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药典01

BAM:

AerobicPlateCount

January2001

BacteriologicalAnalyticalManual

Chapter3

AerobicPlateCount

Authors:

LarryMaturinandJamesT.Peeler

Foradditionalinformation,contactJohnBryce.

ChapterContents

∙ConventionalPlateCountMethod

∙SpiralPlateMethod

∙References

Theaerobicplatecount（APC）isintendedtoindicatethelevelofmicroorganisminaproduct.DetailedproceduresfordeterminingtheAPCoffoodshavebeendevelopedbytheAssociationofOfficialAnalyticalChemists（AOAC）（3）andtheAmericanPublicHealthAssociation（APHA）

（1）.Theconventionalplatecountmethodforexaminingfrozen,chilled,precooked,orpreparedfoods,outlinedbelow,conformstoAOACOfficialMethodsofAnalysis,sec.966.23,withoneproceduralchange（966.23C）.Thesuitablecolonycountingrange（10）is25-250.Theautomatedspiralplatecountmethodfortheexaminationoffoodsandcosmetics（5）,outlinedbelow,conformstoAOACOfficialMethodsofAnalysis,sec.977.27.Forproceduraldetailsofthestandardplatecount,seeref.2.Guidelinesforcalculatingandreportingplatecountshavebeenchangedtoconformwiththeanticipatedchangesinthe16theditionofStandardMethodsfortheExaminationofDairyProducts

（2）andtheInternationalDairyFederation（IDF）procedures（6）.

ConventionalPlateCountMethod

A.Equipmentandmaterials

1.Workarea,leveltablewithamplesurfaceinroomthatisclean,well-lighted（100foot-candlesatworkingsurface）andwell-ventilated,andreasonablyfreeofdustanddrafts.Themicrobialdensityofairinworkingarea,measuredinfalloutpourplatestakenduringplating,shouldnotexceed15colonies/plateduring15minexposure.

2.Storagespace,freeofdustandinsectsandadequateforprotectionofequipmentandsupplies

3.Petridishes,glassorplastic（atleast15x90mm）

4.Pipetswithpipetaids（nomouthpipetting）orpipettors,1,5,and10ml,graduatedin0.1mlunits

5.Dilutionbottles,6oz（160ml）,borosilicate-resistantglass,withrubberstoppersorplasticscrewcaps

6.Pipetandpetridishcontainers,adequateforprotection

7.Circulatingwaterbath,fortemperingagar,thermostaticallycontrolledto45±1°C

8.Incubator,35±1°C;milk,32±1°C

9.Colonycounter,dark-field,Quebec,orequivalent,withsuitablelightsourceandgridplate

10.Tallyregister

11.Dilutionblanks,90±1mlButterfield'sphosphate-buffereddilutionwater（R111）;milk,99±2ml

12.Platecountagar（standardmethods）（M1242）

13.Refrigerator,tocoolandmaintainsamplesat0-5°C;milk,0-4.4°C

14.Freezer,tomaintainfrozensamplesfrom-15to-20°C

15.Thermometers（mercury）appropriaterange;accuracycheckedwithathermometercertifiedbytheNationalInstituteofStandardsandTechnology（NIST）

B.Procedureforanalysisoffrozen,chilled,precooked,orpreparedfoods

Usingseparatesterilepipets,preparedecimaldilutionsof10-2,10-3,10-4,andothersasappropriate,offoodhomogenate（seeChapter13forsamplepreparation）bytransferring10mlofpreviousdilutionto90mlofdiluent.Avoidsamplingfoam.Shakealldilutions25timesin30cm（1ft）arcwithin7s.Pipet1mlofeachdilutionintoseparate,duplicate,appropriatelymarkedpetridishes.Reshakedilutionbottle25timesin30cmarcwithin7sifitstandsmorethan3minbeforeitispipettedintopetridish.Add12-15mlplatecountagar（cooledto45±1°C）toeachplatewithin15minoforiginaldilution.Formilksamples,pouranagarcontrol,pouradilutionwatercontrolandpipetwaterforapipetcontrol.Addagartothelattertwoforeachseriesofsamples.Addagarimmediatelytopetridisheswhensamplediluentcontainshygroscopicmaterials,e.g.,flourandstarch.Pouragaranddilutionwatercontrolplatesforeachseriesofsamples.Immediatelymixsampledilutionsandagarmediumthoroughlyanduniformlybyalternaterotationandback-and-forthmotionofplatesonflatlevelsurface.Letagarsolidify.Invertsolidifiedpetridishes,andincubatepromptlyfor48±2hat35°C.Donotstackplateswhenpouringagarorwhenagarissolidifying.

C.GuidelinesforcalculatingandreportingAPCsinuncommoncases

OfficialMethodsofAnalysis（3）doesnotprovideguidelinesforcountingandreportingplatecounts,whereasStandardMethodsfortheExaminationofDairyProducts,16thed.

（2）presentsdetailedguidelines;foruniformity,therefore,useAPHAguidelinesasmodified（6,8）.Reportallaerobicplatecounts

（2）computedfromduplicateplates.Formilksamples,reportallaerobicplate

（2）countscomputedfromduplicateplatescontaininglessthan25coloniesaslessthan25estimatedcount.Reportallaerobicplatecounts

（2）computedfromduplicateplatescontainingmorethan250coloniesasestimatedcounts.Countsoutsidethenormal25-250rangemaygiveerroneousindicationsoftheactualbacterialcompositionofthesample.Dilutionfactorsmayexaggeratelowcounts（lessthan25）,andcrowdedplates（greaterthan250）maybedifficulttocountormayinhibitthegrowthofsomebacteria,resultinginalowcount.Reportcountslessthan25ormorethan250coloniesasestimatedaerobicplatecounts（EAPC）.Usethefollowingguide:

1.Normalplates（25-250）.Selectspreader-freeplate（s）.Countallcolonyformingunits（CFU）,includingthoseofpinpointsize,onselectedplate（s）.Recorddilution（s）usedandtotalnumberofcoloniescounted.

2.Plateswithmorethan250colonies.WhennumberofCFUperplateexceeds250,foralldilutions,recordthecountsastoonumeroustocount（TNTC）forallbuttheplateclosestto250,andcountCFUinthoseportionsofplatethatarerepresentativeofcolonydistribution.Seeref.2fordetailedguidelines.MarkcalculatedAPCwithEAPCtodenotethatitwasestimatedfromcountsoutside25-250perplaterange（seeD-3）.

3.Spreaders.Spreadingcoloniesareusuallyof3distincttypes:

1）achainofcolonies,nottoodistinctlyseparated,thatappearstobecausedbydisintegrationofabacterialclump;2）onethatdevelopsinfilmofwaterbetweenagarandbottomofdish;and3）onethatformsinfilmofwateratedgeoronsurfaceofagar.Ifplatespreparedfromsamplehaveexcessivespreadergrowthsothat（a）areacoveredbyspreaders,includingtotalareaofrepressedgrowth,exceeds50%ofplatearea,or（b）areaofrepressedgrowthexceeds25%ofplatearea,reportplatesasspreaders.Whenitisnecessarytocountplatescontainingspreadersnoteliminatedby（a）or（b）above,counteachofthe3distinctspreadertypesasonesource.Forthefirsttype,ifonlyonechainexists,countitasasinglecolony.Ifoneormorechainsappeartooriginatefromseparatesources,counteachsourceasonecolony.Donotcounteachindividualgrowthinsuchchainsasaseparatecolony.Types2and3usuallyresultindistinctcoloniesandarecountedassuch.CombinethespreadercountandthecolonycounttocomputetheAPC.

4.PlateswithnoCFU.Whenplatesfromalldilutionshavenocolonies,reportAPCaslessthan1timesthecorrespondinglowestdilutionused.MarkcalculatedAPCwithasterisktodenotethatitwasestimatedfromcountsoutsidethe25-250perplaterange.Whenplate（s）fromasampleareknowntobecontaminatedorotherwiseunsatisfactory,recordtheresult（s）aslaboratoryaccident（LA）.

D.Computingandrecordingcounts（seerefs6,8）

ToavoidcreatingafictitiousimpressionofprecisionandaccuracywhencomputingAPC,reportonlythefirsttwosignificantdigits.RoundofftotwosignificantfiguresonlyatthetimeofconversiontoSPC.Formilksamples,whenplatesforalldilutionshavenocolonies,reportAPCaslessthan25coloniesestimatedcount.Roundbyraisingtheseconddigittothenexthighestnumberwhenthethirddigitis6,7,8,or9andusezerosforeachsuccessivedigittowardtherightfromtheseconddigit.Rounddownwhenthethirddigitis1,2,3,or4.Whenthethirddigitis5,roundupwhentheseconddigitisoddandrounddownwhentheseconddigitiseven.

Examples

CalculatedCount

APC

12,700

13,000

12,400

12,000

15,500

16,000

14,500

14,000

1.Plateswith25-250CFU.

a.CalculatetheAPCasfollows:

where:

N=Numberofcoloniespermlorgofproduct

∑C=Sumofallcoloniesonallplatescounted

n1=Numberofplatesinfirstdilutioncounted

n2=Numberofplatesinseconddilutioncounted

d=Dilutionfromwhichthefirstcountswereobtained

Example

1:

100

1:

1000

232,244

33,28

=537/0.022

=24,409

≈24,000

b.Whencountsofduplicateplatesfallwithinandwithoutthe25-250colonyrange,useonlythosecountsthatfallwithinthisrange.

2.Allplateswithfewerthan25CFU.Whenplatesfrombothdilutionsyieldfewerthan25CFUeach,recordactualplatecountbutrecordthecountaslessthan25 × 1/dwhendisthedilutionfactorforthedilutionfromwhichthefirstcountswereobtained.

Example

Colonies

1:

100

1:

1000

EAPC/ml（g）

18

2

<2,500

0

0

<2,500

3.Allplateswithmorethan250CFU.Whenplatesfromboth2dilutionsyieldmorethan250CFUeach（butfewerthan100/cm2）,estimatetheaerobiccountsfromtheplates（EAPC）nearest250andmultiplybythedilution.

Example

Colonies

1:

100

1:

1000

EAPC/ml（g）

TNTC

640

640,000

TNTC,toonumeroustocount.

EAPC,estimatedaerobicplatecount.

4.Allplateswithspreadersand/orlaboratoryaccident.ReportrespectivelyasSpreader（SPR）,orLaboratoryAccident（LA）.

5.Allplateswithmorethananaverageof100CFUpersqcm.EstimatetheAPCasgreaterthan100timesthehighestdilutionplated,timestheareaoftheplate.Theexamplesbelowhaveanaveragecountof110persqcm.

Example

Colonies/Dilution

1:

100

1:

1000

EAPC/ml（g）

TNT