微生物计数检查法USP61中英对照版

非无菌产品微生物学检查：微生物计数检查法USP61中英对照版<61>MICROBIOLOGICALEXAMINATIONOFNONSTERILEPRODUCTS:MICROBIALENUMENRATIONTESTS非无菌产品微生物学检查：微生物计数检查法INTRODUCTION导言Thetestsdescribedhereafterwillallowquantitativeenumerationofmesophilicbacteriaandfungithatmaygrowunderaerobicconditions.下列所描述的这些检测将使得对在有氧的条件下生长的嗜温性细菌和真菌进行定量计数成为可能。Thetestsaredesignedprimarilytodeterminewhetherasubstanceorpreparationcomplieswithanestablishedspecificationformicrobiologicalquality.Whenusedforsuchpurposes,followtheinstructionsgivenbelow,includingthenumberofsamplestobetaken,andinterprettheresultsasstatedbelow.这些检测重要设计用于测定一种物质或制备品与否符合已确立的微生物质量原则。当用于这类目的时，需遵照下列所给的阐明，涉及待取样品的数量，并且按照下面所述解释成果。Themethodsarenotapplicabletoproductscontainingviablemicroorganismsasactiveingredients.这些办法不合用于以活菌作为活性成分的产品。Alternativemicrobiologicalprocedures,includingautomatedmethods,maybeused,providedthattheirequivalencetothePharmacopeialmethodhasbeendemonstrated.能够使用替代的微生物规程，涉及自动化办法，只要已经证明它们与药典办法具同等作用。GENERALPROCEDURES通用规程Carryoutthedeterminationunderconditionsdesignedtoavoid12microbialcontaminationoftheproducttobeexamined.Theprecautionstakentoavoidcontaminationmustbesuchthattheydonotaffectanymicroorganismsthataretoberevealedinthetest.在通过设计可避免外来微生物污染供试产品的条件下，进行此项测定。用于避免污染的这些防止方法是必须做到，它们不会影响任何试图在此项检查中揭示的微生物。Iftheproducttobeexaminedhasantimicrobialactivity,thisis,insofaraspossible,removedorneutralized.Ifinactivatorsareusedforthispurpose,theirefficacyandtheirabsenceoftoxicityformicroorganismsmustbedemonstrated.如果供试产品含有抗菌活性，则此活性需在尽量的范畴内去除或中和。如果将灭活剂用于这个目的，则必须证明它们的功效和对微生物不具毒性。Ifsurface-activesubstancesareusedforsamplepreparation,theirabsenceoftoxicityformicroorganismsandtheircompatibilitywithanyinactivatorsusedmustbedemonstrated.如果表面活性物质用于样品制备，则必须证明它们对微生物不具毒性以及与所使用的任何灭活剂的兼容性。ENUMERATIONMETHODS计数法UsetheMembraneFiltrationmethodoroneofthePlate-CountMethods,asdirected.TheMost-Probable-Number(MPN)Methodisgenerallytheleastaccuratemethodformicrobialcounts;however,forcertainproductgroupswithverylowbioburden,itmaybethemostappropriatemethod.按规定，使用膜过滤法或多个平板计数法中的一种。液体稀释法（MPN）一般对微生物计数而言是最不精确的办法；然而，对含有非常低生物载荷的特定产品类型，它可能是最适宜的办法。Thechoiceofamethodisbasedonfactorssuchasthenatureoftheproductandtherequiredlimitofmicroorganisms.Themethodchosenmustallowtestingofasufficientsamplesizetojudgecompliancewiththespecification.Thesuitabilityofthechosenmethodmustbeestablished.办法的选择基于某些因素，例如产品的性质和微生物的规定程度。所选的办法必须能够对充足的样本量进行检测，以判断与质量原则的符合性。所选办法的合用性必须被建立。Table1.PreparationandUseofTestMicroorganisms表1.供试微生物的制备和使用GrowthPromotion生长增进SuitabilityofCountingMethodinthePresenceofProduct在产品存在的状况下计数办法的合用性Microorganism微生物PreparationofTestStrain供试菌株的制备TotalAerobicMicrobialCount总好氧微生物计数TotalYeastsandMoldsCount总酵母菌和霉菌计数TotalAerobicMicrobialCount总好氧微生物计数TotalYeastsandMoldsCount总酵母菌和霉菌计数StaphylococcusaureussuchasATCC6538,NCIMB9518,CIP4.83,orNBRC13276金黄色葡萄球菌如：ATCC6538,NCIMB9518,CIP4.83,或NBRC13276Soybean-CaseinDigestAgarorSoybean-CaseinDigestBroth300-35018-24hours大豆酪蛋白消化物琼脂培养基或大豆酪蛋白消化物肉汤培养基300-35018-24小时Soybean-CaseinDigestAgarandSoybean-CaseinDigestBroth≤100cfu300-350≤3days大豆酪蛋白消化物培养基和大豆酪蛋白消化物肉汤培养基≤100cfu300-350≤3天Soybean-CaseinDigestAgar/MPNSoybean-CaseinDigestBroth≤100cfu300-350≤3days大豆酪蛋白消化物培养基/MPN（最大几率数）大豆酪蛋白消化物肉汤培养基≤100cfu300-350≤3天PseudomonasaeruginosasuchasATCC9027,NCIMB8626,CIP82.118,orNBRC13275绿脓杆菌如ATCC9027,NCIMB8626,CIP82.118,或NBRC13275Soybean-CaseinDigestAgarorSoybean-CaseinDigestBroth300-35018-24hours大豆酪蛋白消化物琼脂培养基或大豆酪蛋白消化物肉汤培养基300-35018-24小时Soybean-CaseinDigestAgarandSoybean-CaseinDigestBroth≤100cfu300-350≤3days大豆酪蛋白消化物琼脂培养基和大豆酪蛋白消化物肉汤培养基≤100cfu300-350≤3天Soybean-CaseinDigestAgar/MPNSoybean-CaseinDigestBroth≤100cfu300-350≤3days大豆酪蛋白消化物琼脂培养基/MPN（最大几率数）大豆酪蛋白消化物肉汤培养基≤100cfu300-350≤3天BacillussubtilissuchasATCC6633,NCIMB8054,CIP52.62,orNBRC3134枯草芽孢杆菌如ATCC6633,NCIMB8054,CIP52.62,或NBRC3134Soybean-CaseinDigestAgarorSoybean-CaseinDigestBroth300-35018-24hours大豆酪蛋白消化物琼脂培养基或大豆酪蛋白消化物肉汤培养基300-35018-24小时Soybean-CaseinDigestAgarandSoybean-CaseinDigestBroth≤100cfu300-350≤3days大豆酪蛋白消化物琼脂培养基和大豆酪蛋白消化物肉汤培养基≤100cfu300-350≤3天Soybean-CaseinDigestAgar/MPNSoybean-CaseinDigestBroth≤100cfu300-350≤3days大豆酪蛋白消化物琼脂培养基/MPN（最大几率数）大豆酪蛋白消化物肉汤培养基≤100cfu300-350≤3天CandidaalbicanssuchasATCC10231,NCPF3179,IP48.72,orNBRC1594白色念珠菌如ATCC10231,NCPF3179,IP48.72,或NBRC1594SabouraudDextroseAgarorSabouraudDextroseBroth200-2502-3daysSabouraud（沙氏）葡萄糖琼脂培养基或Sabouraud（沙氏）葡萄糖肉汤培养基Soybean-CaseinDigestAgar≤100cfu300-350≤5days大豆酪蛋白消化物琼脂培养基≤100cfu300-350≤5天SabouraudDextroseAgar≤100cfu200-250≤5daysSabouraud（沙氏）葡萄糖琼脂培养基≤100cfu200-250≤5天Soybean-CaseinDigestAgar≤100cfu300-350≤5daysMPN:notapplicable

大豆酪蛋白消化物琼脂培养基≤100cfu300-350≤5天MPN(最大几率数)：不合用SabouraudDextroseAgar≤100cfu200-250≤5daysSabouraud（沙氏）葡萄糖琼脂培养基≤100cfu200-250≤5天AspergillusnigersuchasATCC16404,IMI149007,IP1431.83,orNBRC9455黑曲霉如ATCC16404,IMI149007,IP1431.83,或NBRC9455SabouraudDextroseAgarorPotato-DextroseAgar200-2505-7days,oruntilgoodsporulationisachievedSabouraud（沙氏）葡萄糖琼脂培养基或马铃薯葡萄糖琼脂培养基

200-250

5-7天,或直到实现良好的产孢Soybean-CaseinDigestAgar≤100cfu300-350≤5days大豆酪蛋白消化物琼脂培养基≤100cfu300-350≤5天SabouraudDextroseAgar≤100cfu200-250≤5daysSabouraud（沙氏）葡萄糖琼脂培养基≤100cfu200-250≤5天Soybean-CaseinDigestAgar≤100cfu300-350≤5daysMPN:notapplicable大豆酪蛋白消化物琼脂培养基≤100cfu300-350≤5天MPN(最大几率数)：不合用SabouraudDextroseAgar≤100cfu200-250≤5daysSabouraud（沙氏）葡萄糖琼脂培养基≤100cfu200-250≤5天GROWTHPROMOTIONTESTANDSUITABILITYOFTHECOUNTINGMETHOD生长增进实验和计数办法的合用性GeneralConsiderations通用考虑因素Theabilityofthetesttodetectmicroorganismsinthepresenceofproducttobetestedmustbeestablished.在供试产品存在的状况下，必须确立检测微生物的实验能力。Suitabilitymustbeconfirmedifachangeintestingperformanceorachangeintheproductthatmayaffecttheoutcomeofthetest,isintroduced.如果引入了可能影响实验成果的在测试性能或产品方面的变更，则必须确认其合用性。PreparationofTestStrains供试菌株的制备Usestandardizedstablesuspensionsofteststrainsorprepareasstatedbelow.Seed-lotculturemaintenancetechniques(seed-lotsystems)areusedsothattheviablemicroorganismsusedforinoculationarenotmorethan5passagesremovedfromtheoriginalmasterseed-lot.GroweachofbacterialandfungalteststrainsseparatelyasdescribedinTable1.使用供试菌株的原则化稳定悬浮液或按下面所述制备。使用菌种保藏技术（种子批系统），方便用于接种的可萌发微生物从最初的主种子批开始传代不超出5次。按照在表1中的描述，分别培养每个细菌和霉菌供试菌株。UseBufferedSodiumChloride-PeptoneSolutionpH7.0orPhosphateBufferSolutionpH7.2tomaketestsuspensions;tosuspendA.nigerspores,0.05%ofpolysorbate80maybeaddedtothebuffer.Usethesuspensionswithin2hours,orwithin24hoursifstoredbetween2oand8o.AsanalternativetopreparingandthendilutingafreshsuspensionofvegetativecellsofA.nigerorB.subtilis,astablesporesuspensionispreparedandthenanappropriatevolumeofthesporesuspensionisusedfortestinoculation.Thestablesporesuspensionmaybemaintainedat2oto8oforavalidatedperiodoftime.使用pH7.0的缓冲氯化钠-蛋白胨溶液，或pH7.2的磷酸盐缓冲液制作供试悬浮液；为使黑曲霉孢子悬浮，能够将0.05%的聚山梨醇酯80加入该缓冲液中。这些悬浮液需在2个小时之内使用，或如果寄存在2o至8o的条件下，则可在24小时之内使用。作为制备然后稀释黑曲霉或枯草杆菌的新鲜体细胞悬浮液的替代办法，可制备稳定的孢子悬浮液，然后将适量的孢子悬浮液用于实验接种。此稳定的孢子悬浮液能够在2o至8o之间于一段通过验证的时间内保存。NegativeControl阴性对照Toverifytestingconditions,anegativecontrolisperformedusingthechosendiluentinplaceofthetestpreparation.Theremustbenogrowthofmicroorganisms.为了确认实验的条件，在制备供试品的场合使用所选的稀释液替代供试品，作阴性对照。其必须没有微生物的增加。GrowthPromotionoftheMedia培养基的生长增进Testeachbatchofready-preparedmediumandeachbatchofmediumpreparedeitherfromdehydratedmediumorfromtheingredientsdescribed.对每一批已经制备好的培养基和每一批从脱水培养基或根据描述的组分制备出来的培养基，进行测试。Inoculateportions/platesofSoybean-CaseinDigestBrothandSoybean-CaseinDigestAgarwithasmallnumber(notmorethan100cfu)ofthemicroorganismsindicatedinTable1,usingaseparateportion/plateofmediumforeach.InoculateplatesofSabouraudDextroseAgarwithasmallnumber(notmorethan100cfu)ofthemicroorganismsindicatedinTable1,usingaseparateplateofmediumforeach.IncubateaccordingtotheconditionsdescribedinTable1.在部分/整个平皿内的大豆酪蛋白消化物肉汤培养基和大豆酪蛋白消化物琼脂培养基中接种少量(不超出100cfu)表1中指定的微生物，每种微生物均使用单独的部分/整个平皿的培养基。在平皿内的Sabouraud（沙氏）葡萄粮琼脂培养基中接种少量(不超出100cfu)表1中指定的微生物，每种均使用一种单独平皿的培养基。按照表１中描述的条件进行培养。Forsolidmedia,growthobtainedmustnotdifferbyafactorgreaterthan2fromthecalculatedvalueforastandardizedinoculum.Forafreshlypreparedinoculum,growthofthemicroorganismscomparabletothatpreviouslyobtainedwithapreviouslytestedandapprovedbatchofmediumoccurs.Liquidmediaaresuitableifclearlyvisiblegrowthofthemicroorganismscomparabletothatpreviouslyobtainedwithapreviouslytestedandapprovedbatchofmediumoccurs.对于固体培养基，所获得的生长与原则化接种体的计算值之间的差别因素决不能不不大于2。对于刚刚制备好的接种体，发生的微生物生长与用以前检查并同意过的培养基批次所得到的微生物生长相称。如果出现了与以前从上一种通过测试并同意的培养基批次获得的微生物生长相称的、清晰可见的、微生物生长，则液体培养基合用。SuitabilityoftheCountingMethodinthePresenceofProduct在存在产品的状况下计数法的合用性PREPARATIONOFTHESAMPLE样品的制备Themethodforsamplepreparationdependsonthephysicalcharacteristicsoftheproducttobetested.Ifnoneoftheproceduresdescribedbelowcanbedemonstratedtobesatisfactory,asuitablealternativeproceduremustbedeveloped.样品制备办法取决于供试品的物理特性。如果下列描述的规程不能够被令人满意地证明，则必须开发一种合用的替代规程。Water-SolubleProducts—Dissolveordilute(usuallya1in10dilutionisprepared)theproducttobeexaminedinBufferedSodiumChloride-PeptoneSolutionpH7.0,PhosphateBufferSolutionpH7.2,orSoybean-CaseinDigestBroth.Ifnecessary,adjusttoapHof6to8.Furtherdilutions,wherenecessary,arepreparedwiththesamediluent.水溶性产品——溶解或稀释（一般制备1:10的稀释液）供试品，于pH值为7.0的缓冲氯化钠-蛋白胨溶液中，pH值为7.2的磷酸盐缓冲液，或大豆酪蛋白消化物肉汤培养基。如有必要，将pH值调节为6至8。在需要时，用相似的稀释剂进一步地稀释。NonfattyProductsInsolubleinWater—Suspendtheproducttobeexamined(usuallya1in10dilutionisprepared)inBufferedSodiumChloride—PeptoneSolutionpH7.0,PhosphateBufferSolutionpH7.2,orSoybean—CaseinDigestBroth.Asurface—activeagentsuchas1gperLofpolysorbate80maybeaddedtoassistthesuspensionofpoorlywettablesubstances.Ifnecessary,adjusttoapHof6to8.Furtherdilutions,wherenecessary,arepreparedwiththesamediluent.不溶于水的非脂肪性供试品——将检测的供试品（一般制备1:10的稀释液）悬浮于pH值为7.0的缓冲氯化钠-蛋白胨溶液，pH值为7.2的磷酸盐缓冲液，或者大豆酪蛋白消化物肉汤培养基中。能够加入某个表面活性剂，例如1克每升的聚山梨酯80，以协助难以与水混合的物质悬浮。如有必要，调节pH值为6至8。在需要时，用相似的稀释剂进一步的稀释。FattyProducts—Dissolveinisopropylmyristatesterilizedbyfiltration,ormixtheproducttobeexaminedwiththeminimumnecessaryquantityofsterilepolysorbate80oranothernoninhibitorysterilesurface-activereagentheated,ifnecessary,tonotmorethan40oor,inexceptionalcases,tonotmorethan45o.Mixcarefullyandifnecessarymaintainthetemperatureinawaterbath.Addasufficientquantityoftheprewarmedchosendiluenttomakea1in10dilutionoftheoriginalproduct.Mixcarefully,whilemaintainingthetemperaturefortheshortesttimenecessaryfortheformationofanemulsion.Furtherserial10-folddilutionsmaybepreparedusingthechosendiluentcontainingasuitableconcentrationofsterilepolysorbate80oranothernoninhibitorysterilesurface-activereagent.脂肪性供试品——溶解于以过滤除菌的豆蔻酸异丙酯，或者将供试品与所需最少量的、通过加热的无菌聚山梨酯80或另一种非抑菌性的无菌表面活性剂进行混合，如有必要，可加热至不超出40o或者，在特殊状况下，至不超出45o。认真混匀，如有必要，在水浴锅里中维持该温度。加入充足数量、通过预热的所选择稀释剂，制成原产品的1:10稀释液。认真混匀，同时维持该温度至形成乳化剂所必需的最短的时间。能够用所选择的含有适宜浓度的无菌聚山梨酯80或者另一种非抑菌性的无菌表面活性剂的稀释液，来制备后续的系列10倍稀释液。FluidsorSolidsinAerosolForm—Asepticallytransfertheproductintoamembranefilterapparatusorasterilecontainerforfurthersampling.Useeitherthetotalcontentsoradefinednumberofmetereddosesfromeachofthecontainerstested.气溶胶与液溶胶——以无菌操作将供试品转移至一种过滤膜设备或一种无菌容器里，以进一步取样。从每个被检测的容器中，使用全部内容物或剂量的规定倍数。

TransdermalPatches—Removetheprotectivecoversheets(“releaseliners”)ofthetransdermalpatchesandplacethem,adhesivesideupwards,onsterileglassorplastictrays.Covertheadhesivesurfacewithasuitablesterileporousmaterial(e.g.,sterilegauze)topreventthepatchesfromstickingtogether,andtransferthepatchestoasuitablevolumeofthechosendiluentcontaininginactivatorssuchaspolysorbate80and/orlecithin.Shakethepreparationvigorouslyforatleast30minutes.贴剂——去除贴剂的防护面（“释放衬板”）并将它们放置在无菌玻璃或塑料托盘上，胶贴剂的一面对上。用适宜的无菌多孔材料（例如：无菌纱布）盖住胶贴面，以避免贴剂粘在一起，并将贴剂转移到所选的含有灭活剂（例如聚山梨醇酯80和/或卵磷脂）的适量稀释剂中。用力摇动供试品最少30分钟。INOCULATIONANDDILUTION接种和稀释Addtothesamplepreparedasdirectedaboveandtoacontrol(withnotestmaterialincluded)asufficientvolumeofthemicrobialsuspensiontoobtainaninoculumofnotmorethan100cfu.Thevolumeofthesuspensionoftheinoculumshouldnotexceed1%ofthevolumeofdilutedproduct.向按上述规定制备的样品中以及向对照制备品（其中无供试物质）中，加入充足体积的微生物悬浮液，以获得一种不多于100cfu的接种体。接种的菌体悬浮液体积应当不超出被稀释产品体积的1%。Todemonstrateacceptablemicrobialrecoveryfromtheproduct,thelowestpossibledilutionfactorofthepreparedsamplemustbeusedforthetest.Wherethisisnotpossibleduetoantimicrobialactivityorpoorsolubility,furtherappropriateprotocolsmustbedeveloped.Ifinhibitionofgrowthbythesamplecannototherwisebeavoided,thealiquotofthemicrobialsuspensionmaybeaddedafterneutralization,dilution,orfiltration.为证明供试品有可接受的微生物回收率，必须使用已制备样品的最低可能稀释倍数来进行检测。当由于抗菌活性或低溶解度而无法做到这一点时，必须进一步开发更适合的规程。如果样品对生长的克制不能以其它方式避免，能够在中和、稀释、或过滤之后，加入等量的微生物悬浮液。NEUTRALIZATION/REMOVALOFANTIMICROBIALACTIVITY抗菌活性的中和/去除ThenumberofmicroorganismsrecoveredfromthepreparedsampledilutedasdescribedinInoculationandDilutionandincubatedfollowingtheproceduredescribedinRecoveryofMicroorganismsinthePresenceofProduct,iscomparedtothenumberofmicroorganismsrecoveredfromthecontrolpreparation.用制备好的样品按接种和稀释项下的描述稀释，并按在产品存在的状况下微生物的回收率项下描述的规程培养，将从中回收的微生物的数量与从对照制备品中回收的微生物的数量进行比较。Ifgrowthisinhibited(reductionbyafactorgreaterthan2),thenmodifytheprocedurefortheparticularenumerationtesttoensurethevalidityoftheresults.Modificationoftheproceduremayinclude,forexample,如果生长被克制（以不不大于2的因素减少），那么修改特定的计数测试规程，以确保成果的有效性。规程的修改能够涉及，例如：(1)

Anincreaseinthevolumeofthediluentorculturemedium稀释剂或培养基体积的增加;(2)

Incorporationofaspecificorgeneralneutralizingagentsintothediluent将某个特定或通用的中和剂整合至稀释剂中;(3)

Membranefiltration膜过滤;or或(4)

Acombinationoftheabovemeasures上述办法的组合.NeutralizingAgents—Neutralizingagentsmaybeusedtoneutralizetheactivityofantimicrobialagents(seeTable2).Theymaybeaddedtothechosendiluentorthemediumpreferablybeforesterilization.Ifused,theirefficacyandtheirabsenceoftoxicityformicroorganismsmustbedemonstratedbycarryingoutablankwithneutralizerandwithoutproduct.中和剂——中和剂能够用于中和抗菌剂（见表2）的活性。最佳在灭菌之前，将它们加入到所选的稀释剂中或培养基中。如果使用，则必须通过进行一种带中和剂而不含产品的空白实验，来论证它们的功效和无毒性。Ifnosuitableneutralizingmethodcanbefound,itcanbeassumedthatthefailuretoisolatetheinoculatedorganismisattributabletothemicrobicidalactivityoftheproduct.Thisinformationservestoindicatethatthearticleisnotlikelytobecontaminatedwiththegivenspeciesofthemicroorganism.However,itispossiblethattheproductinhibitsonlysomeofthemicroorganismsspecifiedherein,butdoesnotinhibitothersnotincludedamongtheteststrainsorthoseforwhichthelatterarenotrepresentative.Then,performthetestwiththehighestdilutionfactorcompatiblewithmicrobialgrowthandthespecificacceptancecriterion.如果没有找到适宜的中和办法，则能够假定未能分离所接种有机体的因素是供试品杀灭微生物活性。这个信息协助指出此物品不太可能被特定微生物种群所污染。然而，有可能供试品只克制这里所列出微生物中的某些，而并不克制未涉及在供试菌株之中其它微生物或者后者对其不具代表性的那些微生物。然后，用与微生物生长和具体接受原则相适应的最高稀释倍数进行实验。RECOVERYOFMICROORGANISMSINTHEPRESENCEOFPRODUCT在产品存在的状况下微生物的回收Foreachofthemicroorganismslisted,separatetestsareperformed.Onlymicroorganismsoftheaddedteststrainarecounted.对所列出的每个微生物，做单独的检测。只计算所加入的供试菌株的微生物。MembraneFiltration—Usemembranefiltershavinganominalporesizenotgreaterthan0.45µm.Thetypeoffiltermaterialischoseninsuchawaythatthebacteria-retainingefficiencyisnotaffectedbythecomponentsofthesampletobeinvestigated.Foreachofthemicroorganismslisted,onemembranefilterisused.膜过滤——使用一种标称孔径不超出0.45µm的膜过滤器。过滤器的材料按下列原则选择，即待检测样品的构成部分不会对细菌保存效率产生影响。对于所列出的每个微生物，单独使用一种膜过滤器。TransferasuitablequantityofthesamplepreparedasdescribedunderPreparationoftheSample,InoculationandDilution,andNeutralization/RemovalofAntimicrobialActivity(preferablyrepresenting1goftheproduct,orlessiflargenumbersofcfuareexpected)tothemembranefilter,filterimmediately,andrinsethemembranefilterwithanappropriatevolumeofdiluent.将按照样品的制备、接种和稀释、和抗菌活性的中和/去除项下描述所制备的适宜数量的样品（最佳相称于1克产品，在cfu数量预计较大时，也可少于此数量）转移至膜过滤器，立刻过滤，并用适量的稀释剂冲洗膜过滤器。Forthedeterminationoftotalaerobicmicrobialcount(TAMC),transferthemembranefiltertothesurfaceoftheSoybean-CaseinDigestAgar.Forthedeterminationoftotalcombinedyeastsandmoldscount(TYMC),transferthemembranetothesurfaceoftheSabouraudDextroseAgar.IncubatetheplatesasindicatedinTable1.Performthecounting.

对于总好氧微生物计数的测定（TAMC），将滤膜转移至大豆酪蛋白消化物琼脂培养基的表面。对于总酵母菌和霉菌联累计数（TYMC）的测定，将膜转移至Sabouraud（沙氏）葡萄糖琼脂培养基的表面。按表1中规定的条件培养这些平皿。进行计数。Plate-CountMethods—Performplate-countmethodsatleastinduplicateforeachmedium,andusethemeancountoftheresult.平板计数法——每种培养基最少进行两次平板计数法，并使用计数成果的平均值。Pour-PlateMethod—ForPetridishes9cmindiameter,addtothedish1mLofthesamplepreparedasdescribedunderPreparationoftheSample,InoculationandDilution,andNeutralization/RemovalofAntimicrobialActivityand15to20mLofSoybean-CaseinDigestAgarorSabouraudDextroseAgar,bothmediamaintainedatnotmorethan45o.IflargerPetridishesareused,theamountofagarmediumisincreasedaccordingly.ForeachofthemicroorganismslistedinTable1,atleasttwoPetridishesareused.倾注培养法——对于直径为9cm的平皿，将按照样品的制备、接种和稀释、抗菌活性的中和/去除项下的描述所制备的样品1mL以及15至20mL大豆酪蛋白消化物琼脂培养基或者Sabouraud（沙氏）葡萄粮琼脂培养基加入至平皿，此两种培养基的温度均维持在不超出45o。如果使用较大的平皿，琼脂培养基的量也需对应地增加。对于表1中所列出的每一种微生物，最少要使用两个平皿。IncubatetheplatesasindicatedinTable1.Takethearithmeticmeanofthecountspermedium,andcalculatethenumberofcfuintheoriginalinoculum.按表1中的批示培养这些平皿。取每培养基计数的算术平均值，计算在最初的接种体中的菌落数（cfu）数量。Surface-SpreadMethod—ForPetridishes9cmindiameter,add15to20mLofSoybean-CaseinDigestAgarorSabouraudDextroseAgaratabout45otoeachPetridish,andallowtosolidify.IflargerPetidishesareused,thevolumeoftheagarisincreasedaccordingly.Drytheplates,forexample,inalaminar-airflowcabinetorinanincubator.ForeachofthemicroorganismslistedinTable1,atleasttwoPetridishesareused.Spreadameasuredvolumeofnotlessthan0.1mLofthesample,preparedasdirectedunder

PreparationoftheSample,InoculationandDilution,andNeutralization/RemovalofAntimicrobialActivityoverthesurfaceofthemedium.IncubateandcountasdirectedforPour-PlateMethod.表面铺展法——对于直径为9cm的平皿，倾入15至20mL、温度大概为45o的大豆酪蛋白消化物琼脂培养基或Sabouraud（沙氏）葡萄粮琼脂培养基至每个平皿中，静置凝固。如果使用较大的平皿，琼脂的数量也需对应地增加。干燥平皿，例如，在层流柜或恒温箱里。对于表1中列出的每个微生物，最少使用两个平皿。将已称量好的体积不少于0.1mL、按照样品的制备、接种和稀释、和抗菌活性的中和/去除项下的规定所制备的样品，铺展在培养基的表面。按照倾注培养法项下的规定培养和计数。Most-Probable-Number(MPN)Method—TheprecisionandaccuracyoftheMPNMethodislessthanthatoftheMembraneFiltrationmethodorthePlate-CountMethod.Unreliableresultsareobtainedparticularlyfortheenumerationofmolds.Forthesereasons,theMPNMethodisreservedfortheenumerationofTAMCinsituationswherenoothermethodisavailable.Iftheuseofthemethodisjustified,proceedasfollows.最大几率数（MPN）法——MPN法的精密度和精确度低于膜过滤法或平板计数法。所获得的霉菌计数的成果特别不可靠。由于这些因素，MPN法被保存用于当没有其它可行办法状况下的总好氧微生物计数。如果有证据支持此办法的使用，则按以下进行。Table2.CommonNeutralizingAgents/MethodsforInterferingSubstances表2.对干扰物质的惯用中和试剂/办法InterferingSubstance干扰物质PotentialNeutralizingAgents/Method潜在中和试剂/办法Glutaraldehyde,mercurials戊二醛，汞制剂Sodiumhydrogensulfite(Sodiumbisulfite)亚硫酸氢钠Phenolics,alcohol,aldehydes,sorbate酚类物质，酒精，醛类，山梨酸Dilution稀释剂Aldehydes醛类Glycine甘氨酸Quaternaryammoniumcompounds(QACs),parahydroxybenzoates(parabens),bisbiguanides季铵盐化合物（QACs），对羟苯甲酸（防腐剂），Lecithin卵磷脂QACs,iodine,parabensQACs，碘，防腐剂Polysorbate聚山梨醇酯Mercurials汞制剂Thioglycollate硫胶质Mercurials,halogens,aldehydes汞制剂，卤素，醛类Thiosulfate硫代硫酸盐EDTA(edetate)EDTA乙二胺四乙酸盐MgorCaions镁或钙离子Table3.Most-Probable-NumberValuesofMicroorganisms表3微生物的最大几率数的值ObservedCombinationsofNumbersofTubesShowingGrowthinEachSet每套显示生长的试管所观察到的数量组合MPNpergorpermLofProduct每克或每毫升产品的最大几率数95%ConfidenceLimits95%置信程度NumberofgormLofProductperTube每管中的产品克或毫升数Prepareaseriesofatleastthreeserial10-folddilutionsoftheproductasdescribedforPreparationoftheSample,InoculationandDilution,andNeutralization/RemovalofAntimicrobialActivity.Fromeachlevelofdilution,threealiquotsof1gor1mLareusedtoinoculatethreetubeswith9to10mLofSoybean-CaseinDigestBroth.Ifnecessaryasurface-activeagentsuchaspolysorbate80,oraninactivatorofantimicrobialagentsmaybeaddedtothemedium.Thus,ifthreelevelsofdilutionareprepared,ninetubesareinoculated.按样品的制备，接种和稀释，以及抗菌活性的中和/移除这些项下的描述，制备系列的、最少持续三级、每级稀释10倍的产品稀释液。从每个水平的稀释液，将1克或者1毫升的三等分试样接种到三管9至10毫升的大豆酪蛋白消化物肉汤培养基。如有必要，像聚山梨醇酯80这样的表面活性剂，或某种抗菌剂的灭活剂能够加入到培养基中。因此，如果制备了三水平的稀释液，则会接种九个试管。Incubatealltubesat30oto35ofornotmorethan3days.Ifreadingoftheresultsisdifficultoruncertainowingtothenatureoftheproducttobeexamined,subcultureinthesamebrothorinSoybean-CaseinDigestAgarfor1to2daysatthesametemperature,andusetheseresults.FromTable3,determinethemostprobablenumberofmicroorganismspergormLoftheproducttobeexamined.培养全部的试管，温度为30o至35o之间，不超出3天。如果由于供试品的性质造成成果的读数很难或不拟定，则在相似温度下，在同样的肉汤培养基或大豆酪蛋白消化物琼脂培养基中放置1至2天进行再次培养，并且使用这些成果。从表3中，拟定每克或每毫升的供试品中微生物的最大几率数。RESULTSANDINTERPRETATION成果和阐明WhenverifyingthesuitabilityoftheMembraneFiltrationmethodorthePlate-CountMethod,ameancountofanyofthetestorganismsnotdifferingbyafactorgreaterthan2fromthevalueofthecontroldefinedinInoculationandDilutionintheabsenceofproductmustbeobtained.WhenverifyingthesuitabilityoftheMPNMethod,thecalculatedvaluefromtheinoculummustbewithin95%confidencelimitsoftheresultsobtainedwiththecontrol.当确认膜过滤法或平板计数法的合用性时，任何供试微生物的平均计数与在没有产品的状况下，从培养和稀释项下规定的对照组的数值差距必须不超出2。当确认最大几率数法的合用性时，从接种体得到的计算值必须是在从对照组获得的成果的95%置信程度以内。TESTINGOFPRODUCTS供试品的测试AmountUsedfortheTest用于测试的数量Unlessotherwisedirected,use10gor10mLoftheproducttobeexaminedtakenwiththeprecautionsreferredtoabove.Forfluidsorsolidsinaerosolform,sample10containers.Fortransdermalpatches,sample10patches.除非另有规定，使用10克或者10毫升供试品，并采用上面提到的防止方法。对于气溶胶形式下的液体或固体，取10个容器作样品。对于贴剂，取10片作样品。Theamounttobetestedmaybereducedforactivesubstancesthatwillbeformulatedinthefollowingconditions:theamountperdosageunit(e.g.,table,capsule,injection)islessthanorequalto1mg,ortheamountpergormL(forpreparationsnotpresentedindoseunits)islessthan1mg.Inthesecases,theamountofsampletobetestedisnotlessthantheamountpresentin10dosageunitsor10gor10mLoftheproduct.对于某些活性物质来说，供试数量能够减少，前提是这些活性物质将会按以下条件组方：每剂量单位（例如，片，胶囊，注射液）数量不大于或等于1毫克，或者每克或毫升中数量（对于不按剂量单位使用的制备品）不大于1毫克。在这些状况下，供试样品数量不不大于目前10剂量单位中存在的数量，或10克或10毫升的供试品。ExaminationoftheProduct产品的检测MEMBRANEFILTRATION膜过滤Useafiltrationapparatusdesignedtoallowthetransferofthefiltertothemedium.PreparethesampleusingamethodthathasbeenshowntobesuitableasdescribedinGrowthPromotionTestandSuitabilityoftheCountingMethod,transfertheappropriateamounttoeachoftwomembranefilters,andfilterimmediately.Washeachfilterfollowingtheprocedureshowntobesuitable.使用能够将滤膜转移至培养基中的过滤设备。按生长增进测试和计数办法的合用性项下描述的、已经证明合用的办法制备样品，转移适宜的量至两个膜过滤器中的每一种，并立刻过滤。按照下列已经证明合用的规程清洗每个过滤器。ForthedeterminationofTAMC,transferoneofthemembranefilterstothesurfaceofSoybean-CaseinDigestAgar.ForthedeterminationofTYMC,transfertheothermembranetothesurfaceofSabouraudDextroseAgar.IncubatetheplateofSoybean-CaseinDigestAgarat30oto35ofor3to5daysandtheplateofSabouraudDextroseAgarat20oto25ofor5to7days.CalculatethenumberofcfupergorpermLofproduct.对于总好氧微生物计数的测定，转移滤膜中的一种至大豆酪蛋白消化物琼脂培养基的表面。对酵母菌和霉菌联累计数的测定，转移另一种滤膜至Sabouraud（沙氏）葡萄糖琼脂培养基的表面。在温度30o至35o之间培养大豆酪蛋白消化物琼脂培养基的平皿3至5天，并在温度20o至25o之间培养Sabouraud（沙氏）葡萄糖琼脂培养基的平皿5至7天。计算每克或每毫升供试品的菌落数（cfu）数量。Whenexaminingtransdermalpatches,separatelyfilter10%ofthevolumeofthepreparationdescribedforPreparationoftheSamplethrougheachoftwosterilefiltermembrances.TransferonemembrancetoSoybean-CaseinDigestAgarforTAMCandtheothermembranetoSabouraudDextroseAgarforTYMC.当检测贴剂时，分别将样品的制备项下描述的制备品数量的10%过滤通过两个无菌滤膜中的一种。为总好氧微生物计数将一种滤膜转移至大豆酪蛋白消化物琼脂培养基，以检测总好氧微生物计数，而将另一种滤膜转移至Sabouraud（沙氏）葡萄糖琼脂培养基，以检测总酵母菌和霉菌联累计数。

PLATE-COUNTMETHODS平板计数法Pour-PlateMethod—PreparethesampleusingamethodthathasbeenshowntobesuitableasdescribedinGrowthPromotionTestandSuitabilityoftheCountingMethod.PrepareforeachmediumatleasttwoPetridishesforeachlevelofdilution.IncubatetheplatesofSoybean-CaseinDigestAgarat30oto35ofor3to5daysandtheplatesofSabouraudDextroseAgarat20oto25ofor5to7days.Selecttheplatescorrespondingtoagivendilutionandshowingthehighestnumberofcolonieslessthan250forTAMCand50forTYMC.Takethearithmeticmeanperculturemediumofthecou