USP38-NF33_C71_无菌检查法-中英对照

1、USP38 NF33<71> STERILITY TESTS<71>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

2、方对应部分进行了协调，不一致的部分用符号()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

3、品的无菌性主要通过对灭菌工艺或者无菌保障程序的验证来完成。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

4、the test.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。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 s

5、terility 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 carryin

6、g out appropriate controls.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

7、of the Growth Promotion 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 w

8、ill also detect aerobic 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

9、天青水溶液（1:1000）1.0mL纯化水1000 mLpH after sterilization: 7.1±0.2.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

10、 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 solutionagain without boiling, and filter while hot through moistened filter paper. Add the resazurin sodium solution, mix, a

11、nd 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 medium is stored, s

12、tore 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

13、, 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.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7.1±0.2。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

14、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°

15、; 35° . For products containing a 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 Te

16、st of Aerobes, Anaerobes, andFungi. Where prescribed 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. Steriliz

17、e as directed above. The pH after sterilization is 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

18、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厌氧条件下培养。pH after sterilization: 7.3±0.2.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

19、to room temperature, and adjust the pH with 1 N sodium 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

20、sterile well-closed container, unless it is intended for immediate use. Do not use 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7.1±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。S

21、oybeanCasein Digest Medium is to be incubated at 22.5° ± 2.5°.大豆-酪胨消化物培养基在22.5±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

22、 be Examined, modify the preparation of Fluid Pancreatic Digest of Casein 17.0 g Papaic Digest of Soybean 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 fo

23、llows. 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 assayed prev

24、iously for itspenicillin- or cephalosporin-inactivating power. NOTESupplemented -lactamase media can also 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）

25、。（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 sterility 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 S

26、taphylococcus 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 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

27、-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, NBRC13276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, N

28、BRC 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

29、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

30、 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 microorganis

31、m: Clostridium sporogenes, Pseudomonas aeruginosa, and Staphylococcus aureus. Inoculate 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) o

32、f the following microorganisms, using a separate portion of medium for each of the 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

33、culture maintenance techniques (seed-lot systems) are used so that the viable microorganisms 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

34、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

35、A稀释剂APREPARATION制备Dissolve 1 g of peptic digest of animal tissue in water 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7.1±0.2。分装入容

36、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 (s

37、ee 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 a

38、rticles containing lecithin or oil, or for devices labeled as “sterilepathway.”向每升稀释剂A中，加入1mL聚山梨酯80，调节pH值至7.1±0.2，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

39、 1 L. Adjust the pH to obtain, after sterilization, a pH of 6.9 ± 0.2. Dispense 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6.9±0.2。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

40、 under Test for Sterility of the Product to be Examined using exactly the same 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, ad

41、d an inoculum of a small number of viable microorganisms (not more than 100 cfu) 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 (

42、for catgut and other surgical sutures for veterinary use: strands) to the culture 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 microorganism

43、s as those described above under Growth Promotion Test of Aerobes, Anaerobes, 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 vi

44、sible growth of microorganisms is obtained after the incubation, visually comparable 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

45、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 p

46、roduct, the product possesses antimicrobial activity that has not been satisfactorily 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

47、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) when 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 Sterilit

48、y 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。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 s

49、pecified in Table 3. If the contents of each article are of sufficient quantity (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

50、 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最少接种量（除另有规定和授权）液体

51、少于1mL每个包装全量140mL每个包装半量，且不少于1mL大于40mL，但不大于100mL20mL大于100mL每个包装内容物的10%，但不得少于20mL液体抗生素1mL需悬浮或乳化的不溶性制剂、乳膏、油膏每个包装不少于200mg内容物固体少于50mg每个包装全量50mg或以上，但不大于300mg每个包装半量，但不少于50mg300mg5g150mg多余5g500mg兽医用肠线和其他外科缝合线一股线的3部分（每个30cm长）外科敷料/棉花/纱布（在包装中）每包装100mg缝合线和其他独立包装的一次性材料材料整体其他医用设备整个设备，切成片或拆开表3批出厂产品的最小检验数量批产品数量最小样品数

52、量（除另有规定和授权）注射剂不多于100个容器10%或4个容器，选较多者多于100个，但不多于500个容器10个容器多于500个容器2%或者20个容器，选较少者大体积注射剂2%或者10个容器，选较少者固体抗生素固体制剂（<5g）20个容器固体制剂（5g）6个容器散装固体见固体原料眼科和其他非注射剂不多于200个容器5%或者2个容器，选较多者多于200个容器10个容器如果该产品存在于单剂量容器中，应用上述用于注射用剂方案兽医用肠线和其他外科缝合线2%或5个包装，选较多者，最多可达20个包装不多于100件10%或4个物品，选较多者多于100但不超过500件10个物品多于500件2%或20个物

53、品，选较少者固体原料最多4个容器每个容器多于4个容器，但不多于50个容器20%或4个容器，选较多者超过50个容器2%或者10个容器，选较多者如果批量大小未知，请使用相关规定的最大数。如果一个容器的内容物足够接种2个培养基，则此表格给出的容器数量为用于全部2个培养基的数量。The test may be carried out using the technique of Membrane Filtration or by Direct Inoculation of the Culture Medium with the product to be examined. Appropriate n

54、egative controls are included. The technique of membrane filtration is used whenever the nature of the product permits; that is, for filterable aqueous preparations, for alcoholic or oily preparations, and for preparations miscible with, or soluble in, aqueous or oily solvents, provided these solven

55、ts do not have an antimicrobial effect in the conditions of the test.无菌检验可以使用膜过滤法或培养基直接接种法进行，应设置适宜的阴性对照。但只要该产品的性质许可，就应使用膜过滤法，也就是说，对水溶性制剂、酒精或油性制剂、易混合或溶解于水或油性溶剂的制剂，即使没有抑菌作用，也均应尽量采用薄膜过滤法进行无菌检查。Membrane Filtration膜过滤法Use membrane filters having a nominal pore size not greater than 0.45 m, in which the e

56、ffectiveness to retain microorganisms has been established. Cellulose nitrate filters, for example, are used for aqueous, oily, and weakly alcoholic solutions; and cellulose acetate filters, for example, are used for strongly alcoholic solutions. Specially adapted filters may be needed forcertain pr

57、oducts (e.g., for antibiotics).使用标称孔径不大于0.45m的膜过滤器，此孔径已知能够有效截留微生物。例如，硝酸纤维素过滤器可用于水、油、烯醇溶液；而醋酸纤维素可用于浓醇溶液。特定产品（例如抗生素）可能需要特别改造过的特殊过滤器。The technique described below assumes that membranes about 50 mm in diameter will be used. If filters of a different diameter are used, the volumes of the dilutions and t

58、he washings should be adjusted accordingly. The filtration apparatus and membrane are sterilized by appropriate means. The apparatus is designed so that the solution to be examined can be introduced and filteredunder aseptic conditions: it permits the aseptic removal of the membrane for transfer to the medium, or it is suitable for carrying out the incubation after adding the medium to the apparatus itself.以下涉及的方法中所使用的均为直径约50mm的滤膜。如果使用不同直径的过滤器，稀释液和冲洗液的体积应当做相应调节。过滤器和滤膜都应该经过灭菌处理。过滤器应该满足