ep_8.02.6.13_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、文档从互联网中收集，已重新修正排版，word格式支持编辑，如有帮助欢迎下载支持。EP 8.004/2010:20613MICROBIOLOGICAL EXAMINATION OF NON-STERILE PRODUCTS: TEST FORSPECIFIED MICRO ORGANISMS。）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-or

2、ganisms 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 giv

3、en 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 Pharma

4、copoeia 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 样品的制备方法参见（If the product to be examined has antimicrobial activity, this is insofar as possible removed or neu

5、tralised as described in general chapter 如果供试品有抗微生物活性，按照（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（，对微生物无毒性并且可

6、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 Iword格式支持编辑，如有帮助欢迎下载支持。文档从互联网中收集，已重新修正排版，word格式支持编

7、辑，如有帮助欢迎下载支持。 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 s

8、uspensions 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

9、-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 for 18-24 h. Grow the test strain for Candida albicans separately on Sabouraud-dextrose agar or in Sabouraud-dextrose broth at 20-25 for 2-3 day

10、s.3-1-1.需氧菌将试验用菌株分别接种于胰酪大豆陈液体培养基或胰酪大豆陈琼脂培养基中 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 8

11、2.118 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 ent erica subsp. enterica serovar Typhimurium, such as ATCC 14028 or, as an alternat

12、ive, Salmonella enterica subsp. ent erica 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, NCPF3179, 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 chlor

13、ide-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 .使用pH为7.0的氯化钠-蛋白陈缓冲液或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 C条件下应在24小时内使用。3-1-2. Clostridia. Use Clostridium sporogenes such as ATCC 11437 (NB

14、RC 14293, NCIMB 12343,文档从互联网中收集，已重新修正排版，word格式支持编辑，如有帮助欢迎下载支持。 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 for 24-48 h. As an alternative to preparing and then diluting down

15、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 for a validated period.3-1-2梭菌 使用梭状芽抱杆菌，例如ATCC 11437 (NBRC 14293,NCIMB 12343, CIP 100651)或 ATCC 19404 (NCTC 532 或 CIP 79.03) 或NBRC 14

16、293,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胞杆菌抱子悬液稀释后用于接种。稳定的胞子悬液可保存 在2-8 C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.

17、 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 M

18、EDIA 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111脱水培养基或III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 按照表(，检测相关培养基的特性。3word格式支持编辑，如有帮助欢迎下载支持。Ta

19、ble - 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 gramnegative 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Growth 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.

20、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 + indicative 生

21、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 Salmonell a enrichment Broth 沙门氏菌琼脂培养基Growth promoting 生长促进Salmonella enterica subsp. enterica serovarTyphimurium or Salmonella enterics subsp. enterica serovar A bony沙门氏菌Inhibitory生长抑制S. aureus金黄色葡萄球菌Xylose, lysine, deoxycholate

22、agar木糖赖氨酸去氧胆酸盐琼脂培Growth promoting + indicative 生长促进和指示作用Salmonella enterica subsp. enterica serovarTyphimurium or Salmonella ent erica subsp. enterica serovar养基Abony沙门氏菌Inhibitory指示作用E. coli 大肠埃希菌Test forPseudomonas Aeruginosa 铜绿假单胞菌的检测Cetrimide agar 滨棕二甲筱琼脂培养基Growth promoting 生长促进P. aeruginosa铜绿假单胞

23、菌Inhibitory生长抑制E. coli大肠埃希菌Test forStaphylococcus 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 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梭状芽胞杆菌Columbia agar 哥伦

24、比亚琼脂培养基Growth promoting 生长促进Cl. Sporogenes梭状芽胞杆菌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5Test for growth promoting properties, liquid media : ino

25、culate a portion of the appropriate medium with a small number (not more than 100 CFU) of the appropriate micro-organism. Incubate at the specified temperature for not more than the shortest period of time specified in the test. Clearly visible growth of the micro-organism comparable to that previou

26、sly 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(<I00 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 p

27、late with a small number (not more than 100 CFU) of the appropriate micro-organism. Incubate at the specified temperature for not more than the shortest period of time specified in the test. Growth of the micro-organism comparable to that previously obtained with a previously tested and approved bat

28、ch 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(100CFU)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 a

29、t the specified temperature for not less than the longest period of time specified in the test. No growth of the test micro-organism occurs.固体或液体培养基的抑制能力检查:将适量的微生物(>100CFU)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 properties9, perform the surface-spread method, inoculating

30、 each plate with a small number (not more than 100 CFU) of the appropriate micro-organism. 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 those previously obtained with a previously

31、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(<I00 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

32、paragraph in section 4. Add each test strain at the time of mixing, in the prescribed growth medium. Inoculate the test strains individually. 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

33、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 prescribed.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

34、检测。Any antimicrobial activity of the product necessitates a modification of the test procedure （see 4-5-3 of general chapter任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

35、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

36、 less than 1 g of the product to be examined as described in general chapter , but using casein soya bean digest broth as the chosen diluent, mix and incubate at 20-25 for a time sufficient to resuscitate the bacteria but not sufficient to encourage multiplication of the organisms （usually 2 h but n

37、ot more than 5 h）.样品的制备和预培养按照（，接种于胰酪大豆陈液体培养基中，20-25C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 for 24-48

38、h. Subculture on plates of violet red bile glucose agar. Incubate at 30-35 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 °C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

39、-1-3. Quantitative test 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 produ

40、ct to be examined. Incubate at 30-35 for 24-48 h. Subculture each of thecultures on a plate of violet red bile glucose agar. Incubate at 30-35 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于适量的肠道菌增菌肉汤培养基中， 30-35 °C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

41、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. Determine from Table 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

42、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Table 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 number of bacteria per gram or millilitre of product 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&l

43、t;104-2. ESCHERICHIA COLI 大肠埃希茵4-2-L 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 or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount (determ

44、ined as described under 3-4) of casein soya bean digest broth, mix and incubate at 30-35 for 18-24 h.样品的制备和增施培养按照(，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陈液体培养基中(符合3-4的要求)，30-35C条件下培养18-24小时。4-2-2. Selection and subculture. Shake the container, transfer 1mL of casein soya bean digest broth to 100mL o

45、f MacConkey broth and incubate at 42-44 for 24-48 h. Subculture on a plate of MacConkey agar at 30-35 for 18-72 h.选择和分离力养振动容器，将1ml胰酪大豆陈液体培养物转移到100ml麦康凯肉汤培养基中， 42-44C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 C条件下培养18-72 小时。4-2-3. Interpretation. Growth of colonies indicates the possible presence of E. coli

46、. 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 沙门菌4-3-1. Sample preparation and pre-incubation. Prepar

47、e the product to be examined as described in general chapter , and use the quantity corresponding to not less than 10 g or 10 inL to inoculate a suitable amount （determined as described under 3-4） of casein soya bean digest broth, mix and incubate at 30-35 for 18-24 h.供试液的制备和增菌培养 按照（，将相当于含供试品10g或10m

48、l的供试液接种于适量的胰酪 大豆族液体培养基中（符合3-4的要求），30-35C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 at 30-35 for 18-24 h. Subculture on plates of xylose, lysine, deoxycholate agar. Incubate a

49、t 30-35 for 18-48 h.选择和分离培养将0.1ml胰酪大豆陈液体培养物转移到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 Salmonella is indicated by the growth of well-developed, red colonies, with or without black centres. This is confirmed by ide

50、ntification 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-L Samp

51、le 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 so

52、ya 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 through a sterile filter membrane and place in 100 mL of casein soya bean digest broth. Incubate at 30-35 for 18-24 h.供试品液的

53、制备和增菌培养按照（，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陈液体培养基中（符合3-4的要求）。当检测透皮贴剂时，取一剂量供试品 按照（4-5-1）中的方法使用无菌滤膜过滤后，接种于100ml胰酪大豆陈液体培养基中，30-35 条件下培养18-24小时。4-4-2. Selection and subculture. Subculture on a plate of cetrimide agar and incubate at 30-35 for 18-72 h.选择和分离培养接种于澳化十六烷基三甲筱琼脂培养基中，3O-35C条件下培养18-72小时4-

54、4-3. Interpretation. Growth of colonies indicates the possible presence of P. aeruginosa. This is confirmed by identification tests. The product complies with the test if colonies are not present or if the confirmatory identification tests are negative.结果分析如果有菌落生长，说明供试品中可能含有铜绿假单胞菌。是否确实含有铜绿假单胞菌, 需做鉴别

55、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铜绿假单胞菌。4-5. STAPHYLOCOCCUS A UREUS 金黄色葡萄球菌4-5-1. 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 or the quantity corresponding to 1 g or I m

56、L 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 through a sterile filter membrane and plac

57、e in 100 mL of casein soya bean digest broth. Incubate at 30-35 for 18-24 h.供试品液的制备和增菌培养按照（，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陈液体培养基中（符合3-4的要求）。当检测透皮贴剂时，取一剂量供试 品按照（4-5-1）中的方法使用无菌滤膜过滤后，接种于100ml胰酪大豆陈液体培养基中，30-35 条件下培养18-24小时。4-5-2. Selection and subculture. Subculture on a plate of mannitol salt a

58、gar and incubate at 30-35 for 18-72 h.选择和分离培养接种于甘露静氯化钠琼脂培养基中，30-35°。条件下培养18-72小时4-5-3. Interpretation. The possible presence of S. aureus is indicated by the growth of yellow/white colonies surrounded by a yellow zone. 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 如果有黄色/白色菌落生长，外围为黄色，表明供试品中可能含有金黄色葡萄球菌。是 否确实含有金黄色葡萄球菌，需做鉴