微生物限度培训课件

61MICROBIALLIMITTESTS微生物限度检查THISCHAPTERPROVIDESTESTSFORTHEESTIMATIONOFTHENUMBEROFVIABLEAEROBICMICROORGANISMSPRESENTANDFORFREEDOMFROMDESIGNATEDMICROBIALSPECIESINPHARMACEUTICALARTICLESOFALLKINDS,FROMRAWMATERIALSTOTHEFINISHEDFORMSANAUTOMATEDMETHODMAYBESUBSTITUTEDFORTHETESTSPRESENTEDHERE,PROVIDEDITHASBEENPROPERLYVALIDATEDASGIVINGEQUIVALENTORBETTERRESULTSINPREPARINGFORANDINAPPLYINGTHETESTS,OBSERVEASEPTICPRECAUTIONSINHANDLINGTHESPECIMENSUNLESSOTHERWISEDIRECTED,WHERETHEPROCEDURESPECIFIESSIMPLY“INCUBATE,”HOLDTHECONTAINERINAIRTHATISTHERMOSTATICALLYCONTROLLEDATATEMPERATUREBETWEEN30AND35,FORAPERIODOF24TO48HOURSTHETERM“GROWTH”ISUSEDINASPECIALSENSEHEREIN,IE,TODESIGNATETHEPRESENCEANDPRESUMEDPROLIFERATIONOFVIABLEMICROORGANISMS本章为估计具有繁殖能力的有氧微生物数量和保证从原料到最终剂型都不含有各种药物条款所指定的微生物种群提供检查方法。若某种自动化方法已经证明能提供等效或更好的检查效果，那么可以取代此处所说的检查方法。在准备和开始检查时，观察样品处理过程的无菌预防措施。除非有另外的说明过程中何处只需“培养”的指导，否则将容器置于温度恒控制在3035之间的空气中2448小时。“生长”在此为专用的标明存在和可能存在的有繁殖能力的微生物的增殖的术语。PREPARATORYTESTING预备检验THEVALIDITYOFTHERESULTSOFTHETESTSSETFORTHINTHISCHAPTERRESTSLARGELYUPONTHEADEQUACYOFADEMONSTRATIONTHATTHETESTSPECIMENSTOWHICHTHEYAREAPPLIEDDONOT,OFTHEMSELVES,INHIBITTHEMULTIPLICATION,UNDERTHETESTCONDITIONS,OFMICROORGANISMSTHATMAYBEPRESENTTHEREFORE,PREPARATORYTOCONDUCTINGTHETESTSONAREGULARBASISANDASCIRCUMSTANCESREQUIRESUBSEQUENTLY,INOCULATEDILUTEDSPECIMENSOFTHEMATERIALTOBETESTEDWITHSEPARATEVIABLECULTURESOFSTAPHYLOCOCCUSAUREUS,ESCHERICHIACOLI,PSEUDOMONASAERUGINOSA,ANDSALMONELLATHISCANBEDONEBYADDING1MLOFNOTLESSTHAN103DILUTIONOFA24HOURBROTHCULTUREOFTHEMICROORGANISMTOTHEFIRSTDILUTIONINPH72PHOSPHATEBUFFER,FLUIDSOYBEANCASEINDIGESTMEDIUM,ORFLUIDLACTOSEMEDIUMOFTHETESTMATERIALANDFOLLOWINGTHETESTPROCEDUREFAILUREOFTHEORGANISMSTOGROWINTHERELEVANTMEDIUMINVALIDATESTHATPORTIONOFTHEEXAMINATIONANDNECESSITATESAMODIFICATIONOFTHEPROCEDUREBY1ANINCREASEINTHEVOLUMEOFDILUENT,THEQUANTITYOFTESTMATERIALREMAININGTHESAME,ORBY2THEINCORPORATIONOFASUFFICIENTQUANTITYOFSUITABLEINACTIVATINGAGENTSINTHEDILUENTS,ORBY3ANAPPROPRIATECOMBINATIONOFMODIFICATIONS1AND2SOASTOPERMITGROWTHOFTHEINOCULATHEFOLLOWINGAREEXAMPLESOFINGREDIENTSANDTHEIRCONCENTRATIONSTHATMAYBEADDEDTOTHECULTUREMEDIUMTONEUTRALIZEINHIBITORYSUBSTANCESPRESENTINTHESAMPLESOYLECITHIN,05ANDPOLYSORBATE20,40ALTERNATIVELY,REPEATTHETESTASDESCRIBEDINTHEPRECEDINGPARAGRAPH,USINGFLUIDCASEINDIGESTSOYLECITHINPOLYSORBATE20MEDIUMTODEMONSTRATENEUTRALIZATIONOFPRESERVATIVESOROTHERANTIMICROBIALAGENTSINTHETESTMATERIALWHEREINHIBITORYSUBSTANCESARECONTAINEDINTHEPRODUCTANDTHELATTERISSOLUBLE,ASUITABLE,VALIDATEDADAPTATIONOFAPROCEDURESETFORTHINTHESECTIONMEMBRANEFILTRATIONUNDERTESTFORSTERILITYOFTHEPRODUCTTOBEEXAMINEDUNDERSTERILITYTESTS71,MAYBEUSED本章所述的检查结果的有效性很大程度上取决于实证的适当性，即实证中所用的检验样品的本身在检验条件下不会抑制可能存在的微生物增殖。因此，正在筹备的检验应该依据基本规则及所要求的条件下进行，分别用单独的葡萄球菌、大肠埃希氏菌、假单胞菌和沙门氏菌属的培养物接种待检验物质的稀释样品。通过下面的方法可以得到向待检验的物质的第一份稀释液中加入毫升的稀释度不低于103的含微生物的小时肉汤培养基后继续后面的检验程序。生物体在相应的培养基中增殖的失败会使部分检查无效，同时也使修改检验方法成为必要的，修改检验方法可通过以下方式1增加稀释液的体积，待检验物质的质量不变；2向稀释液中加入足够量的适当的破坏活性制剂；3方法1和2的适当结合使接种物增殖；下面是一些可能加入到培养基中中和样品中存在的抑制性物质的成分和浓聚物的例子大豆卵磷脂，05；聚山梨醇酯20,40另有一种方法可供选择重复前述段落所述的检验，用液态酪蛋白水解液大豆卵磷脂聚山梨醇酯20培养基验证检验物质中的防腐剂或其他抗菌剂。若产物中含有可抑制性物质且可溶，那么无菌检验的待检产品的无菌检验的膜过滤章节中所述的方法可用。IFINSPITEOFTHEINCORPORATIONOFSUITABLEINACTIVATINGAGENTSANDASUBSTANTIALINCREASEINTHEVOLUMEOFDILUENT,ITISSTILLNOTPOSSIBLETORECOVERTHEVIABLECULTURESDESCRIBEDABOVEANDWHERETHEARTICLEISNOTSUITABLEFOREMPLOYMENTOFMEMBRANEFILTRATION,ITCANBEASSUMEDTHATTHEFAILURETOISOLATETHEINOCULATEDORGANISMISATTRIBUTABLETOTHEBACTERICIDALACTIVITYOFTHEPRODUCTTHISINFORMATIONSERVESTOINDICATETHATTHEARTICLEISNOTLIKELYTOBECONTAMINATEDWITHTHEGIVENSPECIESOFMICROORGANISMMONITORINGSHOULDBECONTINUEDINORDERTOESTABLISHTHESPECTRUMOFINHIBITIONANDBACTERICIDALACTIVITYOFTHEARTICLE如果适当的破坏活性剂的结合及稀释液体积的增加仍然无法重新得到上面所描述的具繁殖能力的培养物及药物中不适合使用薄膜过滤法的地方，可以假设隔离接种生物体的失败是由于产物的灭菌活性。该信息表明该药物不可能受到所给的微生物种群的污染。继续监测以建立药物的抑制和灭菌活性谱。BUFFERSOLUTIONANDMEDIA缓冲液和培养基CULTUREMEDIAMAYBEPREPAREDASFOLLOWS,ORDEHYDRATEDCULTUREMEDIAMAYBEUSEDPROVIDEDTHAT,WHENRECONSTITUTEDASDIRECTEDBYTHEMANUFACTURERORDISTRIBUTOR,THEYHAVESIMILARINGREDIENTSAND/ORYIELDMEDIACOMPARABLETOTHOSEOBTAINEDFROMTHEFORMULASGIVENHEREIN依据下列方法准备培养基，或在厂商或销售商的指示下恢复的脱水培养基与公式中得到的具有类似的成分与收率时，那么也可以使用脱水培养基。INPREPARINGMEDIABYTHEFORMULASSETFORTHHEREIN,DISSOLVETHESOLUBLESOLIDSINTHEWATER,USINGHEAT,IFNECESSARY,TOEFFECTCOMPLETESOLUTION,ANDADDSOLUTIONSOFHYDROCHLORICACIDORSODIUMHYDROXIDEINQUANTITIESSUFFICIENTTOYIELDTHEDESIREDPHINTHEMEDIUMWHENITISREADYFORUSEDETERMINETHEPHAT252依照此处所述公式准备培养基时，将可溶固体溶解在水中，若需要可以加热促进完全溶解，使用时加入足够量的盐酸或氢氧化钠以得到所要求的PH值。确定PH值为252WHEREAGARISCALLEDFORINAFORMULA,USEAGARTHATHASAMOISTURECONTENTOFNOTMORETHAN15WHEREWATERISCALLEDFORINAFORMULA,USEPURIFIEDWATERPH72PHOSPHATEBUFFERSTOCKSOLUTIONDISSOLVE34GOFMONOBASICPOTASSIUMPHOSPHATEINABOUT500MLOFWATERCONTAINEDINA1000MLVOLUMETRICFLASKADJUSTTOPH7201BYTHEADDITIONOFSODIUMHYDROXIDETSABOUT175ML,ADDWATERTOVOLUME,ANDMIXDISPENSEANDSTERILIZESTOREUNDERREFRIGERATIONFORUSE,DILUTETHESTOCKSOLUTIONWITHWATERINTHERATIOOF1TO800,ANDSTERILIZE配方中需要琼脂的地方，使用水分不超过15的琼脂，需要水的地方使用蒸馏水。磷酸缓冲液的PH值为72原液将34G磷酸二氢钾溶解在装有500ML水的1000ML烧瓶中。加入氢氧化钠调节PH值到7201，再加入水定容并搅拌。分配并灭菌。冷冻储藏。使用时，按照1800的比率加水稀释原液后灭菌。MEDIA培养基UNLESSOTHERWISEINDICATED,THEMEDIASHOULDBESTERILIZEDBYHEATINGINANAUTOCLAVESEESTEAMSTERILIZATIONUNDERSTERILIZATION1211,THEEXPOSURETIMEDEPENDINGONTHEVOLUMETOBESTERILIZED除非有其他的指导，否则培养基应该置于高压锅中加热灭菌，暴露的时间由需要灭菌的体积决定。IFLUIDCASEINDIGESTSOYLECITHINPOLYSORBATE20MEDIUMPANCREATICDIGESTOFCASEIN20GSOYLECITHIN5GPOLYSORBATE2040MLWATER960MLDISSOLVETHEPANCREATICDIGESTOFCASEINANDSOYLECITHININ960MLOFWATER,HEATINGINAWATERBATHAT48TO50FORABOUT30MINUTESTOEFFECTSOLUTIONADD40MLOFPOLYSORBATE20MIX,ANDDISPENSEASDESIREDI液态酪蛋白消化液大豆卵磷脂聚山梨醇酯20培养基酪蛋白胰消化液大豆卵磷脂聚山梨醇酯20水将酪蛋白胰消化液和大豆卵磷脂溶解在的水中，在4850的水浴中加热分钟促进溶解。加入的聚山梨醇酯20。混合，按照所要求的分配。IISOYBEANCASEINDIGESTAGARMEDIUMPANCREATICDIGESTOFCASEIN150GPAPAICDIGESTOFSOYBEANMEAL50GSODIUMCHLORIDE50GAGAR150GWATER1000MLPHAFTERSTERILIZATION7302大豆酪蛋白消化液琼脂培养基酪蛋白胰消化液150G大豆粉的PAPAIC消化液50G氯化钠50G琼脂50G水1000ML灭菌后的PH值7302IIIFLUIDSOYBEANCASEINDIGESTMEDIUMPREPAREASDIRECTEDFORSOYBEANCASEINDIGESTMEDIUMUNDERSTERILITYTESTS71液态大豆酪蛋白消化液培养基依照无菌检验的大豆酪蛋白消化液培养基制备IVMANNITOLSALTAGARMEDIUMPANCREATICDIGESTOFCASEIN50GPEPTICDIGESTOFANIMALTISSUE50GBEEFEXTRACT10GDMANNITOL100GSODIUMCHLORIDE750GAGAR150GPHENOLRED0025GWATER1000MLMIX,THENHEATWITHFREQUENTAGITATION,ANDBOILFOR1MINUTETOEFFECTSOLUTIONPHAFTERSTERILIZATION7402甘露醇盐琼脂培养基酪蛋白胰消化液50G动物组织胃蛋白酶消化液50G牛肉提取物10GD甘露醇100G氯化钠750G琼脂150G酚红0025G水1000ML混合，加热并频繁搅拌，加热到沸腾一分钟以促进溶解。灭菌后的PH值为7402VBAIRDPARKERAGARMEDIUMPANCREATICDIGESTOFCASEIN100GBEEFEXTRACT50GYEASTEXTRACT10GLITHIUMCHLORIDE50GAGAR200GGLYCINE120GSODIUMPYRUVATE100GWATER950MLHEATWITHFREQUENTAGITATION,ANDBOILFOR1MINUTESTERILIZE,COOLTOBETWEEN45AND50,ANDADD10MLOFSTERILEPOTASSIUMTELLURITESOLUTION1IN100AND50MLOFEGGYOLKEMULSIONMIXINTIMATELYBUTGENTLY,ANDPOURINTOPLATESPREPARETHEEGGYOLKEMULSIONBYDISINFECTINGTHESURFACEOFWHOLESHELLEGGS,ASEPTICALLYCRACKINGTHEEGGS,ANDSEPARATINGOUTINTACTYOLKSINTOASTERILEGRADUATEDCYLINDERADDSTERILESALINETSTOOBTAINA3TO7RATIOOFEGGYOLKTOSALINEADDTOASTERILEBLENDERCUP,ANDMIXATHIGHSPEEDFOR5SECONDSPHAFTERSTERILIZATION6802BAIRDPARKER琼脂培养基酪蛋白胰消化液100G牛肉提取物50G酵母菌提取物10G氯化锂50G琼脂200G甘氨酸120G丙酮酸钠100G水950ML加热并频繁搅拌，沸腾1分钟。灭菌，冷却至4550后加入10ML的无菌亚碲酸钠溶液（1）和50ML的蛋黄乳浊液。逐渐充分混合后倒入皿中。（准备蛋黄乳浊液时，给整颗蛋的外壳消毒，在无菌条件下敲破蛋壳并将其内的蛋黄倒入一个无菌量筒中，加入无菌的盐试液使蛋黄和盐的比例为37，将该溶液倒入一个无菌的搅拌器并在高速下搅拌5秒种。灭菌后的PH值为6802）VIVOGELJOHNSONAGARMEDIUMPANCREATICDIGESTOFCASEIN100GYEASTEXTRACT50GMANNITOL100GDIBASICPOTASSIUMPHOSPHATE50GLITHIUMCHLORIDE50GGLYCINE100GAGAR160GPHENOLRED250MGWATER1000MLBOILTHESOLUTIONOFSOLIDSFOR1MINUTESTERILIZE,COOLTOBETWEEN45AND50,ANDADD20MLOFSTERILEPOTASSIUMTELLURITESOLUTION1IN100PHAFTERSTERILIZATION7202VIVOGELJOHNSON琼脂培养基酪蛋白胰消化液100G牛肉提取物50G甘露醇100G磷酸二氢钾50G氯化锂50G甘氨酸100G琼脂160G酚红250MG水1000ML煮沸含固体的溶液1分钟。灭菌后冷却至4550，加入20ML的无菌亚碲酸钠溶液（1）。灭菌后的PH值为7202VIICETRIMIDEAGARMEDIUMPANCREATICDIGESTOFGELATIN200GMAGNESIUMCHLORIDE14GPOTASSIUMSULFATE100GAGAR136GCETYLTRIMETHYLAMMONIUMBROMIDECETRIMIDE03GGLYCERIN100MLWATER1000MLDISSOLVEALLSOLIDCOMPONENTSINTHEWATER,ANDADDTHEGLYCERINHEAT,WITHFREQUENTAGITATION,ANDBOILFOR1MINUTETOEFFECTSOLUTIONPHAFTERSTERILIZATION7202VII溴化十六烷基三甲铵琼脂培养基明胶胰消化液200G氯化镁14G硫酸钾100G琼脂136G溴化十六烷基三甲铵03G甘氨酸100ML水1000ML将所有的固体成分溶解在水中，加入甘油。加热，频繁搅拌，沸腾1分钟促进溶解。灭菌后的PH值为7202VIIIPSEUDOMONASAGARMEDIUMFORDETECTIONOFFLUORESCINPANCREATICDIGESTOFCASEIN100GPEPTICDIGESTOFANIMALTISSUE100GANHYDROUSDIBASICPOTASSIUMPHOSPHATE15GMAGNESIUMSULFATEMGSO47H2O15GGLYCERIN100MLAGAR150GWATER1000MLDISSOLVETHESOLIDCOMPONENTSINTHEWATERBEFOREADDINGTHEGLYCERINHEAT,WITHFREQUENTAGITATION,ANDBOILFOR1MINUTETOEFFECTSOLUTIONPHAFTERSTERILIZATION7202VIII探测二氢荧光素的假单胞菌琼脂培养基酪蛋白胰消化液100G动物组织胃蛋白酶消化液100G无水磷酸二氢钾15G硫酸镁MGSO47H2O15G甘氨酸100ML琼脂150G水1000ML在加入甘油前将固体成分溶解在水中。加热并频繁搅拌，沸腾1分钟以促进溶解。灭菌后的PH值为7202IXPSEUDOMONASAGARMEDIUMFORDETECTIONOFPYOCYANINPANCREATICDIGESTOFGELATIN200GANHYDROUSMAGNESIUMCHLORIDE14GANHYDROUSPOTASSIUMSULFATE100GAGAR150GGLYCERIN100MLWATER1000MLDISSOLVETHESOLIDCOMPONENTSINTHEWATERBEFOREADDINGTHEGLYCERINHEAT,WITHFREQUENTAGITATION,ANDBOILFOR1MINUTETOEFFECTSOLUTIONPHAFTERSTERILIZATION7202IX探测绿菌素的假单胞菌琼脂培养基明胶胰消化液200G无水氯化镁14G无水硫酸钾100G琼脂150G甘氨酸100ML水1000ML在加入甘油前将固体成分溶解在水中。加热并频繁搅拌，沸腾1分钟以促进溶解。灭菌后的PH值为7202XFLUIDLACTOSEMEDIUMBEEFEXTRACT30GPANCREATICDIGESTOFGELATIN50GLACTOSE50GWATER1000MLCOOLASQUICKLYASPOSSIBLEAFTERSTERILIZATIONPHAFTERSTERILIZATION6902X液态乳糖培养基牛肉提取物30G明胶胰消化液50G乳糖50G水1000ML灭菌后尽可能迅速的冷却。灭菌后的PH为6902XIFLUIDSELENITECYSTINEMEDIUMPANCREATICDIGESTOFCASEIN50GLACTOSE40GSODIUMPHOSPHATE100GSODIUMACIDSELENITE40GLCYSTINE100MGWATER1000MLFINALPH7002MIX,ANDHEATTOEFFECTSOLUTIONHEATINFLOWINGSTEAMFOR15MINUTESDONOTSTERILIZEXI液态亚硒酸盐胱氨酸培养基酪蛋白胰消化液50G乳糖40G磷酸钠100G亚硒酸钠40GL胱氨酸100MG水1000ML最终PH值7002搅拌并加热以促进溶解。在流动蒸汽中加热15分钟。不需灭菌。XIIFLUIDTETRATHIONATEMEDIUMPANCREATICDIGESTOFCASEIN25GPEPTICDIGESTOFANIMALTISSUE25GBILESALTS10GCALCIUMCARBONATE100GSODIUMTHIOSULFATE300GWATER1000MLHEATTHESOLUTIONOFSOLIDSTOBOILINGONTHEDAYOFUSE,ADDASOLUTIONPREPAREDBYDISSOLVING5GOFPOTASSIUMIODIDEAND6GOFIODINEIN20MLOFWATERTHENADD10MLOFASOLUTIONOFBRILLIANTGREEN1IN1000,ANDMIXDONOTHEATTHEMEDIUMAFTERADDINGTHEBRILLIANTGREENSOLUTIONXII液态连四硫酸盐培养基酪蛋白胰消化液25G动物组织胃蛋白消化液25G胆汁盐10G碳酸钙100G硫代硫酸钠300G水1000ML加热含固体的溶液至沸腾。到使用的时候加入用5G碘化钠和6G碘溶解在20ML的水中制成的溶液。然后加入10ML的亮绿溶液（1）并搅拌。加入亮绿溶液后不能加热培养基。XIIIBRILLIANTGREENAGARMEDIUMYEASTEXTRACT30GPEPTICDIGESTOFANIMALTISSUE50GPANCREATICDIGESTOFCASEIN50GLACTOSE100GSODIUMCHLORIDE50GSUCROSE100GPHENOLRED80MGAGAR200GBRILLIANTGREEN125MGWATER1000MLBOILTHESOLUTIONOFSOLIDSFOR1MINUTESTERILIZEJUSTPRIORTOUSE,MELTTHEMEDIUM,POURINTOPETRIDISHES,ANDALLOWTOCOOLPHAFTERSTERILIZATION6902XIII亮绿琼脂培养基酵母菌提取物30G动物组织胃蛋白消化液50G酪蛋白胰消化液50G乳糖100G氯化钠50G蔗糖100G酚红80MG琼脂200G亮绿125MG水1000ML煮沸含固体的溶液1分钟。使用前先灭菌，溶化培养基后倒入培养皿中冷却。灭菌后的PH值为6902XIVXYLOSELYSINEDESOXYCHOLATEAGARMEDIUMXYLOSE35GLLYSINE50GLACTOSE75GSUCROSE75GSODIUMCHLORIDE50GYEASTEXTRACT30GPHENOLRED80MGAGAR135GSODIUMDESOXYCHOLATE25GSODIUMTHIOSULFATE68GFERRICAMMONIUMCITRATE800MGWATER1000MLFINALPH7402HEATTHEMIXTUREOFSOLIDSANDWATER,WITHSWIRLING,JUSTTOTHEBOILINGPOINTDONOTOVERHEATORSTERILIZETRANSFERATONCETOAWATERBATHMAINTAINEDATABOUT50,ANDPOURINTOPLATESASSOONASTHEMEDIUMHASCOOLEDXIV木糖赖氨酸去氧胆酸盐琼脂培养基木糖35GL赖氨酸50G乳糖75G蔗糖75G氯化钠50G酵母菌提取物30G酚红80MG琼脂135G去氧胆酸钠25G硫代硫酸钠68G柠檬酸铁胺800MG水1000ML最终PH7402加热固体与水的混合物，搅拌直到刚刚达到沸点，不要过度加热或灭菌，立即转入50的水浴中，当培养基冷却时立即倒入培养皿中。XVBISMUTHSULFITEAGARMEDIUMBEEFEXTRACT50GPANCREATICDIGESTOFCASEIN50GPEPTICDIGESTOFANIMALTISSUE50GDEXTROSE50GSODIUMPHOSPHATE40GFERROUSSULFATE300MGBISMUTHSULFITEINDICATOR80GAGAR200GBRILLIANTGREEN25MGWATER1000MLFINALPH7602HEATTHEMIXTUREOFSOLIDSANDWATER,WITHSWIRLING,JUSTTOTHEBOILINGPOINTDONOTOVERHEATORSTERILIZETRANSFERATONCETOAWATERBATHMAINTAINEDATABOUT50,ANDPOURINTOPLATESASSOONASTHEMEDIUMHASCOOLEDXV亚硫酸铋琼脂培养基牛肉提取物50G酪蛋白胰消化液50G动物组织胃蛋白消化液50G葡萄糖50G磷酸钠40G硫酸亚铁300MG亚硫酸铋指示剂80G琼脂200G亮绿25MG水1000ML最终PH7602加热固体与水的混合物，搅拌直到刚刚达到沸点，不要过度加热或灭菌，立即转入50的水浴中，当培养基冷却时立即倒入培养皿中。XVITRIPLESUGARIRONAGARMEDIUMPANCREATICDIGESTOFCASEIN100GPANCREATICDIGESTOFANIMALTISSUE100GLACTOSE100GSUCROSE100GDEXTROSE10GFERROUSAMMONIUMSULFATE200MGSODIUMCHLORIDE50GSODIUMTHIOSULFATE200MGAGAR130GPHENOLRED25MGWATER1000MLPHAFTERSTERILIZATION7302XVI三糖铁琼脂培养基酪蛋白胰消化液100G动物组织胃蛋白消化液100G乳糖100G蔗糖100G葡萄糖10G硫酸亚铁胺200MG氯化钠50G硫代硫酸钠200MG琼脂130G酚红25MG水1000ML灭菌后的PH值为7302XVIIMACCONKEYAGARMEDIUMPANCREATICDIGESTOFGELATIN170GPANCREATICDIGESTOFCASEIN15GPEPTICDIGESTOFANIMALTISSUE15GLACTOSE100GBILESALTSMIXTURE15GSODIUMCHLORIDE50GAGAR135GNEUTRALRED30MGCRYSTALVIOLET10MGWATER1000MLBOILTHEMIXTUREOFSOLIDSANDWATERFOR1MINUTETOEFFECTSOLUTIONPHAFTERSTERILIZATION7102XVIIMACCONKEY琼脂培养基明胶胰消化液170G酪蛋白胰消化液15G动物组织胃蛋白消化液15G乳糖100G胆汁盐混合物15G氯化钠50G琼脂135G中性红30MG水晶紫10MG水1000ML煮沸固体与水的混合物1分钟，以促进溶解。灭菌后的PH值为7102XVIIILEVINEEOSINMETHYLENEBLUEAGARMEDIUMPANCREATICDIGESTOFGELATIN100GDIBASICPOTASSIUMPHOSPHATE20GAGAR150GLACTOSE100GEOSINY400MGMETHYLENEBLUE65MGWATER1000MLDISSOLVETHEPANCREATICDIGESTOFGELATIN,THEDIBASICPOTASSIUMPHOSPHATE,ANDTHEAGARINTHEWATER,WITHWARMING,ANDALLOWTOCOOLJUSTPRIORTOUSE,LIQUEFYTHEGELLEDAGARSOLUTION,ADDTHEREMAININGINGREDIENTS,ASSOLUTIONS,INTHEFOLLOWINGAMOUNTS,ANDMIXFOREACH100MLOFTHELIQUEFIEDAGARSOLUTION5MLOFLACTOSESOLUTION1IN5,2MLOFTHEEOSINYSOLUTION1IN50,AND2MLOFMETHYLENEBLUESOLUTION1IN300THEFINISHEDMEDIUMMAYNOTBECLEARPHAFTERSTERILIZATION7102XVIII列文伊红亚甲基蓝琼脂培养基明胶胰消化液100G磷酸二氢钾20G琼脂150G乳糖100GY伊红400MG亚甲基蓝65MG水1000ML将明胶胰消化液、磷酸二氢钾和琼脂溶解在水中，加热促进溶解后再冷却。使用前，使凝固的琼脂溶液液化，加入下列成分每100ML液化的琼脂溶液5ML的乳糖溶液（体积比15），2ML的伊红Y溶液（体积比150），2ML的亚甲基蓝溶液（体积比1300）。最终的培养基可能不清澈。灭菌后的PH值为7102XIXSABOURAUDDEXTROSEAGARMEDIUMDEXTROSE40GMIXTUREOFEQUALPARTSOFPEPTICDIGESTOFANIMALTISSUEANDPANCREATICDIGESTOFCASEIN10GAGAR15GWATER1000MLMIX,ANDBOILTOEFFECTSOLUTIONPHAFTERSTERILIZATION5602XIX萨布罗右旋糖琼脂培养基右旋糖40G等量的动物组织胃蛋白酶消化液和酪蛋白胰消化液的混合物10G琼脂15G水1000ML搅拌并使溶液沸腾促进溶解。灭菌后的PH值为5602XXPOTATODEXTROSEAGARMEDIUMCOOK300GOFPEELEDANDDICEDPOTATOESIN500MLOFWATERPREPAREDBYDISTILLATION,FILTERTHROUGHCHEESECLOTH,ADDWATERPREPAREDBYDISTILLATIONTOMAKE1000ML,ANDADDTHEFOLLOWINGAGAR15GGLUCOSE20GDISSOLVEBYHEATING,ANDSTERILIZEPHAFTERSTERILIZATION5602FORUSE,JUSTPRIORTOPOURINGTHEPLATES,ADJUSTTHEMELTEDANDCOOLEDTO45MEDIUMWITHSTERILETARTARICACIDSOLUTION1IN10TOAPHOF3501DONOTREHEATTHEPH35MEDIUMXX马铃薯葡萄糖琼脂培养基将300G已削皮并切成块状的马铃薯放在500ML的蒸馏水中煮，然后用纱布过滤，并用蒸馏水稀释到1000ML，再加入下列物质琼脂15G右旋糖20G加热溶解并灭菌。灭菌后的PH为5602使用时，在倒入培养皿前先溶解并冷却到45，向培养基中加入无菌酒石酸溶液使其PH值为3501PH为35的培养基无需再加热。SAMPLING样品PROVIDESEPARATE10MLOR10GSPECIMENSFOREACHOFTHETESTSCALLEDFORINTHEINDIVIDUALMONOGRAPH为在单独专论中的每个测试提供10ML或10G的样品。PROCEDURE方法PREPARETHESPECIMENTOBETESTEDBYTREATMENTTHATISAPPROPRIATETOITSPHYSICALCHARACTERISTICSANDTHATDOESNOTALTERTHENUMBERANDKINDOFMICROORGANISMSORIGINALLYPRESENT,INORDERTOOBTAINASOLUTIONORSUSPENSIONOFALLORPARTOFITINAFORMSUITABLEFORTHETESTPROCEDURESTOBECARRIEDOUTFORASOLIDTHATDISSOLVESTOANAPPRECIABLEEXTENTBUTNOTCOMPLETELY,REDUCETHESUBSTANCETOAMODERATELYFINEPOWDER,SUSPENDITINTHEVEHICLESPECIFIED,ANDPROCEEDASDIRECTEDUNDERTOTALAEROBICMICROBIALCOUNT,ANDUNDERTESTFORSTAPHYLOCOCCUSAUREUSANDPSEUDOMONASAERUGINOSAANDTESTFORSALMONELLASPECIESANDESCHERICHIACOLI准备待测试的样品时要采取适合其自然特点并且不改变其原来所有的微生物数量和种类的方法，目的是得到与要实行的实验过程相配合的全部或部分样品的溶液或悬浮液。对于溶解到可估计但不完全的程度的固体，将该物质磨成一定精细的粉末后悬浮在指定的溶剂中，再依照总有氧微生物计数、葡萄球菌和假单胞菌的检验及沙门氏菌属和大肠杆菌的检验的指导操作。FORAFLUIDSPECIMENTHATCONSISTSOFATRUESOLUTION,ORASUSPENSIONINWATERORAHYDROALCOHOLICVEHICLECONTAININGLESSTHAN30PERCENTOFALCOHOL,ANDFORASOLIDTHATDISSOLVESREADILYANDPRACTICALLYCOMPLETELYIN90MLOFPH72PHOSPHATEBUFFERORTHEMEDIASPECIFIED,PROCEEDASDIRECTEDUNDERTOTALAEROBICMICROBIALCOUNT,ANDUNDERTESTFORSTAPHYLOCOCCUSAUREUSANDPSEUDOMONASAERUGINOSAANDTESTFORSALMONELLASPECIESANDESCHERICHIACOLI对含有真溶液或悬浮液或乙醇含量不少于30的水醇溶剂与在90ML的PH为72的磷酸缓冲液或指定的培养基中能够充分溶解的固体，依照总有氧微生物计数检验、葡萄球菌与假单胞菌检验以及沙门氏菌属与大肠杆菌检验的指导进行操作。FORWATERIMMISCIBLEFLUIDS,OINTMENTS,CREAMS,ANDWAXES,PREPAREASUSPENSIONWITHTHEAIDOFAMINIMALQUANTITYOFASUITABLE,STERILEEMULSIFYINGAGENTSUCHASONEOFTHEPOLYSORBATES,USINGAMECHANICALBLENDERANDWARMINGTOATEMPERATURENOTEXCEEDING45,IFNECESSARY,ANDPROCEEDWITHTHESUSPENSIONASDIRECTEDUNDERTOTALAEROBICMICROBIALCOUNT,ANDUNDERTESTFORSTAPHYLOCOCCUSAUREUSANDPSEUDOMONASAERUGINOSAANDTESTFORSALMONELLASPECIESANDESCHERICHIACOLI对与水不相溶的液体、油膏、乳膏和石蜡，用适当的最小量无菌乳化剂（如聚山梨醇酯的一种）配制悬浮液，可用机械搅拌器搅拌并加热，但不能超过45，如果需要，依照总有氧微生物计数、葡萄球菌与假单胞菌的检验以及沙门氏菌属与大肠杆菌检验的指导进行操作。FORAFLUIDSPECIMENINAEROSOLFORM,CHILLTHECONTAINERINANALCOHOLDRYICEMIXTUREFORAPPROXIMATELY1HOUR,CUTOPENTHECONTAINER,ALLOWITTOREACHROOMTEMPERATURE,PERMITTHEPROPELLANTTOESCAPE,ORWARMTODRIVEOFFTHEPROPELLANTIFFEASIBLE,ANDTRANSFERTHEQUANTITYOFTESTMATERIALREQUIREDFORTHEPROCEDURESSPECIFIEDINONEOFTHETWOPRECEDINGPARAGRAPHS,ASAPPROPRIATEWHERE100GOR100MLOFTHESPECIMEN,WHICHEVERISAPPLICABLE,CANNOTBEOBTAINEDFROM10CONTAINERSINAEROSOLFORM,TRANSFERTHEENTIRECONTENTSFROM10CHILLEDCONTAINERSTOTHECULTUREMEDIUM,PERMITTHEPROPELLANTTOESCAPE,ANDPROCEEDWITHTHETESTONTHERESIDUESIFTHERESULTSOFTHETESTAREINCONCLUSIVEORDOUBTFUL,REPEATTHETESTWITHASPECIMENFROM20MORECONTAINERS对气雾形式的液态样品，将装有样品的容器放入乙醇干冰混合物中冷藏大约1个小时，截开容器使其达到室温，允许气体逸出，或若可行则加热使气体逸出，移出一定量的在前述的两个章节的一个中详细说明的过程所需的检验物质。当不能从10个装有气雾剂形式的容器中得到100G或100ML的样品时，无论哪种都可用，将10个冷藏的容器全部放入培养基中，允许压缩气体逸出，用残留物进行操作。如果检验结果不能决定或可疑，则使用从20个以上的容器中得到的样品重复检验。TOTALAEROBICMICROBIALCOUNT总有氧微生物计数FORSPECIMENSTHATARESUFFICIENTLYSOLUBLEORTRANSLUCENTTOPERMITUSEOFTHEPLATEMETHOD,USETHATMETHODOTHERWISE,USETHEMULTIPLETUBEMETHODWITHEITHERMETHOD,FIRSTDISSOLVEORSUSPEND100GOFTHESPECIMENIFITISASOLID,OR10ML,ACCURATELYMEASURED,IFTHESPECIMENISALIQUID,INPH72PHOSPHATEBUFFER,FLUIDSOYBEANCASEINDIGESTMEDIUM,ORFLUIDCASEINDIGESTSOYLECITHINPOLYSORBATE20MEDIUMTOMAKE100MLFORVISCOUSSPECIMENSTHATCANNOTBEPIPETEDATTHISINITIAL110DILUTION,DILUTETHESPECIMENUNTILASUSPENSIONISOBTAINED,IE,150OR1100,ETC,THATCANBEPIPETEDPERFORMTHETESTFORABSENCEOFINHIBITORYANTIMICROBIALPROPERTIESASDESCRIBEDUNDERPREPARATORYTESTINGBEFORETHEDETERMINATIONOFTOTALAEROBICMICROBIALCOUNTADDTHESPECIMENTOTHEMEDIUMNOTMORETHAN1HOURAFTERPREPARINGTHEAPPROPRIATEDILUTIONSFORINOCULATION对能使用平板计数法的能充分溶解或透明的样品，使用平板计数法，否则使用多管法。无论使用哪种方法，如果样品是固体则将100G样品溶解或悬浮在PH为72的磷酸缓冲液和液态大豆酪蛋白胰消化液培养基或者液态酪蛋白消化液大豆卵磷脂聚山梨醇酯20培养基的混合物中，若样品是液体则准确量取10ML直接加入上述培养基中，最终体积均为100ML。对在初始的稀释度为110时不能用球管吸量的粘性样品，稀释样品直到得到能用球管吸量悬浮液，如稀释度为150或1100等等。总的有氧微生物计数前在无抑制物质（如灭菌剂）的情况下依照预备试验进行检验。准备好适当稀释度的接种物后在一个小时内向培养基中加入样品。PLATEMETHOD平板计数法DILUTEFURTHER,IFNECESSARY,THEFLUIDSOTHAT1MLWILLBEEXPECTEDTOYIELDBETWEEN30AND300COLONIESPIPET1MLOFTHEFINALDILUTIONONTOEACHOFTWOSTERI