1、USP38 NF33STERILITY TESTS无菌检查法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.本检查法已与欧洲药典和日本药局方对应部分进行了协调，不一致的部分用符号(
2、 )标注。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.按药典规定的无菌检验本身并不能确保一批产品无菌或已经灭菌，产品的无菌性主要通过对灭菌工艺或者无菌保障
3、程序的验证来完成。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.无菌检查法系用于检查药
4、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。PRECAUTIONS AGAINST MICROBIAL CONTAMINATION预防微生物污染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 per
5、formed. 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 co
6、ntrols.无菌检查应在无菌条件下进行，为了达到该条件，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的控制。CULTURE MEDIA AND INCUBATION TEMPERATURES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 they comply with the requirements of the Growth Promot
7、ion Test of Aerobes, Anaerobes, and Fungi.无菌检查需制备下表所述培养基，或者是能够符合需气菌、厌氧菌、真菌促生长试验要求的同等的商用培养基。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 bothfungi and aerobic bacteria.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，但其也可用于需气菌培养。大豆-酪胨培养基适合于培养真菌和需气菌。硫乙醇酸盐流体培养基L-胱氨酸 0.5g氯化钠 2.5g水合葡萄糖/无水葡萄糖 5.5/5.0g琼脂 0.75g酵母提取物（水溶的） 5.0g胰酶消化酪蛋白胨 15.0g硫乙醇酸钠 0.5g或硫乙醇酸 0.3 mL新配制的刃天青水溶液（1:1000
9、） 1.0mL纯化水 1000 mLpH after sterilization: 7.10.2.灭菌后 pH：7.1 0.2。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
10、, 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 solutionagain without boiling, and filter while hot through moistened filter paper. Add the resazurin sodium solution, mix, and place the medium in suit
11、able 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 medium is stored, store at a temperature betwe
12、en 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 th
13、e introduction of nonsterile air into the container. Do not 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，并加热至溶解。将硫乙醇酸钠或硫乙醇酸溶解于该溶液，如果需要可加入1mol/L 氢氧化钠溶液，以便在灭菌后该溶液呈 pH 值 7.10.2。如需要过滤，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
14、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。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 225贮藏。如果超过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。Fluid Thioglycollate Medium is to be incubated at 30 35 . For products containing a
15、mercurial preservative that cannot be tested by the membrane filtration method, Fluid Thioglycollate Medium incubated at 20 25 may be used instead of SoybeanCasein Digest Medium provided that it has been validated as described in Growth Promotion Test of Aerobes, Anaerobes, andFungi. Where prescribe
16、d 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, but omitting the agar and the resazurin sodium solution. Sterilize as directed above. The pH after sterilization is
17、7.1 0.2. Heat in a water bath prior to use and incubate at 30 35 under anaerobic conditions.硫乙醇酸盐流体培养基应在 3035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。经需气菌、厌氧菌、真菌促生长试验验证，在2025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，配制的与硫乙醇酸盐流体培养基成分相同，但省略了琼脂和刃天青溶液的培养基，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 pH：7.1 0.2。使用前用水浴加热，置于 3035厌氧条
18、件下培养。pH after sterilization: 7.30.2.大豆-酪胨消化物培养基酪蛋白胰酶消化物 17.0g大豆粉木瓜蛋白酶消化物 3.0g氯化钠 5.0g磷酸氢二钾 2.5g水合葡萄糖/无水葡萄糖 2.5/2.3纯化水 1000mL灭菌后 pH：7.1 0.2Dissolve 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 sodium hydroxi
19、de 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 use. Do not us
20、e the medium for a longer storage period than has been validated.将固体物质置纯化水中，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 7.10.2。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225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。SoybeanCasein Digest Medium is to be incubated at 22.5 2.5.大豆-酪胨消化物培养基在 22.52.5条件
21、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 Pancreatic Digest of Casein 17.0 g Papaic Digest of Soybea
22、n Meal 3.0 g Sodium Chloride 5.0 g Dibasic Potassium Phosphate 2.5 g Dextrose Monohydrate/Anhydrous 2.5/2.3 g Purified Water 1000 mL Thioglycollate Medium and the SoybeanCasein Digest Medium as follows. To the containers of each medium, transfer aseptically a quantity of -lactamase sufficient to ina
23、ctivate 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 assayed previously for itspenicillin- or cephalosporin-inactivating power. NOTESupplemented -lactamase media can als
24、o be used in the membrane filtration test. 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-酪胨培养基的制备方法。向每一种培养基的容器中，以无菌操作转移足够量能灭活供试样品中抗生素的 -内酰胺酶（之前已经对 -内酰胺酶制品灭活青霉素或头孢菌素的能力进行过测定，据此来确定灭活抗生素所需的 -内酰胺酶量） 。 （注：添加 -内酰胺酶的培养基也可以用于膜过滤试验） 。Alternatively (in an area completely separate from that used for steri
25、lity testing), confirm that an appropriate amount of -lactamase is incorporated into the medium, following either method under Method Suitability Test, using less than 100 colony-forming units (cfu) of Staphylococcus aureus (see Table 1) as the challenge. Typical microbial growth of the inoculated c
26、ulture must be observed as a confirmation that the -lactamase concentration is appropriate. 或者（在与无菌试验完全隔离的区域） ，按照验证试验项下的任意一种方法，使用少于 100cfu 的金黄色葡萄球菌（表 1）作为验证菌，来确认该培养基中含有的 -内酰胺酶量是否适宜。必须观测到接种后培养物中出现典型的微生物生长，才能确认 -内酰胺酶量是适宜的。表 1 用于促生长试验和方法验证试验的测试菌株需气菌金黄色葡萄球菌 ATCC 6538, CIP 4.83, NCTC 10788, NCIMB 9518, N
27、BRC13276枯草芽孢杆菌 ATCC 6633, CIP 52.62, NCIMB 8054, NBRC 3134铜绿假单胞菌 ATCC 9027, NCIMB 8626, CIP 82.118, NBRC 13275厌氧菌生孢梭菌 ATCC 19404, CIP 79.3, NCTC 532 or ATCC 11437, NBRC14293真菌白色念珠菌 ATCC 10231, IP 48.72, NCPF 3179, NBRC 1594巴西曲霉 ATCC 16404, IP 1431.83, IMI 149007, NBRC 9455替代微生物是藤黄微球菌。当需要不形成芽孢微生物时，生孢
28、梭菌的替代微生物是普通拟杆菌。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，这些试验应在供试产品检验前完成或者同时进行。Sterility培养基无菌性Incubate portions of the media for 14 days. No growth of microorganisms occurs.取部分检验用培养基，连续培养 14 天，应不得出现微
29、生物生长。Growth Promotion Test of Aerobes, Anaerobes, and Fungi需气菌、厌氧菌和真菌的促生长试验Test each lot of ready-prepared medium and each batch of medium prepared either from dehydrated medium or from ingredients. Suitable strains of microorganisms are indicated in Table 1.每一批配制好的培养基（采用脱水培养基或按配方制备的培养基）均需进行检查，使用的微生
30、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 microorganism: Clostridium sporogenes, Pseudomonas aeruginosa, and Staphylococcus aureus. Ino
31、culate portions of alternative thioglycollate medium with a small number (not more than 100 cfu) of Clostridium sporogenes. Inoculate portions of SoybeanCasein Digest Medium with a small number (not more than 100 cfu) of the following microorganisms, using a separate portion of medium for each of th
32、e following species of microorganism: Aspergillus brasiliensis, Bacillus subtilis, and Candida albicans. Incubate for not more than 3 days in the case of bacteria and not more than 5 days in the case of fungi. Seed lot culture maintenance techniques (seed-lot systems) are used so that the viable mic
33、roorganisms used for inoculation are not more than five passages removed from the original master seed-lot.去取部分硫乙醇酸盐流体培养基，接种少量（不超过 100cfu）下列微生物，每一种微生物均分别接种于单独培养基中：生孢梭菌、铜绿假单胞菌、金黄色葡萄球菌（在替代硫乙醇酸盐的流体培养基中接种少量（不超过 100cfu）生孢梭菌） 。在大豆-酪胨消化物培养基中接种少量（不超过 100cfu）下列微生物，每一种微生物均接种于单独的培养基：黑曲霉、枯草芽孢杆菌、白色念珠菌。细菌培养时间不超过
34、3 天，真菌培养时间不超过 5 天。采用适宜的菌种保藏技术，以确保用于接种的微生物的传代次数不超过 5 代。The media are suitable if a clearly visible growth of the microorganisms occurs.如果可见清晰的微生物生长，则该培养基是符合要求的。DILUTING AND RINSING FLUIDS FOR MEMBRANE FILTRATION用于膜过滤的稀释剂和冲洗液Fluid A稀释剂 APREPARATION制备Dissolve 1 g of peptic digest of animal tissue in wa
35、ter to make 1 L, filter or centrifuge to clarify, if necessary, and adjust to a pH of 7.1 0.2. Dispense into containers, and sterilize using a validated process.将 1g 动物组织胃蛋白酶消化物溶于 1L 水中，如果需要则通过过滤或离心使其澄清，再调节 pH 值至 7.10.2。分装入容器中，并用经过验证的工艺灭菌。PREPARATION FOR PENICILLINS OR CEPHALOSPORINS用于青霉素或头孢菌素的稀释剂制备
36、Aseptically add to the above Preparation, if necessary, a quantity of sterile -lactamase sufficient to inactivate any residual antibiotic activity on the membranes after the solution of the test specimen has been filtered (see Media for Penicillins or Cephalosporins).在供试样品溶液已经过滤之后，如果需要，向上述制备的稀释剂中，以无
37、菌操作加入数量足够灭活滤膜上残余抗生素的 -内酰胺酶（见用于青霉素或头孢菌素的培养基） 。Fluid D稀释剂 DTo each L of Fluid A add 1 mL of polysorbate 80, adjust to a pH of 7.1 0.2, dispense into containers, and sterilize using a validated process. Use this fluid for articles containing lecithin or oil, or for devices labeled as “sterilepathway.”向
38、每升稀释剂 A 中，加入 1mL 聚山梨酯 80，调节 pH 值至 7.10.2，分装入容器中，并使用经过验证的工艺灭菌。此液体用于含有卵磷脂或油脂的样品，或用于标为“无菌通道”的器械。Fluid K稀释剂 KDissolve 5.0 g of peptic digest of animal tissue, 3.0 g of beef extract, and 10.0 g of polysorbate 80 in water to make 1 L. Adjust the pH to obtain, after sterilization, a pH of 6.9 0.2. Dispense
39、 into containers, and sterilize using a validated process.将 5.0g 动物组织胃蛋白酶消化物、3.0 牛肉提取物、10.0g 聚山梨酯 80 溶解于1L 水中。调节 pH 值，使其灭菌后 pH 值为 6.90.2。分装入容器中，并使用经过验证的工艺灭菌。METHOD SUITABILITY TEST方法适用性试验Carry out a test as described below under Test for Sterility of the Product to be Examined using exactly the same
40、 methods, except for the following modifications.严格按照供试产品无菌检查项下的方法进行无菌检查。当使用到以下方法并需要进行方法调整时，需重新进行方法适用性实验。Membrane Filtration膜过滤法After transferring the content of the container or containers to be tested to the membrane, add an inoculum of a small number of viable microorganisms (not more than 100 cf
41、u) to the final portion of sterile diluent used to rinse the filter.在将一个或多个供试容器中的内容物转移到滤膜之后，在最后一次的冲洗液中加入少量（不超过 100cfu）测试菌株。Direct Inoculation直接接种法After transferring the contents of the container or containers to be tested (for catgut and other surgical sutures for veterinary use: strands) to the cul
42、ture medium, add an inoculum of a small number of viable microorganisms (not more than 100 cfu) to the medium.在将一个或多个供试容器（对于兽医用的肠线和其他外科缝合用线：若干股线）中的内容物转移至培养基之后，将少量试验菌（不超过 100cfu）加入培养基中。In both cases use the same microorganisms as those described above under Growth Promotion Test of Aerobes, Anaerobes
43、, and Fungi. Perform a growth promotion test as a positive control. Incubate all the containers containing medium for not more than 5 days.以上两种情况，均使用上述需气菌、厌氧菌、真菌促生长试验项下规定的菌株。同时设置促生长试验作为阳性对照。微生物在相应培养基中的培养时间不超过5 天。If clearly visible growth of microorganisms is obtained after the incubation, visually c
44、omparable to that in the control vessel without product, either the product possesses no antimicrobial activity under the conditions of the test or such activity has been satisfactorily eliminated. The test for sterility may then be carried out without further modification.若培养后可见清晰的微生物生长，外观与未接种样品的对照
45、菌生长类似，则认为该产品在此试验条件下没有抑菌作用，或者认为可能存在的抑菌作用已经被较完全的消除。此时可以认为该方法无需进一步的变更，无菌试验依法检查即可。If clearly visible growth is not obtained in the presence of the product to be tested, visually comparable to that in the control vessels without product, the product possesses antimicrobial activity that has not been sati
46、sfactorily eliminated under the conditions of the test. Modify the conditions in order to eliminate the antimicrobial activity, and repeat the Method Suitability Test.与未接种样品的对照相比，若在供试品存在条件下不能观察到肉眼可见的混浊，则认为该试验条件下不能消除供试品的抑菌作用。需要对实验条件进行调整以消除抗菌活性，并重新进行方法适用性试验。This method suitability is performed (a) whe
47、n the test for sterility has to be carried out on a new product; and (b) whenever there is a change in the experimental conditions of the test. The method suitability may be performed simultaneously with the Test for Sterility of the Product to be Examined.当一个新产品进行无菌试验时和无论何时无菌试验的试验条件发生改变时，则需进行此验证试验。
48、该验证可以与供试产品无菌检查同时进行。TEST FOR STERILITY OF THE PRODUCT TO BE EXAMINED供试产品无菌检查Number of Articles to Be Tested供试品数量Unless otherwise specified elsewhere in this chapter or in the individual monograph, test the number of articles specified in Table 3. If the contents of each article are of sufficient quan
49、tity (see Table 2), they may be divided so that equal appropriate portions are added to each of the specified media. NOTEPerform sterility testing employing two or more of the specified media. If each article does not contain sufficient quantities for each medium, use twice the number of articles indicated in Table 3.除非在本章节的其他部分或在具体的各论中另有规定，供试物品的数量遵照表3 中的规定。如果每个被检验物品的内容物有足够数量（见表 2） ，可以将其分成若干等份，将适当的等份加入到每个指定的培养基。 （注：使用两个或更多指定培养基，来进行无菌试验。 ）如果每个被检验物品的内容物规格不能满足单个培养基的接种量要求时，检验量扩大为表 3 所规定的检验数量的 2 倍。表 2 每种培养基的最少接种量供试品装量
