganisms orthe action of natural fermentative microorgan-

present in the raw materials or productionare used inon processes but those most widely usedfood industry include lactic acid bacteria andSaccharomyces spp.) (see Appendix: Specific

and the principles of test methods).The metabolic products of fermentation processesthe shelf life and enhance the safety ofproducts because of their inhibitory effect on

and spoilage microorganisms. Lactic acidthe pH and act to restrict the

of many natural spoilage organisms such asspp. Some starter culture organismsan inhibitoryother bacteria. Yeasts used for wine andproduction produce organic acids and ethanol,

create an extremely harsh environmentcan grow.The raw materials used as substrates for fermen-reactions e.g. milk, wort and meat are

the growth of manyand spoilage microorganisms if present.and stability of the fermented product is

the growth of theto produce inhibitory

and the rate at which these compoundsproduced. Strict process control is essential tothat the desired fermentation profiles are

achieved. Slow growth or no growth of the fermen-tative microorganisms may allow pathogenic orspoilage microorganisms to predominate resultingin unsafe products or ones that do not meet the .'411required organoleptic qualities.Fermentations are often monitored using simpleindicators of microbial growth such as decreases inpH or sugar concentrations or increases in cellnumbers (turbidity, direct microscopic examination) ,acidity or alcohol concentrations. Fermentationalone can lead to inhibition ofmicrobial growth and,in some cases, to microbial death although this willbe dependent on both the type and concentration ofmetabolic products produced by the fermentingorganisms.The application of microbiological criteria tofermented foods is dictated by the nature of theproduct and its intended use. Criteria are frequent-ly applied to a product at stages during the processe.g. S. aureus in the production of salami, and to thefinal product. Levels of significance of specificorganism groups inion a particular food must beunderstood e.g. psychrotrophic moulds such as somePenicillium spp. can be commercially signif icant forblock matured, vacuum packed cheese if bags arepunctured and the organism grows to spoil theproduct. In this circumstance, a significant contam-ination rate could be as low as one spore on theentire surface of a block of the cheese and a specifi- ,ation with criteria for moulds of d per gramwould be meaningless. Control requires ensuring the vacuum remains intact and the storage temper-ature is maintained.

In some cases the acidity or alcohol content infermented foods may need to be neutralised ordiluted out prior to enumeration or detection ofmicroorganisms as they may interfere with the indi-cator dyes used in some agar test methods e.g. VioletRed Bile Glucose Agar used for the enumeration ofEnterobacteriaceae. In addition, test procedures forthe detection of pathogens should incorporate aresuscitation stage as the effect of pH, acidity oralcohol content in the food may stress the organ- .k.isms.6. Microbiological limits6.1 IntroductionIndiscriminate application of microbiological testingand criteria is not advocated and should be avoided.If, after due practical consideration of the issuesraised and discussed in previous chapters it isconsidered useful to use microbiological tests andcriteria for clearly specified purposes then thefollowing tables of microbiological information mayhelp as a further guide to the use of relevantcriteria.The figures given in the following tables do notrepresent microbiological cr iteria in themselves butshould be used as the basis for the development of

Food Science and Technology Today 11 (3) 1997


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criteria. Use of internationally recognised or equivalent validated industry methods is assumed.

For easy reference, products are listed in thetables by type and technology, and then broadly splitinto groups based on their use e.g. to be cooked,ready-to-eat. A comprehensive list of foods withassociated combinations of technology, packagingand use is not the aim of this document;Guidance on limits and some key points concerning pathogens and indicator organisms relevant toeach food grouping is given. Important toxins arealso noted for some groups.Some of the levels given in the Tables are basedon existing published criteria (see Chapter 7 andPHLS, 1996). Others are derived from industryspecifications, practice and experience. The limitsgiven in these tables are considered by the authorsto be practical, realistic and relevant to the use ofthe food and take account of the potential hazardsassociated with the food and risk to the consumer.t may not be necessary to apply all the tests

listed for a product category and on someoccasions it may be relevant to test foradditional microorganisms and/or toxins.Although it may be desirable to minimise the level ofspecific microorganisms, criteria for organisms infoods that require cooking may be less stringentthan those for ready-to-eat foods. In particular,criteria for Escherichia coli (not verocytotoxigenicE coli - VTEC) and S aureus are often over-specified and seldom justified n terms of food safety orfood spoilage. High levels (>104 cfu/g - PHLS, 1996)of S aureus may be relevant due to their ability toproduce heat resistant enterotoxins although lowlevels of the organism are usually of little significance in foods prior to cooking.Any raw food may be contaminated by pathogenssuch as Salmonella spp., Campylobacter spp.,Listeria monocytogenes and VTEC. Criteriarequiring the absence of pathogens in raw foods aregenerally impractical, particularly in raw meats. Inaddition, food poisoning is regularly associated wi thdrinking raw milk; therefore, the consumption ofraw meat or raw milk is not advocated. The application of HACCP-based systems and GMP to minimisethe presence of such organisms coupled with clearadvice to consumers about how to handle such foodwill help minimise the risk of foodborne illness.Absence of pathogens in products which are eatenraw is desirable but cannot be guaranteed.6.2 Use of tablesFor practical reasons these tables represent broadproduct categories and inevitably some productgroups may not conform to the limits specified forindicator microorganisms. Also, for some multicomponent products e.g. sandwiches containingcooked meat and salad ingredients, the informationin more than one table will need to be considered.

The figures given in the tables are under two headings: GMP (good manufacturing practice) andMaximumGMP values are those expected immediately follow-ingproduction ofthe food undergood manufacturingconditions Occasional samples may exceed theselimits and such results should not be used asrejection criteria without consideration of all thefactors important in an entire sampling plan(Chapters 1 and 3).Maximum values are those regarded as themaximum acceptable at any point in the shelf life ofthe product Higher levels of pathogens indicate apossible health risk whilst higher levels ofindicators signify failure of the process or hygieneprocedures.In many products, organisms may continue to growthroughout shelf life. If Maximum levels areapproached early in the intended life of the product,it is likely to become unacceptable i.e. hazardous orspoiled within its shelf life. This must be taken intoaccount when using these tables.The levels of any specific indicator organism mayvary according to food material and processingconditions. Maximum levels given in the tablesindicating process failure may therefore differ toreflect these variations. For example, productswhich have been processed to achieve a botulinumcook are expected to remain safe throughout theirshelf life; low Maximum criteria are thereforeusually applicable to these products.The values are intended to be applied to arepresentative sample of the food, which in mostcases would be not less than 109 (25g for detection(presence/absence) tests for pathogens) and wouldinclude surface material, core material and aselection of the components in the case of amulti-component product. Ingredients added afterthe processing stage indicated in the tables are notconsidered and must be taken account of separately.Sampling plans and methods are not indicated inthese tables and should be developed and used inline with the considerations discussed in Chapters 3and 4

The European Union and some individualnations currently have legislation containing microbiological criteria applicable to some foodstuffs atdefined points in the production process e.g. formilk, dairy products, shellfish, egg products, mineral waters, minced meat and meat products (Chapter7). It is essential for food businesses to ensure thatproducts conform to relevant legislation.

Food Science nd Technology 1bday 11 (3) 1997 151


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6.3 Index for foods FOO TABLEFOO TABLE Fish- raw C- cold smoked CAlcoholic beverages S - hot smoked JApple juice E - pickled C- marinaded CBaby foods - dried 0 - dried K NBaby foods - canned, pouched, bottled P - cooked JBaby milks - dried 0 canned P-liquid H Flans - savoury JBacon B Flavourings L MBakery goods M Flour KBean sprouts F Fondant MBeer S Fresh herbs FBiscuits M Freshly squeezed juices EBivalve molluscs C&J Fruit - dried K LBottled food P Q -raw EBread dough D - pasteurised EBreakfast cereals L M preserved QBurgersButter

BG

Fruit cocktailFruit dr -nk -heat processed

EE S- preserved Q SCakes M Fruit juice. raw ECanned food P pasteurised ECereals M - freshly squeezed ECereals breakfast M Fruit squashes SCheese GChilled desserts E G Gravadlax CChocolate M Gravy mixes NCoated fish productsCoated meat productsCocoa butterCoconut

ColasComposite foodsConfectionery

I&JI&JRK L

SNM

Ham-cooked-raw_- dry curedHerbs - fresh- in oil

JBQK,L MFQ

( '

Cook - chillCooking oilsCreamCrisps

JRGMIce creamInfant formulasIntermediate moisture foods (IMF)

GO PQ /CrUditesCured meats FB,J Q Jams Q ,Lard RDairy dessertsDairy ice cream GG LemonadeLong life milk SH / ,Dairy products GDairy products - UHT H Margarine RDesser t mixes N Meat - raw Goints, mince, diced) BDesserts J - cooked JDoughs D - canned pasteurised JDrinks - soft S - canned p- fruit E Q - cured B&J- low/no alcohol beers S - fermented JDry cured ham Q - dried K NDry mixes K L -dry cured J&Q- for baby foods 0 - marinaded- smoked JEgg -liquid J Milk G W-.- dried N - Long lifeMilk powders HN - . ;Fat spreads - non dairy R - for babies 0

Food Science nd Technology bday 11 (3) 199752


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FOOD TABLE FOOD TABLEMilk shakeMuesli HLMussels C&JNoodles D&K

Sousvide JSpaghetti D&KSpices K,L&MSugar MNut oilNutsOffalOilsOysterPart-baked productsPart-cooked foods

RLBRCDI

Tagliatelli D&KUHT dairy products HVegetables - heat treated J- raw and prepared F- dried K- preserved Q- frozenPasta D&K- fresh filled I - stir-fry

FF- canned PPasties JPastry D Vll gin olive oil RPickles Q Wme SPies - (meat, fish and/or vegetable) I J

Pizzas I J Yoghurt -live GPork B&J'Pot' snacks N - pasteurised GPouched foods PPoultry - raw (whole, portions, minced)- cooked AJ- canned P 6.4 Abbreviations- marinaded A- smoked J APC: Aerobic Plate CountPowdered desserts N aw Water activityPrawns -raw C B cereus: Bacillus cereusJQ

...... GProcessed cheese C. perfringens: Clostridium perfringens- cooked cfu: Colony Forming UnitsPreserved foods

Pulses - cooked KJQuiches J

DSP: Diarrhetic Shellfish PoisoningE coli: Escherichia coliF03: see Glossary of TermsGMP: Good Manufacturing PracticeRecipe dishes - raw I

I HACCP: Hazard Analysis Critical Control Point(ready meals) - part cooked- heat processed IMF: Intermediate Moisture FoodsJ PRelishesRiceRice products ,...L monocytogenes: Listeria monocytogenesND: Not DetectedPSP: Paralytic Shellfish Poisoning

QKJRoll mop herrings C

S. aureus: Staphylococcus aureusF&I UHT: Intra Heat Treatmentalad vegetables - raw and preparedSalami J&Q V parahaemolyticus: Vibrio parahaemolyticusSandwiches & sandwich fillings F J VTEC: Verocytotoxigenic E coliSauces J&Q Y. enterocolitica: Yersinia enterocoliticaSauce mixesSausages NB IOn: 1 X IOn where n =2,3,4,5,6,7, B etc.Scombroid fish C&JShellfish C&JSmoked salmon CSnacks M&N 6.5 TablesSoft drinks SSoup All countsllevels in the following tables- mixes N represent colony forming units per gram or- canned P per ml unless otherwise specified.Food Science nd Technology Today 11 3) 1997 153


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....@A: RAWPOULTRYProductexamples: Wholebirds,portions,minced/reformedpoultrymeat,marinadedproducts

""'

Storage: FrozenorchilledUse: 'Ibbecooked (PathogensSalmonella Campylobacter andotherpathogensmaybepresent;monitoring theincidencemaybeusefulfortrendanalysis.

OrganismBacterialpathogens GMP MaximumCriteriaforabsencenotgenerallyapplicable. ,/

Indicators&spoilageorganismsAPCcanbeusedtoindicatequality.Generally,highercountsarefoundin minced/reformedpoultrymeatsthanonwholebirdsorportions.Pseudomonadsarealsofrequentlyusedtoindicatequality. (

Organ'APC N VdPseudomonas ~Yeasts(marinadedproducts)GMP


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C: RAW FISH SHELLFISHroduct examples: Whole, fillets, smoked salmon, mussels, oysters, prawns, gravadlax, roll mopherrings, cold smoked, marinaded or pickled (acidified) products

Storage: Frozen or chilledUse: To be cooked or ready-to-eatPathogens & toxinsPathogens may be present; in particular, Vibrio may occur in warm water fish/shellfish. Monitoringthe incidence of pathogenic bacteria may be useful for trend analysis. As filter feeders, bivalve molluscsconcentra te contaminants from the water in which they grow, including bacterial pathogens, viruses andalgal toxins. Monitoring the incidence of bacterial pathogens and algal toxins may be necessary. Presenceof histamine (scombrotoxin) in scombroid fish should also be considered.Use r g n u ~ x i n GMP MaximumTo be cooked Bacterial pathogens Cri ter ia for absence not general ly applicable.Histamine (scombroid fish)


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:DOUGHS,PASTA&BATTERSProductexamples: Breaddough,pastry,part-bakedproducts,spaghetti,tagliatelli,noodlesStorage: Frozenor chilledUse: 1bbecooked

rPathogensSalmonella and Listeria maybepresentoccasionallyinrawproductsbutusuallyonlyatlowlevelsandshouldbekilledbycooking.B cereus sporesand/ortoxinandS aureus toxincouldsurvivecooking.

Organism GMP MaximumB cereus 10S aureus (pastaandbatters) 10 /

Indicators&spoilageorganismsAPCandEnterobacteriaceaelevelsmaybeveryhighandvariable.E coli maybeusedtoindicatehygieneofproduction.Lacticacidbacteriamaybeusedtoindicatespoilage(exceptforsourdoughs).Organism GMP Maximum (E coli 10Lacticacidbacteria


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F:RAWANDPREPAREDVEGETABLES(INCLUDINGSALADVEGETABLES) Productexamples: Crudites,preparedsalads,stir -fryvegetables,blanchedvegetables,freshherbs,beansprouts,sandwichfillingsStorage: Frozen,chilledor ambientUse: 'Ibbecookedor ready-to-eatPJ\thogens Pathogensmaybepresentonrawvegetablesandwashingproceduresareunlikelytoeliminatethem. However,onlylowincidenceandlevelsmaybeexpectedandcriteriaforspecificpathogensarecommonly applied. Use Organism MP Maximum obewashed Bacterialpathogens Criteriaforabsencenot generally applicable. orcooked Prepared Salmonella spp. NDin 25g NDin25g (ready-to-eat) L monocytogenes NDin 25g 103

Indicators&spoilageorganisms HighlevelsofAPCandEnterobacteriaceaelcoliformsareprobableandcouldbederivedfrom thesoilor poorhandling.E coli levelsmaybeusedtomonitorthequalityof irrigationwaterandhygieneof han-dling.Ifvegetablesaretobeusedinfurtherprocessinge.gcanning,monitoringforbacterialspores maybe useful.Visualinspectionis particularlyimportantforbacterialrotsandmouldcontamination. Use Organism MP Maximumo bewashed APe,coliformsl Criteriaforabsencenotgenerallyapplicable

or cooked EnterobacteriaceaePrepared E coli


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@G: MILK CREAM & DAIRY PRODUCTSProduct examples: Liquid milk, cream, cheese, ice cream, butter, dairy desserts, yoghurts and other

/fermented products )Storage: Frozen or chilledUse: Ready-to-eatPathogensSalmonella, Campylobacter, VTEC, Listeria and S. aureus will occasionally be present in raw milk. Otherpathogens and parasites may also occur. Vegetative pathogens will be absent in properly pasteurised milkand cream; testing for Salmonella and Listeria is not conducted routinely in these products. Low levels ofspores and other thermoduric organisms may occasionally be present.

Organism GMP MaximumSalmonella spp. ND in 25ml/g NO in 25ml/gL. monocytogenes NO in 25ml/g 103S. aureus


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H: UHTMILK CREAM " DAIRY PRODUCTS. @Product examples: Long life milks including flavoured products, milk shakes, sterilised milkStorage: AmbientUse: Ready-to-eatPathogensPathogens should be absent.

Organism GMP MmdmumBacterial pathogens Criteria for absence not generally applicableIndicators : spoilage organismsUHT products should be microbiologically stable. Control of the process is essential . Microbiological testingof the finished product is not considered appropriate. Incubation of finished packs followed by examinationfor signs of microbial growth (pH change, organoleptic defects, blown packs) is most commonly used inindustry. Direct inoculation of agar with incubated product or alternative methods such as impedance!conductance and ATP-bioluminescence are also used to detect growth.

Organism GMP MaximumAll No evidence of growth No evidence of growth(after incubation of pack)

REMINDER: Indiscriminate application of microbiological test ing and criteria is not advocated and should be avoided. It may not be nec8BBaryto apply all the tests listed for a product category and on BOrne occasions it may be relevant to test for additional microorganisms and/or toxins.GMP values are those expected immediately following production of the' food under good manufacturin g conditions. Masimum values are thoseregarded as the maximum acceptable at any point in the shelf life of the product (see 6.1 and 6.2). When apply ing microbiological tests and criteria,all elements of the microbiological criterion including the sampling plan mUBt be clearly specified (see chap ters 1 3).

I, PART COOKED FOODS ci)Product examples: Prepared mealzl, pizBas, coated fish and meat products, fresh filled pastasStorage: Frozen or chilledUse: 'Th be cookedPathogensSalmonella Campylobacter and Listeria could be present (particularly from raw meats, poultry and fish).Spores of C. perfringens and B. cereus may also be present and monitoring for trend analysis may beuseful. Monitoring for S. aureus should be considered (or products receiving significant handling duringprocessing e.g. pizzas and pastas.

Organism GMP MaximumBacterial pathogens Criteria for absence not generally applicable

Indicators : spoilage organismsMeals of different components, some perhaps partly cooked, cannot easily be regarded as a single samplelikely to show a consistent microbial flora; APC and Enterobacteriaceae limits are not generally applicable.Individual components of meals may merit separate consideration. E. coli may be used to indicate hygieneof production.

MaximumOrganism GMP


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J: PROCESSED FOODS E:::Product examples: Ready meals, cooked meats and fish products, pies, pasties, quiches, flans,sandwiches, sous vide products, fermented meats, cured meats, desserts, moistbakery products (muffins, crumpets)Storage: Frozen or chilledUse: Ready-to-eat or to be re-heatedPathogens & toxinsPathogens may be present in some raw fermented meat products. Spores of psychrotrophic strains ofClostridium botulinum may present a hazard in products given an extended shelf life at refrigerationtemperatures. Monitoring the incidence of algal toxins may be necessary in molluscs and the presenceof histamine (scombrotoxin) in scombroid fish should also be considered.

Organism MP MaximumSalmonella spp. ND in25g ND in 25gL monocytogenes ND in 25g 103C. perfringens


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K: DRIED FOODS,TO BE COOKED Product examples: Rice,pulses,cereals,grains, flour,vegetables,fruit, pasta coconut,dry mixes,meats,herbs,spicesStorage: AmbientUse: 'lbbecookedPathogens toxinsPathogensmay be present;monitoringmay beusefulfortrend analysis.Sporesmay survivecooking.Heatstable toxinsof S. aureus and Bacillus spp.may notbeinactivatedbycooking.The presenceof mycotoxinsshouldbeconsidered.

OrganismS.aureus (pasta)B cereusGMP


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M: DRIED HEAT PROCESSED FOODSProduct examples: Breakfast cereals, crisps, snacks, confectionery, filled or topped biscuits and cakes,

Storage:herbs, spicesAmbient

(/

Use: Ready-to-eatPathogensBacterial sporeformers are common in herbs and spices. Spores may survive heat processes.

OrganismSalmonella spp. GMPND in 25g MaximumNDin 25gIndicators ; spoilage organismsAPC is normally low (


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: DRIED BABY FOODSexamples: Dry mixes, milk powder

AmbientReady-to-eat after rehydration

athogensbabies are more susceptible to food poisoning than adults, these products are normally made to

stringent criteria. Low microbial limits are applied together with an increased level of sampling. Ifis held at warm temperatures (30-50C) then pathogens may grow to levels causingThis must be considered when determining appropriate criteria as even more stringent limits thanbelow may be applicable.Organism MP MaximumSalmonella spp. ND in 250g* ND in 250g*VTEC ND in 250g* ND in 250g*(products containing beef)C. perfringens


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Q: PRESERVED FOODS BEAT TREATED (


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R NON-DAIRY FATS AND OILSProduct examples: Cooking oils, fat spreads, lard, virgin olive oil, nut oils, nut butters, cocoa butterStorage: Ambient or chilledUse: AnyPathogens toxinsPathogens should be absent in hot refined oils and 100% fat products packed under good hygienicconditions and monitoring is not generally required. Control of product physico-chemical proper ties(emulsion characteristics, salt, pH) and hygienic processing conditions is important for ensuring thesafety of fat spreads and margarines. Monitoring may be required for some cold pressed products.Mould contaminating nuts may produce mycotoxins which can car ry through to the finished product.Raw material nuts should be routinely screened for mycotoxin contamination.

Organism GMP MaximumBacterial pathogens (heat treated) Criteria for absence not generally applicableSalmonella spp. (not heat treated)L monocytogenes ND in 25gND in 25g ND in 25g103Indicators spoilage organismsAPC and Enterobacteriaceae are normally low and may be useful for trend analysis for monitoring processhygiene. Lipolytic organisms may cause rancidity in fat spreads and testing for these organisms may beappropriate. Control of moisture during processing, packing and shelf life is important for solid fatproducts as the presence of moisture can allow spoilage by surface mould growth.

Organism GMP MaximumEnterobacteriaceae (fat spreads)


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7.European Union directivesAnumber ofEuropean directivescontainingmicrobiological criter ia have already been implementedfor a variety of foodstuffs. The following tables(p167-172) are intended to provide an overviewof information on microbiological criteria in theexistingDirectives. 7b ensure the correct contextfor application the criteria .hould e read inconjunction with the appropriate legi.lation.EU Council Directives consulted(* PrincipalUK Statutory Instrument implementingthe Directive)Council Directive 89/437/EEC on hygiene andhealth problems affecting the production and theplacing on the market of egg products. (OfficialJournal of the European Communities22.7.89No.L212/87) amended by Counci l Directives89/662/EEC (OJ No. L395, 30.12.89, p13) and91/684/EEC(OJ No.L376,31.12.91,p 38)* The Egg Products Regulations, 1993, S. I No.1520.Council Directive 91/492/EEC laying down thehealth conditionsforthe productionand placingonthe market of live bivalve molluscs. (OfficialJournal of the European Communit ies 24.9.91No.L268/1).*TheFoodSafety(LiveBivalveMolluscsand otherShellfish)Regulations,1992,S.I No.3164and TheFood Safety (Live Bivalve Molluscs and otherShellfish) (Import Conditions and MiscellaneousAmendments)Regulations,1994,S. I No. 2782.Council Directive 91/4931EEC laying down thehealth conditionsfor the productionand the placingon the market of fishery products. (OfficialJournal of the European Communit ies 24.9.91No.L268/15).*TheFoodSafety(FisheryProducts)Regulations,1992, S. I No 3163and TheFoodSafety(FisheryProducts) (Import Conditions and MiscellaneousAmendments)Regulations,1994,S.I.No. 2783.CommissionDecision93151/EEConthe microbiologicalcriteriaapplicabletothe productionof cookedcrustaceans, and molluscan shellfish. (OfficialJournal ofthe EuropeanCommunities21.1.93NoL13/11).* The Food Safety (Fishery Products) (ImportConditions and Miscellaneous Amendments)Regulations,1994,S. I No.2783.

ouncilDirective921461EEC layingdown'thehealththe productionand placingonthe marketraw milk, heat-treated milk and milk-based(Official Journal of the European

unities14.9.92No. L268/1).TheDairy Products (Hygiene)Regulations, 1996, I No.1086.66 ood Science and echnology 7bday11(3)1997

CouncilDirective801777IEEConthe approximation of the laws of the member states relating to theexploitation and marketing of natural mineralwaters. (Official Journal of the EuropeanCommunit ies30.8.80No.L229/1).*The Natural Mineral Waters Regulations, 1985,S.I. No. 71.CouncilDirective801778/EEC relating to the quality or water intended. for human consumption, inrelationtodrinking water which is bottled orsold in a bottle. (OfficialJournal of the EuropeanCommunities30.8.80No.L229/11).*The DrinkingWater in Containers Regulations, 1994,S.I No.743. CouncilDirective94/65/EClayingdown the requirements forthe productionand placingonthe market /of minced meat and meat preparations. (Official Journal of the European Communities 31.12.94No. L368/10).* The Minced Meat and Meat Preparations(Hygiene)Regulations,1996,S.I No.3205. "Definitions:m= threshold below which all results areconsideredsatisfactory M= acceptabilitythreshold,the result is consideredunsatisfactoryif oneor moreunitsvalues=or>Mn= number ofunits making up the samplec= number of units in the samplegivingvalues

between m and M; the sample beingconsidered acceptable i the values of theother sampleunits are =or


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[;; s

I:l[; s

tI - 'I - '...-..:a

DIRECTIVEEel produces 89 4371EEC sampled in treatmentestablisbmeDts UYe BMIw MoDucs 91/4921EECIDfaMla! forImmedillte human CODllumptioa

Fish produces 9V49 IEEC aDd CommialioaDedIio. 93151/EEC

PRODUCTSAll egg prodUCIS after tteatme:DtU'Ie bivalve moIllJSCS

Coc*ed cnJSIllCClm and moIlusc:an shellfisbWhole products

ShdIcd or shudced productsSbe1led or sbudtcd productsSbe1led or shucked products

SbdIcd or shucked products exceptcnbmeatCrabmc:II

MICROORGANISMSolntonetla spp.

StopIryloe:cus IInII8Mesophilic aerobic bacteriaEnterobacteriDt:eoe

Salmonella spp.E rcJterlc1tJll coliFeccal coIifonnsParI1ytic sheUfish poison (PSP)Dianbdic: she11tisb poisoo (DSP)

StJ1nuJM1la spp.Other plllhogals d toxins thcR:ofMesophilicaerobic: bacteria (30"C)

StophyIococcu.r tIIIm/.I

chmchkI coli (on solid medium)1bermoIoIennt c:olifonns (44"C on solidmcdiwn)Me30philie aerobic: bactma (3O"C)Mesophilicaerobic: bacteria (30"C)

LEVEL per g or ml unlessotherwise specified)absent in 2Sg or mlabsent in IgM-IOOOOOM"'IOO

8bsent in 2Sg


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i IiJCIo 1. . ~. ..l . . n 11i ifIi IIIi~ ;gI i I g:, I ct !

I!iIIIf 9Ii. . ( [

,I1i ~ r II1s e.I,.:f i 'I

!f f. ~ I, f I& irf! t II'. f I f f iI t t I i(ig.

I I I 8 :: 0i:! 1, J i;ji:>I I jf :1' S''' o ir-t ~ ,f , f 1f 1 1i0

, 0

!1

,8 0 ~ Io EiiOj 8,- -l 1: Iii! fI ttllrlUIil illll ri JI ,JIJ r . i ii I rti: .rii: . . i [ ~ U f brl ~Ij:i 11:i IIIt ~ filII rill .... Ill"~~ [ I 'I ' Clili 3::


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DIRECTIVE PRODUCTS MICROORGANISM LEVEL per g or ml unlessotberwise specified) STATUSJACflONMilk prodUe1S 921461EEC: critaia apply 00removal from the proc:asiog estabHsbmeut(contioued)

Powdered milk-bascd products, CoIifunns 30"C 111 0. M-IO. 0=5, c=2 Indicator organisms (Guidelines)

Liquid milk-bascd products CoIifunns 30"C 111 0. M-S, II 'S. c=2 Indicator organisms (Guidelines) Liquid hcat-tmltcd unfermaIted milk-basedproducl5 PlIIIe COlIIIt (21 "C) afu:r product incubationt 6 C I Sdays m=SO 000. M=JOO 000. n-S. e-2 Indicator organisms (Guidelines) Frozm milkbMed producl5 (including itcream) Colifunns 3O C D f = J O M l O O . n = S . ~ 2 Indicator organisms (Guidelines) PIaIe count (30"C) m-J 00 000, M-500 000. n=S c:-2 . PIstcurised milk PIlhogcoic microorpnisms Absent in 25gm-o, M-O, n-S. c=O When: standlUds excceded n ~ g I I I e BIId take approprilde 8dion . Colifonns m-O. M=-S n=S. C 'I . Ptafc COlIIIt (21 "C) after product incubationt 6 C I Sdays RPSO 000. M=SOO 000, n=S c=1 . Butter 6un paslaIriscd milkor cream CoIiforms 30"C 1I'f O. M-IO, 0=5. c:-2 Indicaror orpnisms (Guidelines) Slerilised IIId U f milk P1IIIe count (30"C) after product incubation1t3O"C tOr I days or SS C for 7 days PIO per O.lmI When: stIlldlUds cxc:c:cded investil/*BIId bib approprillle acliOll Raw cuws' milk for drinkinS in that 5llIb: Sa - I la spp. lIbscnl in 25m1. n=S. c=O SropIJyIococcu. tDD eII3 IIP IOO. M-SOO,II 'S, e-2 P1IIIe count (3O C)


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'

DIRECI VE PRODUcrs MICROORGANISM LEVEL (per g or ml unlessotherwise specified) STATIJSlACflONMilk produdl92I46IEEC: requirements for the_nuflodare ofkat-treated milk and mllk-buedprodadl (contbaued)

Pasteurised milk b production ofllCllltreated drinking milk. 1 1* COlII1l (30"C) (in lhe pastaJrised milk)


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DIRECTIVE PRODUCTS MICROORGANISM LEVEL per g or ml uolessotherwise specified) STATUS/ACTIONNatunl minenl waten 8OfT77IEEC MincnIwater l it soura: IIIld thereafter, up toand including the point of sale Colifonns Absent in 250m1 Must meet this stlllldard E.scMrlchilz oli Absent in 250ml Must meet this standard Faecal streptococci Absent in 250m Pseudomonas Q( rugin06O Absent in 250ml Sporulated sulphite reducing anaerobes Absent in 50m Mineral water l it soura: Aerobic plate count (37"C, 24h; 2022"C,72h) Normal for the soura:

MincnI WIlla lit source (within 12 hours afterbottling; watermaintained l it 4"C:to I"C) Aerobic plate count (37C, 24 hour)


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I-tA

[lb

[l:l

5

......

DIRECTIVE PRODUCTS MICROORGANISM LEVEL (per g or ml unlessotherwise specified)STATUS/ACfIONCompulsory crilCria; Vllues >M IreIIIISldisfaclory (M -lICCCpbbilitythreshold IIbove which rr:sWts Ire noJansa- considered58lis ctory wheR M-10m (solid medium) or30m (liquidmedium). S - the miaobic limitvalue Itwhich product mllSt be CXlIISidered toxicortainuld. ..

Minced meat aDd meat prepantioas 9416S1EC Minced IIIClll Aaobi c mesophilic baeteria IIFSoo 000, M-S 000 000,S - SOO 000 000. n-S, e-2

EsdIerldlia coli m=SO, M=SOO, ....s 1:'=2, 5=50 000 SttlphyIOCOCCIU GIImU m=loo,M-S 000, n-S, e-2, 5=50 000 SaImDneIltI Absent in 109, n-S, c:-o Unacc:eptabIe if any SlIIlpIe showspn:salCe in lOR Meat pIqlII1Itions druk::ItIa oil (solid media)cIrerlt:IrIacoli (liquidmedia) DFSOO, M 'S 000, ...s, e-2m=SOO, MooIS 000, ...s, e-2 CornpuIscry c:riaia; VIIues >M l ieWISIlisfadocy t .IIf tUI (solidmcdim:n)~ m U (liquid mccIium) m-SOO, M-s 000, n-S, c;-lm-SOO, M-lS 000, ...s, c;-I . . SaImDneIltI IIbsa1t in II. ...s, Cl 'O u ~ ifany..... showspaencein I I

\ \ I-, '\ " " ..


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PPENDIXSpecific organisms and the principlesof test methodsAerobic plate countEnumeration of microorganisms by the aerobic platecount is one of the most common methods used toindicate the microbiological quality of a food. t canindicate whether a product has been contaminated,temperature abused or spoilt. The temperature andlength of incubation should be defined. The mostcommon incubation regime is 30C for 48 or 72hours for mesophilic organisms, but other temperatures and times may be more appropriate e.g. 55Cfor 48-72 hours for thermophilic organisms or20-25C for up to 5 days for psychrotrophicorganisms. Different temperatures of incubationwill lead to isolation of different flora; many organisms that develop during chilled storage for examplewill not grow well above 30C, and so a lowertemperature of incubation is preferable i.e. 20-25C.Consideration should be given to the methodused for performing an aerobic plate count. Pourplate methods are frequently specified, but microorganisms commonly found in foods may be injured bythe temperature of the molten agar used especiallyif they are already stressed. In these circumstances,surface inoculation methods may be preferable.Anaerobic plate counts can be used for foods thatprovide anaerobic conditions for microbial growthe.g. processed cheese and vacuum packed products.Aeromonas hydrophila and related speciesThe role ofAeromonas in causing food poisoning hasnot been clearly established, but some strains ofcertain species are thought to be capable of causingdiarrhoea. They are aquatic organisms but havebeen isolated from many foods. Aeromonas spp. growat refrigeration temperatures. Enumeration techniques on solid media and enrichment methods toestablish presence or absence are both appropriate.This group of organisms is not normally included inmicrobiological specifications.Bacillus cereus and other Bacillus speciesThe members of the Bacillaceae produce spores aswell as vegetative cells. The spores can surviveadverse conditions such as drying and pasteurisation. These organisms are common in the environment and in many foods. Some strains produceenterotoxins if allowed to grow, for example Bacilluscereus in rice dishes and high moisture flourproducts and members of the B subtilis - licheni-{ormis group in meat and pastry products and meator seafood rice dishes (Kramer Gilbert, 1989). Theemetic toxin of B cereus is particularly heatresistant l26C for 90 minutes) whereas thediarrhoeagenic toxin is inactivated by exposure to56C for 30 minutes. High levels (>105 per gram) arenecessary to produce enough toxin to causeillness. Bacillus species can also cause spoilage.Recovery and enumeration of Bacillus species

can be accomplished on many media, but mediadesigned specifically for presumptive enumerationof B cereus are usually used. These media mayallow resuscitation, growth from spores andpresumptive identification of B cereus if present.Atypical strains of B cereus can occur and couldlead to a false negative result. In some circumstances mesophilic or thermophilic spore countsmay be required; methods incorporate heat treat-ment of the sample prior to spore enumeration atappropriate temperatures. Biochemical tests(including ammonium salt sugars (glucose,

r i n o s e ~ mannitol and xylose) and a nitrate reduction test) can be used to distinguish the most common food poisoning Bacillus species (Roberts et ai1995).Campylobacter speciesCampylobacter jejuni and other thermotolerantcampylobacters are some of the most common causes of diarrhoea but the routes of transmission arestill unclear. Campylobacters are found in theintestinal tracts of many animals, especially poultryand birds, and can therefore contaminate naturalwater and raw milk as well as meat and poultry.These organisms have f s t i d i o ~ s temperature andatmospheric requirements for growth. They do notgrow in foods under normal storage conditions. sthe infectious dose is low the presence of low numbers in ready-to-eat foods is significant. Wheredetection is required, enrichment methods andselective agars are used for their isolation.Clostridium per ringens and sulphite reducingclostridiaClostridium perfringens is a spore-forming anaerobecommonly found in mammalian faeces and soil. Thespores persist in the environment and oftencontaminate raw food materials. Spores may survivecooking and rapid growth may occur if the food is notchilled promptly. If large numbers of the organismare eaten in a food they can sporulate in the gut andproduce an enterotoxin which causes diarrhoea.

Isolation of C. per{ringens on solid mediarequires anaerobic conditions of incubation, andusually the use of selective agents, followed byconfirmation of suspect colonies. Vegetative cells ofC. per{ringens do not survive well in foods at lowtemperatures, and spore counts may be more appropriate on such foods.The sulphite reducing clostridia (SRCs) includethe pathogens C. perfringens and C. botulinum (thecausative organism of botulism). The SRCs may beused as an indicator of plant hygiene and for thepresence of other clostridia that may cause foodpoisoning or food spoilage. There are no routinelaboratory tests for confirming th

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Microbiological Criteria