USP32NF2762.docx
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USP32NF2762.docx
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USP32NF2762
62
MICROBIOLOGICALEXAMINATIONOFNONSTERILEPRODUCTS:
TESTSFORSPECIFIEDMICROORGANISMS
INTRODUCTION
Thetestsdescribedhereafterwillallowdeterminationoftheabsenceof,orlimitedoccurrenceof,specifiedmicroorganismsthatmaybedetectedundertheconditionsdescribed.
Thetestsaredesignedprimarilytodeterminewhetherasubstanceorpreparationcomplieswithanestablishedspecificationformicrobiologicalquality.Whenusedforsuchpurposes,followtheinstructionsgivenbelow,includingthenumberofsamplestobetaken,andinterprettheresultsasstatedbelow.
Alternativemicrobiologicalprocedures,includingautomatedmethods,maybeused,providedthattheirequivalencetothePharmacopeialmethodhasbeendemonstrated.
GENERALPROCEDURES
Thepreparationofsamplesiscarriedoutasdescribedin.
Iftheproducttobeexaminedhasantimicrobialactivity,thisisinsofaraspossibleremovedorneutralizedasdescribedin.
Ifsurface-activesubstancesareusedforsamplepreparation,theirabsenceoftoxicityformicroorganismsandtheircompatibilitywithanyinactivatorsusedmustbedemonstratedasdescribedin.
GROWTH-PROMOTINGANDINHIBITORYPROPERTIESOFTHEMEDIAANDSUITABILITYOFTHETEST
Theabilityofthetesttodetectmicroorganismsinthepresenceoftheproducttobetestedmustbeestablished.Suitabilitymustbeconfirmedifachangeintestingperformanceorachangeintheproductthatmayaffecttheoutcomeofthetestisintroduced.
PreparationofTestStrains
Usestandardizedstablesuspensionsofteststrainsasstatedbelow.Seed-lotculturemaintenancetechniques(seed-lotsystems)areusedsothattheviablemicroorganismsusedforinoculationarenotmorethanfivepassagesremovedfromtheoriginalmasterseed-lot.
aerobicmicroorganisms
GroweachofthebacterialteststrainsseparatelyincontainerscontainingSoybean–CaseinDigestBrothoronSoybean–CaseinDigestAgarat30
to35
for18to24hours.GrowtheteststrainforCandidaalbicansseparatelyonSabouraudDextroseAgarorinSabouraudDextroseBrothat20
to25
for2to3days.
Staphylococcusaureus
suchasATCC6538,NCIMB9518,CIP,orNBRC13276
Pseudomonasaeruginosa
suchasATCC9027,NCIMB8626,CIP,orNBRC13275
Escherichiacoli
suchasATCC8739,NCIMB8545,CIP,orNBRC3972
Salmonellaentericassp.entericaserotypetyphimuriumor,asanalternative,
suchasATCC14028
Salmonellaentericassp.entericaserotypeabony
suchasNBRC100797,NCTC6017,orCIP
Candidaalbicans
suchasATCC10231,NCPF3179,IP,orNBRC1594
UseBufferedSodiumChloride–PeptoneSolutionpHorPhosphateBufferSolutionpHtomaketestsuspensions.Usethesuspensionswithin2hoursorwithin24hoursifstoredat2
to8
.
clostridia
UseClostridiumsporogenessuchasATCC11437(NBRC14293,NCIMB12343,CIP100651)orATCC19404(NCTC532orCIP.GrowtheclostridialteststrainunderanaerobicconditionsinReinforcedMediumforClostridiaat30
to35
for24to48hours.AsanalternativetopreparingandthendilutingdownafreshsuspensionofvegetativecellsofCl.sporogenes,astablesporesuspensionisusedfortestinoculation.Thestablesporesuspensionmaybemaintainedat2
to8
foravalidatedperiod.
NegativeControl
Toverifytestingconditions,anegativecontrolisperformedusingthechosendiluentinplaceofthetestpreparation.Theremustbenogrowthofmicroorganisms.
GrowthPromotionandInhibitoryPropertiesoftheMedia
Testeachbatchofready-preparedmediumandeachbatchofmediumpreparedeitherfromdehydratedmediumorfromingredients.Verifysuitablepropertiesofrelevantmediaasdescribedin.
Table1.GrowthPromoting,Inhibitory,andIndicativePropertiesofMedia
Test/Medium
Property
TestStrains
Testforbile-tolerantGram-negativebacteria
EnterobacteriaEnrichmentBrothMossel
Growthpromoting
E.coli
P.aeruginosa
Inhibitory
S.aureus
VioletRedBileGlucoseAgar
Growthpromoting+Indicative
E.coli
P.aeruginosa
TestforEscherichiacoli
MacConkeyBroth
Growthpromoting
E.coli
Inhibitory
S.aureus
MacConkeyAgar
Growthpromoting+Indicative
E.coli
TestforSalmonella
RappaportVassiliadisSalmonellaEnrichmentBroth
Growthpromoting
Salmonellaentericassp.entericaserotypetyphimuriumor
Salmonellaentericaserotypeabony
Inhibitory
S.aureus
XyloseLysineDeoxycholateAgar
Growthpromoting+Indicative
Salmonellaentericassp.entericaserotypetyphimuriumor
Salmonellaentericaserotypeabony
Indicative
E.coli
TestforPseudomonasaeruginosa
CetrimideAgar
Growthpromoting
P.aeruginosa
Inhibitory
E.coli
TestforStaphylococcusaureus
MannitolSaltAgar
Growthpromoting+Indicative
S.aureus
Inhibitory
E.coli
TestforClostridia
ReinforcedMediumforClostridia
Growthpromoting
Cl.sporogenes
ColumbiaAgar
Growthpromoting
Cl.sporogenes
TestforCandidaalbicans
SabouraudDextroseBroth
Growthpromoting
C.albicans
SabouraudDextroseAgar
Growthpromoting+Indicative
C.albicans
TestforGrowth-PromotingProperties,LiquidMedia—Inoculateaportionoftheappropriatemediumwithasmallnumber(notmorethan100cfu)oftheappropriatemicroorganism.Incubateatthespecifiedtemperaturefornotmorethantheshortestperiodoftimespecifiedinthetest.Clearlyvisiblegrowthofthemicroorganismcomparabletothatpreviouslyobtainedwithapreviouslytestedandapprovedbatchofmediumoccurs.
TestforGrowth-PromotingProperties,SolidMedia—PerformSurface-SpreadMethod(seePlate-CountMethodsunder),inoculatingeachplatewithasmallnumber(notmorethan100cfu)oftheappropriatemicroorganism.Incubateatthespecifiedtemperaturefornotmorethantheshortestperiodoftimespecifiedinthetest.Growthofthemicroorganismcomparabletothatpreviouslyobtainedwithapreviouslytestedandapprovedbatchofmediumoccurs.
TestforInhibitoryProperties,LiquidorSolidMedia—Inoculatetheappropriatemediumwithatleast100cfuoftheappropriatemicroorganism.Incubateatthespecifiedtemperaturefornotlessthanthelongestperiodoftimespecifiedinthetest.Nogrowthofthetestmicroorganismoccurs.
TestforIndicativeProperties—PerformSurface-SpreadMethod(seePlate-CountMethodsunder),inoculatingeachplatewithasmallnumber(notmorethan100cfu)oftheappropriatemicroorganism.Incubateatthespecifiedtemperatureforaperiodoftimewithintherangespecifiedinthetest.Coloniesarecomparableinappearanceandindicationreactionstothosepreviouslyobtainedwithapreviouslytestedandapprovedbatchofmedium.
SuitabilityoftheTestMethod
ForeachnewproducttobetestedperformsamplepreparationasdescribedintherelevantparagraphunderTestingofProducts.Atthetimeofmixing,addeachteststrainintheprescribedgrowthmedium.Inoculatetheteststrainsindividually.Useanumberofmicroorganismsequivalenttonotmorethan100cfuintheinoculatedtestpreparation.
PerformthetestasdescribedintherelevantparagraphunderTestingofProductsusingtheshortestincubationperiodprescribed.
ThespecifiedmicroorganismsmustbedetectedwiththeindicationreactionsasdescribedunderTestingofProducts.
Anyantimicrobialactivityoftheproductnecessitatesamodificationofthetestprocedure(seeNeutralization/RemovalofAntimicrobialActivityunder).
Foragivenproduct,iftheantimicrobialactivitywithrespecttoamicroorganismforwhichtestingisprescribedcannotbeneutralized,thenitistobeassumedthattheinhibitedmicroorganismwillnotbepresentintheproduct.
TESTINGOFPRODUCTS
Bile-TolerantGram-NegativeBacteria
SamplePreparationandPre-Incubation—Prepareasampleusinga1in10dilutionofnotlessthan1goftheproducttobeexaminedasdescribedin,butusingSoybean–CaseinDigestBrothasthechosendiluent,mix,andincubateat20
to25
foratimesufficienttoresuscitatethebacteriabutnotsufficienttoencouragemultiplicationoftheorganisms(usually2hoursbutnotmorethan5hours).
TestforAbsence—Unlessotherwiseprescribed,usethevolumecorrespondingto1goftheproduct,aspreparedinSamplePreparationandPre-Incubation,toinoculateEnterobacteriaEnrichmentBrothMossel.Incubateat30
to35
for24to48hours.SubcultureonplatesofVioletRedBileGlucoseAgar.Incubateat30
to35
for18to24hours.
Theproductcomplieswiththetestifthereisnogrowthofcolonies.
QuantitativeTest—
SelectionandSubculture—InoculatesuitablequantitiesofEnterobacteriaEnrichmentBrothMosselwiththepreparationasdirectedunderSamplePreparationandPre-Incubationand/ordilutionsofitcontainingrespectively0.1g,0.01g,and0.001g(ormL,mL,andmL)oftheproducttobeexamined.Incubateat30
to35
for24to48hours.SubcultureeachoftheculturesonaplateofVioletRedBileGlucoseAgar.Incubateat30
to35
for18to24hours.
Interpretation—Growthofcoloniesconstitutesapositiveresult.Notethesmallestquantityoftheproductthatgivesapositiveresultandthelargestquantitythatgivesanegativeresult.Determinefromtheprobablenumberofbacteria.
Table2.InterpretationofResults
ResultsforEachQuantity
ofProduct
ProbableNumber
ofBacteria
pergormLofProduct
0.1gor
mL
0.01gor
mL
0.001gor
mL
+
+
+
morethan103
+
+
–
lessthan103andmorethan102
+
–
–
lessthan102andmorethan10
–
–
–
lessthan10
Escherichiacoli
SamplePreparationandPre-Incubation—Prepareasampleusinga1in10dilutionofnotlessthan1goftheproducttobeexaminedasdescribedinanduse10mLort
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