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BAM:FoodSampling/PreparationofSampleHomogenate
April2003

BacteriologicalAnalyticalManual Chapter1 FoodSamplingandPreparationofSampleHomogenate

Authors:WallaceH.AndrewsandThomasS.Hammack RevisionHistory:April2003RevisedsectionA.1.a,Salmonellaspeciessamplecollection.

Theadequacyandconditionofthesampleorspecimenreceivedforexaminationareofprimary importance.Ifsamplesareimproperlycollectedandmishandledorarenotrepresentativeofthe sampledlot,thelaboratoryresultswillbemeaningless.Becauseinterpretationsaboutalarge consignmentoffoodarebasedonarelativelysmallsampleofthelot,establishedsampling proceduresmustbeapplieduniformly.Arepresentativesampleisessentialwhenpathogensor toxinsaresparselydistributedwithinthefoodorwhendisposalofafoodshipmentdependson thedemonstratedbacterialcontentinrelationtoalegalstandard. Thenumberofunitsthatcomprisearepresentativesamplefromadesignatedlotofafood productmustbestatisticallysignificant.Thecompositionandnatureofeachlotaffectsthe homogeneityanduniformityofthetotalsamplemass.Theproperstatisticalsamplingprocedure, accordingtowhetherthefoodissolid,semisolid,viscous,orliquid,mustbedeterminedbythe collectoratthetimeofsamplingbyusingtheInvestigationsOperationManual(5).Samplingand sampleplansarediscussedindetailinref.6. Wheneverpossible,submitsamplestothelaboratoryintheoriginalunopenedcontainers.If productsareinbulkorincontainerstoolargeforsubmissiontothelaboratory,transfer representativeportionstosterilecontainersunderasepticconditions.Therecanbeno compromiseintheuseofsterilesamplingequipmentandtheuseofaseptictechnique.Sterilize onepiecestainlesssteelspoons,forceps,spatulas,andscissorsinanautoclaveordry heat oven.Useofapropanetorchordippingtheinstrumentinalcoholandignitingisdangerousand maybeinadequateforsterilizingequipment. Usecontainersthatareclean,dry,leakproof,widemouthed,sterile,andofasizesuitablefor samplesoftheproduct.Containerssuchasplasticjarsormetalcansthatareleak proofmaybe hermeticallysealed.Wheneverpossible,avoidglasscontainers,whichmaybreakand contaminatethefoodproduct.Fordrymaterials,usesterilemetalboxes,cans,bags,orpackets withsuitableclosures.Sterileplasticbags(fordry,unfrozenmaterialsonly)orplasticbottlesare usefulcontainersforlinesamples.Takecarenottooverfillbagsorpermitpuncturebywire closure.Identifyeachsampleunit(definedlater)withaproperlymarkedstripofmaskingtape. Donotuseafeltpenonplasticbecausetheinkmightpenetratethecontainer.Whenever possible,obtainatleast100gforeachsampleunit.Submitopenandclosedcontrolsofsterile containerswiththesample. Deliversamplestothelaboratorypromptlywiththeoriginalstorageconditionsmaintainedas nearlyaspossible.Whencollectingliquidsamples,takeanadditionalsampleasatemperature control.Checkthetemperatureofthecontrolsampleatthetimeofcollectionandonreceiptat thelaboratory.Makearecordforallsamplesofthetimesanddatesofcollectionandofarrival atthelaboratory.Dryorcannedfoodsthatarenotperishableandarecollectedatambient temperaturesneednotberefrigerated.Transportfrozenorrefrigeratedproductsinapproved insulatedcontainersofrigidconstructionsothattheywillarriveatthelaboratoryunchanged. Collectfrozensamplesinprechilledcontainers. Placecontainersinafreezerlongenoughtochillthemthoroughly.Keepfrozensamplessolidly frozenatalltimes.Coolrefrigeratedsamples,exceptshellfishandshellstock,iniceat0 4Cand transporttheminasamplechestwithsuitablerefrigerantcapableofmaintainingthesampleat 04Cuntilarrivalatthelaboratory.Donotfreezerefrigeratedproducts.Unlessotherwise specified,refrigeratedsamplesshouldnotbeanalyzedmorethan36haftercollection.Special conditionsapplytothecollectionandstorageofshucked,unfrozenshellfishandshellstock( 1). Packsamplesofshuckedshellfishimmediatelyincrushedice(notemperaturespecified)until analyzedkeepshellstockabovefreezingbutbelow10C.Examinerefrigeratedshellfishandshell stockwithin6hofcollectionbutinnocasemorethan24haftercollection.Furtherdetailson samplehandlingandshipmentmaybefoundintheInvestigationsOperationManual(5)andthe LaboratoryProceduresManual(3).TheInvestigationsOperationManual(5)containssampling plansforvariousmicroorganisms.Someofthosecommonlyusedarepresentedhere. A. Samplingplans 1. Salmonellaspecies a. Samplecollection Becauseofthecontinuingoccurrenceof Salmonellainfoods,samplingplansfor theseorganismshavereceivedtheattentionofcommitteesofnationaland internationalorganizations(6,7).Eachofthesecommitteeshasrecommended varyingthenumberofsamplesfromaparticularlotoffoodaccordingtothe samplingcategorytowhichafoodisassigned.Generally,theassignmenttoa samplingorfoodcategorydependson1)thesensitivityoftheconsumergroup (e.g.,theaged,theinfirm,andinfants)2)thepossibilitythatthefoodmay haveundergoneasteplethaltoSalmonelladuringthemanufacturingprocessor

Becauseofthecontinuingoccurrenceof Salmonellainfoods,samplingplansfor theseorganismshavereceivedtheattentionofcommitteesofnationaland internationalorganizations(6,7).Eachofthesecommitteeshasrecommended varyingthenumberofsamplesfromaparticularlotoffoodaccordingtothe samplingcategorytowhichafoodisassigned.Generally,theassignmenttoa samplingorfoodcategorydependson1)thesensitivityoftheconsumergroup (e.g.,theaged,theinfirm,andinfants)2)thepossibilitythatthefoodmay haveundergoneasteplethaltoSalmonelladuringthemanufacturingprocessor inthehomeand3)thehistoryofthefood.Theselectionofasamplingplan dependsmainlyonthefirst2criteriacited.Thehistoryofthefoodwouldbe importantindecidingwhethertosample,i.e.,whethertherewasapasthistory ofcontamination.FortheSalmonellasamplingplandiscussedhere,3categories offoodsareidentified. FoodCategoryI.Foodsthatwouldnotnormallybesubjectedtoaprocess lethaltoSalmonellabetweenthetimeofsamplingandconsumptionandare intendedforconsumptionbytheaged,theinfirm,andinfants. FoodCategoryII.Foodsthatwouldnotnormallybesubjectedtoaprocess lethaltoSalmonellabetweenthetimeofsamplingandconsumption. FoodCategoryIII.Foodsthatwouldnormallybesubjectedtoaprocess lethaltoSalmonellabetweenthetimeofsamplingandconsumption. Incertaininstances,itmaynotbepossibletofullyconformtothesamplingplan. NonethelessitisstillimportanttoascertainwhetherornotSalmonellaispresent inthesuspectfood.Therefore,theanalystshouldstilltrytoanalyzeasmany analyticalunitsasisrequiredforthefoodofinterest,i.e.,60analyticalunitsfor CategoryIfoods,30analyticalunitsforCategoryIIfoods,and15analytical unitsforCategoryIIIfoods.Individual25ganalyticalunitsmaybecombinedinto 375gcompositesasdescribedaboveunlessotherwiseindicatedinChapter5or theOMA.Belowareexamplesofsituationsthatmightconfronttheanalyst. 1)Thenumberandweightsofthesampleunitsiscorrect. Eachsampleshouldbemixedtoensurehomogeneitybeforewithdrawinga 25ganalyticalunit.Theanalyticalunitscanbecomposited(fifteen25g unitsintoa375gcomposite),unlessotherwiseindicatedinChapter5orin theOMA.Samplesshouldbepreenrichedata1:9sampletobrothratio. 2)Thenumberofsampleunitsiscorrect,butseveralofthesampleunits havebeendamagedandareunusable. Forexample,fifteen1lbbagsofpastahavearrivedfortesting,but5ofthe bagsaretornandunusable.Inthiscase,theanalystshouldonlysample fromthe10intactbags.Thecontentsofeachintactbagshouldbemixed toensurehomogeneitybeforetheanalyticalunitsarewithdrawn.Sincethe analystneedsone375gcomposite,ten37.5ganalyticalunits,fromthe remaining10intactbags,shouldbeusedtoformthecomposite.The compositeshouldbecombinedwithitspreenrichmentmediumata1:9 sampletobrothratio(375gsample/3375mlpreenrichment)asdirectedin Chapter5ortheOMA. 3)Thenumberofsampleunitsisincorrect,butthetotalweightofthe sampleunit(s)isgreaterthanwhatwouldbenecessarytoperformthe sampleanalysis. Forexample,asingle10lbwheelofcheesehasarrivedfortesting.Since cheeseisaCategoryIIfood,thirty25ganalyticalunitsmustbeanalyzed. Theseanalyticalunitsshouldbetakenrandomlyfromawidevarietyof locationsaroundthewheel.IfSalmonellaispresentinafood,thenthe oddsofdetectingitwillbeenhancediftwo375gcompositesareanalyzed ratherthanasingle25ganalyticalunit,aswouldbethecaseiftheanalyst weretotreattheentirewheelasasinglesample. 4)Thereislesssampleavailablethanisnecessarytoformtherequired numberofcomposites. Forexample,an8oz(226.8g)bagofalmondshasarrivedfortesting. AlmondsareaCategoryIIfood.CategoryIIfoodsrequirethirty25g analyticalunits(750g),soitisimpossibletoanalyzetheamountofalmonds requiredbythesamplingplan.Inthiscase,theanalystshouldanalyzeallof thealmondsata1:9sampletobrothratio(226.8gsample/2041ml preenrichmentmedium). If,intheaboveexample,thetotalweightofthealmondshadbeenless than2composites(750g),butmorethan1composite,thentheanalyst shouldanalyzebothawholeandapartialcomposite.Theanalyticalunits comprisingthesecompositesshouldbetakenrandomlyfromawidevariety oflocationsinthelotofalmonds.Bothcomposites,shouldbepreenriched ata1:9sampletobrothratio. Thissamplingplanappliestothecollectionoffinishedproductsundersurveillance and/orfordeterminationofcomplianceforregulatoryconsideration.Italso appliestothecollectionoffactorysamplesofrawmaterialsinidentifiablelotsof processedunitsand/orfinishedproductswhereregulatoryactionispossible.It doesnotapplytothecollectionofinlineprocesssampleunitsatvariousstages ofmanufacturesincethosesamplesdonotnecessarilyrepresenttheentirelotof foodunderproduction.Theactualtechniquesinvolvedinsamplingarecoveredin theInvestigationsOperationManual(5). Asampleunitconsistsofaminimumof100gandisusuallyaconsumersize containerofproduct.Takesampleunitsatrandomtoensurethatasampleis representativeofthelot.Whenusingsamplecontainers,submitacontrol consistingofoneemptysamplecontainerthathasbeenexposedtothesame conditionsasthoseunderwhichthesamplewascollected.Collectmorethanone sampleunitfromlargeinstitutionalorbulkcontainerswhenthenumberofsample unitsrequiredexceedsthenumberofcontainersinthelot.Asampleunitwill consistofmorethanonecontainerwhencontainersaresmallerthan100g(e.g., four25gcontainerscouldconstituteasampleunit). Thenumbersofsampleunitstobecollectedineachfoodcategoryareas

containerofproduct.Takesampleunitsatrandomtoensurethatasampleis representativeofthelot.Whenusingsamplecontainers,submitacontrol consistingofoneemptysamplecontainerthathasbeenexposedtothesame conditionsasthoseunderwhichthesamplewascollected.Collectmorethanone sampleunitfromlargeinstitutionalorbulkcontainerswhenthenumberofsample unitsrequiredexceedsthenumberofcontainersinthelot.Asampleunitwill consistofmorethanonecontainerwhencontainersaresmallerthan100g(e.g., four25gcontainerscouldconstituteasampleunit). Thenumbersofsampleunitstobecollectedineachfoodcategoryareas follows:FoodCategoryI,60sampleunitsFoodCategoryII,30sampleunits FoodCategoryIII,15sampleunits.Submitallsamplescollectedtothelaboratory foranalysis.Advisethelaboratoryinadvanceofperishablesampleshipments. b. Sampleanalysis ThelaboratorywillanalyzeeachsampleforthepresenceofSalmonellaaccording tomethodsdescribedinthismanual,orinOfficialMethodsofAnalysis(2).Takea 25ganalyticalunitatrandomfromeach100gsampleunit.Whenasampleunit consistsofmorethanonecontainer,asepticallymixthecontentsofeach containerbeforetakingthe25ganalyticalunit.Toreducetheanalytical workload,theanalyticalunitsmaybecomposited.Themaximumsizeofa compositeunitis375gor15analyticalunits.Theminimumnumberofcomposite unitstobetestedforeachfoodcategoryisasfollows:FoodCategoryI,4 compositeunitsFoodCategoryII,2compositeunitsFoodCategoryIII,one compositeunit.Foreach375gcomposite,theentireamountof375gis analyzedforSalmonella. Keeptheremainderofthesampleunitinasterilecontainerforcompliance requirementsaspersection702(b)oftheFederalFood,Drug,andCosmeticAct asamendedthroughFebruary,1993.Refrigerateperishablesamplesandsamples supportingmicrobialgrowth.Ananalyticalcontrolisrequiredforeachsample tested.Thesampledlotisacceptableonlyifanalysesofallcompositeunitsare negativeforSalmonella.Ifoneormorecompositeunitsarepositivefor Salmonella,thelotisrejected,providedthattheanalyticalcontrolisnegativefor Salmonella.Alotwillnotberesampledunlesstheenvironmentalcontrolfor Salmonellaispositive.ForallsamplespositiveforSalmonella,determinethe somaticgroup.SeeChapter5forinformationonfurtherhandlingofthese cultures.Recommendationsforregulatoryactionmaybebasedonthe identificationoftheSalmonellasomaticgroupandwillnotrequiredefinitive serotypingbeforeinitiationofregulatoryaction. c. Imports. Thesesamplingplansapplytoimportedfoodproductsintendedforhuman consumption. d. Classificationoffoodproductsforsamplingpurposes Foodsthathavebeenclassifiedintothe3categoriesdescribedabovefor regulatorysamplingarelistedinthecategoriesaccordingtotheIndustryProduct Codesequenceandnomenclature(4).Listingdoesnotnecessarilymeanthat theseproductsareprobablesourcesofSalmonella.FoodCategoryI.Foods thatwouldnotnormallybesubjectedtoaprocesslethaltoSalmonellabetween thetimeofsamplingandconsumptionandareintendedforconsumptionbythe aged,theinfirm,andinfants.FoodCategoryII.Foodsthatwouldnotnormally besubjectedtoaprocesslethaltoSalmonellabetweenthetimeofsamplingand consumption.Examplesareasfollows:

IndustryProductCode
2 3 5 7 9 Milledgrainproductsnotcookedbeforeconsumption(branandwheat germ) Bread,rolls,buns,sugaredbreads,crackers,custardandcreamfilled sweetgoods,andicings Breakfastcerealsandotherreadytoeatbreakfastfoods Pretzels,chips,andothersnackfoods Butterandbutterproducts,pasteurizedmilkandrawfluidmilkandfluid milkproductsfordirectconsumption,pasteurizedandunpasteurized concentratedliquidmilkproductsfordirectconsumption,driedmilkand driedmilkproductsfordirectconsumption,casein,sodiumcaseinate,and whey

12 Cheeseandcheeseproducts 13 Icecreamfrompasteurizedmilkandrelatedproductsthathavebeen pasteurized,rawicecreammixandrelatedunpasteurizedproductsfor directconsumption 14 Pasteurizedandunpasteurizedimitationdairyproductsfordirect consumption 15 Pasteurizedeggsandeggproductsfrompasteurizedeggs,unpasteurized eggsandeggproductsfromunpasteurizedeggsforconsumptionwithout furthercooking 16 Cannedandcuredfish,vertebrates,andotherfishproductsfreshand frozenrawshellfishandcrustaceanproductsfordirectconsumption smokedfish,shellfish,andcrustaceansfordirectconsumption 17 Meatandmeatproducts,poultryandpoultryproducts,andgelatin (flavoredandunflavoredbulk) 20 Fresh,frozen,andcannedfruitsandjuices,concentrates,andnectars 22 driedfruitsfordirectconsumptionjams,jellies,preserves,andbutters 23 Nuts,nutproducts,edibleseeds,andedibleseedproductsfordirect consumption 24 Vegetablejuices,vegetablesprouts,andvegetablesnormallyeatenraw 26 Oilsconsumeddirectlywithoutfurtherprocessingoleomargarine 27 Dressingsandcondiments(includingmayonnaise),saladdressing,and vinegar 28 Spices,flavors,andextracts

23 Nuts,nutproducts,edibleseeds,andedibleseedproductsfordirect consumption 24 Vegetablejuices,vegetablesprouts,andvegetablesnormallyeatenraw 26 Oilsconsumeddirectlywithoutfurtherprocessingoleomargarine 27 Dressingsandcondiments(includingmayonnaise),saladdressing,and vinegar 28 Spices,flavors,andextracts 29 Softdrinksandwater 30 Beveragebases 31 Coffeeandtea 33 Candy(withandwithoutchocolatewithandwithoutnuts)andchewing gum 34 Chocolateandcocoaproducts 35 Puddingmixesnotcookedbeforeconsumption,andgelatinproducts 36 Syrups,sugars,andhoney 37 Readytoeatsandwiches,stews,gravies,andsauces 38 Soups 39 Preparedsalads 54 Nutrientsupplements,suchasvitamins,minerals,proteins,anddried inactiveyeast FoodCategoryIII:Foodsthatwouldnormallybesubjectedtoaprocesslethal toSalmonellabetweenthetimeofsamplingandconsumption.Examplesareas follows:

IndustryProductCode
2 Wholegrain,milledgrainproductsthatarecookedbeforeconsumption (cornmealandalltypesofflour),andstarchproductsforhumanuse 3 Prepareddrymixesforcakes,cookies,breads,androlls 4 Macaroniandnoodleproducts 16 Freshandfrozenfishvertebrates(exceptthoseeatenraw)freshand frozenshellfishandcrustaceans(exceptrawshellfishandcrustaceansfor directconsumption)otheraquaticanimals(includingfroglegs,marine snails,andsquid) 18 Vegetableproteinproducts(simulatedmeats)normallycookedbefore consumption 24 Freshvegetables,frozenvegetables,driedvegetables,curedand processedvegetableproductsnormallycookedbeforeconsumption 26 Vegetableoils,oilstock,andvegetableshortening 35 Drydessertmixes,puddingmixes,andrennetproductsthatarecooked beforeconsumption 2. Aerobicplatecounts,totalcoliforms,fecalcoliforms,Escherichiacoli(including enteropathogenicstrains),Staphylococcusspp.,Vibriospp.,Shigellaspp., Campylobacterspp.,Yersiniaspp.,Bacilluscereus,andClostridiumperfringens a. Samplecollection Fromanylotoffood,collectten8ozsubsamples(orretailpackages)atrandom. Donotbreakorcutlargerretailpackagestoobtainan8ozsubsample.Collect theintactretailunitasthesubsampleevenifitislargerthan8oz. b. Sampleanalysis. Analyzesamplesasindicatedincurrentcomplianceprograms. B. Equipmentandmaterials 1. Mechanicalblender.Severaltypesareavailable.Useblenderthathasseveral operatingspeedsorrheostat.Theterm"highspeedblender"designatesmixerwith4 canted,sharpedge,stainlesssteelbladesrotatingatbottomof4lobejarat10,000 12,000rpmorwithequivalentshearingaction.Suspendedsolidsarereducedtofine pulpbyactionofbladesandbylobularcontainer,whichswirlssuspendedsolidsinto blades.Waringblender,orequivalent,meetstheserequirements. 2. Sterileglassormetalhighspeedblenderjar,1000ml,withcover,resistantto autoclavingfor60minat121C 3. Balance,withweights2000gcapacity,sensitivityof0.1g 4. Sterilebeakers,250ml,lowform,coveredwithaluminumfoil 5. Sterilegraduatedpipets,1.0and10.0ml 6. Butterfield'sphosphatebuffereddilutionwater(Rll),sterilizedinbottlestoyieldfinal volumeof901ml 7. Sterileknives,forks,spatulas,forceps,scissors,tablespoons,andtonguedepressors (forsamplehandling) C. Receiptofsamples 1. TheofficialfoodsampleiscollectedbytheFDAinspectororinvestigator.As soonasthesamplearrivesatthelaboratory,theanalystshouldnoteitsgeneral physicalcondition.Ifthesamplecannotbeanalyzedimmediately,itshouldbestored asdescribedlater.Whetherthesampleistobeanalyzedforregulatorypurposes,for investigationofafoodborneillnessoutbreak,orforabacteriologicalsurvey,strict adherencetotherecommendationsdescribedhereisessential. 2. Conditionofsamplingcontainer.Checksamplingcontainersforgrossphysical defects.Carefullyinspectplasticbagsandbottlesfortears,pinholes,andpuncture marks.Ifsampleunitswerecollectedinplasticbottles,checkbottlesforfracturesand looselids.Ifplasticbagswereusedforsampling,becertainthattwistwiresdidnot

physicalcondition.Ifthesamplecannotbeanalyzedimmediately,itshouldbestored asdescribedlater.Whetherthesampleistobeanalyzedforregulatorypurposes,for investigationofafoodborneillnessoutbreak,orforabacteriologicalsurvey,strict adherencetotherecommendationsdescribedhereisessential. 2. Conditionofsamplingcontainer.Checksamplingcontainersforgrossphysical defects.Carefullyinspectplasticbagsandbottlesfortears,pinholes,andpuncture marks.Ifsampleunitswerecollectedinplasticbottles,checkbottlesforfracturesand looselids.Ifplasticbagswereusedforsampling,becertainthattwistwiresdidnot puncturesurroundingbags.Anycrosscontaminationresultingfromoneormoreof abovedefectswouldinvalidatethesample,andthecollectingdistrictshouldbe notified(seeC5,below) 3. Labelingandrecords.Becertainthateachsampleisaccompaniedbyacompleted copyoftheCollectionReport(FormFD464)andofficiallysealedwithtape(FD415a) bearingthesamplenumber,collectingofficial'sname,anddate.Assigneachsample unitanindividualunitnumberandanalyzeasadiscreteunitunlessthesampleis compositedasdescribedpreviouslyinthischapter. 4. Adherencetosamplingplan.Mostfoodsarecollectedunderaspecificallydesigned samplingplaninoneofseveralongoingcomplianceprograms.Foodstobeexamined forSalmonella,however,aresampledaccordingtoastatisticallybasedsamplingplan designedexclusivelyforusewiththispathogen.Dependingonthefoodandthetype ofanalysistobeperformed,determinewhetherthefoodhasbeensampledaccording tothemostappropriatesamplingplan. 5. Storage.Ifpossible,examinesamplesimmediatelyuponreceipt.Ifanalysismustbe postponed,however,storefrozensamplesat20Cuntilexamination.Refrigerate unfrozenperishablesamplesat04Cnotlongerthan36h.Storenonperishable, canned,orlowmoisturefoodsatroomtemperatureuntilanalysis. 6. Notificationofcollectingdistrict.Ifasamplefailstomeettheabovecriteriaandis thereforenotanalyzed,notifythecollectingdistrictsothatavalidsamplecanbe obtainedandthepossibilityofarecurrencereduced. D. Thawing Useaseptictechniquewhenhandlingproduct.Beforehandlingoranalysisofsample,clean immediateandsurroundingworkareas.Inaddition,swabimmediateworkareawith commercialgermicidalagent.Preferably,donotthawfrozensamplesbeforeanalysis.If necessarytotemperafrozensampletoobtainananalyticalportion,thawitintheoriginal containerorinthecontainerinwhichitwasreceivedinthelaboratory.Wheneverpossible, avoidtransferringthesampletoasecondcontainerforthawing.Normally,asamplecanbe thawedat25Cwithin18h.Ifrapidthawingisdesired,thawthesampleatlessthan45C fornotmorethan15min.Whenthawingasampleatelevatedtemperatures,agitatethe samplecontinuouslyinthermostaticallycontrolledwaterbath. E. Mixing Variousdegreesofnonuniformdistributionofmicroorganismsaretobeexpectedinany foodsample.Toensuremoreevendistribution,shakeliquidsamplesthoroughlyand,if practical,mixdriedsampleswithsterilespoonsorotherutensilsbeforewithdrawingthe analyticalunitfromasampleof100gorgreater.Usea50ganalyticalunitofliquidordry foodtodetermineaerobicplatecountvalueandmostprobablenumberofcoliforms.Other analyticalunitsizes(e.g.,25gforSalmonella)mayberecommended,dependingonspecific analysistobeperformed.Useanalyticalunitsizeanddiluentvolumerecommendedfor appropriateBacteriologicalAnalyticalManualmethodbeingused.Ifcontentsofpackageare obviouslynothomogeneous(e.g.,afrozendinner),macerateentirecontentsofpackage andwithdrawtheanalyticalunit,or,preferably,analyzeeachdifferentfoodportion separately,dependingonpurposeoftest. F. Weighing Tarehighspeedblenderjarthenasepticallyandaccurately(0.1g)weighunthawed food(iffrozen)intojar.Ifentiresampleweighslessthantherequiredamount,weigh portionequivalenttoonehalfofsampleandadjustamountofdiluentorbrothaccordingly. Totalvolumeinblendermustcompletelycoverblades. G. Blendinganddilutingofsamplesrequiringenumerationofmicroorganisms 1. Allfoodsotherthannutmeathalvesandlargerpieces,andnutmeal.Add450 mlButterfield'sphosphatebuffereddilutionwatertoblenderjarcontaining50g analyticalunitandblend2min.Thisresultsinadilutionof10 1.Makedilutionsof originalhomogenatepromptly,usingpipetsthatdeliverrequiredvolumeaccurately.Do notdeliverlessthan10%oftotalvolumeofpipet.Forexample,donotusepipetwith capacitygreaterthan10mltodeliver1mlvolumesfordelivering0.1mlvolumes,do notusepipetwithcapacitygreaterthan1.0ml.Preparealldecimaldilutionswith90 mlofsterilediluentplus10mlofpreviousdilution,unlessotherwisespecified.Shakeall dilutionsvigorously25timesin30cm(1ft)arcin7s.Notmorethan15minshould elapsefromthetimesampleisblendeduntilalldilutionsareinappropriatemedia. 2. Nutmeathalvesandlargerpieces.Asepticallyweigh50ganalyticalunitintosterile screwcapjar.Add50mldiluent(Gl,above)andshakevigorously50timesthrough 30cmarctoobtain10 0dilution.Letstand35minandshake5timesthrough30cm arctoresuspendjustbeforemakingserialdilutionsandinoculations. 3. Nutmeal.Asepticallyweigh10ganalyticalunitintosterilescrewcapjar.Add90ml ofdiluent(Gl,above)andshakevigorously50timesthrough30cmarctoobtain10 1 dilution.Letstand35minandshake5timesthrough30cmarctoresuspendjust beforemakingserialdilutionsandinoculations.

References
1. AmericanPublicHealthAssociation.1985.LaboratoryProceduresfortheExaminationof SeawaterandShellfish,5thed.APHA,Washington,DC. 2. AOACINTERNATIONAL.1995.OfficialMethodsofAnalysis,16thed.AOACINTERNATIONAL, Arlington,VA. 3. FoodandDrugAdministration.1989.LaboratoryProceduresManual.FDA,Rockville,MD. 4. FoodandDrugAdministration.1978.EDRODataCodesManual.ProductCodes:Human Foods.FDA,Rockville,MD.

1. AmericanPublicHealthAssociation.1985.LaboratoryProceduresfortheExaminationof SeawaterandShellfish,5thed.APHA,Washington,DC. 2. AOACINTERNATIONAL.1995.OfficialMethodsofAnalysis,16thed.AOACINTERNATIONAL, Arlington,VA. 3. FoodandDrugAdministration.1989.LaboratoryProceduresManual.FDA,Rockville,MD. 4. FoodandDrugAdministration.1978.EDRODataCodesManual.ProductCodes:Human Foods.FDA,Rockville,MD. 5. FoodandDrugAdministration.1993.InvestigationsOperationsManual.FDA,Rockville,MD. 6. InternationalCommissiononMicrobiologicalSpecificationsforFoods.1986.Microorganisms inFoods.2.SamplingforMicrobiologicalAnalysis:PrinciplesandSpecificApplications,2nd ed.UniversityofTorontoPress,Toronto,Ontario,Canada. 7. NationalAcademyofSciences.1969.AnEvaluationoftheSalmonellaProblem.National AcademyofSciences,Washington,DC.

HypertextSource:BacteriologicalAnalyticalManual,Edition8,RevisionA,1998.Chapter1.

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