EP 8.0
04/2010:20613
2.6.13. MICROBIOLOGICAL EXAMINATION OF NON-STERILE PRODUCTS: TEST FOR SPECIFIED MICRO-ORGANISMS(3)
非无菌药品的微生物限度检查：特殊微生物的检查
1. INTRODUCTION 导言
The tests described hereafter will allow determination of the absence or limited occurrence of specified micro-organisms that may be detected under the conditions described.
下述检验方法用于检查在描述的试验条件下特定微生物的定性及限度。
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.
检验的主要目的是为了确定是否原料药或制剂符合已建立的微生物限度标准，当用于这一目的时，应按照以下方式（包括取样量），进行并按照下述描述对结果进行分析。
Alternative microbiological procedures, including automated methods, may be used, provided that their equivalence to the Pharmacopoeia method has been demonstrated.
如果可证明某种试验方法（包括自动化分析法）的效果与药典中的方法等同，该方法可作为另一种供选择的试验方法。
2. GENERAL PROCEDURES 一般程序
The preparation of samples is carried out as described in general chapter 2.6.12. 样品的制备方法参见（2.6.12）。
If the product to be examined has antimicrobial activity, this is insofar as possible removed or neutralised as described in general chapter 2.6.12.
如果供试品有抗微生物活性，按照（2.6.12）中描述的方法对其进行中和。
If surface-active substances are used for sample preparation, their absence of toxicity for
micro-organisms and their compatibility with inactivators used must be demonstrated as described in general chapter 2.6.12.
如果样品的制备过程中使用了表面活性剂，其必须满足（2.6.12）中的要求，对微生物无毒性并且可与灭活剂兼容。
3. GROWTH-PROMOTING AND INHIBITORY PROPERTIES OF THE MEDIA, SUITABILITY OF THE TEST AND NEGATIVE CONTROLS 培养基中的生长促进作用和生长抑制作用，试验方法的适应性和阴性对照试验
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The ability of the test to detect micro-organisms in the presence of the product to be tested must be established. Suitability must be confirmed if a change in testing performance, or the product, which may affect the outcome of the test is introduced. 该试验方法要有一定的检测微生物的能力，如果影响试验结果的操作或被测物品发生变化，该变化可能会影响试验结果时，该试验方法的适应性必须确认。
3-1. PREPARATION OF TEST STRAINS 试验用菌株的制备
Use standardised stable suspensions of test strains or prepare them as stated below. Seed lot culture maintenance techniques (seed-lot systems) are used so that the viable micro-organisms used for inoculation are not more than 5 passages removed from the original master seed-lot.
使用标准稳定的试验用菌株的菌悬液或按照以下方法制备，使用种子批传代次数不得超过5代。
3-1-1. Aerobic micro-organisms. Grow each of the bacterial test strains separately in casein soya bean digest broth or on casein soya bean digest agar at 30-35 °C for 18-24 h. Grow the test strain for Candida albicans separately on Sabouraud-dextrose agar or in Sabouraud-dextrose broth at 20-25 °C for 2-3 days.
3-1-1．需氧菌 将试验用菌株分别接种于胰酪大豆胨液体培养基或胰酪大豆胨琼脂培养基中30-35℃培养18-24 h。将白色念珠菌试验菌株接种于沙氏葡萄糖琼脂培养基或沙氏葡萄糖液体培养基中20-25℃ 培养 2-3 天。
– Staphylococcus aureus such as ATCC 6538, NCIMB 9518, CIP 4.83 or NBRC 13276; 金黄色葡萄球菌：例如ATCC 6538, NCIMB 9518, CIP 4.83 或 NBRC 13276;
– Pseudomonas aeruginosa such as ATCC 9027, NCIMB 8626, CIP 82.118 or NBRC 13275; 铜绿假单胞菌：例如ATCC 9027, NCIMB 8626, CIP 82.118 o或 NBRC 13275;
– Escherichia coli such as ATCC 8739, NCIMB 8545, CIP 53.126 or NBRC 3972; 大肠埃希菌：例如ATCC 8739, NCIMB 8545, CIP 53.126 或 NBRC 3972;
– Salmonella enterica subsp. enterica serovar Typhimurium, such as ATCC 14028 or, as an alternative, Salmonella enterica subsp. enterica serovar Abony such as NBRC 100797, NCTC 6017 or CIP 80.39; 沙门氏菌：例如ATCC 14028 或NBRC 100797, NCTC 6017 或CIP 80.39;
– Candida albicans such as ATCC 10231, NCPF 3179, IP 48.72 or NBRC 1594. 白色念珠菌：例如ATCC 10231, NCPF 3179, IP 48.72 或 NBRC 1594.
Use buffered sodium chloride-peptone solution pH 7.0 or phosphate buffer solution pH 7.2 to make test suspensions. Use the suspensions within 2 h or within 24 h if stored at 2-8 °C.
使用pH为7.0的氯化钠-蛋白胨缓冲液或pH为7.2的磷酸盐缓冲液制备试验用菌悬液。应在2小时内使用菌悬液，如果保存在2-8 ℃条件下应在24小时内使用。
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3-1-2. Clostridia. Use Clostridium sporogenes such as ATCC 11437 (NBRC 14293, NCIMB 12343, CIP 100651) or ATCC 19404 (NCTC 532 or CIP 79.03) or NBRC 14293. Grow the clostridial test strain under anaerobic conditions in reinforced medium for clostridia at 30-35 °C for 24-48 h. As an alternative to preparing and then diluting down a fresh suspension of vegetative cells of Cl. sporogenes, a stable spore suspension is used for test inoculation. The stable spore suspension may be maintained at 2-8 °C for a validated period.
3-1-2 梭菌 使用梭状芽孢杆菌，例如
ATCC 11437 (NBRC 14293,NCIMB 12343, CIP 100651) 或 ATCC 19404 (NCTC 532 或 CIP 79.03) 或 NBRC 14293，在厌氧条件下将梭菌试验用菌株接种于增菌培养基中，在30-35℃条件下培养24-48 小时。将新鲜的有活性的梭状芽孢杆菌孢子悬液稀释后用于接种。稳定的孢子悬液可保存在2-8 ℃条件下，在经过验证的贮存期内使用。
3-2. NEGATIVE CONTROL 阴性对照试验
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 micro-organisms. A negative control is also performed when testing the products as described in section 4. A failed negative control requires an investigation. 为检测试验条件是否符合要求，取试验用稀释剂代替供试品做一阴性对照试验，阴性对照试验应无微生物生长。按照第四部分检测供试品时，也要做一阴性对照试验。当阴性对照试验结果不符合要求时，需要进行偏差调查。
3-3. GROWTH PROMOTION AND INHIBITORY PROPERTIES OF THE MEDIA 培养基的生长促进作用和生长抑制作用
Test each batch of ready-prepared medium and each batch of medium prepared either from dehydrated medium or from ingredients.
每一批成品培养基，以及由脱水培养基或由规定的处方配制的培养基都应进行适用性检查。
Verify suitable properties of relevant media as described in Table 2.6.13.-1. 按照表（2.6.13.-1），检测相关培养基的特性。
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Table 2.6.13.-1 – Growth promoting, inhibitory and indicative properties of media
培养基的生长促进作用、生长抑制作用和指示作用
Medium 培养基 Enterobacteria enrichment broth- Mossel 肠道菌增菌肉汤培养基 Violet red bile glucose Agar 紫红胆汁葡萄糖琼脂培养基 MacConkey broth 麦康凯肉汤培养基 MacConkey agar 麦康凯琼脂培养基 Property 特性 Growth promoting 生长促进 Inhibitory 生长抑制 Growth promoting + indicative 生长促进和指示作用 Growth promoting 生长促进 Inhibitory 生长抑制 Growth promoting + indicative 生长促进和指示作用 Test strains 试验菌株 E. coli 大肠埃希菌 P. aeruginosa 铜绿假单胞菌 S. aureus 金黄色葡萄球菌 E. coli 大肠埃希菌 P. aeruginosa 铜绿假单胞菌 E. coli 大肠埃希菌 S. aureus 金黄色葡萄球菌 E. coli 大肠埃希菌 Salmonella enterica subsp. enterica serovar Typhimurium or Salmonella en-terica subsp. enterica serovar Abony 沙门氏菌 S. aureus 金黄色葡萄球菌 Salmonella enterica subsp. enterica serovar Typhimurium or Salmonella enterica subsp. enterica serovar Abony 沙门氏菌 Test for bile-tolerant gram- negative bacteria 耐胆盐革兰氏阴性菌的检测 Test for Escherichia Coli 大肠埃希菌的检测 Rappaport Vassiliadis Salmonella enrichment Broth 沙门氏菌琼脂培养基 Test for Salmonella 沙门氏菌的检测 Xylose, lysine, deoxycholate agar 木糖赖氨酸去氧胆酸盐琼脂培养基 Growth promoting 生长促进 Inhibitory 生长抑制 Growth promoting + indicative 生长促进和指示作用 4 / 18
Inhibitory 指示作用 Test for Pseudomonas Aeruginosa 铜绿假单胞菌的检测 Test for Staphylococcus aureus 金黄色葡萄球菌的检测 Cetrimide agar 溴棕三甲铵琼脂培养基 Growth promoting 生长促进 Inhibitory 生长抑制 Growth promoting + indicative 生长促进和指示作用 Inhibitory 生长抑制剂 Growth promoting 生长促进 Growth promoting 生长促进 Growth promoting 生长促进 Growth promoting + indicative 生长促进和指示作用 E. coli 大肠埃希菌 P. aeruginosa 铜绿假单胞菌 E. coli 大肠埃希菌 S. aureus 金黄色葡萄球菌 E. coli 大肠埃希菌 Cl. Sporogenes 梭状芽孢杆菌 Cl. Sporogenes 梭状芽孢杆菌 C. albicans 白色念珠菌 C. albicans 白色念珠菌 Mannitol salt agar 甘露醇氯化钠琼脂培养基 Reinforced medium for clostridia 增菌培养基 Columbia agar 哥伦比亚琼脂培养基 Sabouraud dextrose Broth 沙氏葡萄糖肉汤培养基 Sabouraud dextrose agar 沙氏葡萄糖琼脂培养基 Test for clostridia 梭菌的检测 Test for Candida albicans 白色念珠菌的检测
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EP 8.0-2.6.13 非无菌药品的微生物限度检查：特殊微生物的检查(中英对照)
