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1、 MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS MICROBIAL ENUMERATION TESTS 非无菌产品微生物限度检查 微生物计数法 1 INTRODUCTION导言导言 The tests described hereafter will allow quantitative enumeration of mesophilic bacteria and fungi that may grow under aerobic conditions 微生物计数法系用于能在有氧条件下生长的嗜温细菌和霉菌的定量计数 The tests a
2、re 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 当本法用于检查非无
3、菌制剂及其原 辅料等是否符合相应的微生物限度标准时 应按照下述规定 进行检验 包括样品的取样量 结果的判断 The methods are not applicable to products containing viable microorganisms as active ingredients 本法不适用于活菌制剂的检查 Alternative microbiological procedures including automated methods may be used provided that their equivalence to the Pharmacopeial me
4、thod has been demonstrated 可使用包括自动化法在内的替代方法 需确认其与药典方法的等同性 2 GENERAL PROCEDURES通用规程通用规程 Carry out the determination under conditions designed to avoid extrinsic microbial contamination of the product to be examined The precautions taken to avoid contamination must be such that they do not affect any
5、microorganisms that are to be revealed in the test 微生物计数环境应能防止外来微生物对供试品的污染 防止污染的措施不能影响供试品中微生物 的检出 备注 检验全过程必须严格遵守无菌操作 防止再污染 单向气流区域 工作台面及环境应定期 进行监测 If the product to be examined has antimicrobial activity this is insofar as possible removed or neutralized If inactivators are used for this purpose thei
6、r efficacy and their absence of toxicity for microorganisms must be demonstrated 如果供试品有抗菌活性 应尽可能去除或中和 若使用了中和剂或灭活剂 需确认其有效性及对 微生物无毒性 If surface active substances are used for sample preparation their absence of toxicity for microorganisms and their compatibility with any inactivators used must be demo
7、nstrated 供试品制备过程中若使用了表面活性剂 需确认其对微生物无毒性以及与所使用的中和剂或灭活 剂的相容性 3 ENUMERATION METHODS计数方法计数方法 Use the Membrane Filtration method or one of the Plate Count Methods as directed The Most Probable Number MPN Method is generally the least accurate method for microbial counts however for certain product groups
8、with very low bioburden it may be the most appropriate method 计数方法包括薄膜过滤法 平皿法和最可能数法 Most Probable Number 简称 MPN MPN 法用 于微生物计数时精确度最差 但用于微生物污染量小的供试品 MPN 法可能是最合适的方法 MPN法不适用于霉菌的检测 仅在供试品总需氧菌数没有适应计数方法的情况下使用 The choice of a method is based on factors such as the nature of the product and the required limit
9、 of microorganisms The method chosen must allow testing of a sufficient sample size to judge compliance with the specification The suitability of the chosen method must be established 供试品检查时 应根据供试品理化特性和微生物限度标准等因素选择计数方法 所选择的计数方 法应能够通过检测足量的供试品 判断与质量标准的符合性 且该计数方法的适用性须经确认 4 GROWTH PROMOTION TEST SUITABI
10、LITY OF THE COUNTING METHOD AND NEGATIVE CONTROLS促生长实验 计数法适用性促生长实验 计数法适用性试验试验以及阴性对照以及阴性对照 4 1 General Considerations一般要求 The ability of the test to detect microorganisms in the presence of product to be tested must be established Suitability must be confirmed if a change in testing performance or a
11、change in the product that may affect the outcome of the test is introduced 在有供试品存在的情况下 所选用的计数方法需能够检测微生物 若检测程序或产品发生变化可 能影响检测结果时 计数方法需重新进行适用性确认 4 2 Preparation of Test Strains试验菌液的制备 Use standardized stable suspensions of test strains or prepare as stated below Seed lot culture maintenance techniques
12、 seed lot systems are used so that the viable microorganisms used for inoculation are not more than five passages removed from the original master seed lot Grow each of the bacterial and fungal test strains separately as described in Table 1 试验菌液 使用试验菌株的标准化稳定悬浮液或按下述规定制备 按表 1 规定程序分别培养各试 验菌液 菌株 采用适宜的菌
13、种保藏技术 种子批系统 试验用菌株传代次数自主种子批 第 0 代 算 起不得超过 5代 Table 1 Preparation and Use of Test Microorganisms Growth Promotion 促生长实验 Suitability of Counting Method in the Presence of Product 计数方法适用性试验 Microorganism 试验菌株 Preparation of Test Strain 试验菌液的制备 Total Aerobic Microbial Count 总需氧菌数计数 Total Yeasts and Molds
14、 Count 总酵母菌和霉菌计数 Total Aerobic Microbial Count 总需氧菌数计数 Total Yeasts and Molds Count 总酵母菌和霉菌 计数 Staphylococcus aureus such as ATCC 6538 NCIMB 9518 CIP 4 83 or NBRC 13276 金黄色葡萄球菌 Soybean Casein Digest Agar or Soybean Casein Digest Broth 30 35 18 24 hours 大豆酪蛋白消化琼脂 培养基或大豆酪蛋白 消化肉汤培养基 培养温度 30 35 培养时间 18
15、24 小时 Soybean Casein Digest Agar and Soybean Casein Digest Broth 100 cfu 30 35 3 days 大豆酪蛋白消化琼脂 培养基和大豆酪蛋白 消化肉汤基 培养温度 30 35 培养时间不超过 3 天 接种量不大于 100cfu Soybean Casein Digest Agar MPN Soybean Casein Digest Broth 100 cfu 30 35 3 days 大豆酪蛋白消化琼脂 培养基 大豆酪蛋白消化肉汤 培养基 MPN法 培养温度 30 35 培养时间不超过 3 天 接种量不大于 100cfu P
16、seudomonas aeruginosa such as ATCC 9027 NCIMB 8626 CIP 82 118 or NBRC 13275 铜绿假单胞菌 Bacillus subtilis such as ATCC 6633 NCIMB 8054 CIP 52 62 or NBRC 3134 枯草芽孢杆菌 Candida albicans such as ATCC 10231 NCPF 3179 IP 48 72 or NBRC 1594 白色念珠菌 Sabouraud Dextrose Agar or Sabouraud Dextrose Broth 20 25 2 3 days 沙氏葡萄糖琼脂培养 基或沙氏葡萄糖肉汤 培养基 培养温度 20 25 培养时间 2 3 天 Soybean Casein Digest Agar 100 cfu 30 35 5 days 大豆酪蛋白消化琼脂 培养基 培养温度 30 35 培养时间不超过 5 天 接种量不大于 100cfu Sabouraud Dextrose Agar 100 cfu 20 25 5 days 沙氏葡萄糖琼脂培养
