61 MICROBIOLOGICAL EXAMINATION OF NONSTERILE

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61 MICROBIOLOGICALEXAMINATIONOFNONSTERILEPRODU CTS:MICROBIAL

ENUMERATIONTESTS

INTRODUCTION

Thetestsdescribedhereafterwillallowquantitativeenumerationofmesophilicbacteriaandfungithat

maygrowunderaerobicconditions.

Thetestsaredesignedprimarilytodeterminewhetherasubstanceorpreparationcomplieswithan

establishedspecificationformicrobiologicalquality.Whenusedforsuchpurposes,followthe

instructionsgivenbelow,includingthenumberofsamplestobetaken,andinterprettheresultsas

statedbelow.

Themethodsarenotapplicabletoproductscontainingviablemicroorganismsasactiveingredients.

Alternativemicrobiologicalprocedures,includingautomatedmethods,maybeused,providedthat

theirequivalencetothePharmacopeialmethodhasbeendemonstrated.

GENERALPROCEDURES

Carryoutthedeterminationunderconditionsdesignedtoavoidextrinsicmicrobialcontaminationof

theproducttobeexamined.Theprecautionstakentoavoidcontaminationmustbesuchthattheydo

notaffectanymicroorganismsthataretoberevealedinthetest.

Iftheproducttobeexaminedhasantimicrobialactivity,thisis,insofaraspossible,removedor

neutralized.Ifinactivatorsareusedforthispurpose,theirefficacyandtheirabsenceoftoxicityfor

microorganismsmustbedemonstrated.

Ifsurfaceactivesubstancesareusedforsamplepreparation,theirabsenceoftoxicityfor

microorganismsandtheircompatibilitywithanyinactivatorsusedmustbedemonstrated.

ENUMERATIONME THODS

UsetheMembraneFiltrationmethodoroneofthePla teCountMethods,asdirected.TheMost

ProbableNumber(MPN)Methodisgenerallytheleastaccuratemethodformicrobialcounts;however,

forcertainproductgroupswithverylowbioburden,itmaybethemostappropriatemethod.

Thechoiceofamethodisbasedonfactorssuchasthenatureoftheproductandtherequiredlimitof

microorganisms.Themethodchosenmustallowtestingofasufficientsamplesizetojudgecompliance

withthespecification.Thesuitabilityofthechosenmethodmustbeestablished.

Changetoread:

GROWTHPROMOTIONTEST,  SUITABILITYOFTHECOUNTINGMET HOD ANDNEGATIVE

CONTROLS

GeneralConsiderations

Theabilityofthetesttodetectmicroorganismsinthepresenceofproducttobetestedmustbe

established.

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Suitabilitymustbeconfirmedifachangeintestingperformanceorachangeintheproductthatmay

affecttheoutcomeofthetest,isintroduced.

PreparationofTestStrains

Usestandardizedstablesuspensionsofteststrainsorprepareasstatedbelow.Seedlotculture

maintenancetechniques(seedlotsystems)areusedsothattheviablemicroorganismsusedfor

inoculationarenotmorethan5passagesremovedfromtheoriginalmasterseedlot.Groweachofthe

bacterialandfungalteststrainsseparatelyasdescribedinTable1.

Table1.PreparationandUseofTestMicroorganisms

Microorganism

Preparationof

Test

Strain

GrowthPromotion

SuitabilityofCountingMethodin

thePresenceofProduct

TotalAerobic

Microbial

Count

Total

Yeasts

andMolds

Count

TotalAerobic

MicrobialCount

Total

Yeasts

andMolds

Count

Staphylococcus

aureussuchas

ATCC6538,

NCIMB9518,

CIP4.83,or

NBRC13276

Soybean–

CaseinDigest

Agaror

Soybean–

CaseinDigest

Broth30 –35

18–24hours

Soybean–

Casein

DigestAgar

and

Soybean–

Casein

DigestBroth

100

cfu30 –35

3days

Soybean–Casein

DigestAgar/MPN

Soybean–Casein

DigestBroth 100

cfu30 –35  3

days

Pseudomonas

aeruginosa

suchasATCC

9027,NCIMB

8626,CIP

82.118,or

NBRC13275

Soybean–

CaseinDigest

Agaror

Soybean–

CaseinDigest

Broth30 –35

18–24hours

Soybean–

Casein

DigestAgar

and

Soybean–

Casein

DigestBroth

100

cfu30 –35

3days

Soybean–Casein

Digest

Agar/MPNSoybean–

CaseinDigest

Broth 100cfu30

–35  3days

Bacillus

subtilissuchas

ATCC6633,

NCIMB8054,

CIP52.62,or

NBRC3134

Soybean–

CaseinDigest

Agaror

Soybean–

CaseinDigest

Broth30 –35

18–24hours

Soybean–

Casein

DigestAgar

and

Soybean–

Casein

DigestBroth

100

cfu30 –35

3days

Soybean–Casein

Digest

Agar/MPNSoybean–

CaseinDigest

Broth 100cfu30

–35  3days

Candida

albicanssuch

asATCC

10231,NCPF

3179,IP48.72,

orNBRC1594

Sabouraud

DextroseAgar

orSabouraud

DextroseBroth

20 –25 2–3

days

Soybean–

Casein

DigestAgar

100cfu

30 –35 

5days

Sabouraud

Dextrose

Agar

100cfu

Soybean–Casein

DigestAgar 100

cfu30 –35  5

daysMPN:not

applicable

Sabouraud

Dextrose

Agar

100cfu

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20 –25

5days

20 –25

5days

Aspergillus

nigersuchas

ATCC16404,

IMI149007,IP

1431.83,or

NBRC9455

Sabouraud

DextroseAgar

orPotato–

DextroseAgar

20 –25 5–7

days,oruntil

good

sporulationis

achieved

Soybean–

Casein

DigestAgar

100cfu

30 –35 

5days

Sabouraud

Dextrose

Agar

100

cfu20 –

25  5

days

Soybean–Casein

DigestAgar 100

cfu30 –35  5

daysMPN:not

applicable

Sabouraud

Dextrose

Agar

100cfu

20 –25

5days

UseBufferedSodiumChloride–PeptoneSolutionpH7.0orPhosphateBufferSolutionpH7.2tomake

testsuspensions;tosuspendA.nigerspores,0.05%ofpolysorbate80maybeaddedtothebuffer.Use

thesuspensionswithin2hours,orwithin24hoursifstoredbetween2 and8 .Asanalternativeto

preparingandthendilutingafreshsuspensionofvegetativecellsofA.nigerorB.subtilis,astable

sporesuspensionispreparedandthenanappropriatevolumeofthesporesuspensionisusedfortest

inoculation.Thestablesporesuspensionmaybemaintainedat2 to8 foravalidatedperiodoftime.

NegativeControl

Toverifytestingconditions,anegativecontrolisperformedusingthechosendiluentinplaceofthe

testpreparation.Theremustbenogrowthofmicroorganisms. Anegativecontrolisalsoperformed

whentestingtheproductsasdescribedunderTestingofProducts.Afailednegativecontrolrequiresan

investigation.

GrowthPromotionoftheMedia

Testeachbatchofreadypreparedmediumandeachbatchofmediumpreparedeitherfrom

dehydratedmediumorfromtheingredientsdescribed.

Inoculateportions/platesofSoybean–CaseinDigestBrothandSoybean–CaseinDigestAgarwitha

smallnumber(notmorethan100cfu)ofthemicroorganismsindicatedinTable1,usingaseparate

portion/plateofmediumforeach.InoculateplatesofSabouraudDex troseAgarwithasmallnumber

(notmorethan100cfu)ofthemicroorganismsindicatedinTable1,usingaseparateplateofmedium

foreach.IncubateaccordingtotheconditionsdescribedinTable1.

Forsolidmedia,growthobtainedmustnotdifferbyafactorgreaterthan2fromthecalculatedvalue

forastandardizedinoculum.Forafreshlypreparedinoculum,growthofthemicroorganisms

comparabletothatpreviouslyobtainedwithapreviouslytestedandapprovedbatchofmediumoccurs.

Liquidmediaaresuitableifclearlyvisiblegrowthofthemicroorganismscomparabletothatpreviously

obtainedwithapreviouslytestedandapprovedbatchofmediumoccurs.

Suit abilityoftheCountingMethodinthePresenceofProduct

PREPARATIONOFTHESAMPLE

Themethodforsamplepreparationdependsonthephysicalcharacteristicsoftheproducttobe

tested.Ifnoneoftheproceduresdescribedbelowcanbedemonstratedtobesatisfactory,asuitable

alternativeproceduremustbedeveloped.

WaterSolubleProducts—Dissolveordilute(usuallya1in10dilutionisprepared)theproducttobe

examinedinBufferedSodiumChloride–PeptoneSolutionpH7.0,PhosphateBufferSol utionpH7.2,or

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Soybean–CaseinDigestBroth.Ifnecessary,adjusttoapHof6to8.Furtherdilutions,wherenecessary,

arepreparedwiththesamediluent.

NonfattyProductsInsolubleinWater—Suspendtheproducttobeexamined(usuallya1in10

dilutionisprepared)inB ufferedSodiumChloride–PeptoneSolutionpH7.0,PhosphateBufferSolutionpH

7.2,orSoybe an–Case inDigestBroth.Asurfaceactiveagentsuchas1gperLofpolysorbate80maybe

addedtoassistthesuspensionofpoorlywettablesubstances.Ifnecessary,adjusttoapHof6to8.

Furtherdilutions,wherenecessary,arepreparedwiththesamediluent.

FattyP roducts—Dissolveinisopropylmyristatesterilizedbyfiltration,ormixtheproducttobe

examinedwiththeminimumnecessaryquantityofsterilepolysorbate80oranothernoninhibitorysterile

surfaceactivereagentheated,ifnecessary,tonotmorethan40 or,inexceptionalcases,tonotmore

than45 .Mixcarefullyandifnecessarymaintainthetemperatureinawaterbath.Addasufficient

quantityoftheprewarmedchosendiluenttomakea1in10dilutionoftheoriginalproduct.Mix

carefully,whilemaintainingthetemperaturefortheshortesttimenecessaryfortheformationofan

emulsion.Furtherserial10folddilutionsmaybepreparedusingthechosendiluentcontainingasuitable

concentrationofsterilepolysorbate80oranothernoninhibitorysterilesurfaceactivereagent.

FluidsorSolidsinAerosolForm—Asepticallytransfertheproductintoamembranefilterapparatusor

asterilecontainerforfurthersampling.Useeitherthetotalcontentsoradefinednumberofmetered

dosesfromeachofthecontainerstested.

TransdermalPatches—Removetheprotectivecoversheets(“releaseliners”)ofthetransdermal

patchesandplacethem,adhesivesideupwards,onsterileglassorplastictrays.Covertheadhesive

surfacewithasuitablesterileporousmaterial(e.g.,sterilegauze)topreventthepatchesfromsticking

together,andtransferthepatchestoasuitablevolumeofthechosendiluentcontaininginactivators

suchaspolysorbate80and/orlecithin.Shakethepreparationvigorouslyforatleast30minutes.

INOCULATIONANDDILUTION

Addtothesamplepreparedasdirectedaboveandtoacontrol(withnotestmaterialincluded)a

sufficientvolumeofthemicrobialsuspensiontoobtainaninoculumofnotmorethanthan100cfu.The

volumeofthesuspensionoftheinoculumshouldnotexceed1%ofthevolumeofdilutedproduct.

Todemonstrateacceptablemicrobialrecoveryfromtheproduct,thelowestpossibledilutionfactorof

thepreparedsamplemustbeusedforthetest.Wherethisisnotpossibleduetoantimicrobialactivity

orpoorsolubility,furtherappropriateprotocolsmustbedeveloped.Ifinhibitionofgrowthbythesample

cannototherwisebeavoided,thealiquotofthemicrobialsuspensionmaybeaddedafterneutralization,

dilution,orfiltration.

NEUTRALIZATION/REMOVALOFANTIMICROBIALACTIVITY

Thenumberofmicroorganismsrecoveredfromthepreparedsampledilutedasdescribedin

InoculationandDilutionandincubatedfollowingtheproceduredescribedinRecoveryofMicroorganisms

inthePresence ofProduct,iscomparedtothenumberofmicroorganismsrecoveredfromthecontrol

preparation.

Ifgrowthisinhibited(reductionbyafactorgreaterthan2),thenmodifytheprocedureforthe

particularenumerationtesttoensurethevalidityoftheresults.Modificationoftheproceduremay

include,forexample,

1.Anincreaseinthevolumeofthediluentorculturemedium;

2.Incorporationofaspecificorgeneralneutralizingagentsintothediluent;

3.Membranefiltration;or

4.Acombinationoftheabovemeasures.

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NeutralizingAgents—Neutralizingagentsmaybeusedtoneutralizetheactivityofantimicrobial

agents(seeTable2).Theymaybeaddedtothechosendiluentorthemediumpreferablybefore

sterilization.Ifused,theirefficacyandtheirabsenceoftoxicityformicroorganismsmustbe

demonstratedbycarryingoutablankwithneutralizerandwithoutproduct.

Table2.CommonNeutralizingAgents/Methodsfor

InterferingSubstances

InterferingSubstance

PotentialNeutralizing

Agents/Method

Glutaraldehyde,mercurials Sodiumhydrogensulfite

(Sodiumbisulfite)

Phenolics,alcohol,aldehydes,sorbate Dilution

Aldehydes Glycine

Quaternaryammoniumcompounds(QACs),parahydroxybenzoates

(parabens),bisbiguanides

Lecithin

QACs,iodine,parabens Polysorbate

Mercurials Thioglycollate

Mercurials,halogens,aldehydes Thiosulfate

EDTA(edetate) MgorCaions

Ifnosuitableneutralizingmethodcanbefound,itcanbeassumedthatthefailuretoisolatethe

inoculatedorganismisattributabletothemicrobicidalactivityoftheproduct.Thisinformationservesto

indicatethatthearticleisnotlikelytobecontaminatedwiththegivenspeciesofthemicroorganism.

However,itispossiblethattheproductinhibitsonlysomeofthemicroorganismsspecifiedherein,but

doesnotinhibitothersnotincludedamongtheteststrainsorthoseforwhichthelatterarenot

representative.Then,performthetestwiththehighestdilutionfactorcompatiblewithmicrobialgrowth

andthespecificacceptancecriterion.

RECOVERYOFMICROORGANISMSINTHEPRESENCEOFPRODUCT

Foreachofthemicroorganismslisted,separatetestsareperformed.Onlymicroorganismsofthe

addedteststrainarecounted.

MembraneFiltration—Usemembranefiltershavinganominalporesizenotgreaterthan0.45µm.The

typeoffiltermaterialischoseninsuchawaythatthebacteriaretainingefficiencyisnotaffectedby

thecomponentsofthesampletobeinvestigated.Foreachofthemicroorganismslisted,onemembrane

filterisused.

TransferasuitablequantityofthesamplepreparedasdescribedunderPreparation oftheSample ,

InoculationandDilution,andNeutralization/Remo valofAntimicrobialActivity(preferablyrepresenting1

goftheproduct,orlessiflargenumbersofcfuareexpected)tothemembranefilter,filter

immediately,andrinsethemembranefilterwithanappropriatevolumeofdiluent.

Forthedeterminationoftotalaerobicmicrobialcount(TAMC),transferthemembranefiltertothe

surfaceoftheSoybean–CaseinDigestAgar.Forthedeterminationoftotalcombinedyeastsandmolds

count(TYMC),transferthemembranetothesurfaceoftheSabouraud DextroseAgar.Incubatethe

platesasindicatedinTable1.Performthecounting.

PlateCountMethods—Performplatecountmethodsatleastinduplicateforeachmedium,anduse

themeancountoftheresult.

PourPlateMethod—ForPetridishes9cmindiameter,addtothedish1mLofthesamplepreparedas

describedunderPreparationoftheSample ,Inoculationand Dilution,andNeutralization/Removalof

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AntimicrobialActivityand15to20mLofSoybean–CaseinDigestAgarorSabouraudDextroseAgar,

bothmediamaintainedatnotmorethan45 .IflargerPetridishesareused,theamountofagar

mediumisincreasedaccordingly.ForeachofthemicroorganismslistedinTable 1,atleasttwoPetri

dishesareused.

IncubatetheplatesasindicatedinTable1.Takethearithmeticmeanofthecountspermedium,and

calculatethenumberofcfuintheoriginalinoculum.

Sur faceSpreadMethod—ForPetridishes9cmindiameter,add15to20mLofSoybean–CaseinDigest

AgarorSabouraudDextroseAgaratabout45 toeachPetridish,andallowtosolidify.IflargerPetri

dishesareused,thevolumeoftheagarisincreasedaccordingly.Drytheplates,forexample,ina

laminarairflowcabinetorinanincubator.ForeachofthemicroorganismslistedinTable1,atleasttwo

Petridishesareused.Spreadameasuredvolumeofnotlessthan0.1mLofthesample,preparedas

directedunderPreparationo ftheSample,Ino culationandDilution,andNeutralization/Removalof

AntimicrobialActivityoverthesurfaceofthemedium.IncubateandcountasdirectedforPo urPlate

Method.

MostProba bleNumber(MPN)Method—TheprecisionandaccuracyoftheMPNMethodislessthan

thatoftheMembraneFiltrationmethodorthePlateCountMetho d.Unreliableresultsareobtained

particularlyfortheenumerationofmolds.Forthesereasons,theMPNMethodisreservedforthe

enumerationofTAMCinsituationswherenoothermethodisavailable.Iftheuseofthemethodis

justified,proceedasfollows.

Prepareaseriesofatleastthreeserial10folddilutionsoftheproductasdescribedforPreparationof

theSa mple,Inoculatio nandDilution, andNeutr alization/RemovalofAntimicrobialActivity.Fromeach

levelofdilution,threealiquotsof1gor1mLareusedtoinoculatethreetubeswith9to10mLof

Soybean–CaseinDigestBroth.Ifnecessaryasurfaceactiveagentsuchaspolysorbate80,oran

inactivatorofantimicrobialagentsmaybeaddedtothemedium.Thus,ifthreelevelsofdilutionare

prepared,ninetubesareinoculated.

Incubatealltubesat30 to35 fornotmorethan3days.Ifreadingoftheresultsisdifficultor

uncertainowingtothenatureoftheproducttobeexamined,subcultureinthesamebrothorin

Soybean–CaseinDigestAgarfor1to2daysatthesametemperature,andusetheseresults.From

Table3,determinethemostprobablenumberofmicroorganismspergormLoftheproducttobe

examined.

Table3.MostProbableNumberVa luesofMicroo rganisms

Observed

Combinations

ofNumbersofTubes

ShowingGrowthinEachSet

MPNpergor

permLof

Product

95%

Confidence

Limits

NumberofgormLofProductperTube

0.1 0.01 0.001

0 0 0 <3 0–9.4

0 0 1 3 0.1–9.5

0 1 0 3 0.1–10

0 1 1 6.1 1.2–17

0 2 0 6.2 1.2–17

0 3 0 9.4 3.5–35

1 0 0 3.6 0.2–17

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Observed

Combinations

ofNumbersofTubes

ShowingGrowthinEachSet

MPNpergor

permLof

Product

95%

Confidence

Limits

NumberofgormLofProductperTube

0.1 0.01 0.001

1 0 1 7.2 1.2–17

1 0 2 11 4–35

1 1 0 7.4 1.3–20

1 1 1 11 4–35

1 2 0 11 4–35

1 2 1 15 5–38

1 3 0 16 5–38

2 0 0 9.2 1.5–35

2 0 1 14 4–35

2 0 2 20 5–38

2 1 0 15 4–38

2 1 1 20 5–38

2 1 2 27 9–94

2 2 0 21 5–40

2 2 1 28 9–94

2 2 2 35 9–94

2 3 0 29 9–94

2 3 1 36 9–94

3 0 0 23 5–94

3 0 1 38 9–104

3 0 2 64 16–181

3 1 0 43 9–181

3 1 1 75 17–199

3 1 2 120 30–360

3 1 3 160 30–380

3 2 0 93 18–360

3 2 1 150 30–380

3 2 2 210 30–400

3 2 3 290 90–990

3 3 0 240 40–990

3 3 1 460 90–1980

3 3 2 1100 200–4000

3 3 3 >1100

RESULTSANDINTERPRETATION

WhenverifyingthesuitabilityoftheMembraneFiltr ationmethodorthePl ateCountMethod,amean

countofanyofthetestorganismsnotdifferingbyafactorgreaterthan2fromthevalueofthecontrol

definedinInoculationa ndDilutio nintheabsenceofproductmustbeobtained.Whenverifyingthe

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suitabilityoftheMPNMethod,thecalculatedvaluefromtheinoculummustbewithin95%confidence

limitsoftheresultsobtainedwiththecontrol.

Iftheabovecriteriacannotbemetforoneofmoreoftheorganismstestedwithanyofthedescribed

methods,themethodandtestconditionsthatcomeclosesttothecriteriaareusedtotesttheproduct.

TESTINGOFP RODUCTS

AmountUsedfortheTest

Unlessotherwisedirected,use10gor10mLoftheproducttobeexaminedtakenwiththe

precautionsreferredtoabove.Forfluidsorsolidsinaerosolform,sample10containers.For

transdermalpatches,sample10patches.

Theamounttobetestedmaybereducedforactivesubstancesthatwillbeformulatedinthefollowing

conditions:theamountperdosageunit(e.g.,tablet,capsule,injection)islessthanorequalto1mg,or

theamountpergormL(forpreparationsnotpresentedindoseunits)islessthan1mg.Inthesecases,

theamountofsampletobetestedisnotlessthantheamountpresentin10dosageunitsor10gor10

mLoftheproduct.

Formaterialsusedasactivesubstanceswherethesamplequantityislimitedorbatchsizeis

extremelysmall(i.e.,lessthan1000mLor1000g),theamounttestedshallbe1%ofthebatchunless

alesseramountisprescribedorjustifiedandauthorized.

Forproductswherethetotalnumberofentitiesinabatchislessthan200(e.g.,samplesusedin

clinicaltrials),thesamplesizemaybereducedtotwounits,oroneunitifthesizeislessthan100.

Selectthesample(s)atrandomfromthebulkmaterialorfromtheavailablecontainersofthe

preparation.Toobtaintherequiredquantity,mixthecontentsofasufficientnumberofcontainersto

providethesample.

ExaminationoftheProduct

MEMBRANEFILTRATION

Useafiltrationapparatusdesignedtoallowthetransferofthefiltertothemedium.Preparethe

sampleusingamethodthathasbeenshowntobesuitableasdescribedinGrowthPromotionTestand

SuitabilityoftheCountingMethod,transfertheappropriateamounttoeachoftwomembranefilters,

andfilterimmediately.Washeachfilterfollowingtheprocedureshowntobesuitable.

ForthedeterminationofTAMC,transferoneofthemembranefilterstothesurfaceofSoybean–Casein

DigestAgar.ForthedeterminationofTYMC,transfertheothermembranetothesurfaceofSabouraud

DextroseAgar.IncubatetheplateofSoybean–CaseinDigestAgarat30 to35 for3to5daysandthe

plateofSabouraudDextroseAgarat20 to25 for5to7days.Calculatethenumberofcfupergorper

mLofproduct.

Whenexaminingtransdermalpatches,separatelyfilter10%ofthevolumeofthepreparation

describedforPrepa rationof theSamplethrougheachoftwosterilefiltermembranes.Transferone

membranetoSoybean–CaseinDigestAgarforTAMCandtheothermembranetoSabour audDex trose

AgarforTYMC.

PLATECOUNTMETHODS

PourPlateMethod—Preparethesampleusingamethodthathasbeenshowntobesuitableas

describedinGrowthPromotionTestandSuitabilityoftheCountingMethod.Prepareforeachmediumat

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leasttwoPetridishesforeachlevelofdilution.IncubatetheplatesofSoybean–CaseinDigestAgarat30

to35 for3to5daysandtheplatesofSabouraudDextroseAga rat20 to25 for5to7days.Select

theplatescorrespondingtoagivendilutionandshowingthehighestnumberofcolonieslessthan250

forTAMCand50forTYMC.Takethearithmeticmeanperculturemediumofthecounts,andcalculate

thenumberofcfupergorpermLofproduct.

Surface SpreadMethod—Preparethesampleusingamethodthathasbeenshowntobesuitableas

describedinGrowthPromotionTestandSuitabilityoftheCountingMethod.PrepareatleasttwoPetri

dishesforeachmediumandeachlevelofdilution.Forincubationandcalculationofthenumberofcfu,

proceedasdirectedforthePourPlateMethod.

MOSTPROBABLENUMBERMETHOD

Prepareanddilutethesampleusingamethodthathasbeenshowntobesuitableasdecribedin

GrowthP rom otionTestandSuitability oftheCountingMethod.Incubatealltubesfor3to5daysat30

to35 .Subcultureifnecessary,usingtheprocedureshowntobesuitable.Recordforeachlevelof

dilutionthenumberoftubesshowingmicrobialgrowth.Determinethemostprobablenumberof

microorganismspergormLoftheproducttobeexaminedfromTable3.

InterpretationoftheResults

Thetotalaerobicmicrobialcount(TAMC)isconsideredtobeequaltothenumberofcfufoundusing

Soybean–CaseinDigestAgar;ifcoloniesoffungiaredetectedonthismedium,theyarecountedaspart

ofTAMC.Thetotalcombinedyeastsandmoldscount(TYMC)isconsideredtobeequaltothenumberof

cfufoundusingSabouraudDextroseAgar;ifcoloniesofbacteriaaredetectedonthismedium,theyare

countedaspartofTYMC.WhentheTYMCisexpectedtoexceedtheacceptancecriterionduetothe

bacterialgrowth,SabouraudDextroseAga rcontainingantibioticsmaybeused.Ifthecountiscarried

outbytheMPNMethod,thecalculatedvalueisTAMC.

Whenanacceptancecriterionformicrobiologicalqualityisprescribed,itisinterpretedasfollows:

cfu:maximumacceptablecount=20;

cfu:maximumacceptablecount=200;

cfu:maximumacceptablecount=2000;

andsoforth.

TherecommendedsolutionsandmediaaredescribedinMicrobiologicalExaminationofNonsterile

Products:TestsforSpecifiedMicroorganisms 62 .

(EntireChapterandrevisionsmarkedforIRA—OfficialMay1,2009)