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1、精选优质文档-倾情为你奉上非无菌产品微生物学检查：微生物计数检查法USP61中英对照版 MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS: MICROBIAL ENUMENRATION TESTS非无菌产品微生物学检查：微生物计数检查法INTRODUCTION 导言The tests described hereafter will allow quantitative enumeration of mesophilic bacteria and fungi that may grow under aerobic conditions.以下所
2、描述的这些检测将使得对在有氧的条件下生长的嗜温性细菌和真菌进行定量计数成为可能。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 tak
3、en, and interpret the results as stated below.这些检测主要设计用于测定一种物质或制备品是否符合已确立的微生物质量标准。当用于此类目的时，需遵照以下所给的说明，包括待取样品的数量，并且按照下面所述解释结果。The methods are not applicable to products containing viable microorganisms as active ingredients.这些方法不适用于以活菌作为活性成分的产品。Alternative microbiological procedures, including automa
4、ted methods, may be used, provided that their equivalence to the Pharmacopeial method has been demonstrated.可以使用替代的微生物规程，包括自动化方法，只要已经证明它们与药典方法具同等作用。GENERAL PROCEDURES 通用规程Carry out the determination under conditions designed to avoid 12 microbial contamination of the product to be examined. The prec
5、autions taken to avoid contamination must be such that they do not affect any microorganisms that are to be revealed in the test.在经过设计可避免外来微生物污染供试产品的条件下，进行此项测定。用于避免污染的这些预防措施是必须做到，它们不会影响任何试图在此项检验中揭示的微生物。If the product to be examined has antimicrobial activity, this is, insofar as possible, removed or
6、 neutralized. If inactivators are used for this purpose, their efficacy and their absence of toxicity for microorganisms must be demonstrated.如果供试产品具有抗菌活性，则此活性需在尽可能的范围内去除或中和。如果将灭活剂用于这个目的，则必须证实它们的功效和对微生物不具毒性。If surface-active substances are used for sample preparation, their absence of toxicity for m
7、icroorganisms and their compatibility with any inactivators used must be demonstrated.如果表面活性物质用于样品制备，则必须证实它们对微生物不具毒性以及与所使用的任何灭活剂的兼容性。ENUMERATION METHODS计数法Use the Membrane Filtration methodor one of the Plate-Count Methods, as directed. The Most-Probable-Number (MPN) Method is generally the least ac
8、curate method for microbial counts; however, for certain product groups with very low bioburden, it may be the most appropriate method.按规定，使用膜过滤法或多个平板计数法中的一种。液体稀释法（MPN）通常对微生物计数而言是最不准确的方法；然而，对具备非常低生物载荷的特定产品类型，它可能是最合适的方法。The choice of a method is based on factors such as the nature of the product and
9、the required limit of microorganisms. The method chosen must allow testing of a sufficient sample size to judge compliance with the specification. The suitability of the chosen method must be established.方法的选择基于某些因素，例如产品的性质和微生物的要求限度。所选的方法必须能够对充足的样本量进行检测，以判断与质量标准的符合性。所选方法的适用性必须被建立。Table 1. Preparatio
10、n and Use of Test Microorganisms表1. 供试微生物的制备和使用Growth Promotion生长促进Suitability of Counting Method in the Presence of Product在产品存在的情况下计数方法的适用性Microorganism微生物Preparation of Test Strain供试菌株的制备Total Aerobic Microbial Count总好氧微生物计数Total Yeasts and Molds Count 总酵母菌和霉菌计数Total Aerobic Microbial Count总好氧微生物
11、计数Total Yeasts and Molds Count总酵母菌和霉菌计数Staphylococcus aureus such as ATCC 6538, NCIMB 9518, CIP 4.83, or NBRC 13276金黄色葡萄球菌如：ATCC 6538, NCIMB 9518, CIP 4.83, 或 NBRC 13276Soybean-Casein Digest Agar or Soybean-Casein Digest Broth 300-350 18-24 hours大豆酪蛋白消化物琼脂培养基或大豆酪蛋白消化物肉汤培养基300-350 18-24 小时Soybean-Cas
12、ein Digest Agar and Soybean-Casein Digest Broth100 cfu 300-3503 days大豆酪蛋白消化物培养基和大豆酪蛋白消化物肉汤培养基100 cfu 300-350 3 天Soybean-Casein Digest Agar/MPN Soybean-Casein Digest Broth100 cfu 300-3503 days大豆酪蛋白消化物培养基/MPN（最大几率数）大豆酪蛋白消化物肉汤培养基100 cfu 300-350 3 天Pseudomonas aeruginosa such as ATCC 9027, NCIMB 8626,CI
13、P 82.118, or NBRC 13275绿脓杆菌如ATCC 9027, NCIMB 8626,CIP 82.118, 或 NBRC 13275Soybean-Casein Digest Agar or Soybean-Casein Digest Broth 300-350 18-24 hours大豆酪蛋白消化物琼脂培养基或大豆酪蛋白消化物肉汤培养基300-350 18-24 小时Soybean-Casein Digest Agar and Soybean-Casein Digest Broth100 cfu 300-3503 days大豆酪蛋白消化物琼脂培养基和大豆酪蛋白消化物肉汤培养基
14、100 cfu 300-3503 天Soybean-Casein Digest Agar/MPN Soybean-Casein Digest Broth100 cfu 300-3503 days大豆酪蛋白消化物琼脂培养基/MPN（最大几率数）大豆酪蛋白消化物肉汤培养基100 cfu 300-3503 天Bacillus subtilis such as ATCC 6633, NCIMB 8054, CIP 52.62, or NBRC 3134枯草芽孢杆菌 如ATCC 6633, NCIMB 8054, CIP 52.62, 或 NBRC 3134Soybean-Casein Digest A
15、gar or Soybean-Casein Digest Broth 300-350 18-24 hours大豆酪蛋白消化物琼脂培养基或大豆酪蛋白消化物肉汤培养基300-350 18-24 小时Soybean-Casein Digest Agar and Soybean-Casein Digest Broth100 cfu 300-3503 days大豆酪蛋白消化物琼脂培养基和大豆酪蛋白消化物肉汤培养基100 cfu 300-3503 天Soybean-Casein Digest Agar/MPN Soybean-Casein Digest Broth100 cfu 300-3503 days
16、大豆酪蛋白消化物琼脂培养基/MPN（最大几率数）大豆酪蛋白消化物肉汤培养基100 cfu 300-3503 天Candida albicans such as ATCC 10231, NCPF 3179, IP 48.72, or NBRC 1594白色念珠菌 如ATCC 10231, NCPF 3179, IP 48.72, 或 NBRC 1594Sabouraud Dextrose Agar or Sabouraud Dextrose Broth 200-250 2-3 daysSabouraud（沙氏）葡萄糖琼脂培养基或Sabouraud（沙氏）葡萄糖肉汤培养基Soybean-Casei
17、n Digest Agar 100 cfu 300-3505 days大豆酪蛋白消化物琼脂培养基100 cfu 300-3505 天Sabouraud Dextrose Agar 100 cfu 200-2505 daysSabouraud（沙氏）葡萄糖琼脂培养基100 cfu 200-2505 天Soybean-Casein Digest Agar100 cfu 300-3505 days MPN: not applicable大豆酪蛋白消化物琼脂培养基100 cfu 300-3505 天 MPN(最大几率数)：不适用Sabouraud Dextrose Agar100 cfu 200-25
18、05 daysSabouraud（沙氏）葡萄糖琼脂培养基100 cfu 200-2505 天Aspergillus niger such as ATCC 16404, IMI , IP 1431.83, or NBRC 9455黑曲霉如ATCC 16404, IMI , IP 1431.83, 或NBRC 9455Sabouraud Dextrose Agar or Potato-Dextrose Agar 200-250 5-7 days, or until good sporulation is achievedSabouraud（沙氏）葡萄糖琼脂培养基或马铃薯葡萄糖琼脂培养基200-25
19、05-7天,或直到实现良好的产孢Soybean-Casein Digest Agar100 cfu 300-3505 days大豆酪蛋白消化物琼脂培养基100 cfu 300-3505 天Sabouraud Dextrose Agar 100 cfu 200-2505 daysSabouraud（沙氏）葡萄糖琼脂培养基100 cfu 200-2505 天Soybean-Casein Digest Agar100 cfu 300-3505 days MPN: not applicable大豆酪蛋白消化物琼脂培养基100 cfu 300-3505 天MPN(最大几率数)：不适用Sabouraud
20、Dextrose Agar100 cfu 200-2505 daysSabouraud（沙氏）葡萄糖琼脂培养基100 cfu 200-2505 天GROWTH PROMOTION TEST AND SUITABILITY OF THE COUNTING METHOD 生长促进试验和计数方法的适用性General Considerations通用考虑因素The ability of the test to detect microorganisms in the presence of product to be tested must be established.在供试产品存在的情况下，必须
21、确立检测微生物的试验能力。Suitability must be confirmed if a change in testing performance or a change in the product that may affect the outcome of the test, is introduced.如果引入了可能影响试验结果的在测试性能或产品方面的变更，则必须确认其适用性。Preparation of Test Strains供试菌株的制备Use standardized stable suspensions of test strains or prepare as st
22、ated below. Seed-lot culture maintenance techniques (seed-lot systems) are used so that the viable microorganisms used for inoculation are not more than 5 passages removed from the original master seed-lot. Grow each of bacterial and fungal test strains separately as described in Table 1.使用供试菌株的标准化稳
23、定悬浮液或按下面所述制备。使用菌种保藏技术（种子批系统），以便用于接种的可萌发微生物从最初的主种子批开始传代不超过5次。按照在表1 中的描述，分别培养每个细菌和霉菌供试菌株。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% of polysorbate 80 may be added to the buffer. Use the suspension
24、s within 2 hours, or within 24 hours if stored between 2o and 8o. As an alternative to preparing and then diluting a fresh suspension of vegetative cells of A. niger or B. subtilis,a stable spore suspension is prepared and then an appropriate volume of the spore suspension is used for test inoculati
25、on. The stable spore suspension may be maintained at 2o to 8o for a validated period of time.使用pH 7.0的缓冲氯化钠-蛋白胨溶液，或pH7.2的磷酸盐缓冲液制作供试悬浮液；为使黑曲霉孢子悬浮，可以将0.05%的聚山梨醇酯80加入该缓冲液中。这些悬浮液需在2个小时之内使用，或如果存放在2o至8o的条件下，则可在24小时之内使用。作为制备然后稀释黑曲霉或枯草杆菌的新鲜体细胞悬浮液的替代方法，可制备稳定的孢子悬浮液，然后将适量的孢子悬浮液用于试验接种。此稳定的孢子悬浮液可以在2o至8o之间于一段经过验证
26、的时间内保存。Negative Control 阴性对照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 microorganisms.为了确认试验的条件，在制备供试品的场所使用所选的稀释液替代供试品，作阴性对照。其必须没有微生物的增长。Growth Promotion of the Media培养基的生长促进Test each batch of re
27、ady-prepared medium and each batch of medium prepared either from dehydrated medium or from the ingredients described.对每一批已经制备好的培养基和每一批从脱水培养基或根据描述的组分制备出来的培养基，进行测试。Inoculate portions/plates of Soybean-Casein Digest Broth and Soybean-Casein Digest Agar with a small number (not more than 100 cfu) of th
28、e microorganisms indicated in Table 1, using a separate portion/plate of medium for each. Inoculate plates of Sabouraud Dextrose Agar with a small number (not more than 100 cfu) of the microorganisms indicated in Table 1, using a separate plate of medium for each. Incubate according to the condition
29、s described in Table 1.在部分/整个平皿内的大豆酪蛋白消化物肉汤培养基和大豆酪蛋白消化物琼脂培养基中接种少量(不超过100cfu) 表1中指定的微生物，每种微生物均使用单独的部分/整个平皿的培养基。在平皿内的Sabouraud（沙氏）葡萄粮琼脂培养基中接种少量(不超过100cfu)表1中指定的微生物，每种均使用一个单独平皿的培养基。按照表中描述的条件进行培养。For solid media, growth obtained must not differ by a factor greater than 2 from the calculated value for a
30、standardized inoculum. For a freshly prepared inoculum, growth of the microorganisms 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 microorganisms comparable to that previously obtained w
31、ith a previously tested and approved batch of medium occurs.对于固体培养基，所获得的生长与标准化接种体的计算值之间的差异因素决不能大于2。对于刚刚制备好的接种体，发生的微生物生长与用此前检验并批准过的培养基批次所得到的微生物生长相当。如果出现了与此前从上一个经过测试并批准的培养基批次获得的微生物生长相当的、清晰可见的、微生物生长，则液体培养基适用。Suitability of the Counting Method in the Presence of Product在存在产品的情况下计数法的适用性PREPARATION OF THE
32、 SAMPLE样品的制备The method for sample preparation depends on the physical characteristics of the product to be tested. If none of the procedures described below can be demonstrated to be satisfactory, a suitable alternative procedure must be developed.样品制备方法取决于供试品的物理特征。如果以下描述的规程不能够被令人满意地证实，则必须开发一个适用的替代规
33、程。Water-Soluble ProductsDissolve 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 Soybean-Casein Digest Broth. If necessary, adjust to a pH of 6 to 8. Further dilutions, where nece
34、ssary, are prepared with the same diluent.水溶性产品溶解或稀释（通常制备1:10的稀释液）供试品，于pH值为7.0的缓冲氯化钠-蛋白胨溶液中，pH值为7.2的磷酸盐缓冲液，或大豆酪蛋白消化物肉汤培养基。如有必要，将pH值调整为6至8。在需要时，用相同的稀释剂进一步地稀释。Nonfatty Products Insoluble in WaterSuspend the product to be examined (usually a 1 in 10 dilution is prepared) in Buffered Sodium ChloridePept
35、one Solution pH 7.0, Phosphate Buffer Solution pH 7.2, or SoybeanCasein Digest Broth. A surfaceactive agent such as 1 g per L of polysorbate 80 may be added to assist the suspension of poorly wettable substances. If necessary, adjust to a pH of 6 to 8. Further dilutions, where necessary, are prepare
36、d with the same diluent.不溶于水的非脂肪性供试品将检测的供试品（通常制备1:10的稀释液）悬浮于pH值为7.0的缓冲氯化钠-蛋白胨溶液，pH值为7.2的磷酸盐缓冲液，或者大豆酪蛋白消化物肉汤培养基中。可以加入某个表面活性剂，比如1克每升的聚山梨酯80，以帮助难以与水混合的物质悬浮。如有必要，调整pH值为6至8。在需要时，用相同的稀释剂进一步的稀释。Fatty ProductsDissolve in isopropyl myristate sterilized by filtration, or mix the product to be examined with th
37、e minimum necessary quantity of sterile polysorbate 80 or another noninhibitory sterile surface-active reagent heated, if necessary, to not more than 40o or, in exceptional cases, to not more than 45o. Mix carefully and if necessary maintain the temperature in a water bath. Add a sufficient quantity
38、 of the prewarmed chosen diluent to make a 1 in 10 dilution of the original product. Mix carefully, while maintaining the temperature for the shortest time necessary for the formation of an emulsion. Further serial 10-fold dilutions may be prepared using the chosen diluent containing a suitable conc
39、entration of sterile polysorbate 80 or another noninhibitory sterile surface-active reagent.脂肪性供试品溶解于以过滤除菌的豆蔻酸异丙酯，或者将供试品与所需最少量的、经过加热的无菌聚山梨酯80或另一种非抑菌性的无菌表面活性剂进行混合，如有必要，可加热至不超过40o或者，在特殊情况下，至不超过45o。仔细混匀，如有必要，在水浴锅里中维持该温度。加入充足数量、经过预热的所选择稀释剂，制成原产品的1:10稀释液。仔细混匀，同时维持该温度至形成乳化剂所必需的最短的时间。可以用所选择的含有适当浓度的无菌聚山梨酯80
40、或者另一种非抑菌性的无菌表面活性剂的稀释液，来制备后续的系列10倍稀释液。Fluids or Solids in Aerosol FormAseptically 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.气溶胶与液溶胶以无菌操作将供试品转移
41、至一个过滤膜设备或一个无菌容器里，以进一步取样。从每个被检测的容器中，使用全部内容物或剂量的规定倍数。Transdermal PatchesRemove the protective cover sheets (“release liners”) of the transdermal patches and place them, adhesive side upwards, on sterile glass or plastic trays. Cover the adhesive surface with a suitable sterile porous material (e.g., s
42、terile gauze) to prevent the patches 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 minutes.贴剂去除贴剂的防护面（“释放衬板”）并将它们放置在无菌玻璃或塑料托盘上，胶贴剂的一面向上。用适当的无
43、菌多孔材料（例如：无菌纱布）盖住胶贴面，以防止贴剂粘在一起，并将贴剂转移到所选的含有灭活剂（例如聚山梨醇酯80和/或卵磷脂）的适量稀释剂中。用力摇动供试品至少30分钟。INOCULATION AND DILUTION接种和稀释Add to the sample prepared as directed above 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 c
44、fu. The volume of the suspension of the inoculum should not exceed 1% of the volume of diluted product.向按上述规定制备的样品中以及向对照制备品（其中无供试物质）中，加入充足体积的微生物悬浮液，以获得一个不多于100 cfu的接种体。接种的菌体悬浮液体积应当不超过被稀释产品体积的1%。To demonstrate acceptable microbial recovery from the product, the lowest possible dilution factor of the
45、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 neutr
46、alization, dilution, or filtration.为证明供试品有可接受的微生物回收率，必须使用已制备样品的最低可能稀释倍数来进行检测。当由于抗菌活性或低溶解度而无法做到这一点时，必须进一步开发更适合的规程。如果样品对生长的抑制不能以其他方式避免，可以在中和、稀释、或过滤之后，加入等量的微生物悬浮液。NEUTRALIZATION/REMOVAL OF ANTIMICROBIAL ACTIVITY抗菌活性的中和/去除The number of microorganisms recovered from the prepared sample diluted as describ
47、ed in Inoculation and Dilution and incubated following the procedure described in Recovery of Microorganisms in the Presence of Product, is compared to the number of microorganisms recovered from the control preparation.用制备好的样品按接种和稀释项下的描述稀释，并按在产品存在的情况下微生物的回收率项下描述的规程培养，将从中回收的微生物的数量与从对照制备品中回收的微生物的数量进行
48、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,如果生长被抑制（以大于2的因素减少），那么修改特定的计数测试规程，以确保结果的有效性。规程的修改可以包括，例如：(1) An increase in the volume of the diluent or culture medium稀释剂或培养基体积的增加;(2) Incorporation of a specific or general neutralizing agents into the diluent将某个特定或通用的中和剂整合至稀释剂中;(3) Membrane filtration膜过滤; or或(4) A combination of the above measures上述方法的组合.Neutralizing AgentsNeutralizin