USP61非无菌产品微生物学检查：微生物计数检查法 中英对照版

USP61非无菌产品微生物学检查：微生物计数检查法 中英对照版 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 <61> 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 taken, 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 automated 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 precautions 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 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 microorganisms 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 accurate 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 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，以判断与质量的符合性。所选方法的适用 性必须被建立。 Table 1. Preparation and Use of Test Microorganisms 表1. 供试微生物的制备和使用 Suitability of Counting Method in the Presence of Product Growth Promotion 在产品存在的情况下计数方法的适用性 生长促进 Preparation of Total Aerobic Total Total Aerobic Total Yeasts and Molds Microorganism Microbial Yeasts Microbial Count总酵母菌和霉菌计 and Molds 数 微生物 Test Strain Count Count Count 总 供试菌株的制备 总好氧微生物计总好氧微生物计酵母菌和 数 数 霉菌计数 Staphylococcus Soybean-Casein Soybean-Casein Soybean-Casein such as Digest Agar or Digest Agar and Digest aureus ATCC 6538, Soybean-Casein Soybean-Casein Agar/MPN NCIMB 9518, CIP Digest Broth Digest Soybean-Casein 004.83, or NBRC -35 18-24 30Broth?100 cfu Digest 00Broth?100 cfu 13276 hours -35?3 days 30 00-35?3 days 30 金黄色葡萄球菌大豆酪蛋白消化大豆酪蛋白消化 如:ATCC 6538, 物琼脂培养基或物培养基和大豆大豆酪蛋白消化NCIMB 9518, CIP 大豆酪蛋白消化酪蛋白消化物肉物培养基/MPN4.83, 或 NBRC 物肉汤培养基汤培养基?100 (最大几率数) 00 00 13276 30-3518-24 小cfu 30-35?3 大豆酪蛋白消化 时 天 物肉汤培养基 ?100 cfu 00 30-35?3 天 Pseudomonas Soybean-Casein Soybean-Casein Soybean-Casein Digest Agar or Digest Agar and Digest aeruginosa such as ATCC Soybean-Casein Soybean-Casein Agar/MPN 9027, NCIMB Digest Broth Digest Soybean-Casein 008626,CIP -35 18-24 30Broth?100 cfu Digest 0082.118, or NBRC Broth?100 cfu hours -35?3 days 30 0013275 -35?3 days 30 大豆酪蛋白消化大豆酪蛋白消化 绿脓杆菌如物琼脂培养基或物琼脂培养基和大豆酪蛋白消化ATCC 9027, 大豆酪蛋白消化大豆酪蛋白消化物琼脂培养基NCIMB 8626,CIP 物肉汤培养基物肉汤培养基/MPN(最大几率 00 82.118, 或 30-3518-24 小?100 cfu 数)大豆酪蛋白 00NBRC 13275 时 30-35?3 天 消化物肉汤培养 基?100 cfu 0030-35?3 天 Bacillus Soybean-Casein Soybean-Casein Soybean-Casein such Digest Agar or Digest Agar and Digest subtilis as ATCC 6633, Soybean-Casein Soybean-Casein Agar/MPN NCIMB 8054, CIP Digest Broth Digest Soybean-Casein 0052.62, or NBRC -35 18-24 Broth?100 cfu 30Digest 00Broth?100 cfu 3134 hours 30-35?3 days 0030-35?3 days 枯草芽孢杆菌 大豆酪蛋白消化大豆酪蛋白消化 如ATCC 6633, 物琼脂培养基或物琼脂培养基和大豆酪蛋白消化NCIMB 8054, CIP 大豆酪蛋白消化大豆酪蛋白消化物琼脂培养基 52.62, 或 NBRC 物肉汤培养基物肉汤培养基/MPN(最大几率 00 30-3518-24 小?100 cfu 数)大豆酪蛋白3134 00时 30-35?3 天 消化物肉汤培养 基?100 cfu 0030-35?3 天 Candida Sabouraud Soybean-Casein Sabouraud Soybean-Casein Sabouraud Dextrose such Dextrose Agar or Digest Agar Dextrose Digest Agar?100 cfu albicans 00as ATCC 10231, Sabouraud ?100 cfu Agar Agar?100 cfu -25?5 days 20 0000NCPF 3179, IP -35?5 days-35?5 days Dextrose Broth 30?100 cfu 30 0000Sabouraud(沙氏)葡萄48.72, or NBRC 大豆酪蛋白消化-25?5 MPN: not 20-25 2-3 days 20 糖琼脂培养基?100 cfu 物琼脂培养基1594 days applicable 00 Sabouraud(沙20-25?5 天 ?100 cfu 00白色念珠菌 如氏)葡萄糖琼脂培Sabouraud大豆酪蛋白消化30-35?5 天 ATCC 10231, 养基或(沙氏)葡物琼脂培养基NCPF 3179, IP Sabouraud(沙氏)萄糖琼脂?100 cfu 0048.72, 或 NBRC 葡萄糖肉汤培养培养基30-35?5 天 1594 基 ?100 cfu MPN(最大几率 0020-25?5 数):不适用 天 Aspergillus Sabouraud Soybean-Casein Sabouraud Soybean-Casein Sabouraud Dextrose such as Dextrose Agar or Digest Dextrose Digest Agar?100 cfu niger 00ATCC 16404, IMI Potato-Dextrose Agar?100 cfu Agar Agar?100 cfu -25?5 days 20 000000149007, IP -25 5-7 -35?5 days Agar 20?100 cfu 3030-35?5 days 00Sabouraud(沙氏)葡萄1431.83, or days, or until -25?5 MPN: not 20 大豆酪蛋白消化糖琼脂培养基?100 cfu good NBRC 9455 days applicable 00物琼脂培养基20-25?5 天 sporulation is Sabouraud黑曲霉如ATCC ?100 cfu 大豆酪蛋白消化achieved 0016404, IMI 30-35?5 天 (沙氏)葡物琼脂培养基149007, IP Sabouraud(沙氏)萄糖琼脂?100 cfu 001431.83, 或葡萄糖琼脂培养培养基30-35?5 天NBRC 9455 基或马铃薯葡萄?100 cfu MPN(最大几率 00糖琼脂培养20-25?5 数):不适用 00 基 20-255-7天 天,或直到实现良 好的产孢 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 stated 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 to make test suspensions; to suspend A. niger spores, 0.05% Solution pH 7.2 of polysorbate 80 may be added to the buffer. Use the suspensions within oo2 hours, or within 24 hours if stored between 2 and 8. 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 inoculation. ooThe stable spore suspension may be maintained at 2 to 8 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 oo些悬浮液需在2个小时之内使用，或如果存放在2至8的条件下，则可在24小 时之内使用。作为制备然后稀释或的新鲜体细胞悬浮液的替代方黑曲霉枯草杆菌 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 oo的孢子悬浮液可以在2至8之间于一段经过验证的时间内保存。 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 ready-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 the microorganisms indicated in , using a separate portion/plate Table 1 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 conditions 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 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养基 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 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 with 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 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程。 Water-Soluble Products—Dissolve or dilute (usually a 1 in 10 dilution is prepared ) the product to be examined in Buffered Sodium or Chloride-Peptone Solution pH 7.0, Phosphate Buffer Solution pH 7.2, . If necessary, adjust to a pH of 6 to 8. Soybean-Casein Digest Broth Further dilutions, where necessary, 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 的中，，或缓冲氯化钠-蛋白胨溶液pH值为7.2的磷酸盐缓冲液大豆酪蛋白消化 。如有必要，将pH值调整为6至8。在需要时，用相同的稀释剂进物肉汤培养基 一步地稀释。 Nonfatty Products Insoluble in Water—Suspend the product to be examined (usually a 1 in 10 dilution is prepared) in Buffered Sodium or Chloride—Peptone Solution pH 7.0, Phosphate Buffer Solution pH 7.2, . A surface—active agent such as 1 g per L Soybean—Casein Digest Broth 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 prepared 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 Products—Dissolve in isopropyl myristate sterilized by filtration, or mix the product to be examined with the minimum necessary quantity of sterile polysorbate 80 or another noninhibitory sterile surface-active oreagent heated, if necessary, to not more than 40 or, in exceptional cases, oto not more than 45. Mix carefully and if necessary maintain the temperature in a water bath. Add a sufficient quantity 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 concentration 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 oo合，如有必要，可加热至不超过40或者，在特殊情况下，至不超过45。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 Form—Aseptically 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，使用全部物或剂量的规定倍 数。 Transdermal Patches—Remove 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., sterile 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 cfu. 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 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 neutralization, 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 described in and incubated following the Inoculation and Dilution procedure described in Recovery of Microorganisms in the Presence of , is compared to the number of microorganisms recovered from the Product 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 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的回收率 收的微生物的数量进行比较。 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 Agents—Neutralizing agents may be used to neutralize the activity of antimicrobial agents (see Table 2). They may be added to the chosen diluent or the medium preferably before sterilization. If used, their efficacy and their absence of toxicity for microorganisms must be demonstrated by carrying out a blank with neutralizer and without product. 中和剂——中和剂可以用于中和抗菌剂(见)的活性。最好在灭菌之前，将表2 它们加入到所选的稀释剂中或培养基中。如果使用，则必须通过进行一个带中和 剂而不含产品的空白试验，来论证它们的功效和无毒性。 If no suitable neutralizing method can be found, it can be assumed that the failure to isolate the inoculated organism is attributable to the microbicidal activity of the product. This information serves to indicate that the article is not likely to be contaminated with the given species of the microorganism. However, it is possible that the product inhibits only some of the microorganisms specified herein, but does not inhibit others not included among the test strains or those for which the latter are not representative. Then, perform the test with the highest dilution factor compatible with microbial growth and the specific acceptance criterion. 如果没有找到合适的中和方法，则可以假定未能分离所接种有机体的原因是供试 品杀灭微生物活性。这个信息帮助指出此物品不太可能被特定微生物种群所污 染。然而，有可能供试品只抑制这里所列出微生物中的某些，而并不抑制未包括 在供试菌株之中其他微生物或者后者对其不具代表性的那些微生物。然后，用与 微生物生长和具体接受标准相适应的最高稀释倍数进行试验。 RECOVERY OF MICROORGANISMS IN THE PRESENCE OF PRODUCT 在产品存在的情况下微生物的回收 For each of the microorganisms listed, separate tests are performed. Only microorganisms of the added test strain are counted. 对所列出的每个微生物，做单独的检测。只计算所加入的供试菌株的微生物。 Membrane Filtration—Use membrane filters having a nominal pore size not greater than 0.45 µm. The type of filter material is chosen in such a way that the bacteria-retaining efficiency is not affected by the components of the sample to be investigated. For each of the microorganisms listed, one membrane filter is used. 膜过滤——使用一个标称孔径不超过0.45 µm的膜过滤器。过滤器的材料按以下 标准选择，即待检测样品的组成部分不会对细菌保留效率产生影响。对于所列出 的每个微生物，单独使用一个膜过滤器。 Transfer a suitable quantity of the sample prepared as described under Preparation of the Sample, Inoculation and Dilution, and Neutralization/Removal of Antimicrobial Activity (preferably representing 1 g of the product, or less if large numbers of cfu are expected) to the membrane filter, filter immediately, and rinse the membrane filter with an appropriate volume of diluent. 将按照项下描述所制备的适样品的制备、接种和稀释、和抗菌活性的中和/去除 当数量的样品(最好相当于1克产品，在cfu数量预计较大时，也可少于此数量) 转移至膜过滤器，立即过滤，并用适量的稀释剂冲洗膜过滤器。 For the determination of total aerobic microbial count (TAMC), transfer the membrane filter to the surface of the Soybean-Casein Digest Agar. For the determination of total combined yeasts and molds count (TYMC), transfer the membrane to the surface of the Sabouraud Dextrose Agar. Incubate the plates as indicated in Table 1. Perform the counting. 对于总好氧微生物计数的测定(TAMC)，将滤膜转移至大豆酪蛋白消化物琼脂培 的表面。对于总酵母菌和霉菌联合计数(TYMC)的测定，将膜转移至养基 Sabouraud(沙氏)葡萄糖琼脂培养基的表面。按表1中规定的条件培养这些平 皿。进行计数。 Plate-Count Methods—Perform plate-count methods at least in duplicate for each medium, and use the mean count of the result. 平板计数法——每种培养基至少进行两次平板计数法，并使用计数结果的平均 值。 Pour-Plate Method—For Petri dishes 9 cm in diameter, add to the dish 1 mL of the sample prepared as described under Preparation of the Sample, , and Neutralization/Removal of Antimicrobial Inoculation and Dilution and 15 to 20 mL of Soybean-Casein Digest Agar or Sabouraud Activity o, both media maintained at not more than 45. If larger Petri Dextrose Agar dishes are used, the amount of agar medium is increased accordingly. For each of the microorganisms listed in Table 1, at least two Petri dishes are used. 倾注培养法——对于直径为9cm的平皿，将按照样品的制备、接种和稀释、抗菌 项下的描述所制备的样品1 mL以及15至20 mL大豆酪蛋白消活性的中和/去除 加入至平皿，此两种化物琼脂培养基或者Sabouraud(沙氏)葡萄粮琼脂培养基o培养基的温度均维持在不超过45。如果使用较大的平皿，琼脂培养基的量也需 相应地增加。对于表1中所列出的每一个微生物，至少要使用两个平皿。 Incubate the plates as indicated in Table 1. Take the arithmetic mean of the counts per medium, and calculate the number of cfu in the original inoculum. 按中的指示培养这些平皿。取每培养基计数的算术平均值，计算在最初的接表1 种体中的菌落数(cfu)数量。 Surface-Spread Method—For Petri dishes 9 cm in diameter, add 15 to 20 omL of Soybean-Casein Digest Agar or Sabouraud Dextrose Agar at about 45 to each Petri dish, and allow to solidify. If larger Peti dishes are used, the volume of the agar is increased accordingly. Dry the plates, for example, in a laminar-airflow cabinet or in an incubator. For each of the microorganisms listed in Table 1, at least two Petri dishes are used. Spread a measured volume of not less than 0.1 mL of the sample, prepared as directed under Preparation of the Sample, Inoculation and Dilution, over the surface of and Neutralization/Removal of Antimicrobial Activitythe medium. Incubate and count as directed for Pour-Plate Method. o表面铺展法——对于直径为9cm的平皿，倾入15至20 mL、温度大约为45的大 或Sabouraud(沙氏)葡萄粮琼脂培养基至每个平皿豆酪蛋白消化物琼脂培养基 中，静置凝固。如果使用较大的平皿，琼脂的数量也需相应地增加。干燥平皿， 例如，在层流柜或恒温箱里。对于表1中列出的每个微生物，至少使用两个平皿。 将已称量好的体积不少于0.1 mL、按照样品的制备、接种和稀释、和抗菌活性 项下的规定所制备的样品，铺展在培养基的表面。按照倾注培养法的中和/去除 项下的规定培养和计数。 Most-Probable-Number (MPN) Method—The precision and accuracy of the MPN is less than that of the method or the Membrane FiltrationMethod Plate-Count Method. Unreliable results are obtained particularly for the enumeration of molds. For these reasons, the MPN Method is reserved for the enumeration of TAMC in situations where no other method is available. If the use of the method is justified, proceed as follows. 最大几率数(MPN)法——MPN法的精密度和准确度低于膜过滤法或平板计数法。 所获得的霉菌计数的结果尤其不可靠。由于这些原因， MPN法被保留用于当没 有其它可行方法情况下的总好氧微生物计数。如果有证据支持此方法的使用，则 按如下进行。 Table 2. Common Neutralizing Agents/Methods for Interfering Substances 表2.对干扰物质的常用中和试剂/方法 Interfering Substance Potential Neutralizing 干扰物质 Agents/Method 潜在中和试剂/方法 Glutaraldehyde, mercurials Sodium hydrogen sulfite (Sodium 戊二醛，汞制剂 bisulfite)亚硫酸氢钠 Phenolics, alcohol, aldehydes, sorbateDilution 稀释剂 酚类物质，酒精，醛类，山梨酸 Aldehydes醛类 Glycine 甘氨酸 Quaternary ammonium compounds (QACs), Lecithin 卵磷脂 parahydroxybenzoates (parabens), bisbiguanides 季铵盐化合物(QACs)，对羟苯甲酸(防腐剂)， QACs, iodine, parabens Polysorbate 聚山梨醇酯 QACs，碘，防腐剂 Mercurials 汞制剂 Thioglycollate 硫胶质 Mercurials, halogens, aldehydes Thiosulfate硫代硫酸盐 汞制剂，卤素，醛类 EDTA (edetate) Mg or Ca ions EDTA乙二胺四乙酸盐 镁或钙离子 Table 3. Most-Probable-Number Values of Microorganisms 表3 微生物的最大几率数的值 Observed Combinations of Numbers of MPN per g or per mL of 95% Confidence Tubes Showing Growth in Each Set Product Limits 每套显示生长的试管所观察到的数量组合 每克或每毫升产品的最95%置信限度 大几率数 Number of g or mL of Product per Tube 每管中的产品克或毫升数 0.1 0.01 0.001 0 0 0 ,3 0-9.4 0 0 1 3 0.1-9.5 0 1 0 3 0.1-10 0 1 1 6.1 1.2-17 0 2 0 6.2 1.2-17 0 3 0 9.4 3.5-35 1 0 0 3.6 0.2-17 1 0 1 7.2 1.2-17 1 0 2 11 4-35 1 1 0 7.4 1.3-20 1 1 1 11 4-35 1 2 0 11 4-35 1 2 1 15 5-38 1 3 0 16 5-38 2 0 0 9.2 1.5-35 2 0 1 14 4-35 2 0 2 20 5-38 2 1 0 15 4-38 2 1 1 20 5-38 2 1 2 27 9-94 2 2 0 21 5-40 2 2 1 28 9-94 2 2 2 35 9-94 2 3 0 29 9-94 2 3 1 36 9-94 3 0 0 23 5-94 3 0 1 38 9-104 3 0 2 64 16-181 3 1 0 43 9-181 3 1 1 75 17-199 3 1 2 120 30-360 3 1 3 160 30-380 3 2 0 93 18-360 3 2 1 150 30-380 3 2 2 210 30-400 3 2 3 290 90-990 3 3 0 240 40-990 3 3 1 460 90-1980 3 3 2 1100 200-4000 3 3 3 ,1100 Prepare a series of at least three serial 10-fold dilutions of the product as described for Preparation of the Sample, Inoculation and Dilution, and Neutralization/Removal of Antimicrobial Activity. From each level of dilution, three aliquots of 1 g or 1 mL are used to inoculate three tubes with 9 to 10 mL of Soybean-Casein Digest Broth. If necessary a surface-active agent such as polysorbate 80, or an inactivator of antimicrobial agents may be added to the medium. Thus, if three levels of dilution are prepared, nine tubes are inoculated. 按样品的制备，接种和稀释，以及抗菌活性的中和/移除这些项下的描述，制备 系列的、至少连续三级、每级稀释10倍的产品稀释液。从每个水平的稀释液， 将1克或者1毫升的三等分试样接种到三管9至10毫升的大豆酪蛋白消化物肉 。如有必要，像聚山梨醇酯80这样的表面活性剂，或某种抗菌剂的灭汤培养基 活剂可以加入到培养基中。因此，如果制备了三水平的稀释液，则会接种九个试 管。 ooIncubate all tubes at 30 to 35 for not more than 3 days. If reading of the results is difficult or uncertain owing to the nature of the product to be examined, subculture in the same broth or in Soybean-Casein Digest for 1 to 2 days at the same temperature, and use these results. From Agar Table 3, determine the most probable number of microorganisms per g or mL of the product to be examined. oo培养所有的试管，温度为30至35之间，不超过3天。如果由于供试品的性质 导致结果的读数很难或不确定，则在相同温度下，在同样的肉汤培养基或大豆酪 中放置1至2天进行再次培养，并且使用这些结果。从表蛋白消化物琼脂培养基 中，确定每克或每毫升的供试品中微生物的最大几率数。 3 RESULTS AND INTERPRETATION结果和说明 When verifying the suitability of the Membrane Filtration method or the , a mean count of any of the test organisms not differing Plate-Count Method by a factor greater than 2 from the value of the control defined in Inoculation and Dilution in the absence of product must be obtained. When verifying the suitability of the MPN Method, the calculated value from the inoculum must be within 95% confidence limits of the results obtained with the control. 当确认膜过滤法或平板计数法的适用性时，任何供试微生物的平均计数与在没有 产品的情况下，从培养和稀释项下规定的对照组的数值差距必须不超过2。当确 认的适用性时，从接种体得到的计算值必须是在从对照组取得的结最大几率数法 果的95%置信限度以内。 TESTING OF PRODUCTS供试品的测试 Amount Used for the Test用于测试的数量 Unless otherwise directed, use 10 g or 10 mL of the product to be examined taken with the precautions referred to above. For fluids or solids in aerosol form, sample 10 containers. For transdermal patches, sample 10 patches. 除非另有规定，使用10克或者10毫升供试品，并采取上面提到的预防措施。对 于气溶胶形式下的液体或固体，取10个容器作样品。对于贴剂，取10片作样品。 The amount to be tested may be reduced for active substances that will be formulated in the following conditions: the amount per dosage unit (e.g., table, capsule, injection) is less than or equal to 1 mg, or the amount per g or mL (for preparations not presented in dose units) is less than 1 mg. In these cases, the amount of sample to be tested is not less than the amount present in 10 dosage units or 10 g or 10 mL of the product. 对于某些活性物质来说，供试数量可以减少，前提是这些活性物质将会按如下条 件组方:每剂量单位(例如，片，胶囊，注射液)数量小于或等于1毫克，或者 每克或毫升中数量(对于不按剂量单位使用的制备品)小于1毫克。在这些情况 下，供试样品数量不小于当前10剂量单位中存在的数量，或10克或10毫升的 供试品。 Examination of the Product产品的检测 MEMBRANE FILTRATION膜过滤 Use a filtration apparatus designed to allow the transfer of the filter to the medium. Prepare the sample using a method that has been shown to be suitable as described in Growth Promotion Test and Suitability of the Counting Method, transfer the appropriate amount to each of two membrane filters, and filter immediately. Wash each filter following the procedure shown to be suitable. 使用能够将滤膜转移至培养基中的过滤设备。按生长促进测试和计数方法的适用 项下描述的、已经证明适用的方法制备样品，转移合适的量至两个膜过滤器中性 的每一个，并立即过滤。按照以下已经证明适用的规程清洗每个过滤器。 For the determination of TAMC, transfer one of the membrane filters to the surface of Soybean-Casein Digest Agar. For the determination of TYMC, transfer the other membrane to the surface of . Sabouraud Dextrose Agar ooIncubate the plate of Soybean-Casein Digest Agar at 30 to 35 for 3 to oo5 days and the plate of Sabouraud Dextrose Agar at 20 to 25 for 5 to 7 days. Calculate the number of cfu per g or per mL of product. 对于总好氧微生物计数的测定，转移滤膜中的一个至大豆酪蛋白消化物琼脂培养 的表面。对酵母菌和霉菌联合计数的测定，转移另一个滤膜至Sabouraud(沙基oo的表面。在温度30至35之间培养大豆酪蛋白消化物琼氏)葡萄糖琼脂培养基oo的平皿3至5天，并在温度20至25之间培养Sabouraud(沙氏)葡萄脂培养基 的平皿5至7天。计算每克或每毫升供试品的菌落数(cfu)数量。 糖琼脂培养基 When examining transdermal patches, separately filter 10% of the volume of the preparation described for Preparation of the Sample through each of two sterile filter membrances. Transfer one membrance to Soybean-Casein Digest Agar for TAMC and the other membrane to Sabouraud for TYMC. Dextrose Agar 当检测贴剂时，分别将样品的制备项下描述的制备品数量的10%过滤通过两个无 菌滤膜中的一个。为总好氧微生物计数将一个滤膜转移至大豆酪蛋白消化物琼脂 ，以检测总好氧微生物计数，而将另一个滤膜转移至Sabouraud(沙氏)培养基 ，以检测总酵母菌和霉菌联合计数。 葡萄糖琼脂培养基 PLATE-COUNT METHODS平板计数法 Pour-Plate Method—Prepare the sample using a method that has been shown to be suitable as described in Growth Promotion Test and Suitability of . Prepare for each medium at least two Petri dishes the Counting Method for each level of dilution. Incubate the plates of Soybean-Casein Digest oo at 30 to 35 for 3 to 5 days and the plates of Sabouraud Dextrose Agarooat 20 to 25 for 5 to 7 days. Select the plates corresponding to a Agar given dilution and showing the highest number of colonies less than 250 for TAMC and 50 for TYMC. Take the arithmetic mean per culture medium of the counts, and calculate the number of cfu per g or per mL of product. 倾注培养法——使用已经证明适用的、在生长促进测试和计数方法的适用性项下 描述的方法制备样品。对于每种培养基，为每个稀释水平准备至少两个培养平皿。 oo在温度30至35之间，培养大豆酪蛋白消化物琼脂培养基的平皿3至5天，并 oo在温度20至25之间，培养的平皿5至7天。选Sabouraud(沙氏)葡萄糖琼脂 择总好氧微生物的数量不超过250个和总酵母和霉菌的数量不超过50的稀释平 板进行计数。取每培养基计数的算术平均值，并计算每克或每毫升供试品的cfu 数量。 Surface-Spread Method—Prepare the sample using a method that has been shown to be suitable as described in Growth Promotion Test and Suitability Prepare at least two Petri dishes for each medium of the Counting Method. and each level of dilution. For incubation and calculation of the number of cfu, proceed as directed for the Pour-Plate Method. 表面铺展法——使用已经证明适用的、在生长促进测试和计数方法的适用性项下 描述的方法制备供试品。为每种培养基和每个稀释水平准备至少两个培养平皿和 不同程度的稀释液。对于培养和cfu数量的计算，直接按进行。 倾注培养法 MOST-PROBABLE-NUMBER METHOD最大几率数法 Prepare and dilute the sample using a method that has been shown to be suitable as described in Growth Promotion Test and Suitability of the oo Incubate all tubes for 3 to 5 days at 30 to 35. Subculture Counting Method. if necessary, using the procedure shown to be suitable. Record for each level of dilution the number of tubes showing microbial growth. Determine the most probable number of microorganisms per g or mL of the product to be examined from Table 3. 使用已经证明适当的、在生长促进测试和计数方法的适用性项下描述的方法制备 oo和稀释样品。在温度30至35之间时，培养所有试管3到5天。如有必要，则 使用已经证实适合的规程再次培养。记录不同稀释水平下显示出微生物生长的试 管数。从表3中确定每克或每毫升供试品中微生物的最大几率数。 Interpretation of the Results结果的说明 The total aerobic microbial count (TAMC) is considered to be equal to the number of cfu found using ; if colonies of fungi Soybean-Casein Digest Agar are detected on this medium, they are counted as part of TAMC. The total combined yeasts and molds count (TYMC) is considered to be equal to the number of cfu found using Sabouraud Dextrose Agar; if colonies of bacteria are detected on this medium, they are counted as part of TYMC.When the TYMC is expected to exceed the acceptance criterion due to the bacterial growth, containing antibiotics may be used. If Sabouraud Dextrose Agar the count is carried out by the MPN Method, the calculated value is TAMC. 总好氧微生物计数(TAMC)被认为等于使用大豆酪蛋白消化物琼脂培养基所发现的cfu数量，如果在该培养基上发现真菌的菌落，则它们作为总好氧微生物计数的一部分来计数。总酵母菌和霉菌联合计数(TYMC)被认为等于使用Sabouraud 所发现的cfu数量，如果在该培养基上发现细菌的菌(沙氏)葡萄粮琼脂培养基 落，则它们作为总酵母菌和霉菌联合计数的一部分来计数。当细菌的生长导致总酵母菌和霉菌联合计数预计超过接受标准时，可以使用含有抗生素的Sabouraud 。如果通过最大几率数法进行计数，则计算出的数值(沙氏)葡萄粮琼脂培养基 就是总好氧微生物计数。 When an acceptance criterion for microbiological quality is prescribed, it is interpreted as follows: 当规定微生物质量的接受标准给出时，其可以理解如下: 1— 10 cfu: maximum acceptable count = 20; 2— 10 cfu: maximum acceptable count = 200; 3— 10 cfu: maximum acceptable count = 2000; and so forth. 1— 10 cfu: 最高可接受计数,20; 2— 10 cfu: 最高可接受计数,200; 3— 10 cfu: 最高可接受计数,2000; 等等。 The recommended solutions and media are described in Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms 62>. < 推荐的溶液和培养基在非无菌产品微生物学检查:指定微生物检查62<>中描述。