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)

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
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。

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。

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
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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 temperature 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 shortest 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 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 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 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检测。

Any antimicrobial activity of the product necessitates a modification of the test procedure (see 4-5-3 of general chapter 2.6.12).
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 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 2.6.12, but using casein soya 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，20-25℃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 bile 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 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. Incubate at 30-35 °C for 24-48 h. Subculture each of the
cultures 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. Determine 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
4-2. ESCHERICHIA COLI 大肠埃希菌
4-2-1. 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 2.6.12, 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 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 digest 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 colonies 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 沙门菌
4-3-1. Sample preparation and pre-incubation. Prepare the product to be examined as described in general chapter 2.6.12, 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 incubate 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 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 Salmonella 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 2.6.12, 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 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，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）。

当检测透皮贴剂时，取一剂量供试品按照（2.6.12 4-5-1）中的方法使用无菌滤膜过滤后，接种于100ml胰酪大豆胨液体培养基中，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 °C for 18-72 h.
选择和分离培养接种于溴化十六烷基三甲铵琼脂培养基中，30-35℃条件下培养18-72小时
4-4-3. 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 AUREUS 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 2.6.12, 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 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，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）。

当检测透皮贴剂时，取一剂量供试品按照（2.6.12 4-5-1）中的方法使用无菌滤膜过滤后，接种于100ml胰酪大豆胨液体培养基中，30-35℃条件下培养18-24 小时。

4-5-2. Selection and subculture. Subculture on a plate of mannitol salt agar and incubate at 30-35 °C for 18-72 h.
选择和分离培养接种于甘露醇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金黄色葡萄球菌。

4-6. CLOSTRIDIA 梭菌
4-6-1. Sample preparation and heat treatment. Prepare a sample using a 1 in 10 dilution (with a
minimum total volume of 20mL) of not less than 2 g or 2 mL of the product to be examined as described in general chapter 2.6.12. Divide the sample into 2 portions of at least 10 mL. Heat 1 portion at 80 °C for 10 min and cool rapidly. Do not heat the other portion.
供试液制备和热处理按照（2.6.12）中的方法取不少于2g或2ml的供试品制成1:10的供试液（供试液至少为20ml），将样品分成两部分，每部分至少为10ml。

其中一份样品80 ℃加热10分钟后迅速冷却，另一份样品不需要加热。

4-6-2. Selection and subculture. Use 10mL or the quantity corresponding to 1 g or 1 mL of the product to be examined of both portions to inoculate suitable amounts (determined
as described under 3-4) of reinforced medium for clostridia. Incubate under anaerobic conditions at
30-35 °C for 48 h. After incubation, make subcultures from each container on Columbia agar and incubate under anaerobic conditions at 30-35 °C for 48-72 h.
选择和分离培养使用10ml相当于1g或1ml的供试品的供试液接种于适量的培养梭菌用增菌培养基中（符合3-4的要求），在厌氧条件下30-35℃培养48小时。

分离培养接种于哥伦比亚培养基中，在厌氧条件下30-35℃培养48-72小时。

4-6-3. Interpretation. The occurrence of anaerobic growth of rods (with or without endospores) giving a negative catalase reaction indicates the presence of clostridia. 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.
结果分析如果厌氧条件下有杆状菌生长（有或无孢子内壁），过氧化氢酶反应结果为阴性，说明供试品中含有梭菌。

4-7. CANDIDA ALBICANS 白色念珠菌
4-7-1. Sample preparation and pre-incubation. Prepare the product to be examined as described in general chapter 2.6.12, and use 10 mL or the quantity corresponding to not less than 1 g or 1 mL to inoculate 100 mL of Sabouraud-dextrose broth and mix. Incubate at 30-35 °C for 3-5 days.
供试液制备和增菌培养按照（2.6.12）中的方法制备，取10ml或相当于含供试品不少于1g或1ml 的供试液，接种于100ml的沙氏葡萄糖肉汤培养基中，30-35℃条件下培养3-5 天。

4-7-2. Selection and subculture. Subculture on a plate of Sabouraud-dextrose agar and incubate at
30-35 °C for 24-48 h.
选择和分离培养接种于沙氏葡萄糖琼脂培养基中，30-35℃条件下培养24-48小时。

4-7-3. Interpretation. Growth of white colonies may indicate the presence of C. albicans. This is confirmed by identification tests. The product complies with the test if such colonies are not present or if the confirmatory identification tests are negative.
结果分析如果有白色菌落生长，说明供试品中含有白色念珠菌。

是否确实含有白色念珠菌，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白色念珠菌。

The following section is given for information. 以下部分作为信息提供。

5. RECOMMENDED SOLUTIONS AND CULTUREMEDIA 推荐使用的稀释剂和培养基
The following solutions and culture media have been found to be satisfactory for the purposes for which they are prescribed in the test for microbial contamination in the Pharmacopoeia. Other media may be used provided that their suitability can be demonstrated.
以下稀释剂和培养基适用于药典中描述的微生物试验，其他培养基在证明其适用性后也可用于微生物试验。

Stock buffer solution. Place 34 g of potassium dihydrogen phosphate in a 1000 mL volumetric flask, dissolve in 500 mL of purified water, adjust to pH 7.2 ± 0.2 with sodium hydroxide, dilute to 1000.0 mL with purified water and mix. Dispense into containers and sterilise. Store at 2-8 °C.
贮备缓冲溶液：将34g磷酸二氢钾加到1000ml的量瓶中，用500ml的纯化水溶解，用盐酸调节pH 为7.2 ± 0.2，纯化水稀释至1000.0 ml，分装灭菌，2-8 ℃条件下贮存。

Phosphate buffer solution pH 7.2. Prepare a mixture of stock buffer solution and purified water (1:800 V/V) and sterilise.
pH 7.2的磷酸盐缓冲液：上述贮备缓冲溶液与纯化水(1:800 V/V)稀释，灭菌。

Buffered sodium chloride-peptone solution pH 7.0 pH为7.0的氯化钠-蛋白胨缓冲液
Potassium dihydrogen phosphate 磷酸二氢钾3.6 g
Disodium hydrogen phosphate dehydrate 二水磷酸氢二钠7.2 g, （equivalent to 0.067 M phosphate 相当于0.067 M磷酸盐）
Sodium chloride 氯化钠4.3 g
Peptone (meat or casein) 蛋白胨（肉类或酪蛋白）1.0 g
Purified water 纯化水1000 mL
Sterilise in an autoclave using a validated cycle.
高压灭菌器中灭菌，灭菌条件已经过验证。

Casein soya bean digest broth 胰酪大豆胨肉汤培养基
Pancreatic digest of casein 胰消化酪素17.0 g
Papaic digest of soya bean 大豆粉木瓜蛋白酶消化物 3.0 g
Sodium chloride 氯化钠 5.0 g
Dipotassium hydrogen phosphate 磷酸氢二钾 2.5 g
Glucose monohydrate 水合葡萄糖 2.5 g
Purified water 纯化水1000 mL
Adjust the pH so that after sterilisation it is 7.3 ± 0.2 at 25 °C. Sterilise in an autoclave using a
validated cycle.
调节酸度使灭菌后在25 ℃条件下pH为7.3 ±0.2 。

高压灭菌器中灭菌，灭菌条件已经过验证。

Casein soya bean digest agar 胰酪大豆胨琼脂培养基
Pancreatic digest of casein 胰消化酪素15.0 g
Papaic digest of soya bean 大豆粉木瓜蛋白酶消化物 5.0 g
Sodium chloride 氯化钠 5.0 g
Agar 琼脂15.0 g
Purified water 纯化水1000 mL
Adjust the pH so that after sterilisation it is 7.3 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.
调节酸度使灭菌后在25 ℃条件下pH为7.3 ±0.2 。

高压灭菌器中灭菌，灭菌条件已经过验证。

Sabouraud-dextrose agar 沙氏葡萄糖琼脂培养基
Dextrose 葡萄糖40.0 g
Mixture of peptic digest of animal tissue and pancreatic digest of casein (1:1) 胰消化酪素和动物组织胃消化物（1:1）的混合物10.0 g
Agar 琼脂15.0 g
Purified water 纯化水1000 mL
Adjust the pH so that after sterilisation it is 5.6 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.
调节酸度使灭菌后在25 ℃条件下pH为5.6 ±0.2 。

高压灭菌器中灭菌，灭菌条件已经过验证。

Potato dextrose agar 马铃薯葡萄糖琼脂培养基
Infusion from potatoes 马铃薯提取物200 g
Dextrose 葡萄糖20.0 g
Agar 琼脂15.0 g
Purified water 纯化水1000 mL
Adjust the pH so that after sterilisation it is 5.6 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.
调节酸度使灭菌后在25 ℃条件下pH为5.6 ±0.2 。

高压灭菌器中灭菌，灭菌条件已经过验证。

Sabouraud-dextrose broth 沙氏葡萄糖液体培养基
Dextrose 葡萄糖20.0 g
Mixture of peptic digest of animal tissue and pancreatic digest of casein (1:1) 胰消化酪素和动物组织胃消化物（1:1）的混合物10.0 g
Purified water 纯化水1000 mL
Adjust the pH so that after sterilisation it is 5.6 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.
调节酸度使灭菌后在25 ℃条件下pH为5.6 ±0.2 。

高压灭菌器中灭菌，灭菌条件已经过验证。

Enterobacteria enrichment broth-Mossel 肠道菌增菌液体培养基
Pancreatic digest of gelatin 胰消化凝胶10.0 g
Glucose monohydrate 水合葡萄糖 5.0 g
Dehydrated ox bile 脱水牛胆汁20.0 g
Potassium dihydrogen phosphate 脱水牛胆汁 2.0 g
Disodium hydrogen phosphate dihydrate 二水磷酸氢二钠8.0 g
Brilliant green 亮绿试液15 mg
Purified water 纯化水1000 mL
Adjust the pH so that after heating it is 7.2 ± 0.2 at 25 °C. Heat at 100 °C for 30 min and cool immediately.
调节酸度使加热后在25 ℃条件下pH为7.2 ±0.2。

100 ℃加热30分钟后立即冷却。

Violet red bile glucose agar 紫红胆盐葡萄糖琼脂培养基
Yeast extract 酵母浸膏 3.0 g
Pancreatic digest of gelatin 胰消化凝胶7.0 g
Bile salts 胆盐 1.5 g
Sodium chloride 氯化钠 5.0 g
Glucose monohydrate 水合葡萄糖10.0 g
Agar 琼脂15.0 g
Neutral red 中性红30 mg
Crystal violet 结晶紫 2 mg
Purified water 纯化水1000 mL
Adjust the pH so that after heating it is 7.4 ± 0.2 at 25 °C. Heat to boiling; do not heat in an autoclave. 调节酸度使加热后在25 ℃条件下pH为7.4 ±0.2。

加热煮沸，不可在高压灭菌器中灭菌。

MacConkey broth 麦康凯液体培养基
Pancreatic digest of gelatin 胰消化凝胶20.0 g
Lactose monohydrate 水合乳糖10.0 g
Dehydrated ox bile 脱水牛胆汁 5.0 g
Bromocresol purple 溴甲酚紫10 mg
Purified water 纯化水1000 mL
Adjust the pH so that after sterilisation it is 7.3 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.
调节酸度使灭菌后在25 ℃条件下pH为7.3 ±0.2。

高压灭菌器中灭菌，灭菌条件已经过验证。

MacConkey agar 麦康凯琼脂培养基
Pancreatic digest of gelatin 胰消化凝胶17.0 g
Peptones (meat and casein) 蛋白胨（肉类或酪蛋白） 3.0 g
Lactose monohydrate 水合乳糖10.0 g
Sodium chloride 氯化钠 5.0 g
Bile salts 胆盐 1.5 g
Agar 琼脂13.5 g
Neutral red 中性红30.0 mg
Crystal violet 结晶紫 1 mg
Purified water 纯化水1000 mL
Adjust the pH so that after sterilisation it is 7.1 ± 0.2 at 25 °C. Boil for 1 min with constant shaking then sterilise in an autoclave using a validated cycle.
调节酸度使灭菌后在25 ℃条件下pH为7.1 ±0.2。

持续振荡煮沸1分钟后，高压灭菌器中灭菌，灭菌条件已经过验证。

Rappaport Vassiliadis Salmonella enrichment broth 沙门菌液体培养基
Soya peptone 大豆蛋白胨 4.5 g
Magnesium chloride hexahydrate 六水氯化镁29.0 g
Sodium chloride 氯化钠8.0 g
Dipotassium phosphate 磷酸氢二钾0.4 g
Potassium dihydrogen phosphate 磷酸二氢钾0.6 g
Malachite green 孔雀石绿0.036 g
Purified water 纯化水1000 mL
Dissolve, warming gently. Sterilise in an autoclave using a validated cycle, at a temperature not exceeding 115 °C. The pH is to be 5.2 ± 0.2 at 25 °C after heating and autoclaving.
缓慢加热使其溶解，高压灭菌器中灭菌，灭菌条件已经过验证，灭菌温度不得超过115 ℃。

调节酸度使灭菌后在25 ℃条件下pH为5.2 ±0.2。

Xylose, lysine, deoxycholate agar 木糖赖氨酸去氧胆酸盐琼脂培养基
Xylose 木糖 3.5 g
L-Lysine L-赖氨酸 5.0 g
Lactose monohydrate 水合乳糖7.5 g
Sucrose 蔗糖7.5 g
Sodium chloride 氯化钠 5.0 g
Yeast extract 酵母浸膏 3.0 g
Phenol red 酚红80 mg
Agar 琼脂13.5 g
Sodium deoxycholate 去氧胆酸钠 2.5 g
Sodium thiosulfate 硫代硫酸钠 6.8 g
Ferric ammonium citrate 柠檬酸铁铵0.8 g
Purified water 纯化水1000 mL
Adjust the pH so that after heating it is 7.4 ± 0.2 at 25 °C. Heat to boiling, cool to 50 °C and pour into Petri dishes. Do not heat in an autoclave.
调节酸度使加热后在25 ℃条件下pH为7.4 ±0.2 。

加热煮沸后冷却至50 ℃。

倒入皮氏培养皿中。

不可在高压灭菌器中灭菌。

Cetrimide agar 溴化十六烷基三甲铵琼脂培养基
Pancreatic digest of gelatin 胰消化凝胶20.0 g
Magnesium chloride 氯化镁 1.4 g
Dipotassium sulfate 硫酸钾10.0 g
Cetrimide 溴化十六烷基三甲铵0.3 g
Agar 琼脂13.6 g
Purified water 纯化水1000 mL
Glycerol 甘油10.0 mL
Heat to boiling for 1 min with shaking. Adjust the pH so that after sterilisation it is 7.2 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.
持续振荡加热煮沸1分钟，调节酸度使灭菌后在25 ℃条件下pH为7.2 ±0.2。

高压灭菌器中灭菌，灭菌条件已经过验证。

Mannitol salt agar 甘露醇氯化钠琼脂培养基
Pancreatic digest of casein 胰消化凝胶 5.0 g
Peptic digest of animal tissue 动物组织胃消化物 5.0 g
Beef extract 牛肉浸膏 1.0 g
D-Mannitol D-甘露醇10.0 g
Sodium chloride 氯化钠75.0 g
Agar 琼脂15.0 g
Phenol red 酚红0.025 g
Purified water 纯化水1000 mL
Heat to boiling for 1 min with shaking. Adjust the pH so that after sterilisation it is 7.4 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.
持续振荡加热煮沸1分钟，调节酸度使灭菌后在25 ℃条件下pH为7.4 ±0.2。

高压灭菌器中灭菌，灭菌条件已经过验证。

Reinforced medium for clostridia 强化梭菌培养基
Beef extract 牛肉浸膏10.0 g
Peptone 蛋白胨10.0 g
Yeast extract 酵母浸膏 3.0 g
Soluble starch 可溶淀粉 1.0 g
Glucose monohydrate 水合葡萄糖 5.0 g
Cysteine hydrochloride 盐酸半胱氨酸0.5 g
Sodium chloride 氯化钠 5.0 g
Sodium acetate 乙酸钠 3.0 g
Agar 琼脂0.5 g
Purified water 纯化水1000 mL
Hydrate the agar, dissolve by heating to boiling with continuous stirring. If necessary, adjust the pH so that after sterilisation it is 6.8 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.
琼脂水合，不断搅拌加热煮沸使溶解。

如有要求，调节酸度使灭菌后在25 ℃条件下pH为6.8 ±0.2 。

高压灭菌器中灭菌，灭菌条件已经过验证。

Columbia agar 哥伦比亚琼脂培养基
Pancreatic digest of casein 胰消化酪素10.0 g
Meat peptic digest 肉胃酶消化物 5.0 g
Heart pancreatic digest 心胰酶消化物 3.0 g
Yeast extract 酵母浸膏 5.0 g
Maize starch 玉米淀粉 1.0 g
Sodium chloride 氯化钠 5.0 g
Agar, according to gelling power 琼脂，以凝胶粉计算10.0-15.0 g。