微生物計數(shù)檢查法USP61中英對照版

非無菌產(chǎn)品微生物學檢查：微生物計數(shù)檢查法USP61中英對照版<61>MICROBIOLOGICALEXAMINATIONOFNONSTERILEPRODUCTS:MICROBIALENUMENRATIONTESTS非無菌產(chǎn)品微生物學檢查：微生物計數(shù)檢查法INTRODUCTION導言Thetestsdescribedhereafterwillallowquantitativeenumerationofmesophilicbacteriaandfungithatmaygrowunderaerobicconditions.下列所描述的這些檢測將使得對在有氧的條件下生長的嗜溫性細菌和真菌進行定量計數(shù)成為可能。Thetestsaredesignedprimarilytodeterminewhetherasubstanceorpreparationcomplieswithanestablishedspecificationformicrobiologicalquality.Whenusedforsuchpurposes,followtheinstructionsgivenbelow,includingthenumberofsamplestobetaken,andinterprettheresultsasstatedbelow.這些檢測重要設計用于測定一種物質或制備品與否符合已確立的微生物質量原則。當用于這類目的時，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.在通過設計可避免外來微生物污染供試產(chǎn)品的條件下，進行此項測定。用于避免污染的這些防止方法是必須做到，它們不會影響任何試圖在此項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去除或中和。如果將滅活劑用于這個目的，則必須證明它們的功效和對微生物不具毒性。Ifsurface-activesubstancesareusedforsamplepreparation,theirabsenceoftoxicityformicroorganismsandtheircompatibilitywithanyinactivatorsusedmustbedemonstrated.如果表面活性物質用于樣品制備，則必須證明它們對微生物不具毒性以及與所使用的任何滅活劑的兼容性。ENUMERATIONMETHODS計數(shù)法UsetheMembraneFiltrationmethodoroneofthePlate-CountMethods,asdirected.TheMost-Probable-Number(MPN)Methodisgenerallytheleastaccuratemethodformicrobialcounts;however,forcertainproductgroupswithverylowbioburden,itmaybethemostappropriatemethod.按規(guī)定，使用膜過濾法或多個平板計數(shù)法中的一種。液體稀釋法（MPN）一般對微生物計數(shù)而言是最不精確的辦法；然而，對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)品的性質和微生物的規(guī)定程度。所選的辦法必須能夠對充足的樣本量進行檢測，以判斷與質量原則的符合性。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生長增進SuitabilityofCountingMethodinthePresenceofProduct在產(chǎn)品存在的狀況下計數(shù)辦法的合用性Microorganism微生物PreparationofTestStrain供試菌株的制備TotalAerobicMicrobialCount總好氧微生物計數(shù)TotalYeastsandMoldsCount總酵母菌和霉菌計數(shù)TotalAerobicMicrobialCount總好氧微生物計數(shù)TotalYeastsandMoldsCount總酵母菌和霉菌計數(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生長增進實驗和計數(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，必須確立檢測微生物的實驗能力。Suitabilitymustbeconfirmedifachangeintestingperformanceorachangeintheproductthatmayaffecttheoutcomeofthetest,isintroduced.如果引入了可能影響實驗成果的在測試性能或產(chǎn)品方面的變更，則必須確認其合用性。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（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始傳代不超出5次。按照在表1中的描述，分別培養(yǎng)每個細菌和霉菌供試菌株。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，能夠將0.05%的聚山梨醇酯80加入該緩沖液中。這些懸浮液需在2個小時之內使用，或如果寄存在2o至8o的條件下，則可在24小時之內使用。作為制備然后稀釋黑曲霉或枯草桿菌的新鮮體細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驗接種。此穩(wěn)定的孢子懸浮液能夠在2o至8o之間于一段通過驗證的時間內保存。NegativeControl陰性對照Toverifytestingconditions,anegativecontrolisperformedusingthechosendiluentinplaceofthetestpreparation.Theremustbenogrowthofmicroorganisms.為了確認實驗的條件，在制備供試品的場合使用所選的稀釋液替代供試品，作陰性對照。其必須沒有微生物的增加。GrowthPromotionoftheMedia培養(yǎng)基的生長增進Testeachbatchofready-preparedmediumandeachbatchofmediumpreparedeitherfromdehydratedmediumorfromtheingredientsdescribed.對每一批已經(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的培養(yǎng)基，進行測試。Inoculateportions/platesofSoybean-CaseinDigestBrothandSoybean-CaseinDigestAgarwithasmallnumber(notmorethan100cfu)ofthemicroorganismsindicatedinTable1,usingaseparateportion/plateofmediumforeach.InoculateplatesofSabouraudDextroseAgarwithasmallnumber(notmorethan100cfu)ofthemicroorganismsindicatedinTable1,usingaseparateplateofmediumforeach.IncubateaccordingtotheconditionsdescribedinTable1.在部分/整個平皿內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的部分/整個平皿的培養(yǎng)基。在平皿內的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平皿的培養(yǎng)基。按照表１中描述的條件進行培養(yǎng)。Forsolidmedia,growthobtainedmustnotdifferbyafactorgreaterthan2fromthecalculatedvalueforastandardizedinoculum.Forafreshlypreparedinoculum,growthofthemicroorganismscomparabletothatpreviouslyobtainedwithapreviouslytestedandapprovedbatchofmediumoccurs.Liquidmediaaresuitableifclearlyvisiblegrowthofthemicroorganismscomparabletothatpreviouslyobtainedwithapreviouslytestedandapprovedbatchofmediumoccurs.對于固體培養(yǎng)基，所獲得的生長與原則化接種體的計算值之間的差別因素決不能不不大于2。對于剛剛制備好的接種體，發(fā)生的微生物生長與用以前檢查并同意過的培養(yǎng)基批次所得到的微生物生長相稱。如果出現(xiàn)了與以前從上一種通過測試并同意的培養(yǎng)基批次獲得的微生物生長相稱的、清晰可見的、微生物生長，則液體培養(yǎng)基合用。SuitabilityoftheCountingMethodinthePresenceofProduct在存在產(chǎn)品的狀況下計數(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值調節(jié)為6至8。在需要時，用相似的稀釋劑進一步地稀釋。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懸浮。如有必要，調節(jié)pH值為6至8。在需要時，用相似的稀釋劑進一步的稀釋。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、通過加熱的無菌聚山梨酯80或另一種非抑菌性的無菌表面活性劑進行混合，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、通過預熱的所選擇稀釋劑，制成原產(chǎn)品的1:10稀釋液。認真混勻，同時維持該溫度至形成乳化劑所必需的最短的時間。能夠用所選擇的含有適宜濃度的無菌聚山梨酯80或者另一種非抑菌性的無菌表面活性劑的稀釋液，來制備后續(xù)的系列10倍稀釋液。FluidsorSolidsinAerosolForm—Asepticallytransfertheproductintoamembranefilterapparatusorasterilecontainerforfurthersampling.Useeitherthetotalcontentsoradefinednumberofmetereddosesfromeachofthecontainerstested.氣溶膠與液溶膠——以無菌操作將供試品轉移至一種過濾膜設備或一種無菌容器里，以進一步取樣。從每個被檢測的容器中，使用全部內容物或劑量的規(guī)定倍數(shù)。

TransdermalPatches—Removetheprotectivecoversheets(“releaseliners”)ofthetransdermalpatchesandplacethem,adhesivesideupwards,onsterileglassorplastictrays.Covertheadhesivesurfacewithasuitablesterileporousmaterial(e.g.,sterilegauze)topreventthepatchesfromstickingtogether,andtransferthepatchestoasuitablevolumeofthechosendiluentcontaininginactivatorssuchaspolysorbate80and/orlecithin.Shakethepreparationvigorouslyforatleast30minutes.貼劑——去除貼劑的防護面（“釋放襯板”）并將它們放置在無菌玻璃或塑料托盤上，膠貼劑的一面對上。用適宜的無菌多孔材料（例如：無菌紗布）蓋住膠貼面，以避免貼劑粘在一起，并將貼劑轉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對照制備品（其中無供試物質）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當不超出被稀釋產(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)來進行檢測。當由于抗菌活性或低溶解度而無法做到這一點時，必須進一步開發(fā)更適合的規(guī)程。如果樣品對生長的克制不能以其它方式避免，能夠在中和、稀釋、或過濾之后，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)量與從對照制備品中回收的微生物的數(shù)量進行比較。Ifgrowthisinhibited(reductionbyafactorgreaterthan2),thenmodifytheprocedurefortheparticularenumerationtesttoensurethevalidityoftheresults.Modificationoftheproceduremayinclude,forexample,如果生長被克制（以不不大于2的因素減少），那么修改特定的計數(shù)測試規(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將某個特定或通用的中和劑整合至稀釋劑中;(3)

Membranefiltration膜過濾;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，則必須通過進行一種帶中和劑而不含產(chǎn)品的空白實驗，來論證它們的功效和無毒性。Ifnosuitableneutralizingmethodcanbefound,itcanbeassumedthatthefailuretoisolatetheinoculatedorganismisattributabletothemicrobicidalactivityoftheproduct.Thisinformationservestoindicatethatthearticleisnotlikelytobecontaminatedwiththegivenspeciesofthemicroorganism.However,itispossiblethattheproductinhibitsonlysomeofthemicroorganismsspecifiedherein,butdoesnotinhibitothersnotincludedamongtheteststrainsorthoseforwhichthelatterarenotrepresentative.Then,performthetestwiththehighestdilutionfactorcompatiblewithmicrobialgrowthandthespecificacceptancecriterion.如果沒有找到適宜的中和辦法，則能夠假定未能分離所接種有機體的因素是供試品殺滅微生物活性。這個信息協(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其不具代表性的那些微生物。然后，用與微生物生長和具體接受原則相適應的最高稀釋倍數(shù)進行實驗。RECOVERYOFMICROORGANISMSINTHEPRESENCEOFPRODUCT在產(chǎn)品存在的狀況下微生物的回收Foreachofthemicroorganismslisted,separatetestsareperformed.Onlymicroorganismsoftheaddedteststrainarecounted.對所列出的每個微生物，做單獨的檢測。只計算所加入的供試菌株的微生物。MembraneFiltration—Usemembranefiltershavinganominalporesizenotgreaterthan0.45μm.Thetypeoffiltermaterialischoseninsuchawaythatthebacteria-retainingefficiencyisnotaffectedbythecomponentsofthesampletobeinvestigated.Foreachofthemicroorganismslisted,onemembranefilterisused.膜過濾——使用一種標稱孔徑不超出0.45μm的膜過濾器。過濾器的材料按下列原則選擇，即待檢測樣品的構成部分不會對細菌保存效率產(chǎn)生影響。對于所列出的每個微生物，單獨使用一種膜過濾器。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)量預計較大時，也可少于此數(shù)量）轉移至膜過濾器，立刻過濾，并用適量的稀釋劑沖洗膜過濾器。Forthedeterminationoftotalaerobicmicrobialcount(TAMC),transferthemembranefiltertothesurfaceoftheSoybean-CaseinDigestAgar.Forthedeterminationoftotalcombinedyeastsandmoldscount(TYMC),transferthemembranetothesurfaceoftheSabouraudDextroseAgar.IncubatetheplatesasindicatedinTable1.Performthecounting.

對于總好氧微生物計數(shù)的測定（TAMC），將濾膜轉移至大豆酪蛋白消化物瓊脂培養(yǎng)基的表面。對于總酵母菌和霉菌聯(lián)累計數(shù)（TYMC）的測定，將膜轉移至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。進行計數(shù)。Plate-CountMethods—Performplate-countmethodsatleastinduplicateforeachmedium,andusethemeancountoftheresult.平板計數(shù)法——每種培養(yǎng)基最少進行兩次平板計數(shù)法，并使用計數(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)基的量也需對應地增加。對于表1中所列出的每一種微生物，最少要使用兩個平皿。IncubatetheplatesasindicatedinTable1.Takethearithmeticmeanofthecountspermedium,andcalculatethenumberofcfuintheoriginalinoculum.按表1中的批示培養(yǎng)這些平皿。取每培養(yǎng)基計數(shù)的算術平均值，計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)量也需對應地增加。干燥平皿，例如，在層流柜或恒溫箱里。對于表1中列出的每個微生物，最少使用兩個平皿。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)法項下的規(guī)定培養(yǎng)和計數(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濾法或平板計數(shù)法。所獲得的霉菌計數(shù)的成果特別不可靠。由于這些因素，MPN法被保存用于當沒有其它可行辦法狀況下的總好氧微生物計數(shù)。如果有證據(jù)支持此辦法的使用，則按以下進行。Table2.CommonNeutralizingAgents/MethodsforInterferingSubstances表2.對干擾物質的慣用中和試劑/辦法InterferingSubstance干擾物質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，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/移除這些項下的描述，制備系列的、最少持續(xù)三級、每級稀釋10倍的產(chǎn)品稀釋液。從每個水平的稀釋液，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，則會接種九個試管。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.當確認膜過濾法或平板計數(shù)法的合用性時，任何供試微生物的平均計數(shù)與在沒有產(chǎn)品的狀況下，從培養(yǎng)和稀釋項下規(guī)定的對照組的數(shù)值差距必須不超出2。當確認最大幾率數(shù)法的合用性時，從接種體得到的計算值必須是在從對照組獲得的成果的95%置信程度以內。TESTINGOFPRODUCTS供試品的測試AmountUsedfortheTest用于測試的數(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。對于氣溶膠形式下的液體或固體，取10個容器作樣品。對于貼劑，取10片作樣品。Theamounttobetestedmaybereducedforactivesubstancesthatwillbeformulatedinthefollowingconditions:theamountperdosageunit(e.g.,table,capsule,injection)islessthanorequalto1mg,ortheamountpergormL(forpreparationsnotpresentedindoseunits)islessthan1mg.Inthesecases,theamountofsampletobetestedisnotlessthantheamountpresentin10dosageunitsor10gor10mLoftheproduct.對于某些活性物質來說，供試數(shù)量能夠減少，前提是這些活性物質將會按以下條件組方：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MEMBRANEFILTRATION膜過濾Useafiltrationapparatusdesignedtoallowthetransferofthefiltertothemedium.PreparethesampleusingamethodthathasbeenshowntobesuitableasdescribedinGrowthPromotionTestandSuitabilityoftheCountingMethod,transfertheappropriateamounttoeachoftwomembranefilters,andfilterimmediately.Washeachfilterfollowingtheprocedureshowntobesuitable.使用能夠將濾膜轉移至培養(yǎng)基中的過濾設備。按生長增進測試和計數(shù)辦法的合用性項下描述的、已經(jīng)證明合用的辦法制備樣品，轉移適宜的量至兩個膜過濾器中的每一種，并立刻過濾。按照下列已經(jīng)證明合用的規(guī)程清洗每個過濾器。ForthedeterminationofTAMC,transferoneofthemembranefilterstothesurfaceofSoybean-CaseinDigestAgar.ForthedeterminationofTYMC,transfertheothermembranetothesurfaceofSabouraudDextroseAgar.IncubatetheplateofSoybean-CaseinDigestAgarat30oto35ofor3to5daysandtheplateofSabouraudDextroseAgarat20oto25ofor5to7days.CalculatethenumberofcfupergorpermLofproduct.對于總好氧微生物計數(shù)的測定，轉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。對酵母菌和霉菌聯(lián)累計數(shù)的測定，轉移另一種濾膜至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.當檢測貼劑時，分別將樣品的制備項下描述的制備品數(shù)量的10%過濾通過兩個無菌濾膜中的一種。為總好氧微生物計數(shù)將一種濾膜轉移至大豆酪蛋白消化物瓊脂培養(yǎng)基，以檢測總好氧微生物計數(shù)，而將另一種濾膜轉移至Sabouraud（沙氏）葡萄糖瓊脂培養(yǎng)基，以檢測總酵母菌和霉菌聯(lián)累計數(shù)。

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