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12.6.1.STERILITY2.6.1无菌检查法Thetestisappliedtosubstances,preparationsorarticleswhich,accordingtothePharmacopoeia,arerequiredtobesterile.However,asatisfactoryresultonlyindicatesthatnocontaminatingmicro-organismhasbeenfoundinthesampleexaminedintheconditionsofthetest.本检查方法适用于按照药典要求应当无菌的原料、制剂或其他物质。但是，如果按照本无菌检查法的结果符合要求，仅表明在该检查条件下未发现微生物污染。PRECAUTIONSAGAINSTMICROBIALCONTAMINATION微生物污染防范Thetestforsterilityiscarriedoutunderasepticconditions.Inordertoachievesuchconditions,thetestenvironmenthastobeadaptedtothewayinwhichthesterilitytestisperformed.Theprecautionstakentoavoidcontaminationaresuchthattheydonotaffectanymicro-organismswhicharetoberevealedinthetest.Theworkingconditionsinwhichthetestsareperformedaremonitoredregularlybyappropriatesamplingoftheworkingareaandbycarryingoutappropriatecontrols.无菌检测试验应在无菌的条件下进行。为了达到这样的条件，检测环境应当与无菌检测的操作要求相适应。避免污染的防范措施应当不对本检查方法进行检测的微生物造成影响（应并不影响用本检查法检测的微生物）。通过对工作区域的适当取样以及进行适当的控制来对无菌检查的工作环境进行例行监测。CULTUREMEDIAANDINCUBATIONTEMPERATURES培养基和培养温度Mediaforthetestmaybepreparedasdescribedbelow,orequivalentcommercialmediamaybeusedprovidedthattheycomplywiththegrowthpromotiontest.应按下面描述的方法制备无菌检查的培养介质，如果满足生长促进试验要求，与本处所述培养基相当的商业化培养基也可以采用（也可采用与本处……）。Thefollowingculturemediahavebeenfoundtobesuitableforthetestforsterility.Fluidthioglycollatemediumisprimarilyintendedforthecultureof2anaerobicbacteria;however,itwillalsodetectaerobicbacteria.Soya-beancaseindigestmediumissuitableforthecultureofbothfungiandaerobicbacteria.下述的培养基已被证明（经证明）适用于无菌检查。硫乙醇酸盐流体培养基主要用于厌氧菌培养，但是，也适用于需氧菌检测。大豆酪蛋白消化物培养基适用于真菌和需氧菌培养。Fluidthioglycollatemedium硫乙醇酸盐流体培养基L-Cystine0.5gL-胱氨酸0.5gAgar0.75g琼脂0.75gSodiumchloride2.5g氯化钠2.5gGlucosemonohydrate/anhydrous5.5g/5.0g葡萄糖一水合物/无水葡萄糖5.5g/5.0gYeastextract(water-soluble)5.0g酵母提取物（水溶性）5.0gPancreaticdigestofcasein15.0g酪蛋白胰酶消化物15.0gSodiumthioglycollateor0.5g硫乙醇酸钠0.5gThioglycollicacid0.3mL硫乙醇酸0.3mlResazurinsodiumsolution(1g/Lofresazurin1.0mLsodium),freshlyprepared刃天青钠溶液（刃天青钠1g/L），新鲜配制WaterR1000mL水R1000mlpHaftersterilisation7.1±0.2灭菌后的pH7.1±0.23MixtheL-cystine,agar,sodiumchloride,glucose,water-solubleyeastextractandpancreaticdigestofcaseinwiththewaterRandheatuntilsolutioniseffected.Dissolvethesodiumthioglycollateorthioglycollicacidinthesolutionand,ifnecessary,add1Msodiumhydroxidesothat,aftersterilisation,thesolutionwillhaveapHof7.1±0.2.Iffiltrationisnecessary,heatthesolutionagainwithoutboilingandfilterwhilehotthroughmoistenedfilterpaper.Addtheresazurinsodiumsolution,mixandplacethemediuminsuitablevesselswhichprovidearatioofsurfacetodepthofmediumsuchthatnotmorethantheupperhalfofthemediumhasundergoneacolourchangeindicativeofoxygenuptakeattheendoftheincubationperiod.Steriliseusingavalidatedprocess.Ifthemediumisstored,storeatatemperaturebetween2°Cand25°Cinasterile,airtightcontainer.Ifmorethantheupperone-thirdofthemediumhasacquiredapinkcolour,themediummayberestoredoncebyheatingthecontainersinawater-bathorinfree-flowingsteamuntilthepinkcolourdisappearsandcoolingquickly,takingcaretopreventtheintroductionofnon-sterileairintothecontainer.Donotusethemediumforalongerstorageperiodthanhasbeenvalidated.将L-胱氨酸、琼脂、氯化钠、葡萄糖、水溶性酵母提取物以及酪蛋白胰酶消化物与水R混合，加热至溶解。将硫乙醇酸钠或硫乙醇酸用上述溶液溶解，必要时用1M氢氧化钠调节pH值，使灭菌后培养基溶液的pH值为7.1±0.2。如需要过滤处理，将溶液在此加热（加热此溶液），但不得煮到沸腾，乘热采用经润湿的滤纸进行过滤。加入刃天青钠溶液，混合均匀，将制备的培养基装入合适的容器中。在该容器中，培养基的表面和高度应具有恰当的比例，以便在灭菌结束后指示氧气摄入的颜色变化不超过培养基的上半部分。采用经验证的工艺灭菌。如果需要保存，将培养基装入无菌、气密容器并在2-25°C之间储存。如果培养基的上面超过1/3的部分已经出现粉红色，将装有培养基的容器采用水浴或自由流动蒸气加热，直到粉红颜色消失，之后快速冷却，注意预防非无菌的气体被引入装培养基的容器，以此进行培养基再生处理。如果培养基保存的时间超过经验证的保存期限，不得使用。（禁止使用超过验证储存期限的培养基）Fluidthioglycollatemediumistobeincubatedat30-35°C.Forproductscontainingamercurialpreservativethatcannotbetestedbythemembrane-filtration4method,fluidthioglycollatemediumincubatedat20-25°Cmaybeusedinsteadofsoya-beancaseindigestmediumprovidedthatithasbeenvalidatedasdescribedingrowthpromotiontest.硫乙醇酸盐流体培养基用于（应）在30-35°C条件下培养。对于含有汞类防腐剂无法采用薄膜过滤法进行检查的产品，如果已按照生长促进试验所述方法验证，硫乙醇酸盐流体培养基可代替替代重复大豆酪蛋白消化物培养基在20-25°C条件下进行培养。Whereprescribedorjustifiedandauthorised,thefollowingalternativethioglycollatemediummaybeused.Prepareamixturehavingthesamecompositionasthatofthefluidthioglycollatemedium,butomittingtheagarandtheresazurinsodiumsolution,steriliseasdirectedabove.ThepHaftersterilisationis7.1±0.2.Heatinawater-bathpriortouseandincubateat30-35°Cunderanaerobicconditions.按照规定或者证明合理并获得主管机构许可时（如果有经批准的规定或正当理由），也可以使用以下替代硫乙醇酸盐流体培养基。配制与硫乙醇酸盐流体培养基成分相同的混合物，但是不包括琼脂和刃天青钠溶液，按照上面说明的方法进行灭菌。灭菌后培养基的pH值为7.1±0.2，使用前采用水浴加热，在厌氧及30-35°C条件下培养。Soya-beancaseindigestmedium大豆-酪蛋白消化物培养基Pancreaticdigestofcasein17.0g酪蛋白胰酶消化物17.0gPapaicdigestofsoya-beanmeal3.0g大豆粉木瓜蛋白酶消化物3.0gSodiumchloride5.0g氯化钠5.0gDipotassiumhydrogenphosphate2.5g磷酸氢二钾2.5gGlucosemonohydrate/anhydrous2.5g/2.3g葡萄糖一水合物/无水葡萄糖2.5g/2.3gWaterR1000mL水R1000ml5pHaftersterilisation7.3±0.2灭菌后pH7.3±0.2DissolvethesolidsinwaterR,warmingslightlytoeffectsolution.Coolthesolutiontoroomtemperature.Add1Msodiumhydroxide,ifnecessary,sothataftersterilisationthesolutionwillhaveapHof7.3±0.2.Filter,ifnecessary,toclarify,distributeintosuitablevesselsandsteriliseusingavalidatedprocess.Storeatatemperaturebetween2°Cand25°Cina