USP71 无菌检查法Sterility Tests - 图文

USP<71>无菌检查法Sterility Tests

无菌检查法系用于检查药典要求无菌的药物、制剂产品和其他物品是否无菌的一种方法。若供试品符合无菌检查法的规定，仅表明供试品在该检验条件下未发现微生物污染。 The test is applied to substances, preparations, or articles which, according to the Pharmacopeia,are required to be sterile. However, a satisfactory result only indicates that no contaminating microorganism has been found in the sample examined under the conditions of the test.

1 预防微生物污染

PrecaotionsAgainst Microbial Contamination

无菌检查应在无菌条件下进行，为了达到条件，检测环境应符合无菌检查的规定。防止污染的措施不得影响供试品中微生物的检出。检测环境应定期抽样监测并进行适当控制。 The test for sterility is carried out under aseptic conditions. In order to achieve such conditions, the test environment has to be adapted to the way in which the sterility test is performed. The precautions taken to avoid contamination are such that they do not affect any microorganisms that are to be revealed in the test. The working conditions in which the tests are performed are monitored regularly by appropriate sampling of the working area and by carrying out appropriate controls. 2 培养基和培养温度

Culture Media and Incubation

无菌检查需制备下表所述培养基，或者是能够符合需氧菌、厌氧菌、真菌促生长试验要求的同等的商用培养基。

Media for the test may be prepared as described below or equivalent commercial media may beused provided that they comply with the requirements of the Growth Promotion Test of Aerobes,Anaerobes, and Fungi.

以下培养基已经被证实适合用于无菌检查。硫乙醇酸盐流体培养基主要用于厌氧菌的培养，但其也可用于需氧菌培养。大豆酪蛋白培养基适合于培养真菌和需氧菌。 The following culture media have been found to be suitable for the test for sterility. FluidThioglycollate Medium is primarily intended for the culture of anaerobic bacteria. However, it will also detect aerobic bacteria. Soybean–Casein Digest Medium is suitable for the culture of both fungi and aerobic bacteria.

将L-胱氨酸、氯化钠、葡萄糖、酵母提取物、酪蛋白胰酶消化物与纯化水混合，并加热至溶解。将硫乙醇酸钠或硫乙醇酸溶解于该溶液，如果需要可再加入1mol/L氢氧化钠溶液，以便在灭菌后该溶液呈pH值7.1±0.2。如需要过滤，再次加热该溶液但不得煮沸，并趁热以润湿滤纸将该溶液过滤。加入刃天青钠溶液，混匀，并将该培养基置于适当容

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器中，该容器应为培养基提供特定的面积/深度比，以使在培养期结束后能明确显示氧气摄入的变色部分不超过培养基的上半部分二分之一。使用经过验证的工艺进行灭菌。如果需要贮存该培养基，应将其置于无菌、气密容器中，在2~25℃贮藏。如果超过上部三分之一的培养基已经呈粉红色，可以用以下方法恢复该培养基功能，但每批培养基仅能恢复一次：在水浴锅中或者自由流动蒸汽中加热该容器，直至粉色消失，并迅速放凉，须小心防止非无菌空气进入到容器中。灭菌后培养基存放时间超过验证期限时，不得使用。

Mix the L-cystine, agar, sodium chloride, dextrose, yeast extract, and pancreatic digest of casein with the purified water, and heat until solution is effected. Dissolve the sodium thioglycollate or thioglycolic acid in the solution and, if necessary, add 1 N sodium hydroxide so that, after sterilization, the solution will have a pH of 7.1 ± 0.2. If filtration is necessary, heat the solution again without boiling, and filter while hot through moistened filter paper. Add the resazurin sodium solution, mix, and place the medium in suitable vessels that provide a ratio of surface to depth of medium such that not more than the upper half of the medium has undergone a color change indicative of oxygen uptake at the end of the incubation period. Sterilize using a validated process. If the mediumis stored, store at a temperature between 2℃ and 25℃ in a sterile, airtight container. If more than the upper one-third of the medium has acquired a pink color, the medium may be restored once by heating the containers in a water-bath or in free-flowing steam until the pink color disappears and by cooling quickly, taking care to prevent the introduction of nonsterile air into the container. Do not use the medium for a longer storage period than has been validated.

硫乙醇酸盐流体培养基 Fluid Thioglycollate Medium L-胱氨酸L-cystine 氯化钠Sodium Chloride 水合葡萄糖/无水葡萄糖Dextrose Monohydrate/Anhydrous 琼脂Agar 酵母提取物（水溶的）Yeast Extract (water-soluble) 胰酶消化酪蛋白胨Pancreatic Digest of Casein 硫乙醇酸钠Sodium Thioglycollate 或硫乙醇酸or Thioglycolic Acid 新配制的刃天青水溶液（1:1000） Resazurin Sodium Solution (1 in 1000),freshly prepared 纯化水Purified Water 灭菌后pH：7.1±0.2 pH after sterilization: 7.1±0.2.

硫乙醇酸盐流体培养基应在30~35℃条件下进行培养。含有汞制剂防腐剂的产品不能使用膜过滤方法检测。经需氧菌、厌氧菌、真菌促生长试验验证，在20~25℃培养时，大豆酪蛋白消化培养基可以替代硫乙醇酸盐流体培养基。经合理授权，不含琼脂和刃天青钠溶液配制的硫乙醇酸盐流体培养基可以替代硫乙醇酸盐流体培养基使用。按上述方法灭菌，灭菌后pH值为7.1±0.2。使用前使用水浴加热，置于30~35℃厌氧条件下培养。

0.5g 2.5g 5.5/5.0g 0.75g 5.0g 15.0g 0.5g 0.3ml 1.0ml 1000ml 2 / 20

Fluid Thioglycollate Medium is to be incubated at 30–35℃. For products containing a mercurialpreservative that cannot be tested by the membrane filtration method, Fluid Thioglycollate Mediumincubated at 20–25℃ may be used instead of Soybean–Casein Digest Medium provided that it hasbeen validated as described in Growth Promotion Test of Aerobes, Anaerobes, and Fungi. Whereprescribed or justified and authorized, the following alternative thioglycollate medium might be used.Prepare a mixture having the same composition as that of the Fluid Thioglycollate Medium, butomitting the agar and the resazurin sodium solution. Sterilize as directed above. The pH aftersterilization is 7.1 ± 0.2. Heat in a water bath prior to use and incubate at 30–35℃ under anaerobicconditions. 大豆酪蛋白消化物培养基 Soybean–Casein Digest Medium 酪蛋白胰酶消化物Pancreatic Digest of Casein 大豆粉木瓜蛋白酶消化物 Papaic Digest of Soybean Meal 氯化钠Sodium Chloride 磷酸氢二钾Dibasic Potassium Phosphate 水合葡萄糖/无水 Dextrose Monohydrate/Anhydrous 纯化水Purified Water 灭菌后pH：7.3±0.2 pH after sterilization: 7.3±0.2.

将干粉培养基置纯化水中，轻微加热使其溶解。溶液放凉至室温，并用1mol/L氢氧化钠溶液调整pH值，使其灭菌后pH值为7.3±0.2。如需要使之澄清，则过滤，分装入适合的容器，并用经过验证的程序灭菌。如果不立刻使用，则保存在2~25℃无菌且密闭良好的容器中。灭菌后培养基存放时间超过验证期限时，不得使用。

Dissolve the solids in the Purified Water, heating slightly to effect a solution. Cool the solution toroom temperature, and adjust the pH with 1 N sodium hydroxide so that, after sterilization, it willhave a pH of 7.3±0.2. Filter, if necessary to clarify, dispense into suitable containers, and sterilizeusing a validated procedure. Store at a temperature between 2℃ and 25℃ in a sterile well-closedcontainer, unless it is intended for immediate use. Do not use the medium for a longer storage periodthan has been validated.

大豆酪蛋白消化物培养基应在22.5℃±2.5℃条件下培养。 Soybean–Casein Digest Medium is to be incubated at 22.5℃± 2.5℃. 2.1用于青霉素和头孢菌素的培养基 Media for Penicillins or Cephalosporins

当无菌检查培养基用于供试产品无菌检查项下的直接接种法检验时，按如下内容变更硫乙醇酸盐流体培养基和大豆酪蛋白培养基的制备方法。向每一种培养基的容器中，

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17.0g 3.0g 5.0g 2.5g 2.5/2.3g 1000ml

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