USP无菌检测法.docx
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USP无菌检测法.docx
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USP无菌检测法
MEDIA
Preparemediaforthetestsasdescribedbelow,ordehydratedformulations
maybeusedprovidedthat,whenreconstitutedasdirectedbythemanufacturer
ordistributor,theymeettherequirementsoftheGrowthPromotionTestof
Aerobes,Anaerobes,andFungi.Mediaaresterilizedusingavalidatedprocess.
Thefollowingculturemediahavebeenfoundtobesuitableforthetestfor
sterility.FluidThioglycollateMediumisprimarilyintendedfortheculture
ofanaerobicbacteria.However,itwillalsodetectaerobicbacteria.Soybean–
CaseinDigestMediumissuitableforthecultureofbothfungiandaerobic
bacteria.
FluidThioglycollateMedium
L-Cystine0.5g
SodiumChloride2.5g
Dextrose(C6H12O6·H2O)5.5/5.0g
Agar,granulated(moisturecontentnot
exceeding15%)0.75g
YeastExtract(water-soluble)5.0g
PancreaticDigestofCasein15.0g
SodiumThioglycollate0.5g
orThioglycolicAcid0.3mL
ResazurinSodiumSolution(1in1000),
freshlyprepared1.0mL
PurifiedWater1000mL
MixtheL-cystine,sodiumchloride,dextrose,yeastextract,and
pancreaticdigestofcaseinwiththepurifiedwater,andheatuntilsolution
iseffected.Dissolvethesodiumthioglycollateorthioglycolicacidinthe
solutionand,ifnecessary,add1Nsodiumhydroxidesothat,after
sterilization,thesolutionwillhaveapHof7.1±0.2.Iffiltrationis
necessary,heatthesolutionagainwithoutboiling,andfilterwhilehot
throughmoistenedfilterpaper.Addtheresazurinsodiumsolution,mix,and
placethemediuminsuitablevesselsthatprovidearatioofsurfacetodepth
ofmediumsuchthatnotmorethantheupperhalfofthemediumhasundergone
acolorchangeindicativeofoxygenuptakeattheendoftheincubation
period.Sterilizeusingavalidatedprocess.Ifthemediumisstored,storeat
atemperaturebetween2and25inasterile,airtightcontainer.Ifmorethan
theupperone-thirdofthemediumhasacquiredapinkcolor,themediummaybe
restoredoncebyheatingthecontainersinawater-bathorinfree-flowing
steamuntilthepinkcolordisappearsandbycoolingquickly,takingcareto
preventtheintroductionofnonsterileairintothecontainer.
FluidThioglycollateMediumistobeincubatedat32.5±2.5.
AlternativeThioglycollateMedium
PrepareamixturehavingthesamecompositionasthatoftheFluid
ThioglycollateMedium,butomittingtheagarandtheresazurinsodium
solution,sterilizeasdirectedabove,andallowtocoolpriortouse.ThepH
aftersterilizationis7.1±0.2.Incubateunderanaerobicconditionsforthe
durationoftheincubationperiod.
AlternativeFluidThioglycollateMediumistobeincubatedat32.5±2.5.
Soybean–CaseinDigestMedium(TSB)
PancreaticDigestofCasein17.0g
PapaicDigestofSoybeanMeal3.0g
SodiumChloride5.0g
DibasicPotassiumPhosphate2.5g
Dextrose(C6H12O6·H2O)2.5/2.3g
PurifiedWater1000mL
DissolvethesolidsinthePurifiedWater,heatingslightlytoeffecta
solution.Coolthesolutiontoroomtemperature,andadjustthepHwith1N
sodiumhydroxidesothat,aftersterilization,itwillhaveapHof7.3±
0.2.Filter,ifnecessarytoclarify,dispenseintosuitablecontainers,and
sterilizeusingavalidatedprocedure.Storeatatemperaturebetween2and25
inasterilewell-closedcontainer,unlessitisintendedforimmediateuse.
Soybean–CaseinDigestMediumistobeincubatedat22.5±2.5.
MediaforPenicillinsorCephalosporins
WheresterilitytestmediaaretobeusedintheDirectInoculationof
theCultureMediummethodunderTestforSterilityoftheProducttobe
Examined,modifythepreparationofFluidThioglycollateMediumandthe
Soybean–CaseinDigestMediumasfollows.Tothecontainersofeachmedium,
transferasepticallyaquantityofb-lactamasesufficienttoinactivatethe
amountofantibioticinthespecimenundertest.Determinethequantityofb-
lactamaserequiredtoinactivatetheantibioticbyusingab-lactamase
preparationthathasbeenassayedpreviouslyforitspenicillin-or
cephalosporin-inactivatingpower.[NOTE—Supplementedb-lactamasemediacan
alsobeusedinthemembranefiltrationtest.]
Alternatively(inanareacompletelyseparatefromthatusedforsterility
testing),confirmthatanappropriateamountofb-lactamaseisincorporated
intothemedium,followingeithermethodunderValidationTest,usingless
than100colony-formingunits(cfu)ofStaphylococcusaureus(seeTable1)as
thechallenge.Typicalmicrobialgrowthoftheinoculatedculturemustbe
observedasaconfirmationthattheb-lactamaseconcentrationisappropriate.
Table1.StrainsoftheTestMicroorganismsSuitableforUseintheGrowth
PromotionTestandtheValidationTest
Aerobicbacteria
Staphylococcusaureus1ATCC6538,CIP4.83,NCTC10788,NCIMB9518
BacillussubtilisATCC6633,CIP52.62,NCIMB8054
Pseudomonasaeruginosa2ATCC9027,NCIMB8626,CIP82.118
Anaerobicbacterium
Clostridiumsporogenes3ATCC19404,CIP79.3,NCTC532orATCC11437
Fungi
CandidaalbicansATCC10231,IP48.72,NCPF3179
AspergillusnigerATCC16404,IP1431.83,IMI149007
1、AnalternativetoStaphylococcusaureusisBacillussubtilis(ATCC
6633).
2、AnalternativemicroorganismisMicrococcusluteus(Kocuriarhizophila),
ATCC9341.
3、AnalternativetoClostridiumsporogenes,whenanonspore-forming
microorganismisdesired,isBacetroidesvulgatus(ATCC8482).
[NOTE—Seed-lotculturemaintenancetechniques(seed-lotsystems)areusedso
thattheviablemicroorganismsusedforinoculationarenotmorethanfive
passagesremovedfromtheoriginalmasterseedlot.]
SuitabilityTests
Themediausedcomplywiththefollowingtests,carriedoutbefore,orin
parallel,withthetestontheproducttobeexamined.
STERILITY
Confirmthesterilityofeachsterilizedbatchofmediumbyincubatinga
portionofthemediaatthespecifiedincubationtemperaturefor14days.No
growthofmicroorganismsoccurs.
GROWTHPROMOTIONTESTOFAEROBES,ANAEROBES,ANDFUNGI
Testeachlotofofready-preparedmediumandeachbatchofmediumprepared
eitherfromdehydratedmediumorfromingredients1.Suitablestrainsof
microorganismsareindicatedinTable1.
InoculateportionsofFluidThioglycollateMediumwithasmallnumber(not
morethan100cfu)ofthefollowingmicroorganisms,usingaseparateportion
ofmediumforeachofthefollowingspeciesofmicroorganism:
Clostridium
sporogenes,Pseudomonasaeruginosa,andStaphylococcusaureus.Inoculate
portionsofAlternativeFluidThioglycollateMediumwithasmallnumber(not
morethan100cfu)ofClostridiumsporogenes.InoculateportionsofSoybean–
CaseinDigestMediumwithasmallnumber(notmorethan100cfu)ofthe
followingmicroorganisms,usingaseparateportionofmediumforeachofthe
followingspeciesofmicroorganism:
Aspergillusniger,Bacillussubtilis,and
Candidaalbicans.Incubatefornotmorethan3daysinthecaseofbacteria
andnotmorethan5daysinthecaseoffungi.
Themediaaresuitableifaclearlyvisiblegrowthofthemicroorganisms
occurs.
STORAGE
Ifpreparedmediaarestoredinunsealedcontainers,theycanbeusedfor1
month,providedthattheyaretestedforgrowthpromotionwithin2weeksof
thetimeofuseandthatcolorindicatorrequirementsaremet.Ifstoredin
tightcontainers,themediacanbeusedfor1year,providedthattheyare
testedforgrowthpromotionwithin3monthsofthetimeofuseandthatthe
colorindicatorrequirementsaremet.
DILUTINGANDRINSINGFLUIDSFORMEMBRANEFILTRATION
FluidA
PREPARATION
Dissolve1gofpepticdigestofanimaltissueinwatertomake1L,filter
orcentrifugetoclarify,ifnecessary,andadjusttoapHof7.1±0.2.
Dispenseintocontainers,andsterilizeusingavalidatedprocess.
PREPARATIONFORPENICILLINSORCEPHALOSPORINS
AsepticallyaddtotheabovePreparation,ifnecessary,aquantityof
sterileb-lactamasesufficienttoinactivateanyresidualantibioticactivity
onthemembranesafterthesolutionofthetestspecimenhasbeenfiltered
(seeMediaforPenicillinsorCephalosporins).
FluidD
ToeachLofFluidAadd1mLofpolysorbate80,adjusttoapHof7.1±
0.2,dispenseintocontainers,andsterilizeusingavalidatedprocess.Use
thisfluidforarticlescontaininglecithinoroil,orfordeviceslabeledas
“sterilepathway.”
FluidK
Dissolve5.0gofpepticdigestofanimaltissue,3.0gofbeefextract,
and10.0gofpolysorbate80inwatertomake1L.AdjustthepHtoobtain,
aftersterilization,apHof6.9±0.2.Dispenseintocontainers,and
sterilizeusingavalidatedprocess.
VALIDATIONTEST
CarryoutatestasdescribedbelowunderTestforSterilityoftheProduct
tobeExaminedusingexactlythesamemethods,exceptforthefollowing
modifications.
MembraneFiltration
Aftertransferringthecontentofthecontainerorcontainerstobetested
(notmorethan100cfu)tothefinalportionofsterilediluentusedtorinse
thefilter.
DirectInoculation
Aftertransferringthecontentsofthecontainerorcontainerstobe
tested(forcatgutandothersurgicalsuturesforveterinaryuse:
strands)to
theculturemedium,addaninoculumofasmallnumberofviablemicroorganisms
(notmorethan100cfu)tothemedium.
Inbothcasesusethesamemicroorganismsasthosedescribedaboveunder
GrowthPromotionTestofAerobes,Anaerobes,andFungi.Performagrowth
promotiontestasapositivecontrol.Incubateallthecontainerscontaining
mediumfornotmorethan5days.
Ifclearly
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