2.6.12(新)微生物限度检测

NON-STERILE PRODUCTS: MICROBIAL ENUMERATION TESTS

2．6.12.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。

2．GENERAL 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 Micro-organism 微生物种类 Preparation of test Growth promotion strian 增菌作用 试验用菌株的制备 Suitability of counting method in the presence of the product 计数方法的适应性 Total yeasts and moulds count 酵母菌和霉菌总数 - Total aerobic Total Total aerobial microbial count yeasts microbial count 总好氧菌数 and 总好氧菌数 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 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 - - 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 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 Sabouraud-dextrose agar ≤100CFU 20-25℃ ≤5days 沙保氏-葡萄糖琼脂培养基 Sabouraud-dextrose agar ≤100CFU 20-25℃ ≤5days 沙保氏-葡萄糖琼脂培养基 Casein soya Sabouraubean digest agar d-dextros≤100CFU e agar ≤30-35℃ ≤100CFU 5days MPN:not 20-25℃ applicable ≤5days 干酪素大豆消沙保氏-化琼脂培养基 葡萄糖琼脂培养基 Casein soya Sabouraubean digest agar d-dextros≤100CFU e agar ≤30-35℃ ≤100CFU 5days MPN:not 20-25℃ applicable ≤5days 干酪素大豆消沙保氏-化琼脂培养基 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 Casein soya bean digest agar ≤100CFU 30-35℃ ≤5days 干酪素大豆消化琼脂培养基 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CFU≤100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 Glutaraldehyde, mercurials 戊二醛，水银剂 Phenolics, alcohol, aldehydes, sorbate 酚醛塑料，乙醇，乙醛，山梨酸酯 Aldehydes 乙醛 Potential neutralising method 中和方法 Sodium hydrogensulphite bisulphite) 亚硫酸氢钠 Dilution 稀释法 Glycine 氨基乙酸 ( sodium Quaternary Ammonium Compounds （QACs）, Lecithin 卵磷脂 parahydroxybenzoates (parabens), bis-biguanides 季铵化合物，尼泊金，双胍类 QACs, iodine, parabens 季铵化合物，碘，尼泊金 Mercurials 水银剂 Mercurials, halogens, aldehydes 水银剂，卤素，乙醛 EDTA (edetate) 乙二胺四乙酸 Polysorbate 聚山梨醇酯 Thioglycollate 2-硫基乙醇 Thiosulphate 硫代硫酸盐 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 larger Petri dishes are used, the amount of agar medium is increased accordingly. For each of the micro-organisms listed in Table 2.6.12.-1, at least 2 Petri dishes are used. Incubate the plates as indicated in Table 2.6.12.-1. Take the arithmetic mean of the counts per medium and calculate the number of CFU in the original inoculum. 在直径为9cm的皮氏培养皿中，每个培养皿中加入1ml供试液和15-20ml的干酪素大豆消化琼脂培养基或沙保氏-葡萄糖琼脂培养基，培养基的温度不超过45℃，供试液的制备方法参照（4-5-1 .4-5-2,4-5-3）。如果用较大的皮氏培养皿，琼脂培养基的用量应相应的增加。对于表2.6.12.-1中列出的每一种微生物，至少进行两个平行试验，按照表中规定的条件培养。取各培养皿计数的算术平均值，计算CFU值。

4-5-4-2-2. Surface-spread method 表面涂布平皿法

For Petri dished 9 cm in diameter, add 15-20 ml of casein soya bean digest agar or Sabouraud-dextrose agar at about 45℃ to each Petri dish and allow to solidify. If larger Petri dishes are used, the volume of the agar is increased accordingly. Dry the plates, for example in a laminar-air-flow cabinet or an incubator. For each of the micro-organisms listed in Table 2.6.12.-1, at least 2 Petri dishes are used. Spread a measured volume of not less than 0.1 ml of the sample prepared as described under 4-5-1 to 4-5-3 over the surface of the medium. Incubate and count as prescribed under 4-5-4-2-1.

在直径为9cm的皮氏培养皿中，每个培养皿中加入大约45℃的15-20ml的干酪素大豆消化琼脂培养基或沙保氏-葡萄糖琼脂培养基，使其凝固。如果用较大的皮氏培养皿，琼脂培养基的用量应相应的增加。培养基的凝固过程可在单向流空气区域或恒温箱中进行。对于表2.6.12.-1中列出的每一种微生物，至少进行两个平行试验。供试液的制备方法参照（4-5-1 .4-5-2,4-5-3），将不少于0.1ml的供试液涂布于培养基表面。按照（4-5-4-2-1）培养计数。

4-5-4-3. Most-probable-number ( MPN ) method. The precision and accuracy of the MPN methed is less than that of the membrane filtration method or the plate-count method. Unreliable results are obtained particularly for the enumeration of moulds. For these reasons the MPN method is reserved for the enumeration of TAMC in situations where no other method is available. If the use of the method is justified, proceed as follows.

最大可能数法 最大可能数法的准确度和精密度均小于薄膜过滤法和平皿计数法。应用此法进行霉菌计数时，其数据并不可靠。因此无其他方法可选择的情况下才应用本方法进行总好氧菌计数。在使用本法时，按照以下过程进行。

Prepare a series of at least 3 serial tenfold dilutions of the products as described under 4-5-1 to 4-5-3. From each level of dilution, 3 aliquots of 1g or 1 ml are used to inoculate 3 tubes with 9-10 ml of casein soya bean digest broth. If necessary, a surface-active agent such as polysorbate 80 or an inactivator of antimicrobial agents may be added to the medium. Thus, if 3 levels of dilution are prepared, 9 tubes are inoculated.

参照（4-5-1—4-5-3）制备一系列至少三个连续的1:10稀释度的供试液。对于每一稀释度，均将相当于1g或1ml供试品的供试液接种于装有9～10 ml 干酪素大豆消化肉汤培养基的三只试管中。如需要可将表面活性剂例如吐温-80或针对抗菌物质的灭活剂加入到培养基中，三个稀释度共接种9支试管。

Incubate all tubes at 30-35℃ for not more than 3 days. If reading of the results is difficult or uncertain owing to the nature of the product to be examined, subculture in the same broth, or in casein soya bean digest agar, for 1-2 days at the same temperature and use these results. Determine the most probable number of micro-orgamisms per gram or millilitre of the product to be examined from Table 2.6.12.-3.

接种后的试管在30～35℃培养 ≤3 天，如果由于供试品的特性试验结果不易读取或结果不确定，可将培养物在相同的温度下相同的培养基或干酪素大豆消化琼脂培养基中转代培养1-2天，记录此时的结果。查阅表2.6.12.-13. 获得供试品每克或每毫升所含微生物的最大可能数。

Table 2.6.12.-3. — Most-probable-number values of miro-organisms 微生物的最大可能数值 Observed combinations of numbers of MPN per gram or per 95 per cent tubes showing growth in each set millilitre of product confidence limits 每一稀释级有微生物生长的试管数 供试品MPN/g或ml 95%置信限 Number of grams or millilitres of product per tube 每个试管中接种的供试品克数或毫升数 0.1 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 0.01 0 0 1 1 2 3 0 0 0 1 1 2 2 3 0 0 0 1 1 1 0.001 0 1 0 1 0 0 0 1 2 0 1 0 1 0 0 1 2 0 1 2 < 3 3 3 6.1 6.2 9.4 3.6 7.2 11 7.4 11 11 15 16 9.2 14 20 15 20 27 0-9.4 0.1-9.5 0.1-10 1.2-17 1.2-17 3.5-35 0.2-17 1.2-17 4-35 1.3-20 4-35 4-35 5-38 5-38 1.5-35 4-35 5-38 4-38 5-38 9-94 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

2 2 2 3 3 0 0 0 1 1 1 1 2 2 2 2 3 3 3 3 0 1 2 0 1 0 1 2 0 1 2 3 0 1 2 3 0 1 2 3 21 28 35 29 36 23 38 64 43 75 120 160 93 150 210 290 240 460 1100 > 1100 5-40 9-94 9-94 9-94 9-94 5-94 9-104 16-181 9-181 17-199 30-360 30-380 18-360 30-380 30-400 90-990 40-990 90-1980 200-400 4-6.RESULTS AND INTERPRETATION 结果分析

When verifying the suitability of the membrane filtration method or the plate-count method, a

mean count of any of the test organisms not differing by a factor greater than 2 from the value of the control defined in 4-5-2 in the absence of the product must be obtained. When verifying the suitability of the MPN method the calculated value from the inoculum must be within 95 per cent confidence limits of the results obtained with the control. 检测薄膜过滤法或平皿计数法的适应性时，供试品的实验结果的平均值与对照组对照；检测最大可能数法的适应性时，供试品的实验结果应在对照组试验结果95%的置信区间范围内。

If the above criteria cannot be met for one or more of the organisms tested with any of the described methods, the method and test conditions that come closest to the criteria are used to test the product.

如果采用以上方法进行的一种或几种微生物试验不能达到以上标准，应选择符合标准的方法和试验条件检测供试品。

5. TESTING OF PRODUCTS 供试品检测

5-1. AMOUNT USED FOR THE TEST 供试品用量

Unless otherwise prescribed, use 10g or 10ml of the product to be examined taken with the precautions referred to above. For fluids or solids in aerosol form, sample 10 containers. For transdermal pathes, sample 10 patches.

除另有规定外，取10g 或10ml 的供试品用于检测，对于内容物为液态或固态的气雾剂、喷雾剂应取10个包装单位，对于透皮吸收剂应取10片供试品。

The amount to be tested may be reduced for active substances that will be formulated in the following conditions: the amount per dosage unit ( e.g. tablet, capsule, injection ) is less than or equal to 1mg or the amount per gram or millilitre ( for preparations not presented in dose units ) is less than 1mg. In these cases, the amount to be tested is not less than the amount present in 10 dosage units or 10g or 10ml of the product.

当药物的有效成分出现以下情况时供试品的用量可相应的减少：每一剂量的有效成分含量少于或相当于1mg ，例如片剂、胶囊剂、注射剂；每克或每毫升供试品中的有效成分含量小于1mg，对于不是单剂量分装的供试品。当出现以上情况时，供试品应取不少于10个剂量或取10g或10ml的供试品。

For materials used as active substances where sample quantity is limited or batch size is extremely small ( i.e. less than 1000ml or 1000g ), the amount tested shall be 1 per cent of the batch unless a lesser amount is prescribed or justified and authorised.

用于活性成分的样品，当取样量受到限制或每一批量极少时（例如每一批量为1000ml或1000g），除另有规定外，取样量应为批量的1%。

For products where the total number of entities in a batch is less than 200 ( e.g. samples used in clinical trials ), the sample size may be reduced to 2 units, or 1 unit if the size is less than 100. 每批药品的总量少于200单位时（例如临床试验用药品），取样量应减少到两单位。当批量少于100单位时，取样量应为1单位。

Select the sample(s) at random from the bulk material or from the available containers of the

preparation. To obtain the required quantity, mix the contents of a sufficient number of containers to provide the sample.

从批产品或制剂包装中随机取样，将充分量的包装单位中的内容物混合后取样以获得规定的供试品量。

5-2. EXAMINATION OF THE PRODUCT 供试品检测

5-2-1. Membrane filtration 薄膜过滤法

Use a filtration apparatus designed to allow the transfer of the filter to the medium. Prepare the sample using a method that has been shown suitable as described in section 4 and transfer the appropriate amount to each of 2 membrane filters and filter immediately. Wash each filter following the procedure shown to be suitable.

选择的薄膜过滤器应能使薄膜转移到培养基中，按照第四部分中的方法制备供试液，立即将供试品转移到过滤器过滤，制备两张滤膜，采用适当的方法冲洗滤膜。

For the determination of TAMC, transfer one of the membrane filters to the surface of casein soya bean digest agar. For the determination of TYMC, transfer the other membrane to the surface of Sabouraud-dextrose agar. Incubate the plate of casein soya bean digest agar at 30-35℃ for 3-5 days and the plate of Sabouraud-dextrose agar at 20-25℃ for 5-7 days. Calculate the number of CFU per gram or per millilitre of product. 为测定总好氧菌数量，将其中一滤膜接种于干酪素大豆消化琼脂培养基表面。为测定酵母菌或霉菌总数，将另一滤膜接种于沙保氏-葡萄糖琼脂培养基表面。接种的干酪素大豆消化琼脂培养基在30-35℃条件下培养3-5天，接种的沙保氏-葡萄糖琼脂培养基在20-25℃条件下培养5-7天。记录供试品的CFU/g 或CFU/ml。

When examing transdermal patches, filter 10 per cent of the volume of the preparation described under 4-5-1 separately through each of 2 sterile filter membranes. Transfer one membrane to casein soya bean digest agar for TAMC and the other membrane to Sabouraud-dextrose agar for TYMC.

当检测透皮吸收剂时，取按照（4-5-1）制备的供试液10%的量用于薄膜过滤，制备两张滤膜。将其中一滤膜接种于干酪素大豆消化琼脂培养基用于测定总好氧菌总数，另一滤膜接种于沙保氏-葡萄糖琼脂培养基用于测定酵母菌或霉菌总数。

5-2-2. Plate-count methods 平皿计数法 5-2-2-1. Pour-plate method 平皿法 Prepare the sample using a method that has been shown to be suitable as described in section 4. Prepare for each medium at least 2 Petri dishes for each level dilution. Inculate the plates of casein soya bean digest agar at 30-35℃ for 3-5 days and the plates of Sabouraud-dextrose agar at 20-25℃ for 5-7 days. Select the plates corresponding to a given dilution and showing the highest number of colonies less than 250 for TAMC and 50 for TYMC. Take the arithmetic mean per culture medium of the counts and calculate the number of CFU per gram or per millilitre of product.

按照第四部分中的方法制备供试液，对于每一稀释级的供试液至少接种于两个皮氏培养皿中。干酪素大豆消化琼脂培养基在30-35℃条件下培养3-5天，沙保氏-葡萄糖琼脂培养基在20-25℃条件下培养5-7天。选择总好氧菌数小于250、总酵母菌或霉菌数小于50的稀释

级作为计算菌数的依据，计算供试品CFU/g或CFU/ml。

5-2-2-2. Surface-spread method 表面涂布法

Prepare the sample using a method that has been shown to be suitable as described in section 4. Prepare at least 2 Petri dishes for each medium and each level of dilution. For incubation and calculation of the number of CFU proceed as described for the pour-plate method.

按照第四部分中的方法制备供试液, 对于每一稀释级的供试液至少接种于两个皮氏培养皿中。参照平皿法中的方法培养并计算CFU值。

5-2-2-3. Most-probable-number method 最大可能数法 Prepare and dilute the sample using a method that has been shown to be suitable as described in section 4. Incubate all tubes at 30-35℃ for 3-5 days. Subculture if necessary, using the procedure shown to be suitable. Record for each level of dilution the number of tubes showing microbial growth. Determine the most probable number of micto-organisms per gram or millilitre of the product to be examined from Table 2.6.12.-3.

按照第四部分中的方法制备供试液，将所有接种后的试管在30-35℃条件下培养3-5天。如果需要，可对培养液进行次培养。记录每一稀释级的试管中微生物的生长情况，参照表2.6.12.-3 计算供试品中微生物的 MPN/g 或/ml 。

5-3. INTERPRETATION OF THE RESULTS 试验结果分析

The total aerobic microbial count (TAMC) is considered to be equal to the number of CFU found using casein soya bean digest agar; if colonies of fungi are detected on this medium, they are counted as part of the TAMC. The total combined yeasts/mould count (TYMC) is considered to be equal to the number of CFU found using Sabouraud-dextrose agar; if colonies of bacteria are detected on this medium, they are counted as part of the TYMC. When the TYMC is expected to exceed the acceptance criterion due to the bacterial growth, Sabouraud-dextrose agar containing antibiotics may be used. If the count is carried out by the MPN method the calculated value is the TAMC.

总好氧菌数为干酪素大豆消化琼脂培养基的CFU值，如果在此培养基中检测到真菌菌落，也计为好氧菌的一部分。酵母菌/霉菌总数为沙保氏-葡萄糖琼脂培养基的CFU值，如果在此培养基中检测到其他的细菌菌落，也计为总酵母菌/霉菌数。如果由于细菌的生长使总酵母菌/霉菌数超出标准值，沙保氏-葡萄糖琼脂培养基中需加入抗生素。如果使用最大可能数法，记录的结果为总好氧菌数。

When an acceptance criterion for microbiological quality is prescribed it is interpreted as follows:微生物的限度标准如下：

1

— 10CFU：maximum acceptable count = 20;

1

10CFU是指最大可接受数为20；

2

— 10CFU：maximum acceptable count = 200;

2

10CFU是指最大可接受数为200；

3

— 10CFU：maximum acceptable count = 2000; and so forth.

10CFU是指最大可接受数为2000；等。

The recommended solutions and media are described in general chapter 2.6.13. 推荐使用的稀释液和培养基参见（2.6.13）。

3