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61MICROBIOLOGICALEXAMINATIONOFNONSTERILEPRODUCTS:MICROBIALENUMERATIONTESTS非无菌产品微生物限度检查：微生物计数法1.INTRODUCTION导言Thetestsdescribedhereafterwillallowquantitativeenumerationofmesophilicbacteriaandfungithatmaygrowunderaerobicconditions.微生物计数法系用于能在有氧条件下生长的嗜温细菌和霉菌的定量计数。Thetestsaredesignedprimarilytodeterminewhetherasubstanceorpreparationcomplieswithanestablishedspecificationformicrobiologicalquality.Whenusedforsuchpurposes,followtheinstructionsgivenbelow,includingthenumberofsamplestobetaken,andinterprettheresultsasstatedbelow.当本法用于检查非无菌制剂及其原、辅料等是否符合相应的微生物限度标准时，应按照下述规定进行检验，包括样品的取样量，结果的判断。Themethodsarenotapplicabletoproductscontainingviablemicroorganismsasactiveingredients.本法不适用于活菌制剂的检查。Alternativemicrobiologicalprocedures,includingautomatedmethods,maybeused,providedthattheirequivalencetothePharmacopeialmethodhasbeendemonstrated.可使用包括自动化法在内的替代方法，需确认其与药典方法的等同性。2.GENERALPROCEDURES通用规程Carryoutthedeterminationunderconditionsdesignedtoavoidextrinsicmicrobialcontaminationoftheproducttobeexamined.Theprecautionstakentoavoidcontaminationmustbesuchthattheydonotaffectanymicroorganismsthataretoberevealedinthetest.微生物计数环境应能防止外来微生物对供试品的污染。防止污染的措施不能影响供试品中微生物的检出。备注：检验全过程必须严格遵守无菌操作，防止再污染。单向气流区域、工作台面及环境应定期进行监测。Iftheproducttobeexaminedhasantimicrobialactivity,thisis,insofaraspossible,removedorneutralized.Ifinactivatorsareusedforthispurpose,theirefficacyandtheirabsenceoftoxicityformicroorganismsmustbedemonstrated.如果供试品有抗菌活性，应尽可能去除或中和。若使用了中和剂或灭活剂，需确认其有效性及对微生物无毒性。Ifsurface-activesubstancesareusedforsamplepreparation,theirabsenceoftoxicityformicroorganismsandtheircompatibilitywithanyinactivatorsusedmustbedemonstrated.供试品制备过程中若使用了表面活性剂，需确认其对微生物无毒性以及与所使用的中和剂或灭活剂的相容性。3.ENUMERATIONMETHODS计数方法UsetheMembraneFiltrationmethodoroneofthePlate-CountMethods,asdirected.TheMost-Probable-Number(MPN)Methodisgenerallytheleastaccuratemethodformicrobialcounts;however,forcertainproductgroupswithverylowbioburden,itmaybethemostappropriatemethod.计数方法包括薄膜过滤法、平皿法和最可能数法（Most-Probable-Number简称MPN)。MPN法用于微生物计数时精确度最差，但用于微生物污染量小的供试品，MPN法可能是最合适的方法。（MPN法不适用于霉菌的检测，仅在供试品总需氧菌数没有适应计数方法的情况下使用。）Thechoiceofamethodisbasedonfactorssuchasthenatureoftheproductandtherequiredlimitofmicroorganisms.Themethodchosenmustallowtestingofasufficientsamplesizetojudgecompliancewiththespecification.Thesuitabilityofthechosenmethodmustbeestablished.供试品检查时，应根据供试品理化特性和微生物限度标准等因素选择计数方法。所选择的计数方法应能够通过检测足量的供试品，判断与质量标准的符合性。且该计数方法的适用性须经确认。4.GROWTHPROMOTIONTEST,SUITABILITYOFTHECOUNTINGMETHODANDNEGATIVECONTROLS促生长实验，计数法适用性试验以及阴性对照4.1.GeneralConsiderations一般要求Theabilityofthetesttodetectmicroorganismsinthepresenceofproducttobetestedmustbeestablished.Suitabilitymustbeconfirmedifachangeintestingperformanceorachangeintheproductthatmayaffecttheoutcomeofthetest,isintroduced.在有供试品存在的情况下，所选用的计数方法需能够检测微生物。若检测程序或产品发生变化可能影响检测结果时，计数方法需重新进行适用性确认。4.2.PreparationofTestStrains试验菌液的制备Usestandardizedstablesuspensionsofteststrainsorprepareasstatedbelow.Seed-lotculturemaintenancetechniques(seed-lotsystems)areusedsothattheviablemicroorganismsusedforinoculationarenotmorethanfivepassagesremovedfromtheoriginalmasterseed-lot.GroweachofthebacterialandfungalteststrainsseparatelyasdescribedinTable1.试验菌液：使用试验菌株的标准化稳定悬浮液或按下述规定制备。按表1规定程序分别培养各试验菌液。菌株：采用适宜的菌种保藏技术（种子批系统），试验用菌株传代次数自主种子批（第0代）算起不得超过5代。Table1.PreparationandUseofTestMicroorganismsGrowthPromotion促生长实验SuitabilityofCountingMethodinthePresenceofProduct计数方法适用性试验Microorganism试验菌株PreparationofTestStrain试验菌液的制备TotalAerobicMicrobialCount总需氧菌数计数TotalYeastsandMoldsCount总酵母菌和霉菌计数TotalAerobicMicrobialCount总需氧菌数计数TotalYeastsandMoldsCount总酵母菌和霉菌计数StaphylococcusaureussuchasATCC6538,NCIMB9518,CIP4.83,orNBRC13276金黄色葡萄球菌Soybean-CaseinDigestAgarorSoybean-CaseinDigestBroth30°-35°18-24hours大豆酪蛋白消化琼脂培养基或大豆酪蛋白消化肉汤培养基培养温度30°-35°培养时间18-24小时Soybean-CaseinDigestAgarandSoybean-CaseinDigestBroth≤100cfu30°-35°≤3days大豆酪蛋白消化琼脂培养基和大豆酪蛋白消化肉汤基培养温度30°-35°培养时间不超过3天接种量不大于100cfuSoybean-CaseinDigestAgar/MPNSoybean-CaseinDigestBroth≤100cfu30°-35°≤3days大豆酪蛋白消化琼脂培养基大豆酪蛋白消化肉汤培养基（MPN法）培养温度30°-35°培养时间不超过3天接种量不大于100cfuPseudomonasaeruginosasuchasATCC9027,NCIMB8626,CIP82.118,orNBRC13275铜绿假单胞菌BacillussubtilissuchasATCC6633,NCIMB8054,CIP52.62,orNBRC3134枯草芽孢杆菌CandidaalbicanssuchasATCC10231,NCPF3179,IP48.72,orNBRC1594白色念珠菌SabouraudDextroseAgarorSabouraudDextroseBroth20°-25°2-3days沙氏葡萄糖琼脂培养基或沙氏葡萄糖肉汤培养基培养温度20°-25°培养时间2-3天Soybean-CaseinDigestAgar≤100cfu30°-35°≤5days大豆酪蛋白消化琼脂培养基培养温度30°-35°培养时间不超过5天接种量不大于100cfuSabouraudDextroseAgar≤100cfu20°-25°≤5days沙氏葡萄糖琼脂培养基培养温度20°-25°培养时间不超过5天接种量不大于100cfuSoybean-CaseinDigestAgar≤100cfu30°-35°≤5daysMPN：notapplicable大豆酪蛋白消化琼脂培养基培养温度30°-35°培养时间不超过5天接种量不大于100cfuMPN法不适用SabouraudDextroseAgar≤100cfu20°-25°≤5days沙氏葡萄糖琼脂培养基培养温度20°-25°培养时间不超过5天接种量不大于100cfuAspergillusnigersuchasATCC16404,IMI149007,IP1431.83,orNBRC9455黑曲霉SabouraudDextroseAgarorPotato-DextroseAgar20°-25°5-7daysoruntilgoodsporulationisachieve沙氏葡萄糖琼脂培养基或马铃薯葡萄糖琼脂培养基培养温度20°-25°培养时间5-7天，或直到获得丰富的孢子UseBufferedSodiumChloride–PeptoneSolutionpH7.0orPhosphateBufferSolutionpH7.2tomaketestsuspensions;tosuspendA.brasiliensisspores,0.05%ofpolysorbate80maybeadd