EP8.0-2.6.13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)

1、04/2010:206132.6.13. 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

2、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 s

3、amples 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

4、. 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 .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 ge

5、neral chapter .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 .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

6、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 i

7、f 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.

8、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 bacteria

9、l 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

10、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 a

11、s 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&

12、#160;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

13、0;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. Us

14、e 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。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 1

15、4293. 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.

16、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

17、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 ne

18、gative 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

19、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。Table 2.6.13.-1

20、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Property 特性Test strains 试验菌株Test for bile-tolerant gram-negative bacteria 耐胆盐革兰氏阴性菌的检测Enterobacteria enrichment broth- Mossel 肠道菌增菌肉汤培养基Grow

21、th promoting 生长促进E. coli 大肠埃希菌P. aeruginosa 铜绿假单胞菌 Inhibitory 生长抑制 S. aureus 金黄色葡萄球菌Violet red bile glucose Agar 紫红胆汁葡萄糖琼脂培养基 Growth promoting  + indicative 生长促进和指示作用 E. coli 

22、大肠埃希菌 P. aeruginosa 铜绿假单胞菌Test for Escherichia Coli 大肠埃希菌的检测MacConkey broth 麦康凯肉汤培养基Growth promoting 生长促进E. coli 大肠埃希菌Inhibitory 生长抑制S. aureus 金黄色葡萄球菌MacConkey agar 麦康凯琼脂培养基 Growth promoting + in

23、dicative 生长促进和指示作用E. coli 大肠埃希菌Test for Salmonella 沙门氏菌的检测Rappaport Vassiliadis Salmonella enrichment Broth 沙门氏菌琼脂培养基Growth promoting 生长促进 Salmonella enterica subsp. enterica serovar Typhimurium or &#

24、160;Salmonella en-terica  subsp. enterica serovar  Abony 沙门氏菌Inhibitory 生长抑制S. aureus 金黄色葡萄球菌Xylose, lysine, deoxycholate agar  木糖赖氨酸去氧胆酸盐琼脂培养基Growth promoting + indicative 生长促进和指示作用 Salmonella&#

25、160;enterica subsp. enterica serovar Typhimurium or  Salmonella enterica  subsp. enterica serovar   Abony 沙门氏菌Inhibitory 指示作用E. coli 大肠埃希菌Test for Pseudomonas Aeruginosa 铜绿假单胞菌的检测Cetrimide a

26、gar 溴棕三甲铵琼脂培养基Growth promoting 生长促进 P. aeruginosa  铜绿假单胞菌Inhibitory 生长抑制E. coli  大肠埃希菌Test for     Staphylococcus aureus 金黄色葡萄球菌的检测Mannitol salt agar 甘露醇氯化钠琼脂培养基Growth promoting + indicative

27、 生长促进和指示作用S. aureus 金黄色葡萄球菌Inhibitory 生长抑制剂 E. coli 大肠埃希菌Test for clostridia  梭菌的检测Reinforced medium for clostridia  增菌培养基Growth promoting 生长促进Cl. Sporogenes 梭状芽孢杆菌Columbia agar 哥伦比亚琼脂培养基Gro

28、wth promoting 生长促进Cl. Sporogenes 梭状芽孢杆菌Test for Candida  albicans 白色念珠菌的检测 Sabouraud dextrose Broth 沙氏葡萄糖肉汤培养基 Growth promoting 生长促进 C. albicans 白色念珠菌 Sabouraud dextrose agar  沙氏葡萄

29、糖琼脂培养基Growth promoting + indicative 生长促进和指示作用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 micro-organism. Incubate at the specified temp

30、erature for not more than the shortest period of time specified in the test. Clearly visible growth of the micro-organism comparable to that previously obtained with a previously tested and approved batch of medium occurs.液态培养基的促生长能力检查：将适量的微生物（<100 CFU）接种于培养基中,在指定温度下培养，培养时间应小于微生物试验时规定的最短时间。微

31、生物的生长清晰可见，并且与已批准合格的培养基中微生物的生长情况类似，说明该液体培养基符合要求。Test for growth promoting properties, solid media: perform the surface-spread method, inoculating each plate with a small number (not more than 100 CFU) of the appropriate micro-organism. Incubate at the specified temperature for not more than the short

32、est period of time specified in the test. Growth of the micro-organism comparable to that previously obtained with a previously tested and approved batch of medium occurs.固态培养基的促生长能力检查：使用表面涂布法，将适量的微生物（<100 CFU）接种于每个平皿上，在指定温度下培养，培养时间应小于微生物试验时规定的最短时间。微生物的生长清晰可见，并且与已批准合格的培养基中微生物的生长情况类似，说明该固培养基符

33、合要求。Test for inhibitory properties, liquid or solid media: inoculate the appropriate medium with at least 100 CFU of the appropriate micro-organism. Incubate at the specified temperature for not less than the longest period of time specified in the test. No growth of the test micro-organism occurs.固

34、体或液体培养基的抑制能力检查：将适量的微生物（>100 CFU）接种于培养基中，在指定温度下培养，培养时间应大于微生物试验时规定的最长时间，微生物不生长。Test for indicative properties: perform the surface-spread method, inoculating each plate with a small number (not more than 100 CFU) of the appropriate micro-organism. Incubate at the specified temperature for a pe

35、riod of time within the range specified in the test. Colonies are comparable in appearance and indication reactions to those previously obtained with a previously tested and approved batch of medium.培养基中指示能力的检查：使用表面涂布平皿法，将适量的微生物（<100 CFU）接种于每个平皿上，在指定温度下培养，培养时间应在微生物试验时规定的时间范围内。菌落的外观和指示反应与已批准合

36、格的培养基的指示反应结果相似，说明该培养基的指示作用符合要求。3-4. SUITABILITY OF THE TEST METHOD 试验方法的适应性For each product to be tested, perform the sample preparation as described in the relevant paragraph in section 4. Add each test strain at the time of mixing, in the prescribed growth medium. Inoculate the test strains indivi

37、dually. Use a number of micro-organisms equivalent to not more than 100 CFU in the inoculated test preparation.对于每一种供试品按照第四部分中相关的方法制备，将每种试验用菌株加入到指定的培养基中，每种试验用菌株分别接种。接种用微生物的量应小于100 CFU。Perform the test as described in the relevant paragraph in section 4 using the shortest incubation period presc

38、ribed. 按照第四部分中相关的方法进行试验，培养时间应为微生物试验时规定的最短时间。The specified micro-organisms must be detected with the indication reactions as described in section 4. 特定的微生物必须按照第四部分的要求进行指示剂反应检测。Any antimicrobial activity of the product necessitates a modification of the test procedure (see 4-5-3 of general chapter ).任

39、何有抗微生物活性的供试品需要对试验过程进行修正（参见 2.6.12中的4-5-3）。 If for a given product the antimicrobial activity with respect to a micro-organism for which testing is prescribed cannot be neutralised, then it is to be assumed that the inhibited micro-organism will not be present in the product.如果某种供试品的抗微生物活性不能中和，那

40、么必须证实在试验过程中其不能表现出抗微生物活性。4. TESTING OF PRODUCTS 供试品4-1. BILE-TOLERANT GRAM-NEGATIVE BACTERIA 耐胆盐革兰氏阴性菌 4-1-1. 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 general chapter , but using casein soy

41、a bean digest broth as the chosen diluent, mix and incubate at 20-25 °C for a time sufficient to resuscitate the bacteria but not sufficient to encourage multiplication of the organisms (usually 2 h but not more than 5 h).样品的制备和预培养  按照（2.6.12）中的方法取不少于1g的供试品制成1:10的供试液， 接种于胰酪大豆胨液体培养基中，2

42、0-25条件下培养，培养时间应使微生物复活而又不能使其繁殖（通常在2-5小时范围内）。4-1-2. Test for absence. Unless otherwise prescribed, use the volume corresponding to 1 g of the product, as prepared in 4-1-1, to inoculate enterobacteria enrichment broth-Mossel. Incubate at 30-35 °C for 24-48 h. Subculture on plates of violet red bi

43、le glucose agar. Incubate at 30-35 °C for 18-24 h. The product complies with the test if there is no growth of colonies.定性试验 除另有规定外，取4-1-1中制备的相当于1g供试品的供试液接种于肠道菌增菌肉汤培养基中，30-35 条件下培养24-48 小时后，培养物接种于紫红胆汁葡萄糖琼脂培养基中，30-35 条件下培养18-24小时。如果无菌落生长，说明供试品中未检出耐胆盐革兰氏阴性菌。4-1-3. Quantitative test

44、 定量试验4-1-3-1. Selection and subculture. Inoculate suitable quantities of enterobacteria enrichment broth-Mossel with the preparation as described under 4-1-1 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. Incu

45、bate at 30-35 °C for 24-48 h. Subculture each of thecultures on a plate of violet red bile glucose agar. Incubate at 30-35 °C for 18-24 h.选择和分离培养  将4-1-1制备供试液或将分别含有0.1 g, 0.01 g和0.001 g (或 0.1 ml, 0.01 ml 和 0.001 ml)供试品的供试液接种

46、于适量的肠道菌增菌肉汤培养基中，30-35 条件下培养24-48 小时后，培养物接种于紫红胆盐葡萄糖琼脂培养基中，30-35 条件下培养18-24小时。4-1-3-2. Interpretation. Growth of colonies constitutes a positive result. Note the smallest quantity of the product that gives a positive result and the largest quantity that gives a negative result. Determin

47、e from Table 2.6.13.-2 the probable number of bacteria.结果分析  如果有菌落生长说明试验结果为阳性，记录产生阳性试验结果使用供试品的最小量和产生阴性结果使用供试品的最大量，参照表2.6.13.-2计算出细菌的数量。Table 2.6.13.-2  Interpretation of results 结果分析Results for each quantity of product每一供试品量的试验结果Probable

48、60;number ofbacteria per gram or millilitre ofproduct每毫升或每克中可能含有细菌的量0.1 g or 0.1 ml0.01 g or 0.01 ml0.001 g or 0.001 ml+-+-+->103<103 and >103<102 and >10<104-2. ESCHERICHIA COLI 大肠埃希菌4-2-1. S

49、ample 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 general chapter , and use 10 mL or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount (determined as described under 3-4) of casein

50、 soya bean digest broth, mix and incubate at 30-35 °C for 18-24 h.样品的制备和增菌培养  按照（2.6.12）中的方法取不少于1g的供试品制成1:10的供试液， 取供试液10ml，或相当于含供试品1g或1ml的供试液，接种于适量的胰酪大豆胨液体培养基中（符合3-4的要求），30-35条件下培养18-24 小时。4-2-2. Selection and subculture. Shake the container, transfer 1mL of casein soya bean dige

51、st broth to 100mL of MacConkey broth and incubate at 42-44 °C for 24-48 h. Subculture on a plate of MacConkey agar at 30-35 °C for 18-72 h.选择和分离培养  振动容器，将1ml胰酪大豆胨液体培养物转移到100ml麦康凯肉汤培养基中，42-44条件下培养24-48 小时后，培养物接种于麦康凯琼脂培养基中，30-35 条件下培养18-72小时。4-2-3. Interpretation. Growth of co

52、lonies indicates the possible presence of E. coli. This is confirmed by identification tests. The product complies with the test if no colonies are present or if the identification tests are negative.结果分析  如果有菌落生长，说明供试品中含有大肠埃希菌。是否确实含有大肠埃希菌，需做鉴别试验。如果无菌落生长或鉴别试验为阴性，说明供试品中不含有大肠埃希菌。4-3. SALMONELLA 沙

53、门菌 4-3-1. Sample preparation and pre-incubation. Prepare the product to be examined as described in general chapter , and use the quantity corresponding to not less than 10 g or 10 mL to inoculate a suitable amount (determined as described under 3-4) of casein soya bean digest broth, mix and in

54、cubate at 30-35 °C for 18-24 h.供试液的制备和增菌培养  按照（2.6.12）中的方法制备样品，将相当于含供试品10g或10ml的供试液接种于适量的胰酪大豆胨液体培养基中（符合3-4的要求），30-35条件下培养18-24 小时。4-3-2. Selection and subculture. Transfer 0.1mL of casein soya bean digest broth to 10 mL of Rappaport Vassiliadis Salmonella enrichment broth and incubate

55、 at 30-35 °C for 18-24 h. Subculture on plates of xylose, lysine, deoxycholate agar. Incubate at 30-35 °C for 18-48 h.选择和分离培养  将0.1ml胰酪大豆胨液体培养物转移到10ml RV沙门增菌液体培养基中，30-35条件下培养18-24小时后，培养物接种于木糖赖氨酸去氧胆酸盐琼脂培养基中，30-35 条件下培养18-48小时。4-3-3. Interpretation. The possible presence of Salmone

56、lla is indicated by the growth of well-developed, red colonies, with or without black centres. This is confirmed by identification tests. The product complies with the test if colonies of the types described are not present or if the confirmatory identification tests are negative.结果分析  如果有红色菌落生

57、长，菌落有或无黑色中心，说明供试品中含有沙门氏菌。是否确实含有沙门氏菌，需做鉴别试验。如果菌落外观不是此种特征或鉴别试验结果为阴性，说明供试品中不含有沙门氏菌。4-4. PSEUDOMONAS AERUGINOSA 铜绿假单胞菌4-4-1. 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 general chapter , and use 10 mL

58、 or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount (determined as described under 3-4) of casein soya bean digest broth and mix. When testing transdermal patches, filter the volume of sample corresponding to 1 patch of the preparation described under 4-5-1 in general chapter 2.6.12 through a sterile filter membrane and place in 100 mL of casein soya bean digest broth. Incubate at 30-35 °C for 18-24 h.供试品液的制备和增菌培养  按照（2.6.12）中的方法取不少于1g的供试品制成1:10的供试液，