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Principles of Microbiological Testing:Methodological Concepts , Classes and

ConsiderationsRussell S. Flowers

Silliker Group Corp.

Relating Microbiological Testing and Microbiological Criteria toRelating Microbiological Testing and Microbiological Criteria toPublic Health GoalsPublic Health Goals

October 31October 31-- November 1, 2005November 1, 2005

Gallaudet University Kellogg Conference CenterGallaudet University Kellogg Conference Center

Washington, DCWashington, DC

Basic Principles of Microbiological Basic Principles of Microbiological Testing of FoodTesting of Food

Collect sample(s) of the food Collect sample(s) of the food Prepare homogenates Prepare homogenates Analyze homogenatesAnalyze homogenates

Direct MicroscopyDirect MicroscopyDetection/Enumeration Detection/Enumeration

•• Quantitative; Colony Counts, MPNQuantitative; Colony Counts, MPN•• Qualitative; enrichment followed by detection Qualitative; enrichment followed by detection

technique and isolation of target organismtechnique and isolation of target organism•• Evidence of growth may be detected by various Evidence of growth may be detected by various

means; visual, biochemical, immunological, genetic means; visual, biochemical, immunological, genetic

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Detection TechnologiesDetection TechnologiesEnzyme immunoassayEnzyme immunoassay

ImmunoImmuno--capturecaptureImmunoImmuno--precipitationprecipitation

Nucleic acid hybridizationNucleic acid hybridizationDNA/RNADNA/RNAPCR & other nucleic amplification methodsPCR & other nucleic amplification methods

ElectrochemicalElectrochemicalEnzymatic amplificationEnzymatic amplificationElectrical separationElectrical separationChromatographic separationChromatographic separation

Quantitative Microbial AnalysesQuantitative Microbial AnalysesLower Limit "Rules of Thumb"Lower Limit "Rules of Thumb"

<10 <10 -- 100 cfu/g MPN100 cfu/g MPN>10 >10 -- 100 cfu/g Viable Counts100 cfu/g Viable Counts>10>1033 -- 101044 cfu/g cfu/g DEFTDEFT>10E>10E44 -- 101055 cfu/g ELISA, Flow Cytometry,cfu/g ELISA, Flow Cytometry,

Quantitative PCRQuantitative PCR>10>1055 -- 101066 cfu/g Direct microscopy, cfu/g Direct microscopy,

SpectrophotometrySpectrophotometry

Sensitivity may be increased by methods Sensitivity may be increased by methods through enrichment of multiple samples at through enrichment of multiple samples at the limit of detection (calculated MPN)the limit of detection (calculated MPN)

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Precision of Colony CountsPrecision of Colony CountsFor Poisson distribution, Variance (s2 ) = Mean

S D xR S D (% ) = 1 0 0 = 1 0 0m e a n x

( )x

SD RSD (%)

± ±400 20.0 5.0 360 - 440200 14.1 7.1 172 - 228100 10.0 10.0 80 - 12050 7.1 14.1 36 - 6425 5.0 20.0 15 - 3510 3.2 31.6 4 - 16

Mean Colony Count

95% Limit

Values

Qualitative DetectionQualitative DetectionEnrichment(s) followed by detection, Enrichment(s) followed by detection, and confirmation (if required)and confirmation (if required)

Confirmation Confirmation •• differential isolation, differential isolation, •• Identification of target organism Identification of target organism

Limit of Detection is dependent on;Limit of Detection is dependent on;Number of samplesNumber of samplesSize of samples Size of samples

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TwoTwo--class Plans (c = 0): class Plans (c = 0): Probabilities of AcceptanceProbabilities of Acceptance

ICMSF, 1986. Microorganisms in Foods, Sampling for microbiological analysis: Principles and applications, University of Toronto Press, Toronto.

Composition of Lot Number of sample units tested % acceptable % defective 5 10 20 60 100

98 2 .90 .82 .67 .30 .13 95 5 .77 .60 .36 .05 .01 90 10 .59 .35 .12 < < 80 20 .17 .11 .01 70 30 .03 .03 < 50 50 .01 < 40 60 < 30 70

Applications of Microbiological Testing at Applications of Microbiological Testing at Various LevelsVarious Levels

Nationally and InternationallyNationally and InternationallyEpidemiological data; e.g., outbreaks, recalls, etc.Epidemiological data; e.g., outbreaks, recalls, etc.Baseline studiesBaseline studiesInternational Trade (SPS)International Trade (SPS)

Industry specificIndustry specificTrade association studiesTrade association studiesRetail surveysRetail surveys

CompanyCompanyAcross different production facilities and linesAcross different production facilities and linesCustomer/supplier (purchase specifications)Customer/supplier (purchase specifications)

Facility/product specificFacility/product specificHACCP HACCP Prerequisite programs (GMP, GHP)Prerequisite programs (GMP, GHP)

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Applications of Microbiological Applications of Microbiological Testing in Food Safety ProgramsTesting in Food Safety Programs

Microbiological testing plays an Microbiological testing plays an essential role in HACCP essential role in HACCP

Hazard analysisHazard analysisProcess validationProcess validationMonitoring of critical ingredients Monitoring of critical ingredients and high risk finished productsand high risk finished productsVerification of CCPs and the Verification of CCPs and the overall process overall process

Applications of Microbiological Applications of Microbiological Testing in Food Safety ProgramsTesting in Food Safety Programs

GMP/GHP Programs GMP/GHP Programs Determine potential for post Determine potential for post process contaminationprocess contaminationEstablish adequacy and frequency Establish adequacy and frequency of cleaning and sanitationof cleaning and sanitationDetect difficult areas to clean and Detect difficult areas to clean and sanitizesanitize

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Applications of Microbiological Applications of Microbiological Testing in Food Safety ProgramsTesting in Food Safety Programs

Compliance testingCompliance testingMandatory regulatory programsMandatory regulatory programsPurchase specificationsPurchase specificationsDocumentation in case of litigationDocumentation in case of litigation

Problem solvingProblem solvingOften microbiological data exists but is Often microbiological data exists but is only being used for acceptance on a given only being used for acceptance on a given unit of production (batch, lot, day)unit of production (batch, lot, day)Trend analysis of this data often identifies Trend analysis of this data often identifies the most likely source of a problem, or pin the most likely source of a problem, or pin points areas for further investigation points areas for further investigation

Microbiological Criteria (Codex)Microbiological Criteria (Codex)

A microbiological criterion defines the acceptability A microbiological criterion defines the acceptability of a product or a food lot, based on the absence or of a product or a food lot, based on the absence or presence, or number of microorganisms including presence, or number of microorganisms including parasites, and/or quantity of theirparasites, and/or quantity of theirtoxins/metabolites, per unit(s) of mass, volume, toxins/metabolites, per unit(s) of mass, volume, area, or lot .area, or lot .

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the food , and the point in the food the food , and the point in the food chain to which it applies chain to which it applies any actions to be taken when the any actions to be taken when the Criterion is not metCriterion is not met

Microbiological Criteria should Microbiological Criteria should specify (CODEX)specify (CODEX)

Microorganisms and reasons for concernMicroorganisms and reasons for concern

Analytical methods to be usedAnalytical methods to be used

Sampling plan and size of analytical unitsSampling plan and size of analytical units

Microbiological limitsMicrobiological limits

Numbers of units to be in conformity Numbers of units to be in conformity

Microbiological Criteria Components

Establishment and Application- CAC / GL 21 - 1997

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Uses of Uses of Microbiological Criteria Microbiological Criteria Assess tAssess the safety of foodhe safety of foodVerifVerifyy//validavalidatete procedures in HACCPprocedures in HACCPDemonstrate aDemonstrate adherencedherence to GMP/GHPto GMP/GHPDemonstrate tDemonstrate the utility (suitability) of a food or he utility (suitability) of a food or ingredient for a particular purposeingredient for a particular purposeEstablish tEstablish the keeping quality (shelfhe keeping quality (shelf--life) of certain life) of certain perishable foodsperishable foodsAs a regulatory tool to drive industry As a regulatory tool to drive industry improvementimprovementAs verification that a Performance Objective or As verification that a Performance Objective or FSO has be FSO has be acheivedacheived

• Standard—a mandatory criterion that is part of a law or ordinance.

• Guideline—an advisory criterion issued by a control authority, industry association, or food producer to indicate what might be expected when best practices are applied.

• Specification—Part of a purchasing agreement between a buyer and supplier of a food; such criteria may be mandatory or advisory according to use.

Types of Acceptance Criteria

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Sampling PlansSampling Plans

Define the probability of Define the probability of detecting a microorganisms or detecting a microorganisms or other hazards in a lotother hazards in a lotNone can ensure the absence None can ensure the absence of a particular hazardof a particular hazardShould be administratively and Should be administratively and economically feasibleeconomically feasible

Types of Microbiological Types of Microbiological Sampling PlansSampling Plans

Attributes plans:

Qualitative analytical results (presence/absence) orquantitative results that have been grouped(e.g. <10 cfu/g, 10 to 100 cfu/g, >100 cfu/g)

Variables plans:

Non-grouped quantitative analytical results

Require distributional assumptions be made

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IndicatorsIndicatorsShould indicate something :Should indicate something :

ContaminationContaminationSurvivalSurvivalRecontaminationRecontaminationGrowthGrowth

Should be easy to determineShould be easy to determineShould behave as pathogen Should behave as pathogen

(growth, survival) when(growth, survival) when used used instead of testing for pathogeninstead of testing for pathogen

Cannot be relied upon as Cannot be relied upon as "proof" that pathogen of "proof" that pathogen of concern is absent concern is absent

Pathogen not measurablePathogen not measurable

Example : < 1 Example : < 1 SalmonellaSalmonella / 10 kg of / 10 kg of dried eggdried egg--productproduct

Enterobacteriaceae are good indicators of Enterobacteriaceae are good indicators of adequate pasteurisation andadequate pasteurisation andcontrol of recontaminationcontrol of recontamination

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Salmonella criterion for dried egg productsSalmonella criterion for dried egg productscase 11 : n = 10 c = 0, 25g case 11 : n = 10 c = 0, 25g

samplessamples

lots containing 1 S. per 83 glots containing 1 S. per 83 gwill be rejected with 95% will be rejected with 95%

probabilityprobabilitylots containing < 1 S. per 7.7 kg

will be accepted with 95% probability

A producer would need to test 565 end-productsto verify that he would meet this criterion

Indicators are measurableIndicators are measurable

Example: Absence of EnterobacteriaceaeExample: Absence of Enterobacteriaceaein 1 g of eggin 1 g of egg--productproduct

a) case 7 : n = 5, c = 2 * (use : biscuit)a) case 7 : n = 5, c = 2 * (use : biscuit)b) case 8 : n = 5, c = 1 (dried egg)b) case 8 : n = 5, c = 1 (dried egg)c) case 9 : n = 10, c = 1 (use : tiramisu)c) case 9 : n = 10, c = 1 (use : tiramisu)

* if adequate heating is assured, no testing is necessary

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Utility and Indicator TestingUtility and Indicator Testing

ExamplesExamplesAerobic plate countAerobic plate countColiforms or Enterobacteriaceae (EB)Coliforms or Enterobacteriaceae (EB)Escherichia coli Escherichia coli Yeast and mold countYeast and mold count

Species or groups or microorganisms whose presence may indicate the potential for the presence of pathogens or the extent to which good manufacturing and hygiene practiceswere adhered to during manufacture

Salmonella Incidence in Relationship Salmonella Incidence in Relationship to to E.coliE.coli Most Probable Number (MPN) Most Probable Number (MPN)

in Raw Prein Raw Pre--formed Meat Pattiesformed Meat PattiesEE.. ccoollii MMPPNN//gg SSaammpplleess

WWiitthhiinn MMPPNNRRaannggee

SSaammpplleess PPoossiittiivveeFFoorr SSaallmmoonneellllaa

WWiitthhiinn MMPPNN RRaannggee

%% PPoossiittiivveeWWiitthhiinn MMPPNN

RRaannggee

<<33 227700 22 00..7733--55 440066 2200 44..99

5511--110000 5544 33 55..66110011--224400 9966 44 44..11

224411--11,,110000 6655 33 44..6611,,110011--1111,,000000 5566 99 1166..11

>>1111,,000000 2255 55 2200..00

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No indicators availableNo indicators availableExample : <1 C. botulinum in 1000 ton of Example : <1 C. botulinum in 1000 ton of

lowlow--acid canned meat productacid canned meat product

Reliance on Reliance on Process Criteria (Process Criteria (botbot cook)cook)

and GMP and GMP

No Microbiological Criteria

To Test or Not to TestTo Test or Not to Test ??Severity of the hazard(s)Severity of the hazard(s)New information linking the food to illnessNew information linking the food to illnessWhether the food isWhether the food is

Commonly involved in diseaseCommonly involved in diseasePrimarily destined for a sensitive populationPrimarily destined for a sensitive populationFrom a country with endemic disease of importance to From a country with endemic disease of importance to

food safetyfood safetyHistory of consistency and compliance History of consistency and compliance Distribution of contaminant(s)Distribution of contaminant(s)

Homogenous, heterogeneous, stratifiedHomogenous, heterogeneous, stratifiedAbility to sampleAbility to sample

Sufficient numbersSufficient numbersRandom samplingRandom sampling

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Depending on the application the Depending on the application the requirement for confidence in the data requirement for confidence in the data

will be different will be different Method performance must be appropriate for the Method performance must be appropriate for the ““purpose intendedpurpose intended”” e.g.e.g.

Industrial process monitoring.Industrial process monitoring.Regulatory screening of foods.Regulatory screening of foods.Industrial process verification.Industrial process verification.Regulatory compliance with criteria.Regulatory compliance with criteria.Forensic investigation.Forensic investigation.

Greater Accuracy &Precisionrequired

AOAC Method Validations Protocols AOAC Method Validations Protocols Suggested for Various ApplicationsSuggested for Various Applications

Process & Product Process & Product monitoringmonitoring

Raw materialsRaw materialsInIn--process testingprocess testing

Process Validation & Process Validation & VerificationVerification

Process validationProcess validationHACCP verificationHACCP verification

Regulatory TestingRegulatory Testing

Single Lab (SLV)Single Lab (SLV)1010--20 samples per 20 samples per matrix/level matrix/level

MultiMulti--Lab Validation Lab Validation (MLV)(MLV)

Similar to SLV but Similar to SLV but with multiple labs with multiple labs and more samplesand more samples

Harmonized Harmonized Collaborative Collaborative Validation (HCV)Validation (HCV)

Validation protocolsValidation protocolsApplicationsApplications

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Method Validation CriteriaMethod Validation CriteriaRuggednessRuggednessInclusivityInclusivityExclusivityExclusivityMatricesMatricesComparison to Comparison to standardstandard

CorrelationCorrelationAgreementAgreement

PerformancePerformanceFalse neg.False neg.False pos.False pos.RepeatabilityRepeatabilityReproducibilityReproducibility

Validation Level # Labs

# Rep Samples/

Lab

Total # Rep

SamplesSensitivity

(% +ve) 95% CISLV 1 20 20 50% 27.6 - 72.4%MLV 2 20 40 50% 34.2 - 65.8%HCV 10 6 60 50% 37.1 - 62.9%

Comparison of Confidence Intervals for 3 Levels of Comparison of Confidence Intervals for 3 Levels of Validation for a Qualitative Method, assuming the Validation for a Qualitative Method, assuming the same performance characteristics in each same performance characteristics in each

Note: CI of HCV would normally be greater than SLV or MLV due to inter-laboratory variance (repeatability), not included in above analysis.

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Factors affecting the Confidence Factors affecting the Confidence (Uncertainty) in Microbiological Data (Uncertainty) in Microbiological Data

Sampling plan, method and sizeSampling plan, method and sizeMethod performance characteristics Method performance characteristics

SensitivitySensitivityRepeatabilityRepeatabilityRuggednessRuggedness

Laboratory performance Laboratory performance Error ratesError ratesReproducibilityReproducibility

Uncertainty in Microbiological Uncertainty in Microbiological AnalysesAnalyses

Types of ErrorTypes of ErrorInherent Inherent -- part of the procedure or method part of the procedure or method Performance Performance -- error associated with laboratory and/or error associated with laboratory and/or analystanalyst

Sources of ErrorSources of ErrorDistribution of microorganisms in matrix Distribution of microorganisms in matrix State of the microorganismsState of the microorganismsSampling planSampling planSampling handlingSampling handlingAnalysisAnalysisReportingReporting

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Distribution of microorganismsDistribution of microorganisms

HomogenousHomogenousHeterogeneousHeterogeneousRandomRandomNonNon--randomrandomStratified Stratified

Example AIncidence = 6 pos. per 100 x 1 lb.

samplesLevel = 6 cells per 100 lbs.

Example BIncidence = 6 pos. per 100 x 1 lb.

samplesLevel = 600 cells per 100 lbs.

ca. 100 cells /clump

Incidence vs. LevelIncidence vs. Level

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Distribution of MicroorganismsDistribution of MicroorganismsHom ogeneous d is trib u tion

A

B

C

Non-hom ogeneous d is tribu tion

Sampling plan Sampling plan (others will cover in detail)(others will cover in detail)

Sample selectionSample selectionrandomrandomstratifiedstratifiedspecified frequencyspecified frequency

Sampling methodSampling methodNumber of samplesNumber of samplesSize of samples (analytical unit)Size of samples (analytical unit)

Note: Statistical considerations of sampling plans generally assume that the method ection is error free

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AnalysisAnalysisMethod Selection and PerformanceMethod Selection and Performance

inherent accuracy and precision of the inherent accuracy and precision of the method assuming all procedures are done method assuming all procedures are done perfectly perfectly

Analyst PerformanceAnalyst Performancetrainingtrainingproficiencyproficiency

Laboratory PerformanceLaboratory Performancequality systemsquality systemsproficiencyproficiency

Method validation and proficiency Method validation and proficiency testing are essential components of a testing are essential components of a

laboratorylaboratory’’s quality systems quality systemand are necessary to determine and are necessary to determine

UncertaintyUncertainty of a microbiological data of a microbiological data resultresult

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Method ValidationMethod Validation

WhyWhyIt sets the performance parameters It sets the performance parameters of the method and demonstrates of the method and demonstrates laboratory competencylaboratory competency

WhenWhenUpon introduction of a new method, Upon introduction of a new method, the revision of a method, or addition the revision of a method, or addition of a new test matrixof a new test matrix

Proficiency TestingProficiency Testing

Laboratory proficiency measures a Laboratory proficiency measures a laboratorylaboratory’’s ability to generate data that is s ability to generate data that is comparable to other laboratories for comparable to other laboratories for identical samples.identical samples.

Analyst proficiency measures a analystAnalyst proficiency measures a analyst’’s s ability to generate data that is comparable ability to generate data that is comparable to other analysts for identical samples.to other analysts for identical samples.

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There are 240 private and hundreds more in-plant and corporate food testing labs in the U.S. Approximately 100 labs participate in the AOAC Split Sample Program. This program allows them to benchmark their proficiency against other labs. The Silliker lab data represents 22% of the test volume, and shows significantly better accuracy than the rest of the population.

0

0.5

1

1.5

2

2.5

3

3.5

% Errors

Salmonella Listeria E. Coli 0157:H7 Total

SillikerOthers

Pathogen Testing ErrorsPathogen Testing ErrorsAOAC Split Sample Program AOAC Split Sample Program (Jan 01 (Jan 01 -- Oct 03)Oct 03)

Quality RequirementsQuality RequirementsSummary and ConclusionsSummary and Conclusions

Microbiology is not an exact science Microbiology is not an exact science Many sources of variance not related to mistakesMany sources of variance not related to mistakesMany manual steps subject to human errorMany manual steps subject to human errorInterpretation of data must consider this variationInterpretation of data must consider this variationProduct specifications and process guidelines should Product specifications and process guidelines should be based on sufficient data to know the coefficient of be based on sufficient data to know the coefficient of variationvariationUncertainty of the analytical result must be considered Uncertainty of the analytical result must be considered when establishing microbiological criteria, including when establishing microbiological criteria, including variance associated with the sampling plan, method of variance associated with the sampling plan, method of analysis and laboratory performance.analysis and laboratory performance.

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Summary and ConclusionsSummary and ConclusionsLimitations of Microbiological TestingLimitations of Microbiological Testing

Often it is not practical to test a sufficient Often it is not practical to test a sufficient number of samples for confidence in lot number of samples for confidence in lot acceptanceacceptanceNonNon--random sampling may cause random sampling may cause incorrect conclusions to be drawnincorrect conclusions to be drawnFinished product testing determines Finished product testing determines outcomes, not causes or controlsoutcomes, not causes or controlsNo feasible sampling plan can ensure No feasible sampling plan can ensure absence of a pathogenabsence of a pathogen

Summary and ConclusionsSummary and ConclusionsUses of Microbiological TestingUses of Microbiological Testing

Establish baseline dataEstablish baseline dataControl ingredientsControl ingredientsIdentify highly contaminated lotsIdentify highly contaminated lotsAssessing control of the environmentAssessing control of the environmentVerify compliance of PO and FSO (within Verify compliance of PO and FSO (within limits of sampling and testing)limits of sampling and testing)Verify control of within HACCP/GHP systemsVerify control of within HACCP/GHP systemsValidate a HACCP/GHP system provides a Validate a HACCP/GHP system provides a desired level of controldesired level of control

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Summary and ConclusionsSummary and ConclusionsIf FSOs or POs are within a range allowing If FSOs or POs are within a range allowing practical application of sampling plans and practical application of sampling plans and testing protocols, then testing protocols, then

Microbiological criteria may be useful to verify Microbiological criteria may be useful to verify acceptability of the lot relative to PO and FSO acceptability of the lot relative to PO and FSO

Across Lot/Batch testing may be useful to Across Lot/Batch testing may be useful to verify that a food safety system or process is verify that a food safety system or process is continuing to function as intended continuing to function as intended