微生物計(jì)數(shù)檢查法USP61中英對(duì)照版

非無(wú)菌產(chǎn)品微生物學(xué)檢查：微生物計(jì)數(shù)檢查法USP61中英對(duì)照版<61>MICROBIOLOGICALEXAMINATIONOFNONSTERILEPRODUCTS:MICROBIALENUMENRATIONTESTS非無(wú)菌產(chǎn)品微生物學(xué)檢查：微生物計(jì)數(shù)檢查法INTRODUCTION導(dǎo)言Thetestsdescribedhereafterwillallowquantitativeenumerationofmesophilicbacteriaandfungithatmaygrowunderaerobicconditions.下列所描述的這些檢測(cè)將使得對(duì)在有氧的條件下生長(zhǎng)的嗜溫性細(xì)菌和真菌進(jìn)行定量計(jì)數(shù)成為可能。Thetestsaredesignedprimarilytodeterminewhetherasubstanceorpreparationcomplieswithanestablishedspecificationformicrobiologicalquality.Whenusedforsuchpurposes,followtheinstructionsgivenbelow,includingthenumberofsamplestobetaken,andinterprettheresultsasstatedbelow.這些檢測(cè)重要設(shè)計(jì)用于測(cè)定一種物質(zhì)或制備品與否符合已確立的微生物質(zhì)量原則。當(dāng)用于這類目的時(shí)，需遵照下列所給的闡明，涉及待取樣品的數(shù)量，并且按照下面所述解釋成果。Themethodsarenotapplicabletoproductscontainingviablemicroorganismsasactiveingredients.這些辦法不合用于以活菌作為活性成分的產(chǎn)品。Alternativemicrobiologicalprocedures,includingautomatedmethods,maybeused,providedthattheirequivalencetothePharmacopeialmethodhasbeendemonstrated.能夠使用替代的微生物規(guī)程，涉及自動(dòng)化辦法，只要已經(jīng)證明它們與藥典辦法具同等作用。GENERALPROCEDURES通用規(guī)程Carryoutthedeterminationunderconditionsdesignedtoavoid12microbialcontaminationoftheproducttobeexamined.Theprecautionstakentoavoidcontaminationmustbesuchthattheydonotaffectanymicroorganismsthataretoberevealedinthetest.在通過(guò)設(shè)計(jì)可避免外來(lái)微生物污染供試產(chǎn)品的條件下，進(jìn)行此項(xiàng)測(cè)定。用于避免污染的這些防止方法是必須做到，它們不會(huì)影響任何試圖在此項(xiàng)檢查中揭示的微生物。Iftheproducttobeexaminedhasantimicrobialactivity,thisis,insofaraspossible,removedorneutralized.Ifinactivatorsareusedforthispurpose,theirefficacyandtheirabsenceoftoxicityformicroorganismsmustbedemonstrated.如果供試產(chǎn)品含有抗菌活性，則此活性需在盡量的范疇內(nèi)去除或中和。如果將滅活劑用于這個(gè)目的，則必須證明它們的功效和對(duì)微生物不具毒性。Ifsurface-activesubstancesareusedforsamplepreparation,theirabsenceoftoxicityformicroorganismsandtheircompatibilitywithanyinactivatorsusedmustbedemonstrated.如果表面活性物質(zhì)用于樣品制備，則必須證明它們對(duì)微生物不具毒性以及與所使用的任何滅活劑的兼容性。ENUMERATIONMETHODS計(jì)數(shù)法UsetheMembraneFiltrationmethodoroneofthePlate-CountMethods,asdirected.TheMost-Probable-Number(MPN)Methodisgenerallytheleastaccuratemethodformicrobialcounts;however,forcertainproductgroupswithverylowbioburden,itmaybethemostappropriatemethod.按規(guī)定，使用膜過(guò)濾法或多個(gè)平板計(jì)數(shù)法中的一種。液體稀釋法（MPN）一般對(duì)微生物計(jì)數(shù)而言是最不精確的辦法；然而，對(duì)含有非常低生物載荷的特定產(chǎn)品類型，它可能是最適宜的辦法。Thechoiceofamethodisbasedonfactorssuchasthenatureoftheproductandtherequiredlimitofmicroorganisms.Themethodchosenmustallowtestingofasufficientsamplesizetojudgecompliancewiththespecification.Thesuitabilityofthechosenmethodmustbeestablished.辦法的選擇基于某些因素，例如產(chǎn)品的性質(zhì)和微生物的規(guī)定程度。所選的辦法必須能夠?qū)Τ渥愕臉颖玖窟M(jìn)行檢測(cè)，以判斷與質(zhì)量原則的符合性。所選辦法的合用性必須被建立。Table1.PreparationandUseofTestMicroorganisms表1.供試微生物的制備和使用GrowthPromotion生長(zhǎng)增進(jìn)SuitabilityofCountingMethodinthePresenceofProduct在產(chǎn)品存在的狀況下計(jì)數(shù)辦法的合用性Microorganism微生物PreparationofTestStrain供試菌株的制備TotalAerobicMicrobialCount總好氧微生物計(jì)數(shù)TotalYeastsandMoldsCount總酵母菌和霉菌計(jì)數(shù)TotalAerobicMicrobialCount總好氧微生物計(jì)數(shù)TotalYeastsandMoldsCount總酵母菌和霉菌計(jì)數(shù)StaphylococcusaureussuchasATCC6538,NCIMB9518,CIP4.83,orNBRC13276金黃色葡萄球菌如：ATCC6538,NCIMB9518,CIP4.83,或NBRC13276Soybean-CaseinDigestAgarorSoybean-CaseinDigestBroth300-35018-24hours大豆酪蛋白消化物瓊脂培養(yǎng)基或大豆酪蛋白消化物肉湯培養(yǎng)基300-35018-24小時(shí)Soybean-CaseinDigestAgarandSoybean-CaseinDigestBroth≤100cfu300-350≤3days大豆酪蛋白消化物培養(yǎng)基和大豆酪蛋白消化物肉湯培養(yǎng)基≤100cfu300-350≤3天Soybean-CaseinDigestAgar/MPNSoybean-CaseinDigestBroth≤100cfu300-350≤3days大豆酪蛋白消化物培養(yǎng)基/MPN（最大幾率數(shù)）大豆酪蛋白消化物肉湯培養(yǎng)基≤100cfu300-350≤3天PseudomonasaeruginosasuchasATCC9027,NCIMB8626,CIP82.118,orNBRC13275綠膿桿菌如ATCC9027,NCIMB8626,CIP82.118,或NBRC13275Soybean-CaseinDigestAgarorSoybean-CaseinDigestBroth300-35018-24hours大豆酪蛋白消化物瓊脂培養(yǎng)基或大豆酪蛋白消化物肉湯培養(yǎng)基300-35018-24小時(shí)Soybean-CaseinDigestAgarandSoybean-CaseinDigestBroth≤100cfu300-350≤3days大豆酪蛋白消化物瓊脂培養(yǎng)基和大豆酪蛋白消化物肉湯培養(yǎng)基≤100cfu300-350≤3天Soybean-CaseinDigestAgar/MPNSoybean-CaseinDigestBroth≤100cfu300-350≤3days大豆酪蛋白消化物瓊脂培養(yǎng)基/MPN（最大幾率數(shù)）大豆酪蛋白消化物肉湯培養(yǎng)基≤100cfu300-350≤3天BacillussubtilissuchasATCC6633,NCIMB8054,CIP52.62,orNBRC3134枯草芽孢桿菌如ATCC6633,NCIMB8054,CIP52.62,或NBRC3134Soybean-CaseinDigestAgarorSoybean-CaseinDigestBroth300-35018-24hours大豆酪蛋白消化物瓊脂培養(yǎng)基或大豆酪蛋白消化物肉湯培養(yǎng)基300-35018-24小時(shí)Soybean-CaseinDigestAgarandSoybean-CaseinDigestBroth≤100cfu300-350≤3days大豆酪蛋白消化物瓊脂培養(yǎng)基和大豆酪蛋白消化物肉湯培養(yǎng)基≤100cfu300-350≤3天Soybean-CaseinDigestAgar/MPNSoybean-CaseinDigestBroth≤100cfu300-350≤3days大豆酪蛋白消化物瓊脂培養(yǎng)基/MPN（最大幾率數(shù)）大豆酪蛋白消化物肉湯培養(yǎng)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）葡萄糖瓊脂培養(yǎng)基或Sabouraud（沙氏）葡萄糖肉湯培養(yǎng)基Soybean-CaseinDigestAgar≤100cfu300-350≤5days大豆酪蛋白消化物瓊脂培養(yǎng)基≤100cfu300-350≤5天SabouraudDextroseAgar≤100cfu200-250≤5daysSabouraud（沙氏）葡萄糖瓊脂培養(yǎng)基≤100cfu200-250≤5天Soybean-CaseinDigestAgar≤100cfu300-350≤5daysMPN:notapplicable

大豆酪蛋白消化物瓊脂培養(yǎng)基≤100cfu300-350≤5天MPN(最大幾率數(shù))：不合用SabouraudDextroseAgar≤100cfu200-250≤5daysSabouraud（沙氏）葡萄糖瓊脂培養(yǎng)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）葡萄糖瓊脂培養(yǎng)基或馬鈴薯葡萄糖瓊脂培養(yǎng)基

200-250

5-7天,或直到實(shí)現(xiàn)良好的產(chǎn)孢Soybean-CaseinDigestAgar≤100cfu300-350≤5days大豆酪蛋白消化物瓊脂培養(yǎng)基≤100cfu300-350≤5天SabouraudDextroseAgar≤100cfu200-250≤5daysSabouraud（沙氏）葡萄糖瓊脂培養(yǎng)基≤100cfu200-250≤5天Soybean-CaseinDigestAgar≤100cfu300-350≤5daysMPN:notapplicable大豆酪蛋白消化物瓊脂培養(yǎng)基≤100cfu300-350≤5天MPN(最大幾率數(shù))：不合用SabouraudDextroseAgar≤100cfu200-250≤5daysSabouraud（沙氏）葡萄糖瓊脂培養(yǎng)基≤100cfu200-250≤5天GROWTHPROMOTIONTESTANDSUITABILITYOFTHECOUNTINGMETHOD生長(zhǎng)增進(jìn)實(shí)驗(yàn)和計(jì)數(shù)辦法的合用性GeneralConsiderations通用考慮因素Theabilityofthetesttodetectmicroorganismsinthepresenceofproducttobetestedmustbeestablished.在供試產(chǎn)品存在的狀況下，必須確立檢測(cè)微生物的實(shí)驗(yàn)?zāi)芰Αuitabilitymustbeconfirmedifachangeintestingperformanceorachangeintheproductthatmayaffecttheoutcomeofthetest,isintroduced.如果引入了可能影響實(shí)驗(yàn)成果的在測(cè)試性能或產(chǎn)品方面的變更，則必須確認(rèn)其合用性。PreparationofTestStrains供試菌株的制備Usestandardizedstablesuspensionsofteststrainsorprepareasstatedbelow.Seed-lotculturemaintenancetechniques(seed-lotsystems)areusedsothattheviablemicroorganismsusedforinoculationarenotmorethan5passagesremovedfromtheoriginalmasterseed-lot.GroweachofbacterialandfungalteststrainsseparatelyasdescribedinTable1.使用供試菌株的原則化穩(wěn)定懸浮液或按下面所述制備。使用菌種保藏技術(shù)（種子批系統(tǒng)），方便用于接種的可萌發(fā)微生物從最初的主種子批開(kāi)始傳代不超出5次。按照在表1中的描述，分別培養(yǎng)每個(gè)細(xì)菌和霉菌供試菌株。UseBufferedSodiumChloride-PeptoneSolutionpH7.0orPhosphateBufferSolutionpH7.2tomaketestsuspensions;tosuspendA.nigerspores,0.05%ofpolysorbate80maybeaddedtothebuffer.Usethesuspensionswithin2hours,orwithin24hoursifstoredbetween2oand8o.AsanalternativetopreparingandthendilutingafreshsuspensionofvegetativecellsofA.nigerorB.subtilis,astablesporesuspensionispreparedandthenanappropriatevolumeofthesporesuspensionisusedfortestinoculation.Thestablesporesuspensionmaybemaintainedat2oto8oforavalidatedperiodoftime.使用pH7.0的緩沖氯化鈉-蛋白胨溶液，或pH7.2的磷酸鹽緩沖液制作供試懸浮液；為使黑曲霉孢子懸浮，能夠?qū)?.05%的聚山梨醇酯80加入該緩沖液中。這些懸浮液需在2個(gè)小時(shí)之內(nèi)使用，或如果寄存在2o至8o的條件下，則可在24小時(shí)之內(nèi)使用。作為制備然后稀釋黑曲霉或枯草桿菌的新鮮體細(xì)胞懸浮液的替代辦法，可制備穩(wěn)定的孢子懸浮液，然后將適量的孢子懸浮液用于實(shí)驗(yàn)接種。此穩(wěn)定的孢子懸浮液能夠在2o至8o之間于一段通過(guò)驗(yàn)證的時(shí)間內(nèi)保存。NegativeControl陰性對(duì)照Toverifytestingconditions,anegativecontrolisperformedusingthechosendiluentinplaceofthetestpreparation.Theremustbenogrowthofmicroorganisms.為了確認(rèn)實(shí)驗(yàn)的條件，在制備供試品的場(chǎng)合使用所選的稀釋液替代供試品，作陰性對(duì)照。其必須沒(méi)有微生物的增加。GrowthPromotionoftheMedia培養(yǎng)基的生長(zhǎng)增進(jìn)Testeachbatchofready-preparedmediumandeachbatchofmediumpreparedeitherfromdehydratedmediumorfromtheingredientsdescribed.對(duì)每一批已經(jīng)制備好的培養(yǎng)基和每一批從脫水培養(yǎng)基或根據(jù)描述的組分制備出來(lái)的培養(yǎng)基，進(jìn)行測(cè)試。Inoculateportions/platesofSoybean-CaseinDigestBrothandSoybean-CaseinDigestAgarwithasmallnumber(notmorethan100cfu)ofthemicroorganismsindicatedinTable1,usingaseparateportion/plateofmediumforeach.InoculateplatesofSabouraudDextroseAgarwithasmallnumber(notmorethan100cfu)ofthemicroorganismsindicatedinTable1,usingaseparateplateofmediumforeach.IncubateaccordingtotheconditionsdescribedinTable1.在部分/整個(gè)平皿內(nèi)的大豆酪蛋白消化物肉湯培養(yǎng)基和大豆酪蛋白消化物瓊脂培養(yǎng)基中接種少量(不超出100cfu)表1中指定的微生物，每種微生物均使用單獨(dú)的部分/整個(gè)平皿的培養(yǎng)基。在平皿內(nèi)的Sabouraud（沙氏）葡萄糧瓊脂培養(yǎng)基中接種少量(不超出100cfu)表1中指定的微生物，每種均使用一種單獨(dú)平皿的培養(yǎng)基。按照表１中描述的條件進(jìn)行培養(yǎng)。Forsolidmedia,growthobtainedmustnotdifferbyafactorgreaterthan2fromthecalculatedvalueforastandardizedinoculum.Forafreshlypreparedinoculum,growthofthemicroorganismscomparabletothatpreviouslyobtainedwithapreviouslytestedandapprovedbatchofmediumoccurs.Liquidmediaaresuitableifclearlyvisiblegrowthofthemicroorganismscomparabletothatpreviouslyobtainedwithapreviouslytestedandapprovedbatchofmediumoccurs.對(duì)于固體培養(yǎng)基，所獲得的生長(zhǎng)與原則化接種體的計(jì)算值之間的差別因素決不能不不大于2。對(duì)于剛剛制備好的接種體，發(fā)生的微生物生長(zhǎng)與用以前檢查并同意過(guò)的培養(yǎng)基批次所得到的微生物生長(zhǎng)相稱。如果出現(xiàn)了與以前從上一種通過(guò)測(cè)試并同意的培養(yǎng)基批次獲得的微生物生長(zhǎng)相稱的、清晰可見(jiàn)的、微生物生長(zhǎng)，則液體培養(yǎng)基合用。SuitabilityoftheCountingMethodinthePresenceofProduct在存在產(chǎn)品的狀況下計(jì)數(shù)法的合用性PREPARATIONOFTHESAMPLE樣品的制備Themethodforsamplepreparationdependsonthephysicalcharacteristicsoftheproducttobetested.Ifnoneoftheproceduresdescribedbelowcanbedemonstratedtobesatisfactory,asuitablealternativeproceduremustbedeveloped.樣品制備辦法取決于供試品的物理特性。如果下列描述的規(guī)程不能夠被令人滿意地證明，則必須開(kāi)發(fā)一種合用的替代規(guī)程。Water-SolubleProducts—Dissolveordilute(usuallya1in10dilutionisprepared)theproducttobeexaminedinBufferedSodiumChloride-PeptoneSolutionpH7.0,PhosphateBufferSolutionpH7.2,orSoybean-CaseinDigestBroth.Ifnecessary,adjusttoapHof6to8.Furtherdilutions,wherenecessary,arepreparedwiththesamediluent.水溶性產(chǎn)品——溶解或稀釋（一般制備1:10的稀釋液）供試品，于pH值為7.0的緩沖氯化鈉-蛋白胨溶液中，pH值為7.2的磷酸鹽緩沖液，或大豆酪蛋白消化物肉湯培養(yǎng)基。如有必要，將pH值調(diào)節(jié)為6至8。在需要時(shí)，用相似的稀釋劑進(jìn)一步地稀釋。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試品——將檢測(cè)的供試品（一般制備1:10的稀釋液）懸浮于pH值為7.0的緩沖氯化鈉-蛋白胨溶液，pH值為7.2的磷酸鹽緩沖液，或者大豆酪蛋白消化物肉湯培養(yǎng)基中。能夠加入某個(gè)表面活性劑，例如1克每升的聚山梨酯80，以協(xié)助難以與水混合的物質(zhì)懸浮。如有必要，調(diào)節(jié)pH值為6至8。在需要時(shí)，用相似的稀釋劑進(jìn)一步的稀釋。FattyProducts—Dissolveinisopropylmyristatesterilizedbyfiltration,ormixtheproducttobeexaminedwiththeminimumnecessaryquantityofsterilepolysorbate80oranothernoninhibitorysterilesurface-activereagentheated,ifnecessary,tonotmorethan40oor,inexceptionalcases,tonotmorethan45o.Mixcarefullyandifnecessarymaintainthetemperatureinawaterbath.Addasufficientquantityoftheprewarmedchosendiluenttomakea1in10dilutionoftheoriginalproduct.Mixcarefully,whilemaintainingthetemperaturefortheshortesttimenecessaryfortheformationofanemulsion.Furtherserial10-folddilutionsmaybepreparedusingthechosendiluentcontainingasuitableconcentrationofsterilepolysorbate80oranothernoninhibitorysterilesurface-activereagent.脂肪性供試品——溶解于以過(guò)濾除菌的豆蔻酸異丙酯，或者將供試品與所需最少量的、通過(guò)加熱的無(wú)菌聚山梨酯80或另一種非抑菌性的無(wú)菌表面活性劑進(jìn)行混合，如有必要，可加熱至不超出40o或者，在特殊狀況下，至不超出45o。認(rèn)真混勻，如有必要，在水浴鍋里中維持該溫度。加入充足數(shù)量、通過(guò)預(yù)熱的所選擇稀釋劑，制成原產(chǎn)品的1:10稀釋液。認(rèn)真混勻，同時(shí)維持該溫度至形成乳化劑所必需的最短的時(shí)間。能夠用所選擇的含有適宜濃度的無(wú)菌聚山梨酯80或者另一種非抑菌性的無(wú)菌表面活性劑的稀釋液，來(lái)制備后續(xù)的系列10倍稀釋液。FluidsorSolidsinAerosolForm—Asepticallytransfertheproductintoamembranefilterapparatusorasterilecontainerforfurthersampling.Useeitherthetotalcontentsoradefinednumberofmetereddosesfromeachofthecontainerstested.氣溶膠與液溶膠——以無(wú)菌操作將供試品轉(zhuǎn)移至一種過(guò)濾膜設(shè)備或一種無(wú)菌容器里，以進(jìn)一步取樣。從每個(gè)被檢測(cè)的容器中，使用全部?jī)?nèi)容物或劑量的規(guī)定倍數(shù)。

TransdermalPatches—Removetheprotectivecoversheets(“releaseliners”)ofthetransdermalpatchesandplacethem,adhesivesideupwards,onsterileglassorplastictrays.Covertheadhesivesurfacewithasuitablesterileporousmaterial(e.g.,sterilegauze)topreventthepatchesfromstickingtogether,andtransferthepatchestoasuitablevolumeofthechosendiluentcontaininginactivatorssuchaspolysorbate80and/orlecithin.Shakethepreparationvigorouslyforatleast30minutes.貼劑——去除貼劑的防護(hù)面（“釋放襯板”）并將它們放置在無(wú)菌玻璃或塑料托盤(pán)上，膠貼劑的一面對(duì)上。用適宜的無(wú)菌多孔材料（例如：無(wú)菌紗布）蓋住膠貼面，以避免貼劑粘在一起，并將貼劑轉(zhuǎn)移到所選的含有滅活劑（例如聚山梨醇酯80和/或卵磷脂）的適量稀釋劑中。用力搖動(dòng)供試品最少30分鐘。INOCULATIONANDDILUTION接種和稀釋Addtothesamplepreparedasdirectedaboveandtoacontrol(withnotestmaterialincluded)asufficientvolumeofthemicrobialsuspensiontoobtainaninoculumofnotmorethan100cfu.Thevolumeofthesuspensionoftheinoculumshouldnotexceed1%ofthevolumeofdilutedproduct.向按上述規(guī)定制備的樣品中以及向?qū)φ罩苽淦罚ㄆ渲袩o(wú)供試物質(zhì)）中，加入充足體積的微生物懸浮液，以獲得一種不多于100cfu的接種體。接種的菌體懸浮液體積應(yīng)當(dāng)不超出被稀釋產(chǎn)品體積的1%。Todemonstrateacceptablemicrobialrecoveryfromtheproduct,thelowestpossibledilutionfactorofthepreparedsamplemustbeusedforthetest.Wherethisisnotpossibleduetoantimicrobialactivityorpoorsolubility,furtherappropriateprotocolsmustbedeveloped.Ifinhibitionofgrowthbythesamplecannototherwisebeavoided,thealiquotofthemicrobialsuspensionmaybeaddedafterneutralization,dilution,orfiltration.為證明供試品有可接受的微生物回收率，必須使用已制備樣品的最低可能稀釋倍數(shù)來(lái)進(jìn)行檢測(cè)。當(dāng)由于抗菌活性或低溶解度而無(wú)法做到這一點(diǎn)時(shí)，必須進(jìn)一步開(kāi)發(fā)更適合的規(guī)程。如果樣品對(duì)生長(zhǎng)的克制不能以其它方式避免，能夠在中和、稀釋、或過(guò)濾之后，加入等量的微生物懸浮液。NEUTRALIZATION/REMOVALOFANTIMICROBIALACTIVITY抗菌活性的中和/去除ThenumberofmicroorganismsrecoveredfromthepreparedsampledilutedasdescribedinInoculationandDilutionandincubatedfollowingtheproceduredescribedinRecoveryofMicroorganismsinthePresenceofProduct,iscomparedtothenumberofmicroorganismsrecoveredfromthecontrolpreparation.用制備好的樣品按接種和稀釋項(xiàng)下的描述稀釋，并按在產(chǎn)品存在的狀況下微生物的回收率項(xiàng)下描述的規(guī)程培養(yǎng)，將從中回收的微生物的數(shù)量與從對(duì)照制備品中回收的微生物的數(shù)量進(jìn)行比較。Ifgrowthisinhibited(reductionbyafactorgreaterthan2),thenmodifytheprocedurefortheparticularenumerationtesttoensurethevalidityoftheresults.Modificationoftheproceduremayinclude,forexample,如果生長(zhǎng)被克制（以不不大于2的因素減少），那么修改特定的計(jì)數(shù)測(cè)試規(guī)程，以確保成果的有效性。規(guī)程的修改能夠涉及，例如：(1)

Anincreaseinthevolumeofthediluentorculturemedium稀釋劑或培養(yǎng)基體積的增加;(2)

Incorporationofaspecificorgeneralneutralizingagentsintothediluent將某個(gè)特定或通用的中和劑整合至稀釋劑中;(3)

Membranefiltration膜過(guò)濾;or或(4)

Acombinationoftheabovemeasures上述辦法的組合.NeutralizingAgents—Neutralizingagentsmaybeusedtoneutralizetheactivityofantimicrobialagents(seeTable2).Theymaybeaddedtothechosendiluentorthemediumpreferablybeforesterilization.Ifused,theirefficacyandtheirabsenceoftoxicityformicroorganismsmustbedemonstratedbycarryingoutablankwithneutralizerandwithoutproduct.中和劑——中和劑能夠用于中和抗菌劑（見(jiàn)表2）的活性。最佳在滅菌之前，將它們加入到所選的稀釋劑中或培養(yǎng)基中。如果使用，則必須通過(guò)進(jìn)行一種帶中和劑而不含產(chǎn)品的空白實(shí)驗(yàn)，來(lái)論證它們的功效和無(wú)毒性。Ifnosuitableneutralizingmethodcanbefound,itcanbeassumedthatthefailuretoisolatetheinoculatedorganismisattributabletothemicrobicidalactivityoftheproduct.Thisinformationservestoindicatethatthearticleisnotlikelytobecontaminatedwiththegivenspeciesofthemicroorganism.However,itispossiblethattheproductinhibitsonlysomeofthemicroorganismsspecifiedherein,butdoesnotinhibitothersnotincludedamongtheteststrainsorthoseforwhichthelatterarenotrepresentative.Then,performthetestwiththehighestdilutionfactorcompatiblewithmicrobialgrowthandthespecificacceptancecriterion.如果沒(méi)有找到適宜的中和辦法，則能夠假定未能分離所接種有機(jī)體的因素是供試品殺滅微生物活性。這個(gè)信息協(xié)助指出此物品不太可能被特定微生物種群所污染。然而，有可能供試品只克制這里所列出微生物中的某些，而并不克制未涉及在供試菌株之中其它微生物或者后者對(duì)其不具代表性的那些微生物。然后，用與微生物生長(zhǎng)和具體接受原則相適應(yīng)的最高稀釋倍數(shù)進(jìn)行實(shí)驗(yàn)。RECOVERYOFMICROORGANISMSINTHEPRESENCEOFPRODUCT在產(chǎn)品存在的狀況下微生物的回收Foreachofthemicroorganismslisted,separatetestsareperformed.Onlymicroorganismsoftheaddedteststrainarecounted.對(duì)所列出的每個(gè)微生物，做單獨(dú)的檢測(cè)。只計(jì)算所加入的供試菌株的微生物。MembraneFiltration—Usemembranefiltershavinganominalporesizenotgreaterthan0.45μm.Thetypeoffiltermaterialischoseninsuchawaythatthebacteria-retainingefficiencyisnotaffectedbythecomponentsofthesampletobeinvestigated.Foreachofthemicroorganismslisted,onemembranefilterisused.膜過(guò)濾——使用一種標(biāo)稱孔徑不超出0.45μm的膜過(guò)濾器。過(guò)濾器的材料按下列原則選擇，即待檢測(cè)樣品的構(gòu)成部分不會(huì)對(duì)細(xì)菌保存效率產(chǎn)生影響。對(duì)于所列出的每個(gè)微生物，單獨(dú)使用一種膜過(guò)濾器。TransferasuitablequantityofthesamplepreparedasdescribedunderPreparationoftheSample,InoculationandDilution,andNeutralization/RemovalofAntimicrobialActivity(preferablyrepresenting1goftheproduct,orlessiflargenumbersofcfuareexpected)tothemembranefilter,filterimmediately,andrinsethemembranefilterwithanappropriatevolumeofdiluent.將按照樣品的制備、接種和稀釋、和抗菌活性的中和/去除項(xiàng)下描述所制備的適宜數(shù)量的樣品（最佳相稱于1克產(chǎn)品，在cfu數(shù)量預(yù)計(jì)較大時(shí)，也可少于此數(shù)量）轉(zhuǎn)移至膜過(guò)濾器，立刻過(guò)濾，并用適量的稀釋劑沖洗膜過(guò)濾器。Forthedeterminationoftotalaerobicmicrobialcount(TAMC),transferthemembranefiltertothesurfaceoftheSoybean-CaseinDigestAgar.Forthedeterminationoftotalcombinedyeastsandmoldscount(TYMC),transferthemembranetothesurfaceoftheSabouraudDextroseAgar.IncubatetheplatesasindicatedinTable1.Performthecounting.

對(duì)于總好氧微生物計(jì)數(shù)的測(cè)定（TAMC），將濾膜轉(zhuǎn)移至大豆酪蛋白消化物瓊脂培養(yǎng)基的表面。對(duì)于總酵母菌和霉菌聯(lián)累計(jì)數(shù)（TYMC）的測(cè)定，將膜轉(zhuǎn)移至Sabouraud（沙氏）葡萄糖瓊脂培養(yǎng)基的表面。按表1中規(guī)定的條件培養(yǎng)這些平皿。進(jìn)行計(jì)數(shù)。Plate-CountMethods—Performplate-countmethodsatleastinduplicateforeachmedium,andusethemeancountoftheresult.平板計(jì)數(shù)法——每種培養(yǎng)基最少進(jìn)行兩次平板計(jì)數(shù)法，并使用計(jì)數(shù)成果的平均值。Pour-PlateMethod—ForPetridishes9cmindiameter,addtothedish1mLofthesamplepreparedasdescribedunderPreparationoftheSample,InoculationandDilution,andNeutralization/RemovalofAntimicrobialActivityand15to20mLofSoybean-CaseinDigestAgarorSabouraudDextroseAgar,bothmediamaintainedatnotmorethan45o.IflargerPetridishesareused,theamountofagarmediumisincreasedaccordingly.ForeachofthemicroorganismslistedinTable1,atleasttwoPetridishesareused.傾注培養(yǎng)法——對(duì)于直徑為9cm的平皿，將按照樣品的制備、接種和稀釋、抗菌活性的中和/去除項(xiàng)下的描述所制備的樣品1mL以及15至20mL大豆酪蛋白消化物瓊脂培養(yǎng)基或者Sabouraud（沙氏）葡萄糧瓊脂培養(yǎng)基加入至平皿，此兩種培養(yǎng)基的溫度均維持在不超出45o。如果使用較大的平皿，瓊脂培養(yǎng)基的量也需對(duì)應(yīng)地增加。對(duì)于表1中所列出的每一種微生物，最少要使用兩個(gè)平皿。IncubatetheplatesasindicatedinTable1.Takethearithmeticmeanofthecountspermedium,andcalculatethenumberofcfuintheoriginalinoculum.按表1中的批示培養(yǎng)這些平皿。取每培養(yǎng)基計(jì)數(shù)的算術(shù)平均值，計(jì)算在最初的接種體中的菌落數(shù)（cfu）數(shù)量。Surface-SpreadMethod—ForPetridishes9cmindiameter,add15to20mLofSoybean-CaseinDigestAgarorSabouraudDextroseAgaratabout45otoeachPetridish,andallowtosolidify.IflargerPetidishesareused,thevolumeoftheagarisincreasedaccordingly.Drytheplates,forexample,inalaminar-airflowcabinetorinanincubator.ForeachofthemicroorganismslistedinTable1,atleasttwoPetridishesareused.Spreadameasuredvolumeofnotlessthan0.1mLofthesample,preparedasdirectedunder

PreparationoftheSample,InoculationandDilution,andNeutralization/RemovalofAntimicrobialActivityoverthesurfaceofthemedium.IncubateandcountasdirectedforPour-PlateMethod.表面鋪展法——對(duì)于直徑為9cm的平皿，傾入15至20mL、溫度大概為45o的大豆酪蛋白消化物瓊脂培養(yǎng)基或Sabouraud（沙氏）葡萄糧瓊脂培養(yǎng)基至每個(gè)平皿中，靜置凝固。如果使用較大的平皿，瓊脂的數(shù)量也需對(duì)應(yīng)地增加。干燥平皿，例如，在層流柜或恒溫箱里。對(duì)于表1中列出的每個(gè)微生物，最少使用兩個(gè)平皿。將已稱量好的體積不少于0.1mL、按照樣品的制備、接種和稀釋、和抗菌活性的中和/去除項(xiàng)下的規(guī)定所制備的樣品，鋪展在培養(yǎng)基的表面。按照傾注培養(yǎng)法項(xiàng)下的規(guī)定培養(yǎng)和計(jì)數(shù)。Most-Probable-Number(MPN)Method—TheprecisionandaccuracyoftheMPNMethodislessthanthatoftheMembraneFiltrationmethodorthePlate-CountMethod.Unreliableresultsareobtainedparticularlyfortheenumerationofmolds.Forthesereasons,theMPNMethodisreservedfortheenumerationofTAMCinsituationswherenoothermethodisavailable.Iftheuseofthemethodisjustified,proceedasfollows.最大幾率數(shù)（MPN）法——MPN法的精密度和精確度低于膜過(guò)濾法或平板計(jì)數(shù)法。所獲得的霉菌計(jì)數(shù)的成果特別不可靠。由于這些因素，MPN法被保存用于當(dāng)沒(méi)有其它可行辦法狀況下的總好氧微生物計(jì)數(shù)。如果有證據(jù)支持此辦法的使用，則按以下進(jìn)行。Table2.CommonNeutralizingAgents/MethodsforInterferingSubstances表2.對(duì)干擾物質(zhì)的慣用中和試劑/辦法InterferingSubstance干擾物質(zhì)PotentialNeutralizingAgents/Method潛在中和試劑/辦法Glutaraldehyde,mercurials戊二醛，汞制劑Sodiumhydrogensulfite(Sodiumbisulfite)亞硫酸氫鈉Phenolics,alcohol,aldehydes,sorbate酚類物質(zhì)，酒精，醛類，山梨酸Dilution稀釋劑Aldehydes醛類Glycine甘氨酸Quaternaryammoniumcompounds(QACs),parahydroxybenzoates(parabens),bisbiguanides季銨鹽化合物（QACs），對(duì)羥苯甲酸（防腐劑），Lecithin卵磷脂QACs,iodine,parabensQACs，碘，防腐劑Polysorbate聚山梨醇酯Mercurials汞制劑Thioglycollate硫膠質(zhì)Mercurials,halogens,aldehydes汞制劑，鹵素，醛類Thiosulfate硫代硫酸鹽EDTA(edetate)EDTA乙二胺四乙酸鹽MgorCaions鎂或鈣離子Table3.Most-Probable-NumberValuesofMicroorganisms表3微生物的最大幾率數(shù)的值ObservedCombinationsofNumbersofTubesShowingGrowthinEachSet每套顯示生長(zhǎng)的試管所觀察到的數(shù)量組合MPNpergorpermLofProduct每克或每毫升產(chǎn)品的最大幾率數(shù)95%ConfidenceLimits95%置信程度NumberofgormLofProductperTube每管中的產(chǎn)品克或毫升數(shù)Prepareaseriesofatleastthreeserial10-folddilutionsoftheproductasdescribedforPreparationoftheSample,InoculationandDilution,andNeutralization/RemovalofAntimicrobialActivity.Fromeachlevelofdilution,threealiquotsof1gor1mLareusedtoinoculatethreetubeswith9to10mLofSoybean-CaseinDigestBroth.Ifnecessaryasurface-activeagentsuchaspolysorbate80,oraninactivatorofantimicrobialagentsmaybeaddedtothemedium.Thus,ifthreelevelsofdilutionareprepared,ninetubesareinoculated.按樣品的制備，接種和稀釋，以及抗菌活性的中和/移除這些項(xiàng)下的描述，制備系列的、最少持續(xù)三級(jí)、每級(jí)稀釋10倍的產(chǎn)品稀釋液。從每個(gè)水平的稀釋液，將1克或者1毫升的三等分試樣接種到三管9至10毫升的大豆酪蛋白消化物肉湯培養(yǎng)基。如有必要，像聚山梨醇酯80這樣的表面活性劑，或某種抗菌劑的滅活劑能夠加入到培養(yǎng)基中。因此，如果制備了三水平的稀釋液，則會(huì)接種九個(gè)試管。Incubatealltubesat30oto35ofornotmorethan3days.Ifreadingoftheresultsisdifficultoruncertainowingtothenatureoftheproducttobeexamined,subcultureinthesamebrothorinSoybean-CaseinDigestAgarfor1to2daysatthesametemperature,andusetheseresults.FromTable3,determinethemostprobablenumberofmicroorganismspergormLoftheproducttobeexamined.培養(yǎng)全部的試管，溫度為30o至35o之間，不超出3天。如果由于供試品的性質(zhì)造成成果的讀數(shù)很難或不擬定，則在相似溫度下，在同樣的肉湯培養(yǎng)基或大豆酪蛋白消化物瓊脂培養(yǎng)基中放置1至2天進(jìn)行再次培養(yǎng)，并且使用這些成果。從表3中，擬定每克或每毫升的供試品中微生物的最大幾率數(shù)。RESULTSANDINTERPRETATION成果和闡明WhenverifyingthesuitabilityoftheMembraneFiltrationmethodorthePlate-CountMethod,ameancountofanyofthetestorganismsnotdifferingbyafactorgreaterthan2fromthevalueofthecontroldefinedinInoculationandDilutionintheabsenceofproductmustbeobtained.WhenverifyingthesuitabilityoftheMPNMethod,thecalculatedvaluefromtheinoculummustbewithin95%confidencelimitsoftheresultsobtainedwiththecontrol.當(dāng)確認(rèn)膜過(guò)濾法或平板計(jì)數(shù)法的合用性時(shí)，任何供試微生物的平均計(jì)數(shù)與在沒(méi)有產(chǎn)品的狀況下，從培養(yǎng)和稀釋項(xiàng)下規(guī)定的對(duì)照組的數(shù)值差距必須不超出2。當(dāng)確認(rèn)最大幾率數(shù)法的合用性時(shí)，從接種體得到的計(jì)算值必須是在從對(duì)照組獲得的成果的95%置信程度以內(nèi)。TESTINGOFPRODUCTS供試品的測(cè)試AmountUsedfortheTest用于測(cè)試的數(shù)量Unlessotherwisedirected,use10gor10mLoftheproducttobeexaminedtakenwiththeprecautionsreferredtoabove.Forfluidsorsolidsinaerosolform,sample10containers.Fortransdermalpatches,sample10patches.除非另有規(guī)定，使用10克或者10毫升供試品，并采用上面提到的防止方法。對(duì)于氣溶膠形式下的液體或固體，取10個(gè)容器作樣品。對(duì)于貼劑，取10片作樣品。Theamounttobetestedmaybereducedforactivesubstancesthatwillbeformulatedinthefollowingconditions:theamountperdosageunit(e.g.,table,capsule,injection)islessthanorequalto1mg,ortheamountpergormL(forpreparationsnotpresentedindoseunits)islessthan1mg.Inthesecases,theamountofsampletobetestedisnotlessthantheamountpresentin10dosageunitsor10gor10mLoftheproduct.對(duì)于某些活性物質(zhì)來(lái)說(shuō)，供試數(shù)量能夠減少，前提是這些活性物質(zhì)將會(huì)按以下條件組方：每劑量單位（例如，片，膠囊，注射液）數(shù)量不大于或等于1毫克，或者每克或毫升中數(shù)量（對(duì)于不按劑量單位使用的制備品）不大于1毫克。在這些狀況下，供試樣品數(shù)量不不大于目前10劑量單位中存在的數(shù)量，或10克或10毫升的供試品。ExaminationoftheProduct產(chǎn)品的檢測(cè)MEMBRANEFILTRATION膜過(guò)濾Useafiltrationapparatusdesignedtoallowthetransferofthefiltertothemedium.PreparethesampleusingamethodthathasbeenshowntobesuitableasdescribedinGrowthPromotionTestandSuitabilityoftheCountingMethod,transfertheappropriateamounttoeachoftwomembranefilters,andfilterimmediately.Washeachfilterfollowingtheprocedureshowntobesuitable.使用能夠?qū)V膜轉(zhuǎn)移至培養(yǎng)基中的過(guò)濾設(shè)備。按生長(zhǎng)增進(jìn)測(cè)試和計(jì)數(shù)辦法的合用性項(xiàng)下描述的、已經(jīng)證明合用的辦法制備樣品，轉(zhuǎn)移適宜的量至兩個(gè)膜過(guò)濾器中的每一種，并立刻過(guò)濾。按照下列已經(jīng)證明合用的規(guī)程清洗每個(gè)過(guò)濾器。ForthedeterminationofTAMC,transferoneofthemembranefilterstothesurfaceofSoybean-CaseinDigestAgar.ForthedeterminationofTYMC,transfertheothermembranetothesurfaceofSabouraudDextroseAgar.IncubatetheplateofSoybean-CaseinDigestAgarat30oto35ofor3to5daysandtheplateofSabouraudDextroseAgarat20oto25ofor5to7days.CalculatethenumberofcfupergorpermLofproduct.對(duì)于總好氧微生物計(jì)數(shù)的測(cè)定，轉(zhuǎn)移濾膜中的一種至大豆酪蛋白消化物瓊脂培養(yǎng)基的表面。對(duì)酵母菌和霉菌聯(lián)累計(jì)數(shù)的測(cè)定，轉(zhuǎn)移另一種濾膜至Sabouraud（沙氏）葡萄糖瓊脂培養(yǎng)基的表面。在溫度30o至35o之間培養(yǎng)大豆酪蛋白消化物瓊脂培養(yǎng)基的平皿3至5天，并在溫度20o至25o之間培養(yǎng)Sabouraud（沙氏）葡萄糖瓊脂培養(yǎng)基的平皿5至7天。計(jì)算每克或每毫升供試品的菌落數(shù)（cfu）數(shù)量。Whenexaminingtransdermalpatches,separatelyfilter10%ofthevolumeofthepreparationdescribedforPreparationoftheSamplethrougheachoftwosterilefiltermembrances.TransferonemembrancetoSoybean-CaseinDigestAgarforTAMCandtheothermembranetoSabouraudDextroseAgarforTYMC.當(dāng)檢測(cè)貼劑時(shí)，分別將樣品的制備項(xiàng)下描述的制備品數(shù)量的10%過(guò)濾通過(guò)兩個(gè)無(wú)菌濾膜中的一種。為總好氧微生物計(jì)數(shù)將一種濾膜轉(zhuǎn)移至大豆酪蛋白消化物瓊脂培養(yǎng)基，以檢測(cè)總好氧微生物計(jì)數(shù)，而將另一種濾膜轉(zhuǎn)移至Sabouraud（沙氏）葡萄糖瓊脂培養(yǎng)基，以檢測(cè)總酵母菌和霉菌聯(lián)累計(jì)數(shù)。

PLATE-COUNTMETHODS平板計(jì)數(shù)法Pour-PlateMethod—PreparethesampleusingamethodthathasbeenshowntobesuitableasdescribedinGrowthPromotionTestandSuitabilityoftheCountingMethod.PrepareforeachmediumatleasttwoPetridishesforeachlevelofdilution.IncubatetheplatesofSoybean-CaseinDigestAgarat30oto35ofor3to5daysandtheplatesofSabouraudDextroseAgarat20oto25ofor5to7days.Selecttheplatescorrespondingtoagivendilutionandshowingthehighestnumberofcolonieslessthan250forTAMCand50forTYMC.Takethearithmeticmeanperculturemediumofthecou