2.6.12(新)微生物限度检测

2.6.12.MICROBIOLOGICAL EXAMINATION OF NON- STERILE PRODUCTS: MICROBIAL ENUMERATION TESTS 26.12.非无菌药品的微生物检测：微生物计数试验 1. INTRODUCTION 导言 The tests described hereafter will allow quantitative enumeration of mesophilic bacteria and fungi which may grow under aerobic conditions. 本试验用于检测在有氧条件下生长的嗜温性细菌和真菌总数。 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. 本试验用于检测原料药或制剂是否符合已建立的微生物限度要求，基于这一目的应遵循以 下指导原则，包括取样量并且按照下述方法分析试验结果。 The methods are not applicable to products containing viable micro-organisms as active ingredients. 此试验方法不适用于含有活性微生物的药品的检测。 Alternative microbiological procedures, including automated methods, may be used, provided that their equivalence to the Pharmacopoeia method has been demonstrated. 倘若已证明某种方法可产生与药典试验方法同样的效果，此方法也可应用，其中包括自动 化方法。 2GENERAL PROCEDURES 一般程序 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 micro-organisms that are to be revealed in the test. 试验应在供试品不被外来微生物污染的条件下进行，用于防止供试品污染的措施不应影响 微生物的检出。 If the product to be examined has antimicrobial activity, this is insofar as possible removed or neutralised. If inactivators are used for this purpose, their efficacy and their absence of toxicity for micro-organisms must be demonstrated. 如果被检测供试品有抗微生物活性，必须进行中和。如果使用灭活剂来消除供试品的抗微 生物活性，必须证明其有效性和不会对微生物产生毒性。 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. 如果在供试品的制备过程中使用了表面活性剂，必须证明其不会对微生物产生毒性并且可 与灭活剂兼容。 3.ENUMERATION METHODS 计数方法 Use the membrane filtration method or the plate-count methods, as prescribed. The most-probable-number(MPN) method is generally the least accurate method for microbial counts, however, for certain product groups with a very low bioburden, it may be the most appropriate method. 使用薄膜过滤法或平皿计数法进行计数。最大可能数法通常测定的结果是不很准确，然而 对于一些微生物生长极少的供试品，最大可能数法是一种比较适用的方法。 The choice of method is based on factors such as the nature of the product and the required limit of micro-organisms. The chosen method must allow testing of a sufficient sample size to judge compliance with the specification. The suitability of the method chosen must be established. 方法的选择基于供试品的性质和微生物限度的要求，使用选择的方法对足够量的供试品检 测，测定其是否符合要求。必须建立选择使用的方法的适应性。 4.GROWTH PROMOTION TEST, SUITABILITY OF THE COUNTING METHOD AND NEGATIVE CONTROLS 培养基的增菌作用试验，计数方法适应性试验，阴性对照试验 4-1 GENERAL CONSIDERATIONS 总则 The ability of the test to detect micro-organisms in the presence of product to be tested must be established. 该试验要有一定的检测微生物的能力。 Suitability must be confirmed if a change in testing performance, or the product, which may affect the outcome of the test is introduced. 如果产生影响试验结果的操作或被测物品发生变化，该试验的适应性必须证实。 4-2.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. Grow each of the bacterial and fungal test strains separately as deacribed in Table 2.6.12.-1.。 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，使用的种子批传代次数不得超 过 5 代。按照表 2.6.12-1.制备细菌和真菌的试验用菌株。 Table 2.6.12.-1.Preparation and use of test micro-organisms 试验用菌株的制备和使用 Growth promotion 增菌作用 Suitability of counting method in the presence of the product 计数方法的适应性 Micro-organism 微生物种类 Preparation of test strian 试验用菌株的制备 Total aerobic microbial count 总好氧菌数 Total yeasts and moulds count 酵母菌和 霉菌总数 Total aerobial microbial count 总好氧菌数 Total yeasts and moulds count 酵母菌和 霉菌总数 Staphylococcus aureus such as:ATCC 6538 NCIMB 9518 CIP 4.83 NBRC 13276 金黄色葡萄球菌，如 ATCC 6538, NCIMB 9518, CIP 4.83, NBRC 13276 Casein soya bean digest agar or casein soya bean digest broth 30-35 18- 24h 干酪素大豆消化琼 脂培养基或干酪素 大豆消化肉汤培养 基 30-35条件下 培养 18-24 小时 Casein soya bean digest agar or casein soya bean digest broth 100CFU 30-35 3days 干酪素大豆消化琼脂 培养基或干酪素大豆 消化肉汤培养基 - Casein soya bean digest agar/MPN casein soya bean digest broth 100CFU 30- 35 3days 干酪素大豆消 化琼脂培养基/ 最大可能数法 或干酪素大豆 消化肉汤培养 基 - Pseudomonas aeruginosa such as:ATCC 9027 NCIMB 8626 CIP82.118 NBRC 13275 铜绿假单胞菌，如 ATCC 9027,NCIMB 8626,CIP82.118,NBRC 13275 Casein soya bean digest agar or casein soya bean digest broth 30-35 18- 24h 干酪素大豆消化琼 脂培养基或干酪素 大豆消化肉汤培养 基 30-35条件下 培养 18-24 小时 Casein soya bean digest agar or casein soya bean digest broth 100CFU 30-35 3days 干酪素大豆消化琼脂 培养基或干酪素大豆 消化肉汤培养基 - Casein soya bean digest agar/MPN casein soya bean digest broth 100CFU 30- 35 3days 干酪素大豆消 化琼脂培养基/ 最大可能数法 或干酪素大豆 消化肉汤培养 基 - Bacillus subtilis such as: ATCC 6633 NCIMB 8054 CIP 52.62 NBRC 3134 枯草芽孢杆菌，如 ATCC 6633,NCIMB 8054,CIP 52.62, NBRC 3134 Casein soya bean digest agar or casein soya bean digest broth 30-35 18- 24h 干酪素大豆消化琼 脂培养基或干酪素 大豆消化肉汤培养 基 30-35条件下 培养 18-24 小时 Casein soya bean digest agar or casein soya bean digest broth 100CFU 30-35 3days 干酪素大豆消化琼脂 培养基或干酪素大豆 消化肉汤培养基 - Casein soya bean digest agar/MPN casein soya bean digest broth 100CFU 30- 35 3days 干酪素大豆消 化琼脂培养基/ 最大可能数法 或干酪素大豆 消化肉汤培养 基 - Candida albicans such as: ATCC 10231 NCPF 3179 IP 48.72 NBRC 1594 白色念珠菌，如 ATCC 10231,NCPF 3179,IP 48.72,NBRC 1594 Sabouraud-dextrose agar or Sabouraud- dextrose broth 20- 25 2-3days 沙保氏-葡萄糖琼脂 培养基或沙保氏-葡 萄糖肉汤培养基 20-25条件下培养 2-3 天 Casein soya bean digest agar 100CFU 30-35 5days 干酪素大豆消化琼脂 培养基 Sabourau d- dextrose agar 100CFU 20-25 5days 沙保氏- 葡萄糖琼 脂培养基 Casein soya bean digest agar 100CFU 30- 35 5days MPN:not applicable 干酪素大豆消 化琼脂培养基 Sabourau d- dextrose agar 100CFU 20-25 5days 沙保氏- 葡萄糖琼 脂培养基 Aspergills niger such as: ATCC 16404 IMI 1431.83 NBRC9455 黑曲霉菌，如 ATCC 16404,IMI 1431.83,NBRC9455 Sabouraud-dextrose agar or potato- dextrose agar 20- 25 5-7days,or until good sporulation is achieved 沙保氏-葡萄糖琼脂 培养基或马铃薯葡 萄糖琼脂培养基 20-25条件下培养 5-7 天或者培养到 形成很好的孢子 Casein soya bean digest agar 100CFU 30-35 5days 干酪素大豆消化琼脂 培养基 Sabourau d- dextrose agar 100CFU 20-25 5days 沙保氏- 葡萄糖琼 脂培养基 Casein soya bean digest agar 100CFU 30- 35 5days MPN:not applicable 干酪素大豆消 化琼脂培养基 Sabourau d- dextrose agar 100CFU 20-25 5days 沙保氏- 葡萄糖琼 脂培养基 Use buffered sodium chloride-peptone solution pH 7.0 or phosphate buffer solution pH 7.2 to make test suspensions; to suspend A. niger spores, 0.05 per cent of polysorbate 80 may be added to the buffer. Use the suspensions within 2h or within 24h if stored at 2-8. As an alternative to preparing and then diluting a fresh suspension of vegetable cells of A.niger or B.subtilis, a stable spore suspension is prepared and then an appropriate volume of the spore suspension may be maintained at 2-8 for a validated period of time. 使用 pH 为 7.0 的氯化钠蛋白胨缓冲溶液或 pH 为 7.2 的磷酸盐缓冲溶液制备试验用菌悬液， 对于黑曲霉孢子悬液需加入 0.05%的吐温-80。当菌悬液贮存在 2-8条件下时，可在 2 小 时或 24 小时内使用。或者先制备新鲜的黑曲霉或枯草芽孢杆菌悬液再将其稀释，适宜量的 稳定的孢子悬液贮存在 2-8 条件下用于验证。 4-3.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 on section 5.A failed negative control requires an investigation. 为检测试验条件是否符合要求，取试验用稀释液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。按照第五部分检测供试品时，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，要进行调查。 4-4.GROWTH PROMOTION OF THE MEDIA 培养基的增菌作用 Test each batch of ready-prepared medium and each batch of medium, prepared either from dehydrated medium or from the ingredients described. 检测每一批培养基，这些培养基由脱水培养基或由规定的成分制得。 Inoculate portions/plates of casein soya bean digest broth and casein soya bean digest agar with a small number ( not more than 100CFU ) of the micro-organisms indicated in Table 2.6.12.-1 , using a separate portion/plate of medium for each. Inoculate plates of Sabouraud-dextrose agar with a small number ( not more than 100CFU ) of the micro-organisms indicated in Table 2.6.12.- 1, using a separate plate of medium for each. Incubate in the conditions described in Table 2.6.12.- 1. 将表 2.6.12.-1.中的菌种（金黄色葡萄球菌、铜绿假单胞菌、枯草芽孢杆菌 100CFU）分 别接种于干酪素大豆消化肉汤和干酪素大豆消化琼脂培养基中，将表 2.6.12.-1.中的菌种 （白色念珠菌、黑曲霉菌100CFU）分别接种于沙保氏-葡萄糖琼脂培养基中，在表中规定 的条件下培养。 For solid media, growth obtained must not differ by a factor greater than 2 from the calculated value for a standardised inoculum. For a freshly prepared inoculum, growth of the micro- organisms comparable to that previously obtained with a previously tested and approved batch of medium occurs. Liquid media are suitable if clearly visible growth of the micro-organisms comparable to that previously obtained with a previously tested and approved batch of medium occurs. 对于固体培养基，如果微生物的生长情况与已批准合格的培养基中微生物的生长情况类似， 说明固体培养基符合要求；对于液体培养基，如果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，说明液体培养基符合要求。 4-5.SUITABILITY OF THE COUTING METHOD IN THE PRESENCE OF PRODUCT 计数方法的适应性 4-5-1.Preparation of the sample. The method for sample preparation depends upon the physical characteristics of the product to be tested. If none of the procedures described below can be demonstrated to be satisfactory, an alternative procedure must be developed. 样品的制备：样品的制备方法依赖于供试品的物理性质。如果对于某种供试品下述方法都 不适合，应建立其他供选择的方法。 Water-soluble products. Dissolve or dilute ( usually a 1 in 10 dilution is prepared ) the product to be examined in buffered sodium chloride-peptone solution pH 7.0, phosphate buffer solution pH 7.2 or casein soya bean digest broth. If necessary, adjust to pH 6-8. Further dilutions, where necessary, are prepared with the same diluent. 水溶性供试品 将供试品溶解或稀释于 pH 为 7.0 的氯化钠蛋白胨缓冲溶液、 pH 为 7.2 的磷 酸盐缓冲溶液或干酪素大豆消化肉汤中，制成 1:10 的供试液。如果需要可将供试液 pH 调 节到 6-8。当需要进一步稀释时，可用同样的稀释液将供试液稀释。 Non-fatty products insoluble in water. Suspend the product to be examined ( ususlly a 1 in 10 dilution is prepared ) in buffered solution chloride-peptone solution pH 7.0, phosphate buffer solution pH 7.2 or casein soya bean digest broth. A surface-active agent such as 1g/l of polysorbate 80 may be added to assist the suspension of poorly wettable substances. If necessary, adjust to pH 6-8. Further dilutions, where necessary, are prepared with the same diluent. 不溶于水的非脂类供试品 将供试品混悬于 pH 为 7.0 的氯化钠蛋白胨缓冲溶液、pH 为 7.2 的磷酸盐缓冲溶液或干酪素大豆消化肉汤中，制成 1:10 的供试液。可加入表面活性剂，例 如 1g/l 的吐温-80。如果需要可将供试液 pH 调节到 6-8。当需要进一步稀释时，可用同样 的稀释液将供试液稀释。 Fatty products. Dissolve in isopropyl myristate, sterilised by filtration or mix the product to be examined with the minimum necessary quantity of sterile polysorbate 80 or another non-inhibitory sterile surface-active agent, heated if necessary to not more than 40, or in exceptional cases to not more than 45. Mix carefully and if necessary maintain the temperature in a water-bath. Add sufficient of the pre-warmed chosen diluent to make a 1 in 10 dilution of the original product. Mix carefully whilst maintaining the temperature for the shortest time necessary for the formation of an emulsion. Further serial tenfold dilutions may be prepared using the chosen diluent containing a suitable concentration of sterile polysorbate 80 or another non-inhibitory sterile surface-active agent. 脂类供试品 将供试品溶于薄膜滤过的无菌十四烷酸异丙酯或将供试品混合于最小量无菌吐 温-80 或其他的表面活性剂中，如需加热，加热温度不得高于 40，特殊情况下加热温度 可升高但不可高于 45。如需要可在水浴条件下保持温度。加入已预热的稀释液制成 1:10 的供试液。小心混合同时保持温度使其在最短的时间内形成乳状液。可使用含有吐温-80 或其他无菌表面活性剂的稀释液将供试液进一步 1:10 稀释。 Fluids or solids in aerosol form. Aseptically transfer the product into a membrane filter apparatus or a sterile container for further sampling. Use either the total contents or a defined number of metered doses from each of the containers tested. 气雾剂、喷雾剂供试品 在无菌条件下将供试品转移到薄膜过滤器中或无菌的容器中用于取 样。取全部供试品或一定量用于试验。 Transdermal patches. Remove the protective cover sheets ( “release liners” ) of the transdermal patches and place them, adhesive side upsides, on sterile glass or plastic trays. Cover the adhesive surface with a sterile porous from sticking together, and transfer the patches to a suitable volume of the chosen diluent containing inactivators such as polysorbate 80 and/or lecithin. Shake the preparation vigorously for at least 30 min. 透皮吸收剂 除去透皮吸收剂的保护性衬垫，具有粘性的一面朝上置于无菌玻璃或塑料盘中， 将无菌的渗透性纱布覆盖于有粘性的一面上。将其转移到适量的含有灭活剂的稀释液中， 如含有吐温-80 或卵磷脂，剧烈振摇至少 30 分钟。 4-5-2.Inoculation and dilution. Add to the sample prepared as described above ( 4-5-1 ) and to a control ( with no test material included ) a sufficient volume of the microbial suspension to obtain an inoculum of not more than 100 CFU. The volume of the suspension of the inoculum should not exceed 1 per cent of the volume of diluted product. 接种和稀释 将按照（ 4-5-1）中的方法制备的供试液接种培养，另制备 CFU100 的微生物 对照组。接种用的供试液的量不应超过供试液总量的 1%。 To demonstrate acceptable microbial recovery from the product, the lowest possible dilution factor of the prepared sample must be used for the test. Where this is not possible due to antimicrobial activity or poor solubility, further appropriate protocols must be developed. If inhibition of growth by the sample cannot otherwise be avoided, the aliquot of the microbial suspension may be added after neutralisation, dilution or filtration. 为了证明供试品中的微生物有较好的复活率，将供试品稀释到最低浓度用于试验。由于供 试品有抗微生物活性或溶解度较低，应建立其他有效的措施。如果供试品的生长抑制作用 无法中和，应采用中和法、稀释法或过滤法处理后再使用。 4-5-3. Neutralisation/removal of antimicrobial activity. The number of micro-organisms recovered from the prepared sample diluted as described in 4-5-2 and incubated following the procedure described in 4-5-4, is compared to the number of micro-organisms recovered from the control preparation. 中和或消除供试品的抗微生物活性 按照（4-5-2 ）稀释的供试液按照（4-5-4 ）培养后，微 生物的复活数量与对照组中微生物的复活数量相比较。 If growth is inhibited ( reduction by a factor greater than 2 ), then modify the procedure for the particular enumeration test to ensure the validity of the results. Modification of the procedure may include, for example, (1) an increase in the volume of the diluent or culture medium, (2) incorporation of specific or general neutralising agents into the diluent, (3) membrance filtration, or (4) a combination of the above measures. 如果微生物的生长受到抑制，为保证结果的有效性更改操作规程以建立合适的计数方法。 操作规程的修改包括：（1）增加稀释液或培养基的用量；（2）稀释剂中加入中和剂； （3）薄膜过滤；（4）以上方法的联合使用等。 Neutralising agents. Neutralising agents may be used to neutralise the activity of antimicrobial agents ( Table 2.6.12.-2 ). They may be added to the chosen diluent or the medium preferably before sterilisation. If used, their efficacy and their absence of toxicity for micro-organisms must be demonstrated by carrying out a blank with neutraliser and without product. 中和剂：如下表 2.6.12.-2.中的中和剂用于中和供试品的抗微生物活性，在稀释液或培养基 灭菌之前将中和剂加到其中。如果使用中和剂，必须做一空白试验以证明中和剂的有效性 和对微生物无毒性，空白试验是指加入中和剂不加供试品的试验。 Table 2.6.12.-2. Common neutralising agents for interfering substances 常用的中和剂 Interfering substance 干扰物质 Potential neutralising method 中和方法 Glutaraldehyde, mercurials 戊二醛，水银剂 Sodium hydrogensulphite ( sodium bisulphite) 亚硫酸氢钠 Phenolics, alcohol, aldehydes, sorbate 酚醛塑料，乙醇，乙醛，山梨酸酯 Dilution 稀释法 Aldehydes 乙醛 Glycine 氨基乙酸 Quaternary Ammonium Compounds （QACs ）, parahydroxybenzoates (parabens), bis-biguanides 季铵化合物，尼泊金，双胍类 Lecithin 卵磷脂 QACs, iodine, parabens 季铵化合物，碘，尼泊金 Polysorbate 聚山梨醇酯 Mercurials 水银剂 Thioglycollate 2-硫基乙醇 Mercurials, halogens, aldehydes 水银剂，卤素，乙醛 Thiosulphate 硫代硫酸盐 EDTA (edetate) 乙二胺四乙酸 Mg2+ or Ca2+ ions 钙或镁离子 If no suitable neutralising method can be found, it can be assumed that the failure to isolate the inoculated organism is attributable to the microbicidal activity of the product. This information serves to indicate that the product is not likely to be contaminated with the given species of the micro-organism. However, it is possible that the product only inhibits some of the micro- organisms specified herein, but does not inhibit others not included amongst the test strains or for which the latter are not representative. Then, perform the test with the highest dilution factor compatible with microbial growth and the specific acceptance criterion. 如果没有合适的中和方法，可以认为由于供试品的抗微生物活性分离不出可存活的微生物。 这也说明供试品不会被已知的试验菌株污染。然而，只能说明供试品能抑制几种微生物的 生长，不能说明其对其他微生物是否有抑制生长的作用。此种情况下，应制备高稀释级的 供试液与特殊可接受的标准对照。 4-5-4.Recovery of micro-organisms in the presence of product. For each of the micro- organisms listed, separate tests are performed. Only micro-organisms of the added test strain are counted. 微生物的复活 对于每一种微生物分别进行试验，只记录待检测菌的数目。 4-5-4-1. Membrane filtration. Use membrane filters having a normal pore size not greater than 0.45um. The type of filter material is chosen such that the bacteria-retaining efficiency is not affected by the components of the sample to be investigated. For each of the micro-organisms listed, one membrane filter is used. 薄膜过滤法 使用标准规格的孔径小于 0.45um 的滤膜，选择的滤膜材料应满足能使细菌有 效截留而不受供试品成分的影响。每进行一次微生物检测使用一个滤膜。 Transfer a suitable amount of the sample prepared as described under 4-5-1 to 4-5-3 ( preferably representing 1g of the product, or less if large numbers of CFU are expected ) to the membrane filter, filter immediately and rinse the membrane filter with an appropriate volume of diluent. 将按照（4-5-1 .4-5-2,4-5-3）制备的供试液（如果要得到较大的 CFU 值，取1g 的供试品） 转移到薄膜过滤器，立即过滤，用适量的稀释液冲洗滤膜。 For the determination of total aerobic microbial count (TAMC), transfer the membrane filter to the surface of casein soya bean digest agar. For the determination of total combined yeasts/moulds count ( TYMC ), transfer the membrane to the surface of Sabouraud-dextrose agar. Incubate the plates as indicated in Table 2.6.12.-1. Perform the counting. 为了测定总好氧菌数，将滤膜转移到干酪素大豆消化琼脂培养基表面；为了测定酵母菌或 霉菌总数，将滤膜转移到沙保氏-葡萄糖琼脂培养基表面。在表 2.6.12.-1.中规定的条件下培 养，计数。 4-5-4-2. Plate-count methods. Perform plate-count methods at least in duplicate for each medium and use the mean count of the result. 平皿计数法 平皿计数法至少进行两个平行试验，两次试验的平均值最为计数结果。 4-5-4-2-1. Pour-plate method 平皿法 For Petri dishes 9 cm in diameter, add to the dish 1 ml of the sample prepared as described under 4-5-1 to 4-5-3 and 15-20 ml of casein soya bean digest agar or Sabourated-dextrose agar, both media being at not more than 45. If