<61>MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS:
MICROBIAL ENUMERATION TESTS
非无菌产品微生物限度检查：微生物计数法
1.INTRODUCTION导言
The tests described hereafter will allow quantitative enumeration of mesophilic bacteria and fungi that may grow under aerobic conditions.
微生物计数法系用于能在有氧条件下生长的嗜温细菌和霉菌的定量计数。
The tests are designed primarily to determine whether a substance or preparation complies with an established specification for microbiological quality. When ud for such purpos, follow the instructions given below, including the number of samples to be taken, and interpret the results as stated below.
当本法用于检查非无菌制剂及其原、辅料等是否符合相应的微生物限度标准时，应按照下述规定进行检验，包括样品的取样量，结果的判断。
The methods are not applicable to products containing viable microorganisms as active ingredients.
本法不适用于活菌制剂的检查。
Alternative microbiological procedures, including automated methods, may be ud, provided that their equivalence to the Pharmacopeial method has been demonstrated.
可使用包括自动化法在内的替代方法，需确认其与药典方法的等同性。
2.GENERAL PROCEDURES通用规程
Carry out the determination under conditions designed to avoid extrinsic 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.
微生物计数环境应能防止外来微生物对供试品的污染。防止污染的措施不能影响供试品中微生物的检出。
备注：检验全过程必须严格遵守无菌操作，防止再污染。单向气流区域、工作台面及环境应定期进行监测。
If the product to be examined has antimicrobial activity, this is, insofar as possible, removed or neutralized. If inactivators are ud for this purpo, their efficacy and their abnce of toxicity for microorganisms must be demonstrated.
如果供试品有抗菌活性，应尽可能去除或中和。若使用了中和剂或灭活剂，需确认其有效性及对微生物无毒性。
If surface-active substances are ud for sample preparation, their abnce of toxicity for microorganisms and their compatibility with any inactivators ud must be demonstrated.
供试品制备过程中若使用了表面活性剂，需确认其对微生物无毒性以及与所使用的中和剂或灭活剂的相容性。
3.ENUMERATION METHODS计数方法
U the Membrane Filtration method or 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.
计数方法包括薄膜过滤法、平皿法和最可能数法（Most-Probable-Number 简称MPN)。MPN法用于微生物计数时精确度最差，但用于微生物污染量小的供试品，MPN法可能是最合适的方法。
（MPN法不适用于霉菌的检测，仅在供试品总需氧菌数没有适应计数方法的情况下使用。）
The choice of a method is bad on factors such as the nature of the product and the required limit of microorganisms. The method chon must allow testing of a sufficient sample size to judge compliance with the specification. The suitability of the chon method must be established.
供试品检查时，应根据供试品理化特性和微生物限度标准等因素选择计数方法。所选择的计数方法应能够通过检测足量的供试品，判断与质量标准的符合性。且该计数方法的适用性须经确认。
4.GROWTH PROMOTION TEST, SUITABILITY OF THE COUNTING METHOD AND
NEGATIVE CONTROLS促生长实验，计数法适用性试验以及阴性对照
4.1.General Considerations一般要求
The ability of the test to detect microorganisms in the prence of product to be tested must be established. 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.
在有供试品存在的情况下，所选用的计数方法需能够检测微生物。若检测程序或产品发生变化可能影响检测结果时，计数方法需重新进行适用性确认。
4.2.Preparation of Test Strains试验菌液的制备
U standardized stable suspensions of test strains or prepare as stated below. Seed-lot culture maintenance techniques (ed-lot systems) are ud so that the viable microorganisms ud for inoculation are not more than five passages removed from the original master ed-lot. Grow each of the bacterial and fungal test strains parately as described in Table 1.
试验菌液：使用试验菌株的标准化稳定悬浮液或按下述规定制备。按表1规定程序分别培养各试验菌液。
菌株：采用适宜的菌种保藏技术（种子批系统），试验用菌株传代次数自主种子批（第0代）算起不得超过5代。
Table 1. Preparation and U of Test Microorganisms
Growth Promotion 促生长实验Suitability of Counting Method in the Prence of Product
计数方法适用性试验
Microorganism 试验菌株Preparation of Test
Strain
试验菌液的制备
Total Aerobic
Microbial Count
总需氧菌数计数
Total Yeasts and Molds
Count
总酵母菌和霉菌计数
Total Aerobic
Microbial Count
总需氧菌数计数
Total Yeasts and
Molds Count
总酵母菌和霉菌
计数
Staphylococcus aureus such as A TCC 6538,
NCIMB 9518, CIP 4.83, or NBRC 13276 金黄色葡萄球菌Soybean-Cain Digest
Agar or Soybean-Cain
Digest Broth
30°-35°
18-24 hours
大豆酪蛋白消化琼脂
培养基或大豆酪蛋白
消化肉汤培养基
培养温度30°-35°
培养时间18-24 小时
Soybean-Cain
Digest Agar and
Soybean-Cain
Digest Broth
≤100 cfu
30°-35°
≤3 days
大豆酪蛋白消化琼脂
培养基和大豆酪蛋白
消化肉汤基
培养温度30°-35°
培养时间不超过3天
接种量不大于100cfu
函授本科有用吗
Soybean-Cain Digest
Agar/MPN Soybean-
Cain Digest Broth
≤100 cfu
30°-35°
≤3 days
大豆酪蛋白消化琼脂
培养基
大豆酪蛋白消化肉汤
培养基（MPN法）
培养温度30°-35°
培养时间不超过3天
接种量不大于100cfu
Pudomonas aeruginosa such as ATCC 9027, NCIMB 8626,CIP 82.118, or NBRC 13275
铜绿假单胞菌Bacillus subtilis such as ATCC 6633, NCIMB 8054, CIP 52.62, or NBRC 3134 枯草芽孢杆菌
Candida albicans such as ATCC 10231, NCPF 3179, IP 48.72, or NBRC 1594
白色念珠菌Sabouraud Dextro
Agar or Sabouraud
Dextro Broth
20°-25°
2-3 days
沙氏葡萄糖琼脂培养
基或沙氏葡萄糖肉汤
培养基
圣诞快乐的英语培养温度 20°-25°
培养时间 2-3天
Soybean-Cain
Digest Agar
≤100 cfu
30°-35°
≤5 days
大豆酪蛋白消化琼脂
培养基
培养温度30°-35°
培养时间不超过5天
接种量不大于100cfu
Sabouraud Dextro
Agar ≤100 cfu
20°-25°
≤5 days
finley
沙氏葡萄糖琼脂培养
基
培养温度20°-25°
培养时间不超过5天
接种量不大于100cfu
Soybean-Cain Digest
Agar
≤100 cfu
30°-35°
≤5 days
MPN：not applicable
大豆酪蛋白消化琼脂
培养基
培养温度30°-35°
培养时间不超过5天
接种量不大于100cfu
MPN法不适用
Sabouraud
Dextro Agar
carey
安装预算员培训
≤100 cfu
20°-25°
≤5 days
沙氏葡萄糖琼脂
培养基
培养温度20°-
25°
培养时间不超过
5天
接种量不大于
100cfu
Aspergillus niger such as ATCC 16404, IMI 149007, IP 1431.83, or NBRC 9455
nor黑曲霉Sabouraud Dextro Agar or Potato-Dextro Agar
20°-25°
大白菜的英文5-7 days or until good sporulation is achieve 沙氏葡萄糖琼脂培养基或马铃薯葡萄糖琼脂培养基
kink
培养温度 20°-25°
培养时间5-7天，或直到获得丰富的孢子
U Buffered Sodium Chloride–Peptone Solution pH 7.0 or Phosphate Buffer Solution pH 7.2 to make test suspensions; to suspend A. brasiliensis spores, 0.05% of polysorbate 80 may be added to the buffer.explode
U the suspensions within 2 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. brasiliensis or B. subtilis, a stable spore suspension is prepared and then an appropriate volume of the spore suspension is ud for test inoculation. The stable spore suspension may be maintained at 2 to 8 for a validated period of time.
菌液制备：取表1各试验菌株的新鲜培养物，用PH7.0的氯化钠-蛋白胨缓冲液或PH7.2的磷酸缓冲液制备混悬液。制备黑曲霉孢子悬液，需在缓冲液中加入0.05%的聚山梨酯80。菌液制备后若在室温下放置，应在2小时内使用；若保存在2~8℃，可在24小时内使用。
黑曲霉和枯草杆菌新鲜细胞的混悬液，可用其孢子混悬液替代用于试验。该孢子混悬液可保存在2~8℃，在验证过的储存期限内使用。
4.3.Negative Control阴性对照
To verify testing conditions, a negative control is performed using the chon diluent in place of the test preparation. There must be no growth of microorganisms. A negative control is also performed when testing the products as described under Testing of Products. A failed negative control requires an investigation.
为确认试验条件是否符合要求，需用选择的稀释剂代替供试品进行阴性对照。阴性对照应无菌生长。如阴性对照有菌生长，应进行偏差调查。
供试品检测时也需要进行阴性对照。
4.4.Growth Promotion of the Media培养基促生长试验（培养基适用性检查/培养基灵敏度检查）
Test each batch of ready-prepared medium and each batch of medium prepared either from dehydrated medium or from the ingredients described.
微生物计数用的制备培养基、脱水培养基或按处方配置的培养基，每批均需进行适用性检查
Inoculate portions/plates of Soybean–Cain Digest Broth and Soybean–Cain Digest Agar with a small number (not more than 100 cfu) of the microorganisms indicated in Table 1, using a parate portion/plate of medium for each. Inoculate plates of Sabouraud Dextro Agar with a small number (not more than 100 cfu) of the microorganisms indicated in Table 1, using a parate plate of medium for each. Incubate according to the conditions described in Table 1.
按表1规定，接种不大于100cfu的指定菌株至TSA或TSB或SDA培养基。在表1规定的条件下培养。对于TSA或TSB培养基，每一菌株使用单独的培养基或培养基上单独的区域。对于SDA培养基，每一菌株使用单独的培养基。
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.
我自己用英语怎么说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.
被检固体培养基上的菌落数与参比培养基（标准培养物）上的菌落相比，差异因子不能大于2（50%~200%的范围内）；被检新鲜制备的培养物与参比培养基（之前检测并批准批次的培养基，）比较，应有菌生长；被检液体培养基管与参比培养基管（之前检测并批准批次的培养基，）比较，试验菌应生长良好。
备注：2005年11月，美国药典会和欧洲药典会在芝加哥会议上签署了微生物检查法协调案，从2007年开始，ＵＳＰ、ＥＰ、ＪＰ逐渐执行协调一致的新的微生物检查法，新方法的重要环节是培养基适用性检查，规定使用的每批培养基必须和之前验证过的培养基进行比较试验。
2008年，在《中国药典》2010年版的增修订过程中，借鉴欧美药典，引入了培养基适用性检查以保障微生物限度检查结果的准确性和可靠性。但是，在药典修订过程中，对于欧美药典中的“之前验证过的培养基”的采纳与否，存在很大争议，主要集中在两点：一是，我国药品微生物实验室小而分散，数以千计的企业和基层药检所难以有效地进行参比培养基的验证、评价工作；二是，在监督检查中，难以有效评估企业微生物实验室对参比培养基的验证情况。经过第九届药典委员会微生物专业委员会的反复讨论，提出以统一的、经过充分评价和验证过的对照培养基替代需要由每个实验室分散评价的“之前
验证过的培养基”。微生物实验室以对照培养基进行培养基适用性比较试验，中国食品药品检定研究院承担对照培养基的研制任务。（《中国药典》2010年版对照培养基的研制与应用，中国药事2012年第26卷第8期847页）
4.5.Suitability of the Counting Method in the Prence of Product 计数方法适用性检测
4.5.1.PREPARATION OF THE SAMPLE供试品制备
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.
根据供试品的理化性质，采用适宜的方法进行供试品制备。如以下制备方法均不适用，可采用
其他合适的方法。
Water-Soluble Products— Dissolve 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–Cain Digest Broth. If necessary, adjust to a pH of 6 to 8. Further dilutions, where necessary, are prepared with the same diluent.
水溶性供试品——取供试品，用PH7.0 氯化钠-蛋白胨缓冲液，或PH7.2磷酸盐缓冲液，或TSB溶解或稀释成1:10供试品溶液。若需要，调节供试品溶液PH值至6~8.必要时，用同一稀释液进一步稀释（10倍系列稀释）。
Nonfatty Products Insoluble in Water— Suspend the product to be examined (usually a 1 in 10 dilution is prepared) in Buffered Sodium Chloride–Peptone Solution pH 7.0, Phosphate Buffer Solution pH 7.2, or Soybean–Cain Digest Broth. A surface-active 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 prepared with the same diluent.
水不溶性非脂类供试品——取供试品，用PH7.0 氯化钠-蛋白胨缓冲液，或PH7.2磷酸盐缓冲液，或TSB配制成1:10供试品液。分散性差的供试品，可在稀释液中加入表面活性剂如1g/L的聚山梨酯80. 若需要，调节供试品溶液PH值至6~8.必要时，用同一稀释液进一步稀释（10倍系列稀释）。
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 reagent heated, if necessary, to not more than 40 or, in exceptional cas, to not more than 45. Mix carefully and if necessary maintain the temperature in a water bath. Add a sufficient quantity of the prewarmed chon 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 rial 10-fold dilutions may be prepared using the chon diluent containing a suitable concentration of sterile polysorbate 80 or another noninhibitory sterile surface-active reagent.
油脂类供试品——取供试品，加入经无菌过滤的十四酸异丙酯，或与最少量（并能使供试品乳化）的无菌聚山梨酯80或其他无抑菌性的无菌表面活性剂混合，必要时加热，温度一般不得超过40℃（特殊情况，不得超过45℃）。小心混合，如若必要，可在水浴中进行，然后加入预热的稀释剂，将供试品进行1:10的稀释。保温，小心混合，并在最短时间内形成乳状液。必要时，用含上述无菌表面活性剂的稀释剂进一步稀释（10倍系列稀释）。
Fluids or Solids in Aerosol Form— Aptically transfer the product into a membrane filter apparatus or
a sterile container for further sampling. U either the total contents or a defined number of metered
dos from each of the containers tested.
固体或液体气溶胶——无菌操作，将供试品转移至膜过滤装置或无菌容器，进一步取样。从每一被测容器中取全部内容物或剂量的倍数进行检测。
Transdermal Patches— Remove the protective cover sheets (“relea 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 chon diluent containing inactivators such as polysorbate 80 and/or lecithin. Shake the preparation vigorously for at least 30 minutes.
透皮贴剂——取供试品，去掉保护层（即释放衬板），将粘贴面朝上放置在无菌玻璃或塑料器皿上，在粘贴面上覆盖一层适宜的无菌多孔材料（如无菌纱布），防止贴膏剂粘在一起。将处理好的贴膏剂放入盛有适量体积并含灭活剂如聚山梨酯80和/或卵磷脂的容器中。剧烈振摇至少30分钟。
4.5.2.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
