62 MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS: TESTS FOR SPECIFIED
MICROORGANISMS
非无菌产品微生物限度检查：控制菌（USP38）
1.INTRODUCTION导言
The tests described hereafter will allow determination of the abnce of, or limited occurrence of, specified microorganisms that may be detected under the conditions described.
控制菌检查法系用于在规定的试验条件下，检查供试品中是否存在特定的微生物。
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是否符合相应的微生物限度标准时，应按下列规定进行检验，，包括样品的取样量，结果的判断.
Alternative microbiological procedures, including automated methods, may be ud, provided that their equivalence to the Pharmacopeial method has been demonstrated.
可以使用包括自动化法在内的方法，需确认与药典方法的等同性.
2.GENERAL PROCEDURES通用规程
The preparation of samples is carried out as described in Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests 61.
供试品制备，同USP<61>
If the product to be examined has antimicrobial activity, this is insofar as possible removed or neutralized as described in Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests 61.
若供试品有抗菌活性，应尽可能中和或去除，中和或去除的方法同USP<61>
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 as described in Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests 61.
若供试品制备过程中使用了表面活性剂，应确认其对微生物的无毒性以及与所用的中和剂/灭活剂的相容性，同USP<61>
3.GROWTH-PROMOTING AND INHIBITORY PROPERTIES OF THE MEDIA, SUITABILITY
OF THE TEST AND NEGATIVE CONTROLS 培养基适用性检查，控制菌检查方法的适用性
确认，阴性对照
The ability of the test to detect microorganisms in the prence of the 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，所建立的方法应能检测微生物。若检测程序或产品发生变化可能影响检测结果时，控制菌检查方法应重新进行适用性试验。
3.1.Preparation of Test Strains试验菌液的制备
U standardized stable suspensions of test strains 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.
使用菌株标准化稳定的悬浮液。采用适宜的菌种保藏技术（种子批系统），确保接种的培养物自主种子批开始传代不超过5代。
3.1.1.AEROBIC MICROORGANISMS需氧菌
Grow each of the bacterial test strains parately in containers containing Soybean–Cain Digest Broth or on Soybean–Cain Digest Agar at 30° to 35° for 18 to 24 hours. Grow the test strain for Candida albicans parately on Sabouraud Dextro Agar or in Sabouraud Dextro Broth at 20° to 25° for 2 to 3 days.
将各试验菌株分别接种于TSB或TSA培养基，30~35℃培养18~24小时。将白色念珠菌接种于SDA或SDB培养基， 20~25℃培养2~3天。
android系统Staphylococcus aureus金黄色葡萄球菌such as A TCC 6538, NCIMB 9518, CIP 4.83, or
NBRC 13276
Pudomonas aeruginosa铜绿假单胞菌such as ATCC 9027, NCIMB 8626, CIP 82.118, or
NBRC 13275
猫鼠大战Escherichia coli大肠埃希菌such as ATCC 8739, NCIMB 8545, CIP 53.126, or
NBRC 3972
Salmonella enterica subsp. enterica rovar
Typhimurium or, as an alternative,
肠道沙门氏菌肠道亚种鼠伤寒血清型（伤寒沙门氏
菌），或
such as ATCC 14028
Salmonella enterica subsp. enterica rovar Abony
肠道沙门氏菌肠道亚种阿邦尼血清型（阿邦尼沙门氏菌清型）such as NBRC 100797, NCTC 6017, or CIP 80.39
Candida albicans 白色念珠菌such as A TCC 10231, NCPF 3179, IP 48.72, or NBRC 1594
U Buffered Sodium Chloride–Peptone Solution pH 7.0 or Phosphate Buffer Solution pH 7.2 to make test suspensions. U the suspensions within 2 hours or within 24 hours if stored at 2° to 8°.
上述培养物用PH7.0的氯化钠-蛋白胨缓冲液或PH7.2磷酸盐缓冲液配制成适宜浓度的菌悬液。
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。
3.1.2.CLOSTRIDIA梭菌
U Clostridium sporogenes such as ATCC 11437 (NBRC 14293, NCIMB 12343, CIP 100651) or ATCC 19404 (NCTC 532 or CIP 79.3). Grow the clostridial test strain under anaerobic conditions in Reinforced Medium for Clostridia at 30° to 35° for 24 to 48 hours. As an alternative to preparing and then diluting down a fresh suspension of vegetative cells of Cl. sporogenes, a stable spore suspension is ud for test inoculation. The stable spore suspension may be maintained at 2° to 8°for a validated period.
梭菌，菌种ATCC 11437 (NBRC 14293, NCIMB 12343, CIP 100651) or ATCC 19404 (NCTC 532 or CIP 79.3)
鼻翼宽将梭菌接种于梭菌增菌培养基中，置厌氧条件下30~35℃培养24~48小时。稳定的梭菌孢子混悬液可
替代新鲜的菌悬液。孢子混悬液可保存在2~8℃，在验证过的期限内使用。
3.2.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，应用所选择的稀释剂替代供试品做阴性对照试验。阴性对照应无菌生长。若有菌生长，应进行偏差调查。
供试品检查时，也需要做阴性对照。
3.3. Growth Promotion and Inhibitory Properties of the Media 培养基的促生长和抑制能力试验（适用性
检查）
Test each batch of ready-prepared medium and each batch of medium prepared either from dehydra
ted medium or from ingredients. Verify suitable properties of relevant media as described in Table 1.
已制备培养基，干燥的培养基或按照组分配制的培养基，每一批都需要进行适用性检查，检查项目包括促生长能力，抑制能力及指示特性的检查。各培养基的检查项目及所用菌株见表1. Table 1 . Growth Promoting, Inhibitory, and Indicative Properties of Media
培养基促生长能力，抑制能力和指示特性
Test/Medium 培养基 Property 项目
Test Strains 试验菌株 Test for bile-tolerant Gram-negative bacteria 耐胆盐革兰阴性菌
Enterobacteria Enrichment Broth Mosl
肠道菌增菌液体培养基 Growth promoting 促生长能力 E. coli 大肠埃希菌 P . aeruginosa 铜绿假单胞菌 Inhibitory 抑制能力
S. aureus 金黄色葡萄球菌 Violet Red Bile Gluco Agar 紫红胆盐葡萄糖琼脂培养基 Growth promoting +Indicative
促生长能力+指示特性 E. coli 大肠埃希菌 P . aeruginosa 铜绿假单胞菌
Test for Escherichia coli 大肠埃希菌
MacConkey Broth 麦康凯液体培养基 Growth promoting 促生长能力
E. coli 大肠埃希菌 Inhibitory 抑制能力 S. aureus 金黄色葡萄球菌
MacConkey Agar 麦康凯琼脂培养基 Growth promoting +Indicative 促生长能力+指示特性
E. coli 大肠埃希菌 Test for Salmonella 沙门氏菌
Rappaport Vassiliadis Salmonella Enrichment Broth
RV 沙门菌增菌液体培养基
Growth promoting 促生长能力 Salmonella enterica subsp. Enterica
rovar Typhimurium or
肠道沙门氏菌肠道亚种鼠伤寒血清
型（伤寒沙门氏菌），或 Salmonella enterica subsp. enterica rovar Abony 肠道沙门氏菌肠道亚种阿邦尼血清
型（阿邦尼沙门氏菌，血清型）经典粤语歌曲
Inhibitory 抑制能力 S. aureus 金黄色葡萄球菌
Xylo Lysine Deoxycholate Agar 木糖赖氨酸脱氧胆酸盐琼脂培养基 Growth promoting +Indicative
促生长能力+指示特性
Salmonella enterica subsp. Enterica
rovar Typhimurium or
肠道沙门氏菌肠道亚种鼠伤寒血清
型（伤寒沙门氏菌），或 Salmonella enterica subsp. enterica rovar Abony
肠道沙门氏菌肠道亚种阿邦尼血清
型（阿邦尼沙门氏菌，血清型）
Test for Pudomonas aeruginosa 铜绿假单胞菌
Cetrimide Agar 溴化十六烷基三甲铵琼脂培养基 Growth promoting 促生长能力
P . aeruginosa 铜绿假单胞菌 Inhibitory 抑制能力 E. coli 大肠埃希菌
Test for Staphylococcus aureus 金黄色葡萄球菌
Mannitol Salt Agar 甘露醇盐琼脂培养基 Growth promoting +Indicative 促生长能力+指示特性 S. aureus 金黄色葡萄球菌 Inhibitory 抑制能力 E. coli 大肠埃希菌
Test for Clostridia 梭菌
Reinforced Medium for Clostridia 梭菌增菌培养基
Growth promoting 促生长能力 Cl. Sporogenes 生孢梭菌 Columbia Agar 哥伦比亚琼脂培养基 Growth promoting 促生长能力 Cl. Sporogenes 生孢梭菌
Test for Candida albicans白色念珠菌
Sabouraud Dextro Broth沙氏葡萄
糖液体培养基
Growth promoting促生长能力 C. albicans白色念珠菌
Sabouraud Dextro Agar沙氏葡萄糖琼脂培养基Growth promoting +Indicative
促生长能力+指示特性 C. albicans
白色念珠菌
Test for Growth-Promoting Properties, Liquid Media— Inoculate a portion of the appropriate medium with a small number (not more than 100 cfu) of the appropriate microorganism. Incubate at the specified temperature for not more than the shortest period of time specified in the test. Clearly visible growth of the microorganism comparable to that previously obtained with a previously tested and approved batch of medium occurs.
液体培养基的促生长能力检查：
接种不大于100cfu的试验菌至液体培养基，在相应控制菌检查规定的培养温度及不大于规定的最短培养时间下培养。与参比培养基比较（之前检测并批准的培养基），菌落生长良好。
Test for Growth-Promoting Properties, Solid Media— Perform Surface-Spread Method (e Plate-Co我的歌声里歌词
unt Methods under Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests 61, inoculating each plate with a small number (not more than 100 cfu) of the appropriate microorganism. Incubate at the specified temperature for not more than the shortest period of time specified in the test. Growth of the microorganism comparable to that previously obtained with a previously tested and approved batch of medium occurs.
固体培养基的促生长能力检查，按涂布法（详见USP61）操作：
接种不大于100cfu的试验菌，在相应控制菌检查规定的培养温度及不大于规定的最短培养时间下培养。与参比培养基比较（之前检测并批准的培养基），菌落大小，形态特征应一致。
Test for Inhibitory Properties, Liquid or Solid Media— Inoculate the appropriate medium with at least 100 cfu of the appropriate microorganism. Incubate at the specified temperature for not less than the longest period of time specified in the test. No growth of the test microorganism occurs.
液体或固体培养基的抑制能力检查——接种不少于100cfu的试验菌。在相应控制菌检查规定的培养温度及不小于规定的最长培养时间下培养。试验菌应不得生长。
Test for Indicative Properties— Perform Surface-Spread Method (e Plate-Count Methods under Mi
crobiological Examination of Nonsterile Products: Microbial Enumeration Tests 61, inoculating each plate with a small number (not more than 100 cfu) of the appropriate microorganism. Incubate at the specified temperature for a period of time within the range specified in the test. Colonies are comparable in appearance and indication reactions to tho previously obtained with a previously tested and approved batch of medium.
培养基的指示特性检查，按涂布法（详见USP61）操作：
接种不大于100cfu的试验菌。在相应控制菌检查规定的培养温度和培养时间范围内培养。参照之前检测和批准的培养基，试验菌生长的菌落大小，形态特性，指示剂反应应一致。
3.4.Suitability of the Test Method控制菌检查方法的适用性试验
For each new product to be tested perform sample preparation as described in the relevant paragraph under Testing of Products. At the time of mixing, add each test strain in the prescribed growth medium.
Inoculate the test strains individually. U a number of microorganisms equivalent to not more than 100 cfu in the inoculated test preparation.
根据“供试品检查”项下的要求制备供试品溶液。混合步骤，分别接种不大于100cfu的各试验菌至规定的培养基中。
Perform the test as described in the relevant paragraph under Testing of Products using the shortest
incubation period prescribed.
按“供试品检查”项下要求操作，并在规定的最短培养时间内培养。
The specified microorganisms must be detected with the indication reactions as described under Testing of Products.
应能检出所加控制菌“供试品检查”项下描述的反应特征。
Any antimicrobial activity of the product necessitates a modification of the test procedure (e Neutralization/Removal of Antimicrobial Activity under Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests 61
若供试品有抗菌活性，需调整方法，对供试品进行中和或灭活，具体方法详见USP 61
陈慥
四川省高考分数线For a given product, if the antimicrobial activity with respect to a microorganism for which testing is prescribed cannot be neutralized, then it is to be assumed that the inhibited microorganism will not be prent in the product.
如果经过试验确证供试品对某微生物的抑菌活性无法中和，那么可假设被抑制的微生物将不会存在于该供试品中。
4.TESTING OF PRODUCTS供试品检查
陕西方言顺口溜4.1.Bile-Tolerant Gram-Negative Bacteria耐胆盐革兰阴性菌
Sample Preparation and Pre-Incubation— Prepare a sample using a 1 in 10 dilution of not less than 1 g of the product to be examined as described in Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests 61, but using Soybean–Cain Digest Broth as the chon diluent, mix, and incubate at 20°to 25°for a time sufficient to resuscitate the bacteria but not sufficient to encourage multiplication of the organisms (usually 2 hours but not more than 5 hours).
供试液制备和预培养：
取不少于1g供试品，TSB作为稀释剂，按1:10稀释得到供试品溶液，其他操作同USP 61，混匀，在20~
25℃培养，培养时间应使供试品中的细菌复苏但不增值（通常适宜的培养时间为2小时，最长不得多于5小时）
4.1.1.Test for Abnce— 定性检测
Unless otherwi prescribed, u the volume corresponding to 1 g of the product, as prepared in Sample Preparation and Pre-Incubation, to inoculate Enterobacteria Enrichment Broth Mosl.
Incubate at 30° to 35° for 24 to 48 hours. Subculture on plates of Violet Red Bile Gluco Agar.
Incubate at 30° to 35° for 18 to 24 hours. The product complies with the test if there is no growth of colonies.
除另有规定外，取相当于1g样品的上述预培养物，接种至适宜体积的增菌培养基（经方法适用性试验确定），在30~35℃培养24~48小时后，划线接种至紫红胆盐葡萄糖琼脂培养基上进行分离培养，30~35℃培养18~24小时。如果平板上无菌落生长，判断供试品未检出耐胆盐革兰阴性菌。
4.1.2.Quantitative Test—定量检测
Selection and Subculture— Inoculate suitable quantities of Enterobacteria Enrichment Broth Mosl
with the preparation as directed under Sample Preparation and Pre-Incubation and/or dilutions of it containing respectively 0.1 g, 0.01 g, and 0.001 g (or 0.1 mL, 0.01 mL, and 0.001 mL) of the product to be examined. Incubate at 30 to 35 for 24 to 48 hours. Subculture each of the cultures on a plate of Violet Red Bile Gluco Agar. Incubate at 30° to 35° for 18 to 24 hours.
选择和分离培养——取按“供试品制备”和“预培养”项下制备的相当于0.1g, 0.01g 0.001g(0.1
