EP802613非无菌药品的微生物限度检查特殊微生物的检查(中英对照).doc

EP802613非无菌药品的微生物限度检查特殊微生物的检查(中英对照) EP8.004/xx:206132.6.13.MICROBIOLOGICAL EXAMINATIONOF NON-STERILE PRODUCTS:TEST FORSPECIFIED MICRO-ORGANISMS (3)非无菌药品的微生物限度检查特殊微生物的检查1.INTRODUCTION导言The testsdescribed hereafterwill allowdetermination of the absenceor limitedourrence ofspecified micro-organisms thatmay be detected underthe conditionsdescribed.下述检验方法用于检查在描述的试验条件下特定微生物的定性及限度。 The tests are designedprimarily todetermine whethera substanceor preparationplies with an establishedspecification for microbiological quality.When usedfor suchpurposes,follow theinstructions givenbelow,including thenumber of samples to be taken,and interpretthe resultsas statedbelow.检验的主要目的是为了确定是否原料药或制剂符合已建立的微生物限度标准，当用于这一目的时，应按照以下方式（包括取样量），进行并按照下述描述对结果进行分析。 Alternative microbiologicalprocedures,including automatedmethods,may beused,provided that their equivalenceto thePharmacopoeia methodhas beendemonstrated.如果可证明某种试验方法（包括自动化分析法）的效果与药典中的方法等同，该方法可作为另一种供选择的试验方法。 2.GENERAL PROCEDURES一般程序The preparationofsamplesis carriedout as described in general chapter2.6.12.样品的制备方法参见（2.6.12）。 If the product to be examinedhas antimicrobial activity,this isinsofar aspossible removedor neutralisedas described in general chapter2.6.12.如果供试品有抗微生物活性，按照（2.6.12）中描述的方法对其进行中和。 If surface-active substancesare usedfor sample preparation,their absenceof toxicityformicro-organisms and their patibilitywith inactivatorsused must be demonstratedas described in general chapter2.6.12.如果样品的制备过程中使用了表面活性剂，其必须满足（2.6.12）中的要求，对微生物无毒性并且可与灭活剂兼容。 3.GROWTH-PROMOTING AND INHIBITORY PROPERTIES OF THE MEDIA,SUITABILITY OFTHE TESTAND NEGATIVE CONTROLS培养基中的生长促进作用和生长抑制作用，试验方法的适应性和阴性对照试验The abilityof the test todetect micro-organisms in the presence of the product to be testedmustbeestablished.Suitability mustbe confirmedif achange intesting performance,or the product,which mayaffect theoute of the testis introduced.该试验方法要有一定的检测微生物的能力，如果影响试验结果的操作或被测物品发生变化，该变化可能会影响试验结果时，该试验方法的适应性必须确认。 3-1.PREPARATION OFTEST STRAINS试验用菌株的制备Use standardisedstable suspensionsof test strains orprepare themas statedbelow.Seed lotculture maintenancetechniques(seed-lot systems)are usedso that the viablemicro-organisms usedfor inoculationare not more than5passages removedfrom theoriginal masterseed-lot.使用标准稳定的试验用菌株的菌悬液或按照以下方法制备，使用种子批传代次数不得超过5代。 3-1-1.Aerobic micro-organisms.Grow eachof thebacterial test strains separatelyin casein soya bean digest broth or oncasein soya bean digestagar at30-35C for18-24h.Grow the teststrainfor Candida albicans separatelyon Sabouraud-dextrose agaror inSabouraud-dextrose brothat20-25C for2-3days.3-1-1需氧菌将试验用菌株分别接种于胰酪大豆胨液体培养基或胰酪大豆胨琼脂培养基中30-35培养18-24h。 将白色念珠菌试验菌株接种于沙氏葡萄糖琼脂培养基或沙氏葡萄糖液体培养基中20-25培养2-3天。 Staphylocous aureussuch as ATCC6538,NCIMB9518,CIP4.83or NBRC13276;金黄色葡萄球菌例如ATCC6538,NCIMB9518,CIP4.83或NBRC13276;Pseudomonas aeruginosasuch asATCC9027,NCIMB8626,CIP82.118or NBRC13275;铜绿假单胞菌例如ATCC9027,NCIMB8626,CIP82.118o或NBRC13275;Escherichia colisuch asATCC8739,NCIMB8545,CIP53.126or NBRC3972;大肠埃希菌例如ATCC8739,NCIMB8545,CIP53.126或NBRC3972;Salmonella enterica subsp.enterica serovarTyphimurium,such asATCC14028or,as analternative,Salmonella enterica subsp.enterica serovarAbony such as NBRC100797,NCTC6017or CIP80.39;沙门氏菌例如ATCC14028或NBRC100797,NCTC6017或CIP80.39;Candida albicanssuchasATCC10231,NCPF3179,IP48.72or NBRC1594.白色念珠菌例如ATCC10231,NCPF3179,IP48.72或NBRC1594.Use bufferedsodium chloride-peptone solution pH7.0or phosphatebuffer solutionpH7.2to maketest suspensions.Use thesuspensions within2horwithin24h ifstored at2-8C.使用pH为7.0的氯化钠-蛋白胨缓冲液或pH为7.2的磷酸盐缓冲液制备试验用菌悬液。 应在2小时内使用菌悬液，如果保存在2-8条件下应在24小时内使用。 3-1-2.Clostridia.Use Clostridiumsporogenes suchasATCC11437(NBRC14293,NCIMB12343,CIP100651)or ATCC19404(NCTC532or CIP79.03)or NBRC14293.Grow theclostridial teststrain under anaerobic conditionsin reinforcedmedium for clostridia at30-35C for24-48h.As analternative topreparing andthen dilutingdown afresh suspensionof vegetativecells of Cl.sporogenes,a stablespore suspensionis usedfor testinoculation.The stablespore suspensionmay bemaintained at2-8C fora validatedperiod.3-1-2梭菌使用梭状芽孢杆菌，例如ATCC11437(NBRC14293,NCIMB12343,CIP100651)或ATCC19404(NCTC532或CIP79.03)或NBRC14293，在厌氧条件下将梭菌试验用菌株接种于增菌培养基中，在30-35条件下培养24-48小时。 将新鲜的有活性的梭状芽孢杆菌孢子悬液稀释后用于接种。 稳定的孢子悬液可保存在2-8条件下，在经过验证的贮存期内使用。 3-2.NEGATIVECONTROL阴性对照试验To verifytesting conditions,a negative control isperformed using the chosendiluent inplace of the test preparation.There mustbe nogrowth ofmicro-organisms.A negativecontrol isalso performedwhen testingthe productsas described in section4.A failednegativecontrolrequires aninvestigation.为检测试验条件是否符合要求，取试验用稀释剂代替供试品做一阴性对照试验，阴性对照试验应无微生物生长。 按照第四部分检测供试品时，也要做一阴性对照试验。 当阴性对照试验结果不符合要求时，需要进行偏差调查。 3-3.GROWTH PROMOTIONANDINHIBITORYPROPERTIESOFTHEMEDIA培养基的生长促进作用和生长抑制作用Test eachbatch ofready-prepared mediumand eachbatch ofmedium preparedeither fromdehydrated mediumor fromingredients.每一批成品培养基，以及由脱水培养基或由规定的处方配制的培养基都应进行适用性检查。 Verify suitableproperties ofrelevant mediaas described in Table2.6.13.-1.按照表（2.6.13.-1），检测相关培养基的特性。 Table2.6.13.-1Growth promoting,inhibitory andindicative propertiesof media培养基的生长促进作用、生长抑制作用和指示作用Medium培养基Property特性Test strains试验菌株Test forbile-tolerant gram-negative bacteria耐胆盐革兰氏阴性菌的检测Enterobacteria enrichmentbroth-Mossel肠道菌增菌肉汤培养基Growth promoting生长促进E.coli大肠埃希菌P.aeruginosa铜绿假单胞菌Inhibitory生长抑制S.aureus金黄色葡萄球菌Violet red bile glucoseAgar紫红胆汁葡萄糖琼脂培养基Growth promoting+indicative生长促进和指示作用E.coli大肠埃希菌P.aeruginosa铜绿假单胞菌Test forEscherichia Coli大肠埃希菌的检测MacConkey broth麦康凯肉汤培养基Growth promoting生长促进E.coli大肠埃希菌Inhibitory生长抑制S.aureus金黄色葡萄球菌MacConkey agar麦康凯琼脂培养基Growth promoting+indicative生长促进和指示作用E.coli大肠埃希菌Test forSalmonella沙门氏菌的检测Rappaport VassiliadisSalmonella enrichmentBroth沙门氏菌琼脂培养基Growth promoting生长促进Salmonella entericasubsp.enterica serovarTyphimurium orSalmonella en-tericasubsp.enterica serovarAbony沙门氏菌Inhibitory生长抑制S.aureus金黄色葡萄球菌Xylose,lysine,deoxycholate agarGrowth promoting+indicative生长促进和指示作用Salmonella entericasubsp.enterica serovarTyphimurium orSalmonella ent木糖赖氨酸去氧胆酸盐琼脂培养基ericasubsp.enterica serovarAbony沙门氏菌Inhibitory指示作用E.coli大肠埃希菌Test forPseudomonas Aeruginosa铜绿假单胞菌的检测Cetrimide agar溴棕三甲铵琼脂培养基Growth promoting生长促进P.aeruginosa铜绿假单胞菌Inhibitory生长抑制E.coli大肠埃希菌Test forStaphylocous aureus金黄色葡萄球菌的检测Mannitol salt agar甘露醇氯化钠琼脂培养基Growth promoting+indicative生长促进和指示作用S.aureus金黄色葡萄球菌Inhibitory生长抑制剂E.coli大肠埃希菌Test for clostridia梭菌的检测Reinforced medium forclostridia增菌培养基Growth promoting生长促进Cl.Sporogenes梭状芽孢杆菌Columbia agar哥伦比亚琼脂培养基Growth promoting生长促进Cl.Sporogenes梭状芽孢杆菌Test forCandidaalbicans白色念珠菌的检测Sabouraud dextroseBroth沙氏葡萄糖肉汤培养基Growth promoting生长促进C.albicans白色念珠菌Sabouraud dextrose agar沙氏葡萄糖琼脂培养基Growth promoting+indicative生长促进和指示作用C.albicans白色念珠菌Test forgrowth promotingproperties,liquid media:inoculate aportion of the appropriate medium with a smallnumber(not more than100CFU)of the appropriate micro-organism.Incubate atthe specifiedtemperature fornot morethan theshortest periodof timespecified in the test.Clearly visiblegrowth of the micro-organism parableto thatpreviously obtainedwith apreviously testedand approvedbatch ofmedium ours.液态培养基的促生长能力检查将适量的微生物（100CFU）接种于培养基中,在指定温度下培养，培养时间应小于微生物试验时规定的最短时间。 微生物的生长清晰可见，并且与已批准合格的培养基中微生物的生长情况类似，说明该液体培养基符合要求。 Test forgrowth promotingproperties,solid media:perform thesurface-spread method,inoculating eachplate with a smallnumber(not morethan100CFU)of theappropriate micro-organism.Incubate atthe specifiedtemperature fornot morethan theshortest periodof timespecified in the test.Growth of the micro-organism parableto thatpreviously obtainedwithapreviously testedand approvedbatch ofmedium ours.固态培养基的促生长能力检查使用表面涂布法，将适量的微生物（100CFU）接种于培养基中，在指定温度下培养，培养时间应大于微生物试验时规定的最长时间，微生物不生长。 Test forindicative properties:perform thesurface-spread method,inoculating eachplate witha smallnumber(not morethan100CFU)oftheappropriatemicro-organism.Incubate atthe specifiedtemperature fora periodof timewithin therange specifiedin the test.Colonies areparable inappearance andindication reactionsto thosepreviously obtainedwithapreviously testedand approvedbatch ofmedium.培养基中指示能力的检查使用表面涂布平皿法，将适量的微生物（10310310104-2.ESCHERICHIA COLI大肠埃希菌4-2-1.Sample preparationand pre-incubation.Prepare asample using a1in10dilution ofnot lessthan1g oftheproduct tobe examined asdescribedin general chapter2.6.12,and use10mL orthe quantitycorresponding to1g or1mL to inoculate asuitable amount(determined asdescribed under3-4)of casein soya bean digest broth,mix and incubate at30-35C for18-24h.样品的制备和增菌培养按照（2.6.12）中的方法取不少于1g的供试品制成1:10的供试液，取供试液10ml，或相当于含供试品1g或1ml的供试液，接种于适量的胰酪大豆胨液体培养基中（符合3-4的要求），30-35条件下培养18-24小时。 4-2-2.Selection andsubculture.Shake thecontainer,transfer1mL of casein soya bean digest broth to100mL of MacConkey broth and incubate at42-44C for24-48h.Subculture ona plateofMacConkey agar at30-35C for18-72h.选择和分离培养振动容器，将1ml胰酪大豆胨液体培养物转移到100ml麦康凯肉汤培养基中，42-44条件下培养24-48小时后，培养物接种于麦康凯琼脂培养基中，30-35条件下培养18-72小时。 4-2-3.Interpretation.Growth ofcolonies indicatesthe possiblepresence ofE.coli.This isconfirmed byidentification tests.The productplies withthetestif nocolonies arepresent or if theidentification tests are negative.结果分析如果有菌落生长，说明供试品中含有大肠埃希菌。 是否确实含有大肠埃希菌，需做鉴别试验。 如果无菌落生长或鉴别试验为阴性，说明供试品中不含有大肠埃希菌。 4-3.SALMONELLA沙门菌4-3-1.Sample preparationand pre-incubation.Prepare theproducttobeexamined asdescribedin generalchapter2.6.12,and usethe quantitycorresponding tonot lessthan10g or10mL toinoculate asuitable amount(determined asdescribed under3-4)of casein soya bean digest broth,mix and incubate at30-35C for18-24h.供试液的制备和增菌培养按照（2.6.12）中的方法制备样品，将相当于含供试品10g或10ml的供试液接种于适量的胰酪大豆胨液体培养基中（符合3-4的要求），30-35条件下培养18-24小时。 4-3-2.Selection andsubculture.Transfer0.1mL of caseinsoya bean digestbroth to10mL ofRappaport VassiliadisSalmonella enrichmentbrothandincubate at30-35C for18-24h.Subculture onplates ofxylose,lysine,deoxycholate agar.Incubate at30-35C for18-48h.选择和分离培养将0.1ml胰酪大豆胨液体培养物转移到10ml RV沙门增菌液体培养基中，30-35条件下培养18-24小时后，培养物接种于木糖赖氨酸去氧胆酸盐琼脂培养基中，30-35条件下培养18-48小时。 4-3-3.Interpretation.The possiblepresence ofSalmonella isindicated bythe growthof well-developed,red colonies,with orwithout blackcentres.This isconfirmed byidentification tests.The productplies withthetestif coloniesofthetypes describedare not present orif the confirmatory identification tests are negative.结果分析如果有红色菌落生长，菌落有或无黑色中心，说明供试品中含有沙门氏菌。 是否确实含有沙门氏菌，需做鉴别试验。 如果菌落外观不是此种特征或鉴别试验结果为阴性，说明供试品中不含有沙门氏菌。 4-4.PSEUDOMONAS AERUGINOSA铜绿假单胞菌4-4-1.Sample preparationand pre-incubation.Prepare asample using a1in10dilution ofnot lessthan1g oftheproducttobeexamined asdescribedingeneralchapter2.6.12,and use10mL orthe quantitycorresponding to1g or1mL toinoculate asuitable amount(determined asdescribed under3-4)of caseinsoyabean digestbrothand mix.When testingtransdermal patches,filter thevolume ofsample corresponding to1patch ofthe preparationdescribed under4-5-1ingeneralchapter2.6.12through asterile filtermembrane andplace in100mL of caseinsoyabeandigestbroth.Incubate at30-35C for18-24h.供试品液的制备和增菌培养按照（2.6.12）中的方法取不少于1g的供试品制成1:10的供试液，取供试液10ml，或相当于含供试品1g或1ml的供试液，接种于适量的胰酪大豆胨液体培养基中（符合3-4的要求）。 当检测透皮贴剂时，取一剂量供试品按照（2.6.124-5-1）中的方法使用无菌滤膜过滤后，接种于100ml胰酪大豆胨液体培养基中，30-35条件下培养18-24小时。 4-4-2.Selection andsubculture.Subculture ona plateof cetrimideagar andincubateat30-35C for18-72h.选择和分离培养接种于溴化十六烷基三甲铵琼脂培养基中，30-35条件下培养18-72小时4-4-3.Interpretation.Growth ofcolonies indicatesthe possiblepresence ofP.aeruginosa.This isconfirmed byidentification tests.The productplies withthetestif colonies are notpresent orif theconfirmatory identification tests arenegative.结果分析如果有菌落生长，说明供试品中可能含有铜绿假单胞菌。 是否确实含有铜绿假单胞菌，需做鉴别试验。 如果无菌落生长或鉴别试验为阴性，说明供试品中不含有铜绿假单胞菌。 4-5.STAPHYLOCOCCUS AUREUS金黄色葡萄球菌4-5-1.Sample preparationand pre-incubation.Prepare asample using a1in10dilution ofnot lessthan1g oftheproducttobeexamined asdescribedingeneralchapter2.6.12,and use10mL orthe quantitycorresponding to1g or1mL toinoculate asuitable amount(determined asdescribed under3-4)of caseinsoyabeandigestbrothand mix.When testingtransdermal patches,filter thevolume ofsample correspondingto1patch ofthe preparationdescribed under4-5-1ingeneralchapter2.6.12through asterile filtermembrane andplace in100mL ofcaseinsoyabeandigestbroth.Incubate at30-35C for18-24h.供试品液的制备和增菌培养按照（2.6.12）中的方法取不少于1g的供试品制成1:10的供试液，取供试液10ml，或相当于含供试品1g或1ml的供试液，接种于适量的胰酪大豆胨液体培养基中（符合3-4的要求）。 当检测透皮贴剂时，取一剂量供试品按照（2.6.124-5-1）中的方法使用无菌滤膜过滤后，接种于100ml胰酪大豆胨液体培养基中，30-35条件下培养18-24小时。 4-5-2.Selection andsubculture.Subculture ona plateof mannitolsaltagarandincubateat30-35C for18-72h.选择和分离培养接种于甘露醇氯化钠琼脂培养基中，30-35条件下培养18-72小时。 4-5-3.Interpretation.The possiblepresenceofS.aureus isindicated bythe growthof yellow/white coloniessurrounded bya yellowzone.This isconfirmed byidentification tests.The productplies withthetestif coloniesofthetypes describedare notpresent orif theconfirmatory identification testsarenegative.结果分析如果有黄色/白色菌落生长，外围为黄色，表明供试品中可能含有金黄色葡萄球菌。 是否确实含有金黄色葡萄球菌，需做鉴别试验。 如果菌落外观不是此种特征或鉴别试验结果为阴性，说明供试品中不含有金黄色葡萄球菌。 4-6.CLOSTRIDIA梭菌4-6-1.Sample preparationand heattreatment.Prepare asample using a1in10dilution(withaminimum totalvolume of20mL)ofnot lessthan2g or2mL oftheproducttobeexaminedasdescribedingeneralchapter2.6.12.Divide thesample into2portions ofatleast10mL.Heat1portion at80C for10min andcool rapidly.Do notheat theother portion.供试液制备和热处理按照（2.6.12）中的方法取不少于2g或2ml的供试品制成1:10的供试液（供试液至少为20ml），将样品分成两部分，每部分至少为10ml。 其中一份样品80加热10分钟后迅速冷却，另一份样品不需要加热。 4-6-2.Selection andsubculture.Use10mL orthe quantitycorrespondingto1g or1mL oftheproducttobeexamined ofboth portionstoinoculatesuitable amounts(determined asdescribedunder3-4)of reinforcedmediumforclostridia.Incubate underanaerobic conditions at30-35C for48h.After incubation,make subculturesfrom eachcontainer onColumbia agarandincubateunderanaerobionditionsat30-35C for48-72h.选择和分离培养使用10ml相当于1g或1ml的供试品的供试液接种于适量的培养梭菌用增菌培养基中（符合3-4的要求），在厌氧条件下30-35培养48小时。 分离培养接种于哥伦比亚培养基中，在厌氧条件下30-35培养48-72小时。 4-6-3.Interpretation.The ourrenceof anaerobicgrowthofrods(with orwithout endospores)giving anegative catalasereaction indicatesthe presenceof clostridia.This isconfirmed byidentificationtests.The productplies withthetestif coloniesofthetypes describedare notpresent orif theconfirmatory identificationtestsarenegative.结果分析如果厌氧条件下有杆状菌生长（有或无孢子内壁），过氧化氢酶反应结果为阴性，说明供试品中含有梭菌。 4-7.CANDIDA ALBICANS白色念珠菌4-7-1.Sample preparationand pre-incubation.Prepare theproducttobeexaminedasdescribedingeneralc