1、04/2010:206132.6.13. MICROBIOLOGICAL EXAMINATION OF NON-STERILE PRODUCTS: TEST FOR SPECIFIED MICRO-ORGANISMS(3) 非無菌藥品的微生物限度檢查：特殊微生物的檢查1. INTRODUCTION 導言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.下述檢驗方法用于檢查在描述的試驗條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.檢驗的主要目的是為了確定是否原料藥或制劑符合已建立的微生物限度標準，當用于這一目的時，應按照以下方式（包括取樣量），進行并按照下述描述對結果進行分析。Alternative microbiological procedures, including automated methods, may be used, provided that their equivalence to the Pharmacopoeia method has been demonstrated

4、. 如果可證明某種試驗方法（包括自動化分析法）的效果與藥典中的方法等同，該方法可作為另一種供選擇的試驗方法。2. GENERAL PROCEDURES 一般程序The preparation of samples is carried out as described in general chapter .樣品的制備方法參見（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 .如果供試品有抗微生物活性，按照（2.6.12）中描述的方法對其進行中和。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 .如果樣品的制備過程中使用了表面活性劑，其必須滿足（2.6.12）中的要求，對微生

6、物無毒性并且可與滅活劑兼容。3. GROWTH-PROMOTING AND INHIBITORY PROPERTIES OF THE MEDIA, SUITABILITY OF THE TEST AND NEGATIVE CONTROLS 培養基中的生長促進作用和生長抑制作用，試驗方法的適應性和陰性對照試驗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.該試驗方法要有一定的檢測微生物的能力，如果影響試驗結果的操作或被測物品發生變化，該變化可能會影響試驗結果時，該試驗方法的適應性必須確認。3-1. PREPARATION OF TEST STRAINS 試驗用菌株的制備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.使用標準穩定的試驗用菌株的菌懸液或按照以下方法制備，使用種子批傳代次數不得超過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 將試驗用菌株分別接種于胰酪大豆胨液體培養基或胰酪大豆

10、胨瓊脂培養基中30-35培養18-24 h。將白色念珠菌試驗菌株接種于沙氏葡萄糖瓊脂培養基或沙氏葡萄糖液體培養基中20-25 培養 2-3 天。 Staphylococcus aureus such as ATCC 6538, NCIMB 9518, CIP 4.83 or NBRC 13276;金黃色葡萄球菌：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;銅綠假單胞菌：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;大腸埃希菌：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;沙門氏菌：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為7.0的氯化鈉-蛋白胨緩沖液或pH為7.2的磷酸鹽緩沖液制備試驗用菌懸液。應在2小時內使用菌懸液，如果保存在2-8 條件下應在24小時內使用。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 使用梭狀芽孢桿菌，例如ATCC 11437 (NBRC 14293,NCIMB 12343, CIP 100651) 或 ATCC 19404 (NCTC 532 或 CIP 79.03) 或 NBRC 14293，在厭氧條件下將梭菌

17、試驗用菌株接種于增菌培養基中，在30-35條件下培養24-48 小時。將新鮮的有活性的梭狀芽孢桿菌孢子懸液稀釋后用于接種。穩定的孢子懸液可保存在2-8 條件下，在經過驗證的貯存期內使用。3-2. NEGATIVE CONTROL 陰性對照試驗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.為檢測試驗條件是否符合要求，取試驗用稀釋劑代替供試品做一陰性對照試驗，陰性對照試驗應無微生物生長。按照第四部分檢測供試品時，也要做一陰性對照試驗。當陰性對照試驗結果不符合要求時，需要進行偏差調查。3-3. GROWTH PROMOTION AND INHIBITORY PROPERTIES OF THE MEDIA 培養基

19、的生長促進作用和生長抑制作用 Test each batch of ready-prepared medium and each batch of medium prepared either from dehydrated medium or from ingredients. 每一批成品培養基，以及由脫水培養基或由規定的處方配制的培養基都應進行適用性檢查。Verify suitable properties of relevant media as described in Table 2.6.13.-1.按照表（2.6.13.-1），檢測相關培養基的特性。Table 2.6.13.-1

20、Growth promoting, inhibitory and indicative properties of media培養基的生長促進作用、生長抑制作用和指示作用Medium 培養基Property 特性Test strains 試驗菌株Test for bile-tolerant gram-negative bacteria 耐膽鹽革蘭氏陰性菌的檢測Enterobacteria enrichment broth- Mossel 腸道菌增菌肉湯培養基Grow

21、th promoting 生長促進E. coli 大腸埃希菌P. aeruginosa 銅綠假單胞菌 Inhibitory 生長抑制 S. aureus 金黃色葡萄球菌Violet red bile glucose Agar 紫紅膽汁葡萄糖瓊脂培養基 Growth promoting  + indicative 生長促進和指示作用 E. coli 

22、大腸埃希菌 P. aeruginosa 銅綠假單胞菌Test for Escherichia Coli 大腸埃希菌的檢測MacConkey broth 麥康凱肉湯培養基Growth promoting 生長促進E. coli 大腸埃希菌Inhibitory 生長抑制S. aureus 金黃色葡萄球菌MacConkey agar 麥康凱瓊脂培養基 Growth promoting + in

23、dicative 生長促進和指示作用E. coli 大腸埃希菌Test for Salmonella 沙門氏菌的檢測Rappaport Vassiliadis Salmonella enrichment Broth 沙門氏菌瓊脂培養基Growth promoting 生長促進 Salmonella enterica subsp. enterica serovar Typhimurium or &#

24、160;Salmonella en-terica  subsp. enterica serovar  Abony 沙門氏菌Inhibitory 生長抑制S. aureus 金黃色葡萄球菌Xylose, lysine, deoxycholate agar  木糖賴氨酸去氧膽酸鹽瓊脂培養基Growth promoting + indicative 生長促進和指示作用 Salmonella&#

25、160;enterica subsp. enterica serovar Typhimurium or  Salmonella enterica  subsp. enterica serovar   Abony 沙門氏菌Inhibitory 指示作用E. coli 大腸埃希菌Test for Pseudomonas Aeruginosa 銅綠假單胞菌的檢測Cetrimide a

26、gar 溴棕三甲銨瓊脂培養基Growth promoting 生長促進 P. aeruginosa  銅綠假單胞菌Inhibitory 生長抑制E. coli  大腸埃希菌Test for     Staphylococcus aureus 金黃色葡萄球菌的檢測Mannitol salt agar 甘露醇氯化鈉瓊脂培養基Growth promoting + indicative

27、 生長促進和指示作用S. aureus 金黃色葡萄球菌Inhibitory 生長抑制劑 E. coli 大腸埃希菌Test for clostridia  梭菌的檢測Reinforced medium for clostridia  增菌培養基Growth promoting 生長促進Cl. Sporogenes 梭狀芽孢桿菌Columbia agar 哥倫比亞瓊脂培養基Gro

28、wth promoting 生長促進Cl. Sporogenes 梭狀芽孢桿菌Test for Candida  albicans 白色念珠菌的檢測 Sabouraud dextrose Broth 沙氏葡萄糖肉湯培養基 Growth promoting 生長促進 C. albicans 白色念珠菌 Sabouraud dextrose agar  沙氏葡萄

29、糖瓊脂培養基Growth promoting + indicative 生長促進和指示作用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.液態培養基的促生長能力檢查：將適量的微生物（<100 CFU）接種于培養基中,在指定溫度下培養，培養時間應小于微生物試驗時規定的最短時間。微

31、生物的生長清晰可見，并且與已批準合格的培養基中微生物的生長情況類似，說明該液體培養基符合要求。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.固態培養基的促生長能力檢查：使用表面涂布法，將適量的微生物（<100 CFU）接種于每個平皿上，在指定溫度下培養，培養時間應小于微生物試驗時規定的最短時間。微生物的生長清晰可見，并且與已批準合格的培養基中微生物的生長情況類似，說明該固培養基符

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、體或液體培養基的抑制能力檢查：將適量的微生物（>100 CFU）接種于培養基中，在指定溫度下培養，培養時間應大于微生物試驗時規定的最長時間，微生物不生長。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.培養基中指示能力的檢查：使用表面涂布平皿法，將適量的微生物（<100 CFU）接種于每個平皿上，在指定溫度下培養，培養時間應在微生物試驗時規定的時間范圍內。菌落的外觀和指示反應與已批準合

36、格的培養基的指示反應結果相似，說明該培養基的指示作用符合要求。3-4. SUITABILITY OF THE TEST METHOD 試驗方法的適應性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.對于每一種供試品按照第四部分中相關的方法制備，將每種試驗用菌株加入到指定的培養基中，每種試驗用菌株分別接種。接種用微生物的量應小于100 CFU。Perform the test as described in the relevant paragraph in section 4 using the shortest incubation period presc

38、ribed. 按照第四部分中相關的方法進行試驗，培養時間應為微生物試驗時規定的最短時間。The specified micro-organisms must be detected with the indication reactions as described in section 4. 特定的微生物必須按照第四部分的要求進行指示劑反應檢測。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試品需要對試驗過程進行修正（參見 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.如果某種供試品的抗微生物活性不能中和，那

40、么必須證實在試驗過程中其不能表現出抗微生物活性。4. TESTING OF PRODUCTS 供試品4-1. BILE-TOLERANT GRAM-NEGATIVE BACTERIA 耐膽鹽革蘭氏陰性菌 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).樣品的制備和預培養  按照（2.6.12）中的方法取不少于1g的供試品制成1:10的供試液， 接種于胰酪大豆胨液體培養基中，2

42、0-25條件下培養，培養時間應使微生物復活而又不能使其繁殖（通常在2-5小時范圍內）。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.定性試驗 除另有規定外，取4-1-1中制備的相當于1g供試品的供試液接種于腸道菌增菌肉湯培養基中，30-35 條件下培養24-48 小時后，培養物接種于紫紅膽汁葡萄糖瓊脂培養基中，30-35 條件下培養18-24小時。如果無菌落生長，說明供試品中未檢出耐膽鹽革蘭氏陰性菌。4-1-3. Quantitative test

44、 定量試驗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.選擇和分離培養  將4-1-1制備供試液或將分別含有0.1 g, 0.01 g和0.001 g (或 0.1 ml, 0.01 ml 和 0.001 ml)供試品的供試液接種

46、于適量的腸道菌增菌肉湯培養基中，30-35 條件下培養24-48 小時后，培養物接種于紫紅膽鹽葡萄糖瓊脂培養基中，30-35 條件下培養18-24小時。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.結果分析  如果有菌落生長說明試驗結果為陽性，記錄產生陽性試驗結果使用供試品的最小量和產生陰性結果使用供試品的最大量，參照表2.6.13.-2計算出細菌的數量。Table 2.6.13.-2  Interpretation of results 結果分析Results for each quantity of product每一供試品量的試驗結果Probable

48、60;number ofbacteria per gram or millilitre ofproduct每毫升或每克中可能含有細菌的量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 大腸埃希菌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.樣品的制備和增菌培養  按照（2.6.12）中的方法取不少于1g的供試品制成1:10的供試液， 取供試液10ml，或相當于含供試品1g或1ml的供試液，接種于適量的胰酪大豆胨液體培養基中（符合3-4的要求），30-35條件下培養18-24 小時。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.選擇和分離培養  振動容器，將1ml胰酪大豆胨液體培養物轉移到100ml麥康凱肉湯培養基中，42-44條件下培養24-48 小時后，培養物接種于麥康凱瓊脂培養基中，30-35 條件下培養18-72小時。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.結果分析  如果有菌落生長，說明供試品中含有大腸埃希菌。是否確實含有大腸埃希菌，需做鑒別試驗。如果無菌落生長或鑒別試驗為陰性，說明供試品中不含有大腸埃希菌。4-3. SALMONELLA 沙

53、門菌 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.供試液的制備和增菌培養  按照（2.6.12）中的方法制備樣品，將相當于含供試品10g或10ml的供試液接種于適量的胰酪大豆胨液體培養基中（符合3-4的要求），30-35條件下培養18-24 小時。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.選擇和分離培養胰酪大豆胨液體培養物轉移到10ml RV沙門增菌液體培養基中，30-35條件下培養18-24小時后，培養物接種于木糖賴氨酸去氧膽酸鹽瓊脂培養基中，30-35 條件下培養18-48小時。4-3-3. Interpretation. The possible presence of Salmonella is indica

56、ted 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.結果分析  如果有紅色菌落生長，菌落有或無黑色中心，說

57、明供試品中含有沙門氏菌。是否確實含有沙門氏菌，需做鑒別試驗。如果菌落外觀不是此種特征或鑒別試驗結果為陰性，說明供試品中不含有沙門氏菌。4-4. PSEUDOMONAS AERUGINOSA 銅綠假單胞菌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 or the quant

58、ity 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 thro

59、ugh a sterile filter membrane and place in 100 mL of casein soya bean digest broth. Incubate at 30-35 °C for 18-24 h.供試品液的制備和增菌培養  按照（2.6.12）中的方法取不少于1g的供試品制成1:10的供試液， 取供試液10ml，或相當于含供試品1g或1ml的供試液，接種于適量的胰酪大豆胨液體培養基中（符合3-4的要求）。當檢測透皮貼劑時，取一劑量供試品按照（2.6.12  4-5-1）中的方法使用無菌濾膜過濾后，接種于10

60、0ml胰酪大豆胨液體培養基中，30-35條件下培養18-24 小時。 4-4-2. Selection and subculture. Subculture on a plate of cetrimide agar and incubate at 30-35 °C for 18-72 h.選擇和分離培養  接種于溴化十六烷基三甲銨瓊脂培養基中，30-35條件下培養18-72小時4-4-3. Interpretation. Growth of colonies indicates the possible presence of P. aeruginosa

61、. 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.結果分析  如果有菌落生長，說明供試品中可能含有銅綠假單胞菌。是否確實含有銅綠假單胞菌，需做鑒別試驗。如果無菌落生長或鑒別試驗為陰性，說明供試品中不含有銅綠假單胞菌。4-5. STAPHYLOCOCCUS AUREUS 金黃色葡萄球菌4-5-1. Sample

62、 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 soya

63、 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.供試品液的制備和增菌培養  按照（2.6.12）中的方法取不少于1g的供試品制成1:10的供試液， 取供試液10ml，或相當于含供試品1g或1ml的供試液，接種于適量的胰酪大豆胨液體培養基中（符合3-4的要求）。當檢測透皮貼劑時，取一劑量供試品按照（2.6.12  4-5-1）中的方法使用無菌濾膜過濾后，接種于100ml胰酪大豆胨液體培養基中，30-35條件下培養18-24 小時。4-5-2. Selection and subculture. Su