EP 8.0-2.6.13 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

EP 8.004/2010:206132.6.13. MICROBIOLOGICAL EXAMINATION OF NON-STERILE PRODUCTS: TEST FOR SPECIFIED 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 tests described hereafter will allow determination of the absence or limited occurrence of specified micro-organisms that may be detected under the conditions described.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 designed primarily to determine whether a substance or preparation complies with an established specification for microbiological quality. When used for such purposes, follow the instructions given below, including the number of samples to be taken, and interpret the results as stated below.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 microbiological procedures, including automated methods, may be used, provided that their equivalence to the Pharmacopoeia method has been demonstrated. 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 preparation of samples is carried out as described in general chapter 2.6.12.样品的制备方法参见（2.6.12）。If the product to be examined has antimicrobial activity, this is insofar as possible removed or neutralised as described in general chapter 2.6.12.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 substances are used for sample preparation, their absence of toxicity for micro-organisms and their compatibility with inactivators used must be demonstrated as described in general chapter 2.6.12.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 OF THE TEST AND 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 ability of the test to detect micro-organisms in the presence of the product to be tested must be established. Suitability must be confirmed if a change in testing performance, or the product, which may affect the outcome of the test is 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 OF TEST STRAINS 试验用菌株的制备Use standardised stable suspensions of test strains or prepare them as stated below. Seed lot culture maintenance techniques (seed-lot systems) are used so that the viable micro-organisms used for inoculation are not more than 5 passages removed from the original master seed-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 each of the bacterial test strains separately in casein soya bean digest broth or on casein soya bean digest agar at 30-35 C for 18-24 h. Grow the test strain for Candida albicans separately on Sabouraud-dextrose agar or in Sabouraud-dextrose broth at 20-25 C for 2-3 days.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。 Staphylococcus aureus such as ATCC 6538, NCIMB 9518, CIP 4.83 or NBRC 13276;金黄色葡萄球菌：例如ATCC6538,NCIMB9518,CIP4.83或NBRC13276; Pseudomonas aeruginosa such as ATCC 9027, NCIMB 8626, CIP 82.118 or NBRC 13275;铜绿假单胞菌：例如ATCC9027,NCIMB8626,CIP82.118o或NBRC13275; Escherichia coli such as ATCC 8739, NCIMB 8545, CIP 53.126 or NBRC 3972;大肠埃希菌：例如ATCC8739,NCIMB8545,CIP53.126或NBRC3972; Salmonella enterica subsp. enterica serovar Typhimurium, such as ATCC 14028 or, as an alternative, Salmonella enterica subsp. enterica serovar Abony such as NBRC 100797, NCTC 6017 or CIP 80.39;沙门氏菌：例如ATCC14028或NBRC100797,NCTC6017或CIP80.39; Candida albicans such as ATCC 10231, NCPF 3179, IP 48.72 or NBRC 1594.白色念珠菌：例如ATCC10231,NCPF3179,IP48.72或NBRC1594.Use buffered sodium chloride-peptone solution pH 7.0 or phosphate buffer solution pH 7.2 to make test suspensions. Use the suspensions within 2 h or within 24 h if stored at 2-8 C.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 Clostridium sporogenes such as ATCC 11437 (NBRC 14293, NCIMB 12343, CIP 100651) or ATCC 19404 (NCTC 532 or CIP 79.03) or NBRC 14293. Grow the clostridial test strain under anaerobic conditions in reinforced medium for clostridia at 30-35 C for 24-48 h. As an alternative to preparing and then diluting down a fresh suspension of vegetative cells of Cl. sporogenes, a stable spore suspension is used for test inoculation. The stable spore suspension may be maintained at 2-8 C for a validated period.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. NEGATIVE CONTROL 阴性对照试验To verify testing conditions, a negative control is performed using the chosen diluent in place of the test preparation. There must be no growth of micro-organisms. A negative control is also performed when testing the products as described in section 4. A failed negative control requires an investigation.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 PROMOTION AND INHIBITORY PROPERTIES OF THE MEDIA 培养基的生长促进作用和生长抑制作用 Test each batch of ready-prepared medium and each batch of medium prepared either from dehydrated medium or from ingredients. 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 suitable properties of relevant media as described in Table 2.6.13.-1.按照表（2.6.13.-1），检测相关培养基的特性。 18 / 18Table 2.6.13.-1 Growth promoting, inhibitory and indicative properties of 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Teststrains试验菌株Testforbile-tolerantgram-negativebacteria耐胆盐革兰氏阴性菌的检测Enterobacteriaenrichmentbroth-Mossel肠道菌增菌肉汤培养基Growthpromoting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VioletredbileglucoseAgar紫红胆汁葡萄糖琼脂培养基Growthpromoting+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TestforEscherichiaColi大肠埃希菌的检测MacConkeybroth麦康凯肉汤培养基Growthpromoting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MacConkeyagar麦康凯琼脂培养基Growthpromoting+indicative生长促进和指示作用E.coli大肠埃希菌TestforSalmonella沙门氏菌的检测RappaportVassiliadisSalmonellaenrichmentBroth沙门氏菌琼脂培养基Growthpromoting生长促进Salmonellaentericasubsp. entericaserovarTyphimuriumorSalmonellaen-tericasubsp.entericaserovarAbony沙门氏菌Inhibitory生长抑制S.aureus金黄色葡萄球菌Xylose,lysine,deoxycholateagar木糖赖氨酸去氧胆酸盐琼脂培养基Growthpromoting+indicative生长促进和指示作用Salmonellaentericasubsp. entericaserovarTyphimuriumorSalmonellaentericasubsp.entericaserovar Abony沙门氏菌Inhibitory指示作用E.coli大肠埃希菌TestforPseudomonasAeruginosa铜绿假单胞菌的检测Cetrimideagar溴棕三甲铵琼脂培养基Growthpromoting生长促进P.aeruginosa 铜绿假单胞菌Inhibitory生长抑制E.coli 大肠埃希菌TestforStaphylococcus aureus金黄色葡萄球菌的检测Mannitolsaltagar甘露醇氯化钠琼脂培养基Growthpromoting+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Testforclostridia梭菌的检测Reinforcedmediumforclostridia增菌培养基Growthpromoting生长促进Cl.Sporogenes梭状芽孢杆菌Columbiaagar哥伦比亚琼脂培养基Growthpromoting生长促进Cl.Sporogenes梭状芽孢杆菌TestforCandidaalbicans白色念珠菌的检测SabourauddextroseBroth沙氏葡萄糖肉汤培养基Growthpromoting生长促进C.albicans白色念珠菌Sabourauddextroseagar沙氏葡萄糖琼脂培养基Growthpromoting+indicative生长促进和指示作用C.albicans白色念珠菌Test for growth promoting properties, liquid media : inoculate a portion of the appropriate medium with a small number (not more than 100 CFU) of the appropriate micro-organism. Incubate at the specified temperature for not more than the shortest period of time specified in the test. Clearly visible growth of the micro-organism comparable to that previously obtained with a previously tested and approved batch of medium occurs.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 for growth promoting properties, solid media: perform the surface-spread method, inoculating each plate with a small number (not more than 100 CFU) of the appropriate micro-organism. Incubate at the specified temperature for not more than the shortest period of time specified in the test. Growth of the micro-organism comparable to that previously obtained with a previously tested and approved batch of medium occurs.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 for indicative properties: perform the surface-spread method, inoculating each plate with a small number (not more than 100 CFU) of the appropriate micro-organism. Incubate at the specified temperature for a period of time within the range specified in the test. Colonies are comparable in appearance and indication reactions to those previously obtained with a previously tested and approved batch of medium.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 preparation and pre-incubation. Prepare a sample using a 1 in 10 dilution of not less than 1 g of the product to be examined as described in general chapter 2.6.12, and use 10 mL or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount (determined as described under 3-4) of casein soya bean digest broth, mix and incubate at 30-35 C for 18-24 h.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 and subculture. Shake the container, transfer 1mL of casein soya bean digest broth to 100mL of MacConkey broth and incubate at 42-44 C for 24-48 h. Subculture on a plate of MacConkey agar at 30-35 C for 18-72 h.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 of colonies indicates the possible presence of E. coli. This is confirmed by identification tests. The product complies with the test if no colonies are present or if the identification 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 preparation and pre-incubation. Prepare the product to be examined as described in general chapter 2.6.12, and use the quantity corresponding to not less than 10 g or 10 mL to inoculate a suitable amount (determined as described under 3-4) of casein soya bean digest broth, mix and incubate at 30-35 C for 18-24 h.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 and subculture. Transfer 0.1mL of casein soya bean digest broth to 10 mL of Rappaport Vassiliadis Salmonella enrichment broth and incubate at 30-35 C for 18-24 h. Subculture on plates of xylose, lysine, deoxycholate agar. Incubate at 30-35 C for 18-48 h.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 possible presence of Salmonella is indicated by the growth of well-developed, red colonies, with or without black centres. This is confirmed by identification tests. The product complies with the test if colonies of the types described are not present or if 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 preparation and pre-incubation. Prepare a sample using a 1 in 10 dilution of not less than 1 g of the product to be examined as described in general chapter 2.6.12, and use 10 mL or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount (determined as described under 3-4) of casein soya bean digest broth and mix. When testing transdermal patches, filter the volume of sample corresponding to 1 patch of the preparation described under 4-5-1 in general chapter 2.6.12 through a sterile filter membrane and place in 100 mL of casein soya bean digest broth. Incubate at 30-35 C for 18-24 h.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 and subculture. Subculture on a plate of cetrimide agar and incubate at 30-35 C for 18-72 h.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 of colonies indicates the possible presence of P. aeruginosa. This is confirmed by identification tests. The product complies with the test if colonies are not present or if the confirmatory identification tests are negative.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 preparation and pre-incubation. Prepare a sample using a 1 in 10 dilution of not less than 1 g of the product to be examined as described in general chapter 2.6.12, and use 10 mL or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount (determined as described under 3-4) of casein soya bean digest broth and mix. When testing transdermal patches, filter the volume of sample corresponding to 1 patch of the preparation described under 4-5-1 in general chapter 2.6.12 through a sterile filter membrane and place in 100 mL of casein soya bean digest broth. Incubate at 30-35 C for 18-24 h.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 and subculture. Subculture on a plate of mannitol salt agar and incubate at 30-35 C for 18-72 h. 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 possible presence of S. aureus is indicated by the growth of yellow/white colonies surrounded by a yellow zone. This is confirmed by identification tests. The product complies with the test if colonies of the types described are not present or if the confirmatory identification tests are negative.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