美国药典34版71无菌检测中文.docx

1、美国药典34版71无菌检测中文71 STERILITY TESTS 无菌检测Portions of this general chapter have been harmonized with the corresponding texts of the European Pharmacopeia and/or the Japanese Pharmacopeia. Those portions that are not harmonized are marked with symbols to specify this fact. 此通则的各部分已经与欧洲药典和/或日本药典的对应部分做了协调。

2、不一致的部分用符号来标明。These Pharmacopeial procedures are not by themselves designed to ensure that a batch of product is sterile or has been sterilized. This is accomplished primarily by validation of the sterilization process or of the aseptic processing procedures.这些药典规定程序自身的设计不能确保一批产品无菌或已经灭菌。这主要是通过灭菌工艺或者无

3、菌操作程序的验证来完成。The test is applied to substance, 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.检测应用于根据药典

4、要求无菌的如物质、药剂或商品。而令人满意的结果只包括在这样的检测条件下被测样品的完全无微生物污染。Precautions against microbial contamination微生物污染的预防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 pre

5、cautions 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.无菌测试要在无菌

6、条件下进行。为了达到这样的条件，测试环境要适应于无菌测试的要求。为了避免微生物污染而采取的预防措施不能有可检出的微生物污染。进行测试的环境要进行定期的取样检测并采取适宜的控制措施。Culture media and incubation temperature培养基和培养温度Media for the test may be prepared as described below or equivalent commercial media may be used provided that comply with the requirements of the Growth Promotio

7、n Test of Aerobes, Anaerobes, and Fungi.用于无菌测试的培养基可以按下述方法配制或采用满足好氧菌，厌氧菌和真菌促生长要求的物质的等效商业培养基。The following culture media have been found to be suitable for the test for sterility. Fluid Thioglycollate Medium is primarily intended for the culture of anaerobic bacteria. However, it will also detect aero

8、bic bacteria. SoybeanCasein Digest Medium is suitable for the culture of both fungi and aerobic bacteria.下面的培养基已经被证实适合进行无菌检测。巯基醋酸盐液体培养基主要用于厌氧菌的培养。但其也用于检测好氧菌。大豆酪蛋白消化物培养基适合于培养霉菌和好氧菌。Fluid Thioglycollate Medium 巯基醋酸盐液体培养基L-Cystine L-胱氨酸0.5gSodium Chloride氯化钠2.5gDextrose Monohydrate/Anhydrous葡萄糖水化物/无水葡萄

9、糖5.5/5.0gAgar 琼脂0.75gYeast Extract (water-soluble)酵母提取物(水溶性)5.0gPancreatic Digest of Casein酪蛋白胰酶消化物15.0gSodium Thioglycollate巯基乙酸钠0.5gor Thioglycolic Acid或者巯基乙酸0.3mlResazurin Sodium Solution (1 in 1000),freshly prepared刃天青钠溶液(1比1000)，新配制1.0mlPurified Water 纯化水1000mlpH after sterilization:7.10.2 灭菌后p

10、H：7.10.2Mix the L-cystine, 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 steril

11、ization, 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 medi

12、um 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 medium is stored, store at a temperature between 2 and 25 in a sterile, airtight container. If more than t

13、he 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

14、use the medium for a longer storage period than has been validated.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，并加热至实现溶解。将巯基乙酸钠或者巯基乙酸溶解于该溶液，如果需要可再加入1N氢氧化钠，以便在灭菌后该溶液呈pH值7.1 0.2。如需要则过滤，再次加热该溶液但不得煮沸，并趁热以湿润滤纸将该溶液过滤。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，以使在培养期末表明氧气摄入的变色部分不超过培养基的上半部分。使用经过验证的工艺进行灭菌。如果需要储存该培养

15、基，将其置于无菌、气密容器中，在2至25之间储藏。如果超过上部三分之一的培养基已经呈粉色，可以用以下方法恢复该培养基一次：在水浴锅中或者自由流动蒸气中加热该容器，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。禁止使用超过验证储存期限的培养基。Fluid Thioglycollate Medium is to be incubated at30-35.For products containing a mercurial preservative that cannot be tested by the membrane filtration method, Fluid Thiog

16、lycollate Medium incubated at 20-25may be used instead of Soybean-Casein Digest Medium provided that it has been validated in Growth Promotion Test of Aerobes, Anaerobes, and Fungi. Where prescribed or justified and authorized, the following alternative thioglycollate medium might be used. Prepare a

17、 mixture having the same composition as that of the Fluid Thioglycollate Medium, but omitting the agar and the resazurin sodium solution. Sterilize as described above. The pH after sterilization is 7.10.2. Heat in a water bath prior to use and incubate at 30-35under anaerobic conditions.巯基醋酸盐液体培养基将在

18、30-35条件下进行培养。如果产品含有不能用滤膜法检测的防腐剂，在20-25培养的巯基醋酸盐液体培养可以代替通过好氧菌、厌氧菌和真菌促生长实验验证的大豆-酪蛋白消化物培养基。如果有规定或经授权的正当理由，下面的替代巯基醋酸盐培养基也可以使用。配制与巯基醋酸盐液体培养基成分相同，但省略了琼脂和刃天青钠溶液的混合物，按上述方法灭菌。灭菌后pH值为7.1 0.2。使用前水浴加热，在厌氧条件下培养。SoybeanCasein Digest Medium 大豆-酪蛋白消化物培养基Pancreatic Digest of Casein酪蛋白胰酶消化物17.0 gPapaic Digest of Soybe

19、an Meal大豆粉木瓜蛋白酶消化物3.0 gSodium Chloride氯化钠5.0 gDibasic Potassium Phosphate磷酸氢二钾2.5 gDextrose (C6H12O6H2O)葡萄糖2.5/2.3 gPurified Water纯化水1000 mL灭菌后pH7.30.2Dissolve the solids in the Purified Water, heating slightly to effect a solution. Cool the solution to room temperature, and adjust the pH with 1 N s

20、odium hydroxide so that, after sterilization, it will have a pH of 7.3 0.2. Filter, if necessary to clarify, dispense into suitable containers, and sterilize using a validated procedure. Store at a temperature between 2 and 25 in a sterile well-closed container, unless it is intended for immediate u

21、se. Do not use the medium for a longer storage period than has been validated.将固体物质溶解于纯净水，轻微加热以实现溶解。放凉溶液至室温，并用1N氢氧化钠调整pH值，以便在灭菌后其pH值呈7.3 0.2。过滤，如需要则使之澄清，分装入适合的容器，并用经过验证的程序消毒。如果不立刻使用，则在2到25之间以无菌且密闭良好的容器保存。禁止使用超过验证储存时间的培养基。SoybeanCasein Digest Medium is to be incubated at 22.5 2.5 .大豆-酪蛋白消化物培养基将在22.5

22、2.5 条件下培养。Media for Penicillins or Cephalosporins 用于青霉素和头孢菌素的培养基Where sterility test media are to be used in the Direct Inoculation of the Culture Medium method under Test for Sterility of the Product to be Examined, modify the preparation of Fluid Thioglycollate Medium and the SoybeanCasein Digest

23、Medium as follows. To the containers of each medium, transfer aseptically a quantity of-lactamase sufficient to inactivate the amount of antibiotic in the specimen under test. Determine the quantity of-lactamase required to inactivate the antibiotic by using a -lactamase preparation that has been as

24、sayed previously for its penicillin- or cephalosporin-inactivating power. NOTESupplemented-lactamase media can also be used in the membrane filtration test. Alternatively (in an area completely separate from that used for sterility testing), confirm that an appropriate amount of -lactamase is incorp

25、orated into the medium, following either method under Validation Test, using less than 100 colony-forming units (cfu) of Staphylococcus aureus (see Table 1) as the challenge. Typical microbial growth of the inoculated culture must be observed as a confirmation that the -lactamase concentration is ap

26、propriate. 当无菌检查培养基用于供试产品无菌检查项下的培养基直接接种法时，按如下内容变更巯基醋酸盐液体培养基和大豆-酪蛋白消化物培养基的制备方法。向每一种培养基的容器中，以无菌操作转移足够灭活供试样品中所存在抗生素的-内酰胺酶。使用此前已经对其青霉素或头孢菌素灭活能力进行了测定的-内酰胺酶配制品，来测定灭活该抗生素所必需的-内酰胺酶数量。注意：补充的-内酰胺酶培养基也可以用于膜过滤试验 或者（在与无菌试验所用场所彻底隔离的区域中），按照验证试验项下的任意一种方法，使用少于100个菌落（cfu）的金黄色葡萄球菌（见表1）作为验证菌，来确认适当数量的-内酰胺酶已经被整合到该培养基中。必须

27、观测到接种后培养物中出现典型微生物生长，才能确认-内酰胺酶浓度是适当的。Table 1. Strains of the Test Microorganisms Suitable for Use in the Growth Promotion Test and theValidation Test 表1 适合用于生长促进试验和验证试验中的试验微生物的菌株Aerobic bacteria好氧菌Staphylococcus aureus 金黄色葡萄球菌ATCC 6538, CIP 4.83, NCTC 10788, NCIMB 9518NBRC 13276Bacillus subtilis枯草芽孢杆

28、菌ATCC 6633, CIP 52.62, NCIMB 8054, NBRC 3134Pseudomonas aeruginosa 绿脓杆菌ATCC 9027, NCIMB 8626, CIP 82.118, NBRC 13275Anaerobic bacterium厌氧菌Clostridium sporogenes 产芽胞梭状芽胞杆菌ATCC 19404, CIP 79.3, NCTC 532 or ATCC 11437, NBRC14293Fungi霉菌Candida albicans白色念珠菌ATCC 10231, IP 48.72, NCPF 3179, NBRC 1594Asper

29、gillus niger黑曲霉ATCC 16404, IP 1431.83, IMI 149007, NBRC94551 An alternative microorganism is Kocuria rhizophila(micrococcus luteus)ATCC 9341.替代微生物是藤黄微球菌(micrococcus luteus)，ATCC 93412 An alternative to Clostridium sporogenes, when a nonspore-forming microorganism is desired, is Bacetroides vulgatus

30、(ATCC 8482). 当需要不形成芽孢微生物时，产芽胞梭状芽胞杆菌的替代微生物是Bacetroides vulgatus (ATCC 8482)The media used comply with the following tests, carried out before, or in parallel, with the test on the product to be examined.所使用的培养基须符合下列试验，这些试验应在检验供试产品之前或者同时进行。STERILITY 无菌状态Confirm the sterility of each sterilized batch o

31、f medium by incubating a portion of the media at the specified incubation temperature for 14 days. No growth of microorganisms occurs.通过在指定培养温度下将一部分培养基培养14天，来确认每一批已灭菌培养基的无菌状态。不得出现微生物生长。GROWTH PROMOTION TEST OF AEROBES, ANAEROBES, and FUNGI 好氧菌、厌氧菌、霉菌的生长促生长试验Test each lot of ready-prepared medium and

32、 each batch of medium prepared either from dehydrated medium or from ingredients 1 . Suitable strains of microorganisms are indicated in Table 1.检查每一批已经配制好的培养基和每一批用脱水培养基或配料制备的培养基 1 。适当微生物菌株见表1。Inoculate portions of Fluid Thioglycollate Medium with a small number (not more than 100 cfu) of the following microorganisms, using a separate portion of medium for each of the following species of microorga