MICROBIAL LIMIT TESTS微生物限度测试.doc

MICROBIAL LIMIT TESTS微生物限度测试This chapter provides tests for the estimation of the number of viable aerobic microorganisms present and for freedom from designated microbial species in pharmaceutical articles of all kinds, from raw materials to the finished forms. An automated method may be substituted for the tests presented here, provided it has been properly validated as giving equivalent or better results. In preparing for and in applying the tests, observe aseptic precautions in handling the specimens.本章提供了评估各类药物（从原料到制剂）中需气性微生物存在数量及是否存在某些特定微生物的方法。在已得到验证表明具有同等或更好的结果的情况下，可用一种自动化的方法代替本章阐述的方法。在准备及使用这些方法时，应注意采取无菌措施处理样品。Unless otherwise directed, where the procedure specifies simply incubate ,hold the container in air that is thermostatically controlled at a temperature between 30 and 35 for a period of 24 to 48 hours. The term growth is used in a special sense herein, i.e., to designate the presence and presumed proliferation of viable microorganisms.除非另有说明，在注明直接培养的方法中，将容器置于温度稳定控制在30和35范围内的空气中24小时至48小时。Growth这个术语在这里具有特定的含义，即指有需气性微生物存在且在繁殖。Preparatory Testing准备性试验The validity of the results of the tests set forth in this chapter rests largely upon the adequacy of a demonstration that the test specimens to which they are applied do not, of themselves, inhibit themultiplication, under the test conditions, of microorganisms that may be present. Therefore,preparatory to conducting the tests on a regular basis and as circumstances require subsequently,inoculate diluted specimens of the material to be tested with separate viable cultures of Staphylococcus aureus（金黄色葡萄球菌）, Escherichia coli（大肠杆菌）, Pseudomonas aeruginosa（假单胞铜绿菌）, and Salmonella（沙门氏菌）. This can be done by adding 1 ml of not less than 10-3 dilution of a 24-hour broth culture of the microorganism to the first dilution (in pH 7.2 Phosphate Buffer, Fluid Soybean-Casein Digest Medium（液体胰酶大豆培养基）, or Fluid Lactose Medium（液体乳糖培养基）) of the test material and following the test procedure. Failure of the organism(s) to grow in the relevant medium invalidates that portion of the examination and necessitates a modification of the procedure by (1) an increase in the volume of diluent, the quantity of test material remaining the same, or by (2) the incorporation of a sufficient quantity of suitable inactivating agent(s) in the diluents, or by (3) an appropriate combination of modifications (1) and (2) so as to permit growth of the inocula.本章所提供的测试方法的测试结果的有效性主要依赖于是否能准确的表明在试验条件下被测样品本身不会抑制可能存在的微生物的繁殖。因而，在按照常规的方法进行试验之前，要将分离出的金黄色葡萄球菌、大肠杆菌、假单胞铜绿菌、沙门氏菌的培养物加到该物质稀释后的样品中培养。可以通过按照测试方法，将1ml经过24小时培养的被稀释到低于10-3个的微生物的培养基加到被测样品的第9级稀释液（在磷酸盐缓冲液、液体胰酶大豆培养基或液体乳糖培养基）中。如果在上述培养基中没有微生物生长，则那部分试验无效，就需要对方法进行调整：(1)在被测物量保持不变的情况下，增加稀释液的量，或者(2)在稀释液中加入足够量适宜的灭活剂，或者(3)将(1) 和(2)两种方法适当的结合使得培养物能够生长。The following are examples of ingredients and their concentrations that may be added to the culture medium to neutralize inhibitory substances present in the sample: soy lecithin, 0.5%; and polysorbate 20, 4.0%. Alternatively, repeat the test as described in the preceding paragraph, using Fluid Casein Digest Soy Lecithin polysorbate 20 Medium to demonstrate neutralization of preservatives or other antimicrobial agents in the test material. Where inhibitory substances are contained in the product and the latter is soluble, a suitable, validated adaptation of a procedure set forth in the section Membrane Filtration Method under Test Procedures under Sterility Testsmay be used.下面列出了一些能够加到培养基中能中和存在于样品中的抑制性物质的成分及其浓度：大豆卵磷脂，0.5；聚山梨酯20，4.0。另外，可以用液体胰酶大豆聚山梨酯20培养基按上面所说的重复测试，直到表明被测样品中的防腐剂或其它抗菌剂被中和。 在含有抑制性物质产品可溶的情况下，可采用经过验证的、收载在Membrane Filtration Method under Test Procedures under Sterility Tests中的方法。If in spite of the incorporation of suitable inactivating agents and a substantial increase in the volume of diluent it is still not possible to recover the viable cultures described above and where the article is not suitable for employment of membrane filtration, it can be assumed that the failure to isolate the inoculated organism is attributable to the bactericidal activity of the product. This information serves to indicate that the article is not likely to be contaminated with the given species of microorganism. Monitoring should be continued in order to establish the spectrum of inhibition and bactericidal activity of the article.如果尽管采用了加入适意的灭活剂及大幅度增加稀释剂的量还不能使上述的活性培养物恢复而该物质又不适用合使用薄膜过滤法时，可以假设不能将被培养的微生物分理出来是由于产品的杀菌作用。从这一信息可以得出该物质不可能会受到某种特定的微生物污染。应继续进行监测以建立该物质的抑菌谱合杀菌能力。Buffer Solution and Media缓冲液和培养基Culture media may be prepared as follows, or dehydrated culture media may be used provided that,when reconstituted as directed by the manufacturer or distributor, they have similar ingredients and/or yield media comparable to those obtained from the formulas given herein.可以按下述方法制备培养基，或使用与下面给出的配方成分相同的脱水培养基，假如按照生产者或供应商的指导重新配制所得的培养基可以获得与相同效果的培养基的话。 In preparing media by the formulas set forth herein, dissolve the soluble solids in the water, using heat, if necessary, to effect complete solution, and add solutions of hydrochloric acid or sodium hydroxide in quantities sufficient to yield the desired pH in the medium when it is ready for use. Determine the pH at 25。在按照下面提供的配方制备培养基时，将可溶解的固体溶解在水里，如需要可以加热，以使彻底溶解，加入足够量的盐酸溶液或氢氧化钠溶液使培养基的pH值符合使用时的要求。在25时pH测定pH值。Where agar is called for in a formula, use agar that has a moisture content of not more than 15%.当配方中需使用琼脂时，应使用含水量不大于15的琼脂。Where water is called for in a formula, use Purified Water.当配方中需使用水时，应使用纯化水。pH 7.2 PHOSPHATE BUFFER磷酸盐缓冲液（pH 7.2）Stock Solution Dissolve 34 g of monobasic potassium phosphate in about 500 ml of water contained in a 1000-ml volumetric flask. Adjust to pH 7.2 by the addition of sodium hydroxide TS (about 175 ml), add water to volume, and mix. Dispense and sterilize. Store under refrigeration.For use, dilute the Stock Solution with water in the ratio of 1 to 800, and sterilize.储备液：将34 g磷酸二氢钾置于1000ml容量瓶中用500ml水溶解。加入氢氧化钠试液（约175ml）将pH值调整至7.20.1，用水稀释至刻度，混匀。分装后灭菌。储存于冰箱内。临用时将储备液用水稀释至1800，灭菌。MEDIA培养基Unless otherwise indicated, the media should be sterilized by heating in an autoclave (see Steam Sterilization under Sterilization, the exposure time depending on the volume to be sterilized.除非另有注明，培养基应在高压灭菌器中热压灭菌(见灭菌项下的蒸气灭菌（Steam Sterilization under Sterilization）),灭菌时间取决于被灭菌物的容积。I. Fluid Casein Digest Soy Lecithin Polysorbate 20 Medium液体胰酪大豆卵磷脂聚山梨酯20培养基Pancreatic Digest of Casein 20 g消化蛋白胰酵素分解物 Soy Lecithin 5 g大豆卵磷脂Polysorbate 20 40 ml聚山梨酯20Water 960 ml水Dissolve the pancreatic digest of casein and soy lecithin in 960 ml of water, heating in a water bath at 48to 50for about 30 minutes to effect solution. Add 40 ml of polysorbate 20. Mix, and dispense as desired.将消化蛋白胰酵素分解物和大豆卵磷脂溶解在960 ml 水中，置于48 50的水浴中使完全溶解，加入40 ml聚山梨酯20，混匀，按需要分装。II. Soybean Casein Digest Agar Medium胰酶大豆琼脂培养基Pancreatic Digest of Casein 15.0 g消化蛋白胰酵素分解物Papaic Digest of Soybean Meal 5.0 g大豆酸解物Sodium Chloride 5.0 g氯化钠Agar 15.0 g琼脂Water 1000 ml水pH after sterilization: 7.3 0.2.灭菌后pH值在7.3 0.2。III. Fluid Soybean Casein Digest Medium液体大豆胰酪培养基Prepare as directed for Soybean-Casein Digest Medium under Sterility Tests 按无菌检验项下的大豆胰酪培养基（Soybean-Casein Digest Medium）项下制备。IV. Mannitol Salt Agar Medium甘露醇盐琼脂Pancreatic Digest of Casein 5.0 g消化蛋白胰酵素分解物Peptic Digest of Animal Tissue 5.0 g动物组织酸解物Beef Extract 1.0 g牛肉浸出物D-Mannitol 10.0 gD-甘露醇Sodium Chloride 75.0 g氯化钠Agar 15.0 g琼脂IV. Mannitol Salt Agar Medium甘露醇盐琼脂培养基Phenol Red 0.025 g酚红Water 水 1000 mlMix, then heat with frequent agitation, and boil for 1 minute to effect solution.pH after sterilization: 7.4 0.2.混合然后边加热边不停搅拌，煮沸1分钟使完全溶解。灭菌后pH值应为 7.4 0.2。V. Baird Barker Agar MediumBaird Barker琼脂培养基Pancreatic Digest of Casein 10.0 g消化蛋白胰酵素分解物Beef Extract 5.0 g牛肉浸出物Yeast Extract 1.0 g酵母浸出物Lithium Chloride 5.0 g氯化锂Agar 20.0 g琼脂Glycine 12.0 g甘氨酸Sodium Pyruvate 10.0 g丙酮酸钠Water 950 ml水Heat with frequent agitation, and boil for 1 minute. Sterilize, cool to between 45and 50 and add10 ml of sterile potassium tellurite solution (1 in 100) and 50 ml of egg-yolk emulsion. Mix intimately but gently, and pour into plates. (Prepare the egg-yolk emulsion by disinfecting the surface of whole shell eggs, aseptically cracking the eggs, and separating out intact yolks into a sterile graduated cylinder. Add sterile saline TS to obtain a 3 to 7 ratio of egg yolk to saline. Add to a sterile blender cup, and mix at high speed for 5 seconds.)pH after sterilization: 6.8 0.2.边加热边不停搅拌，煮沸1分钟。灭菌，冷却至4550，加入10 ml无菌亚鍗酸钾溶液（1100）和50 ml卵黄增菌剂。立即轻轻混合，然后倒入培养皿中。（卵黄增菌剂制备：将蛋壳完整的鸡蛋消毒，在无菌条件下将鸡蛋打破，将完整的卵黄分离出来置于一无菌的研钵中，再加入无菌的生理盐水至卵黄与生理盐水呈3：7的比例。置于一无菌混合杯中，高速混合5秒钟）。灭菌后pH值应为 6.8 0.2。VI. Vogel Johnson Agar MediumVogel Johnson琼脂培养基Pancreatic Digest of Casein 10.0 g消化蛋白胰酵素分解物Yeast Extract 5.0 g酵母浸出物Mannitol 10.0 g甘露醇Dibasic Potassium Phosphate 5.0 g 磷酸氢二钾Lithium Chloride 5.0 g氯化锂Glycine 10.0 g甘氨酸Agar 16.0 g琼脂Phenol Red 25.0 mg酚红Water 1000 ml水Boil the solution of solids for 1 minute. Sterilize, cool to between 45and 50and add 20 ml of sterile potassium tellurite solution (1 in 100). pH after sterilization: 7.20.2.将溶液煮沸1分钟。灭菌，冷却至4550，加入20 ml的无菌亚鍗酸钾溶液（1100）。灭菌后pH值应为 7.2 0.2。VII. Cetrimide Agar Medium溴棕三甲铵琼脂培养基Pancreatic Digest of Gelatin 20.0 g消化蛋白胰酵素分解物Magnesium Chloride 1.4 g氯化镁Potassium Sulfate 10.0 g硫酸钾Agar 13.6 g琼脂Cetyl Trimethylammonium Bromide (Cetrimide) 0.3 g溴棕三甲铵Glycerin 10.0 ml甘氨酸Water 1000 ml水Dissolve all solid components in the water, and add the glycerin. Heat, with frequent agitation, and boil for 1 minute to effect solution.pH after sterilization: 7.2 0.2.将所有的固体溶解在水中，再加入甘氨酸。加热并不停搅拌，煮沸1分钟得澄清溶液。灭菌后pH值应为 7.2 0.2。VIII. Pseudomonas Agar Medium for Detection of Fluorescin假单孢菌琼脂培养基（用于检测抑荧光素）Pancreatic Digest of Casein 10.0 g消化蛋白胰酶分解物Peptic Digest of Animal Tissue 10.0 g动物组织酸解物Anhydrous Dibasic Potassium Phosphate 1.5 g无水磷酸氢二钾Magnesium Sulfate (MgSO4：H2O) 1.5 g硫酸镁(MgSO4：H2O)Glycerin 10.0 ml甘氨酸Agar 15.0 g琼脂Water 1000 ml水Dissolve the solid components in the water before adding the glycerin. Heat, with frequent agitation,and boil for 1 minute to effect solution.pH after sterilization: 7.2 0.2.将固体成分在水中溶解，然后加入甘氨酸。加热并不停搅拌，煮沸1分钟即得澄清溶液。灭菌后pH值应为 7.2 0.2。IX. Pseudomonas Agar Medium for Detection of Pyocyanin假单孢菌琼脂培养基（用于检测绿脓菌素）Pancreatic Digest of Gelatin 20.0 g明胶胰酶分解物Anhydrous Magnesium Chloride 1.4 g无水氯化镁Anhydrous Potassium Sulfate 10.0 g无水硫酸钾Agar 15.0 g琼脂IX. Pseudomonas Agar Medium for Detection of Pyocyanin假单孢菌琼脂培养基（用于检测绿脓菌素）Glycerin 10.0 ml甘氨酸Water 1000 ml水Dissolve the solid components in the water before adding the glycerin. Heat, with frequent agitation,and boil for 1 minute to effect solution.pH after sterilization: 7.2 ?0.2.将固体成分在水中溶解，然后加入甘氨酸。加热并不停搅拌，煮沸1分钟即得澄清溶液。灭菌后pH值应为 7.2 0.2。X. Fluid Lactose Medium液体乳糖培养基Beef Extract 3.0 g牛肉浸出物Pancreatic Digest of Gelatin 5.0 g明胶胰酶分解物Lactose 5.0 g乳糖Water 1000 ml水Cool as quickly as possible after sterilization.pH after sterilization: 6.90.2.灭菌后尽快冷却。灭菌后pH值应为6.9 0.2。XI. Fluid Selenite Cystine Medium液体亚硒酸盐胱氨酸培养基Pancreatic Digest of Casein 5.0 g消化蛋白胰酶分解物Lactose 4.0 g乳糖Sodium Phosphate 10.0 g磷酸钠Sodium Acid Selenite 4.0 g亚硒酸钠L-Cystine 10.0 mgL-胱氨酸Water 1000 ml水Final pH: 7.0 0.2.最后pH值为7.0 0.2。Mix, and heat to effect solution. Heat in flowing steam for 15 minutes. Do not sterilize.混合，加热至澄清溶液。再流通蒸气中加热15分钟，不要灭菌。XII. Fluid Tetrathionate Medium液体连四硫酸盐培养基Pancreatic Digest of Casein 2.5 g消化蛋白胰酶分解物Peptic Digest of Animal Tissue 2.5 g动物组织酸解物Bile Salts 1.0 g胆盐Calcium Carbonate 10.0 g碳酸钙Sodium Thiosulfate 30.0 g硫代硫酸钠Water 1000 ml水Heat the solution of solids to boiling. On the day of use, add a solution prepared by dissolving 5 g of potassium iodide and 6 g of iodine in 20 ml of water. Then add 10 ml of a solution of brilliant green (1 in 1000), and mix. Do not heat the medium after adding the brilliant green soluti