药典无菌检测法
2005年版中国药典无菌检查法
2006－07
16
批产品出厂最少检验数量
供试品 注射剂 ≤100 100＜N≤500 ＞500 10%或4个（取较多者） 10个 2%或20个（取较少者）
批产量N（个）
每种培养基最少检验数量
大体积注射剂（>100ml)
眼用及其他非注射产品 ≤200
2%或10个（取较少者）
5%或2个（取较多者） 10个
取规定量，混合，加入适量的聚山梨酯 80或其它适宜的乳化剂及稀释剂使其乳化， 接种至培养基中。或直接接种至含聚山梨 酯80或其它适宜乳化剂的培养基中。
2006－07
24
直接接种法9种供试品的处理和接种 ⑥敷料供试品 取规定数量，以无菌操作拆开包装， 于不同部位分别剪取约100mg或 1cm×3cm的供试品，接种于足以浸没 供试品的适量培养基中。 ⑦肠线、缝合线等供试品 肠线、缝合线及其它一次性使用的医 用材料按规定量取最小包装，无菌拆开 包装，接种于足以浸没供试品的适量培 养基中。
时，培养基用量可在2000ml以上。
薄膜过滤法为100ml/管或50 ml/管．
2006－07
10
无菌检查培养基
培养基的适用性检查
培养基的无菌性检查 培养基的灵敏度检查 符合规定的培养基方可供试品的无菌检 查试验。
2006－07
11
培养基的无菌性检查
每批培养基随机取不少于5支（瓶），培养14天， 应无菌生长。
2006－07 5
洁净度检查
单向流空气区、工作台面及环境应定期按 《医药工业洁净室（区）悬浮粒子、浮游菌和沉降 菌的测试方法》的现行国家标准进行洁净度验证。 隔离系统按相关的要求进行验证，其内部环境的
洁净度须符合无菌检查的要求。
美国药典USP3171无菌检查法双语版
美国药典USP3171⽆菌检查法双语版美国药典USP31-NF26⽆菌检查法《71》.doc71 STERILITY TESTS ⽆菌检查法Portions of this general chapter have been harmonized with the corresponding texts of the European Pharmacopeia and/or the Japanese Pharmacopeia. Those portions that are not harmonized are marked with symbols () to specify this fact.此通则的各部分已经与欧洲药典和/或⽇本药典的对应部分做了协调。
不⼀致的部分⽤符号（）来标明。
The following procedures are applicable for determining whether a Pharmacopeial article purporting to be sterile complies with the requirements set forth in the individual monograph with respect to the test for sterility. Pharmacopeial articles are to be tested by the Membrane Filtration method under Test for Sterility of the Product to be Examined where the nature of the product permits. If the membrane filtration technique is unsuitable, use the Direct Inoculation of the Culture Medium method under Test for Sterility of the Product to be Examined. All devices, with the exception of Devices with Pathways Labeled Sterile, are tested using the Direct Inoculation of the Culture Medium method. Provisions for retesting are included under Observation and Interpretation of Results.下⾯这些步骤适⽤于测定是否某个⽤于⽆菌⽤途的药品是否符合其具体的各论中关于⽆菌检查的要求。
药品无菌检查课件
• 药品无菌检查概述 • 药品无菌检查的方法与流程 • 药品无菌检查的实验操作 • 药品无菌检查的常见问题与解决方案 • 药品无菌检查的案例分析
目录
药品无菌检查概述
定义与目的
定义
药品无菌检查是指对药品中是否 含有微生物的检查，特别是致病 性微生物的检查。
目的
确保药品在生产和处理过程中没 有受到微生物污染，保障药品的 安全性和有效性。
常见问题三：实验操作失误问题
药品无菌检查的案例分析
案例一：某注射液的无菌检查
总结词
操作规范、结果准确
详细描述
某注射液的无菌检查过程严格遵循中国药典规定，采用薄膜过滤法进行检验。 通过控制实验条件，确保了检验结果的准确性和可靠性。
案例二：某胶囊剂的无菌检查
总结词
高效检测、无菌保证
详细描述
某胶囊剂的无菌检查采用了直接接种法，该方法具有较高的检测效率和无菌保证。 在操作过程中，严格控制实验条件，确保了结果的准确性和可靠性。
药品无菌检查的重要性
保障公众健康
无菌药品用于治疗疾病和预防感 染，如果药品中含有微生物，将
给患者带来健康风险。
维护药品质量
微生物污染可能导致药品变质、药 效降低或产生不良反应，因此无菌 检查是保证药品质量的重要环节。
符合法规要求
各国药品监管机构要求对药品进行 无菌检查，确保药品符合相关法规 和标准。
案例三：某原料药的无菌检查
总结词
全面检测、排除污染
详细描述
某原料药的无菌检查采用了多种方法进行全面检测，包括直接接种法和薄膜过滤法等。在操作过程中， 严格控制实验条件，确保了结果的准确性和可靠性，有效排除了污染的可能性。
THANKS
无菌检查法 版中国药典
无菌检查法-2015版中国药典无菌检查法系用于检查药典要求无菌的药品、生物制品、医疗器具、原料、辅料、及其他品种是否无菌的一种方法。
若供试品符合无菌检查法的规定,仅表明了供试品在该检验条件下未发现微生物污染。
无菌检查应在环境洁净度B 级背景下的局部A 级洁净度的单向流空气区域内或隔离系统中进行，其全过程应严格遵守无菌操作，防止微生物污染，防止污染的措施不得影响供试品中微生物的检出。
单向流空气区、工作台面及环境应定期按《医药工业洁净室（区）悬浮粒子、浮游菌和沉降菌的测试方法》的现行国家标准进行洁净度确认。
隔离系统应定期按相关的要求进行验证，其内部环境的洁净度须符合无菌检查的要求。
日常检验还需对试验环境进行监控。
无菌检查人员必须具备微生物专业知识，并经过无菌技术的培训。
培养基硫乙醇酸盐流体培养基主要用于厌氧菌的培养，也可用于需气菌培养；胰酪大豆胨液体培养基适用于真菌和需气菌的培养。
培养基的制备及培养条件培养基可按以下处方制备，亦可使用按该处方生产的符合规定的脱水培养基。
配制后应采用验证合格的灭菌程序灭菌。
制备好的培养基应保存在2～25℃、避光的环境，若保存于非密闭容器中，一般在3周内使用；若保存于密闭容器中，一般可在一年内使用。
1. 硫乙醇酸盐流体培养基酪胨(胰酶水解) 15.0g 酵母浸出粉 5.0g葡萄糖 5.0g 氯化钠 2.5gL-胱氨酸0.5g 新配制的0.1% 刃天青溶液1.0ml硫乙醇酸钠0.5g 琼脂0.75g(或硫乙醇酸) （0.3 ml) 纯化水1000ml除葡萄糖和刃天青溶液外，取上述成分混合，微温溶解，调节pH 为弱碱性，煮沸，滤清，加入葡萄糖和刃天青溶液，摇匀，调节pH 值使灭菌后为7.1±0.2。
分装至适宜的容器中，其装量与容器高度的比例应符合培养结束后培2养基氧化层（粉红色）不超过培养基深度的1/2。
灭菌。
在供试品接种前，培养基氧化层的高度不得超过培养基深度的1/5，否则，须经100℃水浴加热至粉红色消失（不超过20 分钟），迅速冷却，只限加热一次，并防止被污染。
无菌检查(薄膜过滤法)SOP
无菌检查SOP（薄膜过滤法）1.目的为规范无菌检查方法及无菌检查全过程的操作，最大限度防止试验操作过程造成的污染，确保结果准确。
2.范围本SOP适用于生物制品的中间产品（包括原液、纯化产物、半成品、生产中的液体、辅料等）、成品的无菌检查。
只要供试品性状允许，应采用薄膜过滤法。
3.定义无菌检查法系用于检查药典要求无菌的生物制品、医疗器具、原料、辅料及其他品种是否无菌的一种方法。
若供试品符合无菌检查法的规定，仅表明了供试品在该检验条件下未发现微生物污染。
4.职责4.1.QC负责本规程的起草、修订、培训及执行；4.2.QA、QC组长、质量管理部经理负责本规程的审核；4.3.质量总监负责批准本规程；4.4.QA负责本规程执行的监督。
5.引用标准《中华人民共和国药典》2020年版三部6.材料6.1.无菌检查用物品、物料要求：所有物品、物料进入无菌室前，均应经过高压蒸汽灭菌（121℃40分钟，放灭菌指示卡）或180℃干烤2小时或其它经过验证的方法消毒后传入无菌室；如不能用前者消毒方式消毒的物品，须经消毒液浸泡、擦拭后传入无菌室。
6.2.无菌检查环境要求：应在环境洁净度万级下的局部洁净度百级的单向流空气区域内或隔离系统中进行，其全过程应严格遵守无菌操作，防止微生物污染，防止污染的措施不得影响供试品中微生物的检出。
6.3.仪器、设备与设施集菌仪、显微镜、台式灭菌器、无菌试验室（万级）、超净工作台（万级背景下的局部百级）：每月环境检测（沉降菌、浮游菌、尘埃粒子）合格后，方可使用。
20-25℃培养房或培养箱、30-35℃培养房或培养箱。
6.4.试验用具：洁净工作服（洁净底服、十万级工作服、万级工作服、鞋套）、工作鞋、口罩及帽子；无菌医用手套或一次性乳胶医用手套：试验前及试验过程中用消毒液浸泡消毒；封闭式集菌培养器（双针头、三联及单针头、三联）：薄膜孔径不大于0.45μm，直径约为 50mm。
根据供试品及其溶剂的特性选择滤膜材质。
中国药品检验标准操作规范2010年版104无菌检查法
无菌检查法无菌检查法系用于检查药典要求无菌的药品、医疗器具、原料、辅料及其他品种是否无菌的一种方法。
若供试品符合无菌检查法的规定，仅表明了供试品在该检验条件下未发现微生物污染。
无菌检查应在环境洁净度10000级下的局部洁净度100级的单向流空气区域内或隔离系统中进行，其过程必须严格遵守无菌操作，防止微生物污染，防止污染的措施不得影响供试品中微生物的检出。
单向流空气区、工作台面及环境应定期按《医药工业洁净室（区）悬浮粒子、浮游菌和沉降菌的测试方法》的现行国家标准进行洁净度验证。
隔离系统按相关的要求进行验证，其内部环境的洁净度须符合无菌检查的要求。
日常验证还需对试验环境进行监控。
菌检查人员必须具备微生物专业知识，并经过无菌技术的培训。
第一部分准备及保障1 洁净室（区）的要求无菌检查的所有操作均需在严格控制微生物污染的环境下进行，操作环境的无菌保障程度将直接影响无菌检查结果，为了保证无菌检查用洁净室（区）环境的稳定性，确保检查结果的可靠性，对洁净室（区）的环境质量采取合理的控制措施和评价方法是必要的。
无菌加查应在环境洁净度10000级下的局部洁净度100级的单向流空气区域内或隔离系统中进行，其过程必须严格遵守无菌操作，防止微生物污染。
单向流空气区、工作台面及环境应定期按《医药工业洁净室（区）悬浮粒子、浮游菌和沉降菌的测试方法》的现行国家标准进行洁净度验证。
隔离系统按相关的要求进行验证，其内部环境的洁净度须符合无菌检查的要求。
洁净室（区）应采光良好、避免潮湿。
远离厕所及污染区。
由1~2个缓冲间和操作间组成（操作间和缓冲间的门不应直对），操作间与缓冲间之间应具备灭菌功能的样品传递箱。
在缓冲间内应有拖鞋、无菌衣放置架和挂钩等，缓冲间可设手消毒设备，不应放置培养箱和其他杂物；洁净室（区）内应六面光滑平整，能耐受清洗消毒。
墙壁与地面、天花板连接处应呈凹弧形，无缝隙，不留死角。
操作间和缓冲间内不应安装下水道。
无菌操作室应具有空气除菌过滤的单向流空气装置，操作区洁净度100级或放置同等级别的超净工作台，室内温度控制18~26℃，相对湿度40%~60%。
2020版药典《1101无菌检查法》学习笔记
2020版药典《1101无菌检查法》学习笔记1101无菌检查法无菌检查法系……未发现微生物污染。
无菌检查应在无菌条件下进行，试验环境必须达到无菌检查的要求，检验全过程应严格逄守无菌操作，防止微生物污染，防止污染的措施不得影响供试品中微生物的检出。
单向流空气区域、工作台面及受控环境应定期按医药工业洁净室（区）悬浮粒子、浮游菌和沉降菌的测试方法的现行国家标准进行洁净度确认。
隔离系统应定期按相关的要求进行验证，其内部环境的洁净度须符合无菌检查的要求。
日常检验需对试验环境进行监测。
学习：“应在无菌条件下进行”，这句话此版药典仍是如此表述，没有实力改造的情况下，C+A级送风环境应该还能再次维持一个药典周期吧。
“受控”两个字为新增，对于检验洁净环境，受控主要的关注指标应该是悬浮粒子、浮游菌和沉降菌，当然其它如压差、温湿度等的指标和监测频度也应满足9205《药品洁净实验室微生物监测和控制指导原则》的要求。
培养基……（未变化，略）。
培养基的制备及培养条件培养基可按以下处方制备，亦可使用按该处方生产的符合规定的脱水培养基或商品化的预制培养基。
配制后应采用验证合格的灭菌程序灭菌。
制备好的培养基若不即时使用，应置于无菌密闭容器中，在2~25°C 、避光的环境下保存，并在经验证的保存期内使用。
学习：由原来的的“成品培养基”调整为“商品化的预制培养基”，更易于理解。
应注意最后标红的调整内容。
不再强调“非密闭-三周”、“密闭-一年”的规定，便于实践掌握并防止浪费，更适合国情；当然，你最好有一个稳健的验证过程和结果，以支持你的封闭条件和保存周期。
1. 硫乙酵酸盐流体培养基……葡萄糖／无水葡萄糖5.5g/5.0g 刃天青溶液1. 0ml…………分装至适宜的容器中，其装量与容器高度的比例应符合培养结束后培养基氧化层（粉红色）不超过培养基深度的1/2 。
灭菌。
在供试品接种前，培养基氧化层的高度不得超过培养基深度的1/3 ,。
美国药典无菌检查法
detecting Mycoplasmas. Test the effectiveness of the cells to be used by applying the procedure shown below and inocu-lating not more than 100 cfu or ccu microorganisms of suit-able reference strains of M. hyorhinis and M. orale. The cells are suitable if both reference strains are detected. The indi-cator cells must be subcultured without an antibiotic before use in the test.Test MethodNOTE—The following is provided for information.SOLUTIONSPhosphate Buffered Saline—2.0 M Monobasic Potassium Phosphate—Dissolve 13.61 g of anhydrous monobasic potassium phosphate in 50 mL of water.2.0 M Dibasic Potassium Phosphate—Dissolve 17.42 g of anhydrous dibasic potassium phosphate in 50 mL of water.Phosphate Buffered Saline Solution (pH 7.4)—Combine 3.6 mL of 2.0 M Monobasic Potassium Phosphate, 16.4 mL of 2.0 M Dibasic Potassium Phosphate, 8g of sodium chloride, and 1 L of water. Mix thoroughly. Adjust the pH if necessary.Bisbenzimide Stock Solution—Dissolve 5 mg of bisben-zimide in water, and dilute with the same solvent to 100 mL. Store in the dark.Bisbenzimide Working Solution—Immediately before use, dilute 100 m L of Bisbenzimide Stock Solution with Phos-phate Buffered Saline Solution (pH 7.4) to 100 mL.Phosphate-Citrate Buffer Solution pH 5.5—Mix 56.85 mL of a 28.4-g/L solution of anhydrous disodium hydrogen phosphate and 43.15 mL of a 21-g/L solution of citric acid.M ETHOD1.Seed the indicator cell culture at a suitable density (forexample, 2×104 to 2×105 cells/mL, 4×103 to 2.5 ×104 cells/cm2) that will yield confluence after3 days of growth. Inoculate 1 mL of the product to beexamined into the cell culture vessel, and incubate at36±1°.2.After at least 3 days of incubation, when the cells havegrown to confluence, make a subculture on cover slips in suitable containers or on some other surface (for ex-ample, chambered slides) suitable for the test proce-dure. Seed the cells at low density so that they reach50% confluence after 3–5 days of incubation. Com-plete confluence impairs visualization of Mycoplasmasafter staining and must be avoided.3.Remove the medium and rinse the indicator cells withphosphate buffered saline, pH 7.4, then add a suitablefixing solution (a freshly prepared mixture of 1 volume of acetic acid, glacial, TS and 3 volumes of methanol, is suitable when bisbenzimide is used for staining).4.Remove the fixing solution and wash the cells with ster-ile Purified Water. Dry the slides completely if they areto be stained more than 1 hour later (particular care isneeded for staining of slides after drying owing to arti-facts that may be produced).5.Add a suitable DNA stain and allow standing for a suit-able time (bisbenzimide working solution and a stand-ing time of 10 minutes are suitable).6.Remove the stain and rinse the monolayer with PurifiedWater.7.Mount each coverslip, where applicable (a mixture ofequal volumes of glycerol and Phosphate-Citrate BufferSolution pH 5.5 is suitable for mounting). Examine byfluorescence (for bisbenzimide stain a 330 nm/380 nm excitation filter and an LP 440 nm barrier filter are suit-able) at 400× magnification or greater.pare the microscopic appearance of the test cul-tures with that of the negative and positive controls,examining for extranuclear fluorescence. Mycoplasmas produce pinpoints or filaments over the indicator cellcytoplasm. They may also produce pinpoints and fila-ments in the intercellular spaces. Multiple microscopicfields are examined according to the protocol establish-ed during validation.Interpretation of ResultsThe product to be examined complies with the test if flu-orescence typical of Mycoplasmas is not present. The test is invalid if the positive controls do not show fluorescence typ-ical of Mycoplasmas. The test is invalid if the negative con-trols show fluorescence typical of Mycoplasmas.á71ñ STERILITY TESTSu Portions of this general chapter have been harmonized with the corresponding texts of the European Pharmacopeia and/or the Japanese Pharmacopeia. Those portions that are not harmonized are marked with symbols (uu) to specify this fact.uThese Pharmacopeial procedures are not by themselves designed to ensure that a batch of product is sterile or has been sterilized. This is accomplished primarily by validation of the sterilization process or of the aseptic processing pro-cedures.The test is applied to substances, preparations, or articles which, according to the Pharmacopeia, are required to be ster-ile. However, a satisfactory result only indicates that no con-taminating microorganism has been found in the sample ex-amined under the conditions of the test.PRECAUTIONS AGAINST MICROBIALCONTAMINATIONThe test for sterility is carried out under aseptic condi-tions. In order to achieve such conditions, the test environ-ment has to be adapted to the way in which the sterility test is performed. The precautions taken to avoid contamination are such that they do not affect any microorganisms that are to be revealed in the test. The working conditions in which the tests are performed are monitored regularly byUSP 37Microbiological Tests / á71ñ Sterility Tests 71appropriate sampling of the working area and by carrying out appropriate controls.CULTURE MEDIA AND INCUBATIONTEMPERATURESMedia for the test may be prepared as described below or equivalent commercial media may be used provided that they comply with the requirements of the Growth Promotion Test of Aerobes, Anaerobes, and Fungi.The following culture media have been found to be suita-ble for the test for sterility. Fluid Thioglycollate Medium is pri-marily intended for the culture of anaerobic bacteria. How-ever, it will also detect aerobic bacteria. Soybean–Casein Di-gest Medium is suitable for the culture of both fungi and aerobic bacteria.Fluid Thioglycollate MediumL-Cystine0.5 g Sodium Chloride 2.5 g Dextrose Monohydrate/Anhydrous 5.5/5.0 gAgar0.75 g Yeast Extract (water-soluble) 5.0 g Pancreatic Digest of Casein15.0 g Sodium Thioglycollate0.5 gor Thioglycolic Acid0.3 mL Resazurin Sodium Solution (1 in 1000),freshly prepared 1.0 mL Purified Water1000 mLpH after sterilization: 7.1±0.2.Mix the L-cystine, agar, sodium chloride, dextrose, yeast extract, and pancreatic digest of casein with the purified wa-ter, and heat until solution is effected. Dissolve the sodium thioglycollate or thioglycolic acid in the solution and, if nec-essary, add 1N sodium hydroxide so that, after sterilization, the solution will have a pH of 7.1 ± 0.2. If filtration is neces-sary, heat the solution again without boiling, and filter while hot through moistened filter paper. Add the resazurin so-dium solution, mix, and place the medium in suitable ves-sels that provide a ratio of surface to depth of medium such that not more than the upper half of the medium has un-dergone a color change indicative of oxygen uptake at the end of the incubation period. Sterilize using a validated process. If the medium is stored, store at a temperature be-tween 2° and 25° in a sterile, airtight container. If more than the upper one-third of the medium has acquired a pink col-or, the medium may be restored once by heating the con-tainers in a water-bath or in free-flowing steam until the pink color disappears and by cooling quickly, taking care to prevent the introduction of nonsterile air into the container. Do not use the medium for a longer storage period than has been validated.Fluid Thioglycollate Medium is to be incubated at 30°–35°. For products containing a mercurial preservative that cannot be tested by the membrane filtration method, Fluid Thiogly-collate Medium incubated at 20°–25° may be used instead of Soybean–Casein Digest Medium provided that it has been va-lidated as described in Growth Promotion Test of Aerobes, Anaerobes, and Fungi. Where prescribed or justified and au-thorized, the following alternative thioglycollate medium might be used. Prepare a mixture having the same composi-tion as that of the Fluid Thioglycollate Medium, but omitting the agar and the resazurin sodium solution. Sterilize as di-rected above. The pH after sterilization is 7.1 ± 0.2. Heat in a water bath prior to use and incubate at 30°–35° under anaerobic conditions.Soybean–Casein Digest MediumPancreatic Digest of Casein17.0 g Papaic Digest of Soybean Meal 3.0 g Sodium Chloride 5.0 g Dibasic Potassium Phosphate 2.5 g Dextrose Monohydrate/Anhydrous 2.5/2.3 g Purified Water1000 mLpH after sterilization: 7.3±0.2.Dissolve the solids in the Purified Water, heating slightly to effect a solution. Cool the solution to room temperature, and adjust the pH with 1N sodium hydroxide so that, after sterilization, it will have a pH of 7.3 ± 0.2. Filter, if necessary to clarify, dispense into suitable containers, and sterilize us-ing a validated procedure. Store at a temperature between 2° and 25° in a sterile well-closed container, unless it is in-tended for immediate use. Do not use the medium for a longer storage period than has been validated.Soybean–Casein Digest Medium is to be incubated at 22.5± 2.5°.u Media for Penicillins or CephalosporinsWhere sterility test media are to be used in the Direct Inoc-ulation of the Culture Medium method under Test for Sterility of the Product to be Examined, modify the preparation of Flu-id Thioglycollate Medium and the Soybean–Casein Digest Me-dium as follows. To the containers of each medium, transfer aseptically a quantity of b-lactamase sufficient to inactivate the amount of antibiotic in the specimen under test. Deter-mine the quantity of b-lactamase required to inactivate theantibiotic by using a b-lactamase preparation that has been assayed previously for its penicillin- or cephalosporin-inacti-vating power. [N OTE—Supplemented b-lactamase media can also be used in the membrane filtration test.] Alternatively (in an area completely separate from that used for sterility testing), confirm that an appropriate amount of b-lactamase is incorporated into the medium, fol-lowing either method under Method Suitability Test, using less than 100 colony-forming units (cfu) of Staphylococcus aureus (see Table 1) as the challenge. Typical microbial growth of the inoculated culture must be observed as a con-firmation that the b-lactamase concentration is appropri-ate.u72á71ñ Sterility Tests / Microbiological Tests USP 37Table 1. Strains of the Test Microorganisms Suitable for Use in the Growth Promotion Test and the Method Suitability Test Aerobic bacteriaStaphylococcus aureus ATCC 6538, CIP 4.83,NCTC 10788, NCIMB9518, NBRC 13276 Bacillus subtilis ATCC 6633, CIP 52.62,NCIMB 8054, NBRC3134Pseudomonas aeruginosa u1u ATCC 9027, NCIMB 8626, CIP 82.118, NBRC 13275Anaerobic bacteriumClostridium sporogenes u2u ATCC 19404, CIP 79.3, NCTC 532 or ATCC 11437, NBRC 14293FungiCandida albicans ATCC 10231, IP 48.72,NCPF 3179, NBRC 1594Aspergillus brasiliensis (Aspergillus Niger)ATCC 16404, IP 1431.83, IMI 149007, NBRC 9455u1An alternative microorganism is Kocuria rhizophila (Micrococcus luteus) ATCC 9341.uu2An alternative to Clostridium sporogenes, when a nonspore-forming mi-croorganism is desired, is Bacteroides vulgatus (ATCC 8482).uThe media used comply with the following tests, carried out before, or in parallel, with the test on the product to be examined.SterilityIncubate portions of the media for 14 days. No growth of microorganisms occurs.Growth Promotion Test of Aerobes,Anaerobes, and FungiTest each lot of ready-prepared medium and each batch of medium prepared either from dehydrated medium or from ingredients. Suitable strains of microorganisms are in-dicated in Table 1.Inoculate portions of Fluid Thioglycollate Medium with a small number (not more than 100 cfu) of the following mi-croorganisms, using a separate portion of medium for each of the following species of microorganism: Clostridium sporo-genes, Pseudomonas aeruginosa, and Staphylococcus aur-eus. u Inoculate portions of alternative thioglycollate medium with a small number (not more than 100 cfu) of Clostridiumsporogenes.u Inoculate portions of Soybean–Casein DigestMedium with a small number (not more than 100 cfu) of the following microorganisms, using a separate portion of medi-um for each of the following species of microorganism: As-pergillus brasiliensis, Bacillus subtilis, and Candida albicans. In-cubate for not more than 3 days in the case of bacteria and not more than 5 days in the case of fungi. Seed lot culture maintenance techniques (seed-lot systems) are used so that the viable microorganisms used for inoculation are not more than five passages removed from the original master seed-lot.The media are suitable if a clearly visible growth of the microorganisms occurs.u DILUTING AND RINSING FLUIDS FORMEMBRANE FILTRATIONFluid APREPARATIONDissolve 1g of peptic digest of animal tissue in water to make 1 L, filter or centrifuge to clarify, if necessary, and ad-just to a pH of 7.1 ± 0.2. Dispense into containers, and steri-lize using a validated process.PREPARATION FOR PENICILLINS OR CEPHALOSPORINSAseptically add to the above Preparation, if necessary, a quantity of sterile b-lactamase sufficient to inactivate any re-sidual antibiotic activity on the membranes after the solu-tion of the test specimen has been filtered (see Media for Penicillins or Cephalosporins).Fluid DTo each L of Fluid A add 1 mL of polysorbate 80, adjust to a pH of 7.1 ± 0.2, dispense into containers, and sterilize us-ing a validated process. Use this fluid for articles containing lecithin or oil, or for devices labeled as “sterile pathway.”Fluid KDissolve 5.0 g of peptic digest of animal tissue, 3.0 g of beef extract, and 10.0 g of polysorbate 80 in water to make 1 L. Adjust the pH to obtain, after sterilization, a pH of 6.9 ±0.2. Dispense into containers, and sterilize using a validated process.uMETHOD SUITABILITY TESTCarry out a test as described below under Test for Sterility of the Product to be Examined using exactly the same meth-ods, except for the following modifications.Membrane FiltrationAfter transferring the content of the container or contain-ers to be tested to the membrane, add an inoculum of a small number of viable microorganisms (not more than 100 cfu) to the final portion of sterile diluent used to rinse the filter.Direct InoculationAfter transferring the contents of the container or contain-ers to be tested (for catgut and other surgical sutures for veterinary use: strands) to the culture medium, add an inoc-ulum of a small number of viable microorganisms (not more than 100 cfu) to the medium.In both cases use the same microorganisms as those de-scribed above under Growth Promotion Test of Aerobes, Anae-robes, and Fungi. Perform a growth promotion test as a posi-USP 37Microbiological Tests / á71ñ Sterility Tests 73tive control. Incubate all the containers containing medium for not more than 5 days.If clearly visible growth of microorganisms is obtained af-ter the incubation, visually comparable to that in the control vessel without product, either the product possesses no an-timicrobial activity under the conditions of the test or such activity has been satisfactorily eliminated. The test for sterili-ty may then be carried out without further modification.If clearly visible growth is not obtained in the presence of the product to be tested, visually comparable to that in the control vessels without product, the product possesses anti-microbial activity that has not been satisfactorily eliminated under the conditions of the test. Modify the conditions in order to eliminate the antimicrobial activity, and repeat the Method Suitability Test.This method suitability is performed (a) when the test for sterility has to be carried out on a new product; and (b) whenever there is a change in the experimental conditions of the test. The method suitability may be performed simul-taneously with the Test for Sterility of the Product to be Exam-ined.TEST FOR STERILITY OF THE PRODUCT TOBE EXAMINEDu Number of Articles to Be Tested Unless otherwise specified elsewhere in this chapter or in the individual monograph, test the number of articles speci-fied in Table 3. If the contents of each article are of sufficient quantity (see Table 2), they may be divided so that equal appropriate portions are added to each of the specified me-dia. [N OTE—Perform sterility testing employing two or more of the specified media.] If each article does not contain suffi-cient quantities for each medium, use twice the number of articles indicated in Table 3.uTable 2. Minimum Quantity to be Used for Each MediumQuantity per Container Minimum Quantity to be Used (unless otherwise justified andauthorized)LiquidsLess than 1 mL The whole contents of each container 1–40 mL Half the contents of each container,but not less than 1 mLGreater than 40 mL, and notgreater than 100 mL20 mLGreater than 100 mL10% of the contents of the container,but not less than 20 mLAntibiotic liquids 1 mLInsoluble preparations, creams, and ointments to be suspen-ded or emulsified Use the contents of each container to provide not less than 200 mgSolidsLess than 50 mg The whole contents of each container50 mg or more, but less than 300 mg Half the contents of each container, but not less than 50 mg300 mg–5 g150 mg Greater than 5 g500 mgCatgut and other surgical su-tures for veterinary use 3 sections of a strand (each 30-cmlong)Table 2. Minimum Quantity to be Used for Each Medium(Continued)Quantity per ContainerMinimum Quantity to be Used(unless otherwise justified andauthorized)u Surgical dressing/cotton/gauze (in packages)100 mg per packageSutures and other individuallypackaged single-use materialThe whole deviceOther medical devices The whole device, cut into pieces ordisassembleduTable 3. Minimum Number of Articles to be Tested in Relationto the Number of Articles in the BatchNumber of Items in theBatch*Minimum Number of Itemsto be Tested for EachMedium (unless otherwisejustified and authorized)**Parenteral preparationsNot more than 100 containers10% or 4 containers, whichever isthe greaterMore than 100 but not more than500 containers10 containersMore than 500 containers2% or 20 containers, whichever islessu For large-volume parenterals2% or 10 containers, whichever islessAntibiotic solidsPharmacy bulk packages (<5 g)20 containersPharmacy bulk packages (³5 g) 6 containersBulks and blends See Bulk solid productsuOphthalmic and other noninjectable preparationsNot more than 200 containers5% or 2 containers, whichever isthe greaterMore than 200 containers10 containersIf the product is presented in theform of single-dose containers,apply the scheme shown abovefor preparations for parenteraluse.Catgut and other surgical suturesfor veterinary use2% or 5 packages, whichever isthe greater,up to a maximum total of 20packagesu Not more than 100 articles10% or 4 articles, whichever isgreaterMore than 100, but not morethan 500 articles10 articlesMore than 500 articles2% or 20 articles, whichever islessuBulk solid productsUp to 4 containers Each containerMore than 4 containers, but notmore than 50 containers20% or 4 containers, whichever isgreaterMore than 50 containers2% or 10 containers, whichever isgreater* If the batch size is unknown, use the maximum number of items prescri-bed.** If the contents of one container are enough to inoculate the two media,this column gives the number of containers needed for both the media to-gether.74á71ñ Sterility Tests / Microbiological Tests USP 37The test may be carried out using the technique of Mem-brane Filtration or by Direct Inoculation of the Culture Medium with the product to be examined. Appropriate negative controls are included. The technique of membrane filtration is used whenever the nature of the product permits; that is, for filterable aqueous preparations, for alcoholic or oily preparations, and for preparations miscible with, or soluble in, aqueous or oily solvents, provided these solvents do not have an antimicrobial effect in the conditions of the test.Membrane FiltrationUse membrane filters having a nominal pore size not greater than 0.45 m m, in which the effectiveness to retain microorganisms has been established. Cellulose nitrate fil-ters, for example, are used for aqueous, oily, and weakly al-coholic solutions; and cellulose acetate filters, for example, are used for strongly alcoholic solutions. Specially adapted filters may be needed for certain products (e.g., for antibiot-ics).The technique described below assumes that membranes about 50 mm in diameter will be used. If filters of a different diameter are used, the volumes of the dilutions and the washings should be adjusted accordingly. The filtration ap-paratus and membrane are sterilized by appropriate means. The apparatus is designed so that the solution to be exam-ined can be introduced and filtered under aseptic condi-tions: it permits the aseptic removal of the membrane for transfer to the medium, or it is suitable for carrying out the incubation after adding the medium to the apparatus itself.AQUEOUS SOLUTIONSIf appropriate, transfer a small quantity of a suitable, ster-ile diluent such as u Fluid A (see Diluting and Rinsing Fluids forMembrane Filtration)u onto the membrane in the apparatusand filter. The diluent may contain suitable neutralizing sub-stances and/or appropriate inactivating substances, for ex-ample, in the case of antibiotics.Transfer the contents of the container or containers to be tested to the membrane or membranes, if necessary, after diluting to the volume used in the Method Suitability Test with the chosen sterile diluent, but using not less than the quantities of the product to be examined prescribed in Ta-bles 2 and 3. Filter immediately. If the product has antimi-crobial properties, wash the membrane not less than three times by filtering through it each time the volume of the chosen sterile diluent used in the Method Suitability Test. Do not exceed a washing cycle of five times 100 mL per filter, even if during method suitability it has been demonstrated that such a cycle does not fully eliminate the antimicrobial activity. Transfer the whole membrane to the culture medi-um or cut it aseptically into two equal parts, and transfer one half to each of two suitable media. Use the same vol-ume of each medium as in the Method Suitability Test. Alter-natively, transfer the medium onto the membrane in the ap-paratus. Incubate the media for not less than 14 days.SOLUBLE SOLIDSUse for each medium not less than the quantity prescri-bed in Tables 2 and 3 of the product dissolved in a suitable solvent, such as the solvent provided with the preparation, Sterile Water for Injection, sterile saline, or a suitable sterile solution such as u Fluid A (Diluting and Rinsing Fluids for Mem-brane Filtration),uand proceed with the test as described above for Aqueous Solutions using a membrane appropriate to the chosen solvent.OILS and OILY SOLUTIONSUse for each medium not less than the quantity of the product prescribed in Tables 2 and 3. Oils and oily solutions of sufficiently low viscosity may be filtered without dilution through a dry membrane. Viscous oils may be diluted as necessary with a suitable sterile diluent such as isopropyl myristate shown not to have antimicrobial activity in the conditions of the test. Allow the oil to penetrate the mem-brane by its own weight, and then filter, applying the pres-sure or suction gradually. Wash the membrane at least three times by filtering through it each time about 100 mL of a suitable sterile solution such as u Fluid A (see Diluting and Rinsing Fluids for Membrane Filtration)ucontaining a suitable emulsifying agent at a concentration shown to be appropri-ate in the Method Suitability Test, for example polysorbate 80 at a concentration of 10g per L u(Fluid K)u. Transfer the membrane or membranes to the culture medium or media, or vice versa, as described above for Aqueous Solutions, and incubate at the same temperatures and for the same times.OINTMENTS and CREAMSUse for each medium not less than the quantities of the product prescribed in Tables 2 and 3. Ointments in a fatty base and emulsions of the water-in-oil type may be diluted to 1% in isopropyl myristate as described above, by heating, if necessary, to not more than 40°. In exceptional cases it may be necessary to heat to not more than 44°. Filter as rapidly as possible, and proceed as described above for Oils and Oily Solutions.u PREFILLED SYRINGESFor prefilled syringes without attached sterile needles, ex-pel the contents of each syringe into one or two separate membrane filter funnels or into separate pooling vessels pri-or to transfer. If a separate sterile needle is attached, directly expel the syringe contents as indicated above, and proceed as directed for Aqueous Solutions. Test the sterility of the needle, using Direct Inoculation under Method Suitability Test.SOLIDS FOR INJECTION OTHER THAN ANTIBIOTICSConstitute the test articles as directed on the label, and proceed as directed for Aqueous Solutions or Oils and Oily Solutions, whichever applies. [N OTE—If necessary, excess di-USP 37Microbiological Tests / á71ñ Sterility Tests 75luent can be added to aid in the constitution and filtration of the constituted test article.]ANTIBIOTIC SOLIDS FOR INJECTIONPharmacy Bulk Packages, <5g—From each of 20 con-tainers, aseptically transfer about 300 mg of solids, into a sterile 500-mL conical flask, dissolve in about 200 mL of Flu-id A (see Diluting and Rinsing Fluids for Membrane Filtration), and mix; or constitute, as directed in the labeling, each of 20 containers and transfer a quantity of liquid or suspen-sion, equivalent to about 300 mg of solids, into a sterile 500-mL conical flask, dissolve in about 200 mL of Fluid A, and mix. Proceed as directed for Aqueous Solutions or Oils and Oily Solutions, whichever applies.Pharmacy Bulk Packages, ³5g—From each of 6 con-tainers, aseptically transfer about 1g of solids into a sterile 500-mL conical flask, dissolve in about 200 mL of Fluid A, and mix; or constitute, as directed in the labeling, each of 6 containers and transfer a quantity of liquid, equivalent to about 1g of solids, into a sterile 500-mL conical flask, dis-solve in about 200 mL of Fluid A, and mix. Proceed as direc-ted for Aqueous Solutions.ANTIBIOTIC SOLIDS, BULKS, and BLENDS Aseptically remove a sufficient quantity of solids from the appropriate amount of containers (see Table 2), mix to ob-tain a composite, equivalent to about 6g of solids, and transfer to a sterile 500-mL conical flask. Dissolve in about 200 mL of Fluid A, and mix. Proceed as directed for Aqueous Solutions.STERILE AEROSOL PRODUCTSFor fluid products in pressurized aerosol form, freeze the containers in an alcohol-dry ice mixture at least at –20° for about 1 hour. If feasible, allow the propellant to escape be-fore aseptically opening the container, and transfer the con-tents to a sterile pooling vessel. Add 100 mL of Fluid D to the pooling vessel, and mix gently. Proceed as directed for Aqueous Solutions or Oils and Oily Solutions, whichever ap-plies.DEVICES WITH PATHWAYS LABELED STERILE Aseptically pass not less than 10 pathway volumes of Fluid D through each device tested. Collect the fluids in an appro-priate sterile vessel, and proceed as directed for Aqueous Sol-utions or Oils and Oily Solutions, whichever applies.In the case of sterile, empty syringes, draw sterile diluent into the barrel through the sterile needle, if attached, or through a sterile needle attached for the purpose of the test, and express the contents into a sterile pooling vessel. Pro-ceed as directed above.uDirect Inoculation of the Culture MediumTransfer the quantity of the preparation to be examined prescribed in Tables 2 and 3 directly into the culture medi-um so that the volume of the product is not more than 10% of the volume of the medium, unless otherwise prescribed. If the product to be examined has antimicrobial activity, carry out the test after neutralizing this with a suitable neu-tralizing substance or by dilution in a sufficient quantity of culture medium. When it is necessary to use a large volume of the product, it may be preferable to use a concentrated culture medium prepared in such a way that it takes into ac-count the subsequent dilution. Where appropriate, the con-centrated medium may be added directly to the product in its container.OILY LIQUIDSUse media to which have been added a suitable emulsify-ing agent at a concentration shown to be appropriate in the Method Suitability Test, for example polysorbate 80 at a con-centration of 10g per L.OINTMENTS and CREAMSPrepare by diluting to about 1 in 10 by emulsifying with the chosen emulsifying agent in a suitable sterile diluent such as u Fluid A (see Diluting and Rinsing Fluids for Membrane Filtration).uTransfer the diluted product to a medium not containing an emulsifying agent.Incubate the inoculated media for not less than 14 days. Observe the cultures several times during the incubation pe-riod. Shake cultures containing oily products gently each day. However, when Fluid Thioglycollate Medium is used for the detection of anaerobic microorganisms, keep shaking or mixing to a minimum in order to maintain anaerobic condi-tions.CATGUT and OTHER SURGICAL SUTURES FORVETERINARIAN USEUse for each medium not less than the quantities of the product prescribed in Tables 2 and 3. Open the sealed pack-age using aseptic precautions, and remove three sections of the strand for each culture medium. Carry out the test on three sections, each 30-cm long, which have been cut off from the beginning, the center, and the end of the strand. Use whole strands from freshly opened cassette packs. Transfer each section of the strand to the selected medium. Use sufficient medium to cover adequately the material to be tested (20 mL to 150 mL).u SOLIDSTransfer a quantity of the product in the form of a dry sol-id (or prepare a suspension of the product by adding sterile diluent to the immediate container), corresponding to not less than the quantity indicated in Tables 2 and 3. Transfer the material so obtained to 200 mL of Fluid Thioglycollate Medium, and mix. Similarly, transfer the same quantity to 200 mL of Soybean–Casein Digest Medium, and mix. Proceed as directed above.76á71ñ Sterility Tests / Microbiological Tests USP 37。
中国药典2010无菌检查法
装管每基养培丁马良改� lm51于少不量装管每基养培体流盐酸醇乙硫�时同�%01的积体基 养培于大得不�积体品试供的种接合符应量用的基养培中器容个每�外定规有另除。 ) 1:2为 比之数瓶或数支种接的基养培种 2�中基养培丁马良改和基养培体流盐酸醇乙硫至种接别分 量等 �品试供量定规取。品试供的查检菌无行进法滤过膜薄用法无于用适法种接接直 法种接接直�2 。查检菌无的头针菌无的带配所中装包行进法种接接直用采应时同 。作操法方下项品试供性溶水按后然 �滤过即立�匀混�内器容菌无的液释稀量适含至合混或�滤过接直�解溶剂溶的示所签标 或液释稀人吸可要需若�物容内的中器射注将�量定规取 品试供器射注的物药有装 。作操法方的下项品试供剂制性溶水非或液溶水照后然�中器容菌无至 移转液溶品试供将并�器容启开菌无再后剂射抛放释�孔小一钻端上器容在速迅作操菌无以 。时小 1约冻冷室冰的 ℃ 02-少至置器容各将�量定规取 品试供剂雾�喷�气菌无 。作操法方的下项品试供性溶水非照基养培入加及洗冲膜滤后滤过 。滤过液溶品试供为作相水层下取�置静�取萃摇振分充�液释稀的 lm001于少不入加中液溶 品试供的中酯丙异酸烷四十菌无的量适有含于�品试供的滤过法无然仍对。滤过速迅热趁并 � ℃ 44过超得不度温但 �热加当适可要需果如 。解溶分充品试供使 �摇振烈剧 �中酯丙异酸烷 四十菌无的量适至合混 �量定规取 品试供剂油性黏和剂膏的酯丙异酸烷四十于溶可 。作操法 方的下项品试供液溶水照他其。基养培的 08酯梨山聚含不或含入加。次 3于少不般一数次洗 冲�膜滤洗冲液洗冲的 08酯梨山聚� 1� % 1.0含用。滤过即立�合混分充�内器容菌无的 lm003液释稀的剂化乳宜适他其或 08酯梨山聚含于溶合混 �量定规取 品试供性溶水非 。作操法方的下项品试供液 溶水照后然 �溶复明说签标按或解溶液释稀的宜适加 �量定规取 品试供体固性溶水 。 lm001基养培丁马良改 入加中器滤一另� lm001各基养培体流盐酸醇乙硫入加中器滤个 2�后洗冲。验试证验法方 同法方洗冲、量洗冲的用所�次 3于少不般一数次洗冲�膜滤洗冲液洗冲用须�剂腐防含或 用作菌抑有具品试供如。滤过即立�匀混�内器容菌无的液释稀量适含至移转部全或�滤过 接直者上以 lm5为量装品试供�瓶�支每�滤过即立�匀混�内器容菌无的 lm001液释稀宜 适含至移转部全�者下以及 lm5为量装品试供�瓶�支每�量定规取 品试供液溶水 。伤损受物生微的上膜滤免避以� lm000l 过超得不量洗冲总� lm001为般一量洗冲 次每膜滤张每�膜滤洗冲液洗冲用要需若�后滤过膜薄经液溶品试供。面表膜滤个整盖粗液 洗冲及液溶品试供持保意注应�率效滤过大最的膜滤挥发为。燥干分充应前用使在器滤过和 膜滤其�品试供类油。膜滤湿润以�滤过液洗冲的量少将先前滤过液溶品试供性溶水 。性整完的后前滤过在膜滤证保应�时用使。质材膜滤择选性特的剂溶 其及品试供据根。 mm05为约径直 mμ54.0于大不应径孔膜滤�器滤过膜薄式闭封用采 法滤过膜薄�1 。行进法方列下按�外定规有另除 基养培种接及理处品试供 。物容内出取�器容开启作操菌无 按再�气空菌无入导内器容向�头针的器滤过菌除有带如�材器菌无的宜适用可�度空真的 定一有内器容品试供果如。毒梢底彻行进面表器容品试供对液毒消的宜适用�时作操 。同相法方的证验与应件条验检和法方查检的用采所查检菌无 的品试供 。法滤过膜薄用采应�许允状性品试供要只。法种接接直和法滤过膜薄括包法查检菌无
无菌检查法-2015版中国药典
无菌检查法-2015版中国药典无菌检查法系用于检查药典要求无菌的药品、生物制品、医疗器具、原料、辅料、及其他品种是否无菌的一种方法。
若供试品符合无菌检查法的规定,仅表明了供试品在该检验条件下未发现微生物污染。
无菌检查应在环境洁净度B 级背景下的局部A 级洁净度的单向流空气区域内或隔离系统中进行，其全过程应严格遵守无菌操作，防止微生物污染，防止污染的措施不得影响供试品中微生物的检出。
单向流空气区、工作台面及环境应定期按《医药工业洁净室（区）悬浮粒子、浮游菌和沉降菌的测试方法》的现行国家标准进行洁净度确认。
隔离系统应定期按相关的要求进行验证，其内部环境的洁净度须符合无菌检查的要求。
日常检验还需对试验环境进行监控。
无菌检查人员必须具备微生物专业知识，并经过无菌技术的培训。
培养基硫乙醇酸盐流体培养基主要用于厌氧菌的培养，也可用于需气菌培养；胰酪大豆胨液体培养基适用于真菌和需气菌的培养。
培养基的制备及培养条件培养基可按以下处方制备，亦可使用按该处方生产的符合规定的脱水培养基。
配制后应采用验证合格的灭菌程序灭菌。
制备好的培养基应保存在2～25℃、避光的环境，若保存于非密闭容器中，一般在3周内使用；若保存于密闭容器中，一般可在一年内使用。
1. 硫乙醇酸盐流体培养基酪胨(胰酶水解) 15.0g 酵母浸出粉 5.0g葡萄糖 5.0g 氯化钠 2.5gL-胱氨酸0.5g 新配制的0.1% 刃天青溶液1.0ml硫乙醇酸钠0.5g 琼脂0.75g(或硫乙醇酸) （0.3 ml) 纯化水1000ml除葡萄糖和刃天青溶液外，取上述成分混合，微温溶解，调节pH 为弱碱性，煮沸，滤清，加入葡萄糖和刃天青溶液，摇匀，调节pH 值使灭菌后为7.1±0.2。
分装至适宜的容器中，其装量与容器高度的比例应符合培养结束后培2养基氧化层（粉红色）不超过培养基深度的1/2。
灭菌。
在供试品接种前，培养基氧化层的高度不得超过培养基深度的1/5，否则，须经100℃水浴加热至粉红色消失（不超过20 分钟），迅速冷却，只限加热一次，并防止被污染。
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无菌检查法系用于检查药典要求无菌的药品、医疗器具、原料、辅料及其他品种是否无菌的一种方法。
若供试品符合无菌检查法的规定，仅表明了供试品在该检验条件下未发现微生物污染。
无菌检查应在环境洁净度10000级下的局部洁净度100级的单向流空气区域内或隔离系统中进行，其全过程中必须严格遵守无菌操作，防止微生物污染。
单向流空气区、工作台面及环境应定期按《医药工业洁净室（区）悬浮粒子、浮游菌和沉降菌的测试方法》的现行国家标准进行洁净度验证。
隔离系统按相关的要求进行验证，其内部环境的洁净度须符合无菌检查的要求。
培养基培养基的制备培养基可按以下处方制备，也可使用按该处方生产的符合规定的脱水培养基。
配制后应采用验证合格的灭菌程序灭菌。
制备好的培养基应保存在2～25℃、避光的环境。
培养基若保存于非密闭容器中，一般在三周内使用；若保存于密闭容器中，一般可在一年内使用。
∙1、硫乙醇酸盐流体培养基（用于培养好氧菌、厌气菌）酪胨（胰酶水解）15g；氯化钠 2.5g；葡萄糖5g；新配制的0.1%刃天青溶液 1.0ml；L -胱氨酸0.5g（或新配制的0.2%亚甲蓝溶液0.5ml）；硫乙醇酸钠0.5g；琼脂0.75g（或硫乙醇酸0.3ml）；水1000ml；酵母浸出粉5g除葡萄糖和刃天青溶液外，取上述成分混合，微温溶解后，调节pH为弱碱性，煮沸，滤清，加入葡萄糖和刃天青溶液，摇匀，调节pH值使灭菌后为7.1±0.2，分装至适宜的容器中，其装量与容器高度的比例应符合培养结束后培养基氧化层（粉红色）不超过培养基深度的1/2。
灭菌。
在供试品接种前，培养基氧化层的高度不得超过培养基深度的1/5，否则，须经100℃水浴加热至粉红色消失（不超过20分钟）迅速冷却，只限加热一次，并应防止被污染。
硫乙醇酸盐流体培养基置30～35℃培养。
∙2、改良马丁培养基（用于培养真菌）胨5g；磷酸氢二钾1g；酵母浸出粉2g；硫酸镁0.5g；葡萄糖20g；水1000ml除葡萄糖外，取上述成分混合，微温溶解，调节pH值约为 6.8，煮沸，加入葡萄糖溶解后，摇匀，滤清，调节pH值使灭菌后为 6.4±0.2，分装，灭菌。
改良马丁培养基置23～28℃培养。
∙3、选择性培养基按上述硫乙醇酸盐流体培养基或改良马丁培养基的处方及制法，在培养基灭菌或使用前加入适量的中和剂或表面活性剂，如对氨基苯甲酸（用于磺胺类供试品）、聚山梨酯8 0（用于非水溶性供试品）或β-内酰胺酶（用于β-内酰胺类供试品）等。
中和剂或表面活性剂的用量应通过验证。
∙4、营养肉汤培养基胨10g；牛肉浸出粉 3.0g；氯化钠5g；葡萄糖 5.0g；水1000ml取上述成分混合，微温溶解，调节p H为弱碱性，煮沸，滤清，调节pH值使灭菌后为7.2±0.2，分装，灭菌。
∙5、营养琼脂培养基按上述营养肉汤培养基的处方及制法，加入14.0g琼脂，调节pH值使灭菌后为7.2±0. 2，分装，灭菌。
∙6、0.5%葡萄糖肉汤培养基（用于硫酸链霉素等抗生素的无菌检查）胨10g；葡萄糖5g；氯化钠5g；牛肉浸出粉 3.0g；水1000ml除葡萄糖外取上述成分混合，微温溶解后，调节pH为弱碱性，煮沸，加葡萄糖溶解后，摇匀，滤清，调节pH值使灭菌后为7.2±0.2，分装，灭菌。
∙7、改良马丁琼脂培养基按改良马丁培养基的处方及制法，加入14.0g琼脂，调节PH值使灭菌后为 6.4±0.2，分装，灭菌。
培养基的适用性检查无菌检查用的硫乙醇酸盐流体培养基及改良马丁培养基等应符合培养基的无菌性检查及灵敏度检查的要求。
本检查可在供试品的无菌检查前或与供试品的无菌检查同时进行。
无菌性检查每批培养基随机不少于5支（瓶），培养14天，应无菌生长。
灵敏度检查。
培养基灵敏度检查法1.菌种：培养基灵敏度检查所用的菌株传代次数不得超过5代（从菌种保存中心获得的冷冻干燥菌种为第0代），并采用适宜的菌种保藏技术，以保证试验菌株的生物学特性。
∙金黄色葡萄球菌（Staphylococcus aureus）[CMCC（B）26 003]∙铜绿假单胞菌（Pseudomonas aeruginosa）[CMCC（B）10 104]∙枯草芽孢杆菌（Bacillus subtilis ）[CMCC（B）63 501]∙生孢梭菌（Clostridium sporogenes）[CMCC（B）64 941]∙白色念珠菌（Candida albicans）[CMCC（F）98 001]∙黑曲霉（Aspergillus niger ）[CMCC（F）98 003]菌液制备：接种金黄色葡萄球菌、铜绿假单胞菌、枯草芽孢杆菌的新鲜培养物至营养肉汤获营养琼脂培养基中，接种生孢梭菌的新鲜培养物至硫乙醇酸盐流体培养基中，30～35℃培养18～24小时；接种白色念株菌的新鲜培养物至改良马丁培养基或改良马丁培养基中，23～28℃培养24～48小时，上述培养物用0.9%无菌氯化钠溶液制成每1ml含菌数小于100cf u（菌落形成单位）的菌悬液。
接种黑曲霉的新鲜培养物至改良马丁琼脂斜面培养基上，23～28℃培养5～7天，加入3～5ml0.9%无菌氯化钠溶液，将孢子洗脱。
然后，吸出孢子悬液（用管口带有薄的无菌棉花或纱布能过滤菌丝的无菌毛细吸管）至无菌试管内，用0.9%无菌氯化钠溶液制成每1ml含孢子数小于100cfu的孢子悬液。
培养基接种：取每管装量为12ml的硫乙醇酸盐流体培养基9支，分别接种小于100cf u的金黄色葡萄球菌、铜绿假单胞菌、枯草芽孢杆菌、生孢梭菌各2支，另1支不接种作为空白对照，培养3天；取每管装量为9ml的改良马丁培养基5支，分别接种小于100cf u的白色念株菌、黑曲霉各2支，另1支不接种作为空白对照，培养5天。
逐日观察结果。
结果判断：空白对照管应无菌生长，若加菌的培养基管菌生长良好，判该培养基的灵敏度检查符合规定。
稀释液、冲洗液及其制备方法稀释液、冲洗液配制后应采用验证合格的灭菌程序灭菌。
1.0.1%蛋白胨水溶液：取蛋白胨 1.0g，加水1000ml，微温溶解，滤清，调节PH值至7.1±0.2，分装，灭菌。
2.PH7.0氯化钠-蛋白胨缓冲液：取磷酸二氢钾3.56g、磷酸氢二钠7.23g、氯化钠4.3 0g、蛋白胨 1.0g，加水1000ml，微温溶解，滤清，分装，灭菌。
如需要，可在上述稀释液或冲洗液的灭菌前或灭菌后加入表面活性剂或中和剂等。
方法验证或试验当建立药品的无菌检查法时，应进行方法的验证，以证明所采用的方法适合于该药品的无菌检查。
若药品的组分或原检验条件发生改变时，检查方法应重新验证。
验证时，按“供试品的无菌检查”的规定及下列要求进行操作。
对每一对试验菌应逐一进行验证。
菌种及菌液制备：同培养基灵敏度检查。
薄膜过滤法：将规定量的供试品薄膜过滤法过滤，冲洗，在最后一次的冲洗液中加入小于100cf u的试验菌，过滤。
取出滤膜接种至硫乙醇酸盐流体培养基或改良马丁培养基中，或将培养基加至滤筒内。
另取一装有同体积培养基的容器，加入等量试验菌，作为对照。
按规定温度培养3～5天。
各试验菌同法操作。
直接接种法：取符合直接接种法培养基用量要求的硫乙醇酸盐流体培养基8管，分别接入小于100cf u的金黄色葡萄球菌、铜绿假单胞菌、枯草芽孢杆菌、生孢梭菌各2管；取符合直接接种法培养基用量要求的改良马丁培养基4管，分别接入小于100cf u的白色念株菌、黑曲霉各2管。
其中1管接入规定量的供试品，另1管作为对照品，按规定的温度培养3～5天。
结果判断：与对照管比较，如含供试品各容器中的试验菌均生长良好，则供试品的该检验条件下无抑菌作用或其抑菌作用可以忽略不计，照此检查法和检查条件进行供试品的无菌检查。
如含供试品的任一容器中微生物生长微弱、缓慢或不生长，则供试品的该检验量在该检验条件下有抑菌作用，可采用增加冲洗量，或增加培养基的用量，或使用中和剂或灭活剂如β-内酰胺酶、对氨基苯甲酸，或更换滤膜品种等方法，消除供试品的抑菌作用，并重新进行方法验证。
方法验证试验夜可与供试品的无菌检查同时进行。
供试品的无菌检查检验数量：检验数量是指一次试验所用供试品最小包装容器的数量。
除另有规定外，出厂产品按表1规定；上市产品监督检查按表2、表3规定。
表1、表2、表3中最少检验数量不包括阳性对照试验的供试品用量。
一般情况下，供试品无菌检查若采用薄膜过滤法，应增加1/2的最小检验数量作阳性对照用；若采用直接接种法，应增加供试品1支（或瓶）作阳性对照用。
检验量：使指一次试验所用的供试品总量（g或ml）。
除另有规定外，每份培养基接种供试品的量按表2、表3规定。
采用直接接种法时，若每支（瓶）供试品的装量按规定足够接种两份培养基，则应分别接种硫乙醇酸盐流体培养基和改良马丁培养基。
采用薄膜过滤法时，检验量应不少于直接接种法的供试品总接种量，只要供试品特性允许，应将所有容器内的全部内容物过滤。
阳性对照：应根据供试品特性选择阳性对照菌：无抑菌作用及抗革兰阴性菌为主的供试品以大肠埃希菌为对照菌；抗厌氧菌的供试品，以生孢梭菌为对照菌；抗真菌的供试品，以白色念株菌为对照菌。
阳性对照试验的菌液制备同培养基灵敏度检查（大肠埃希菌的菌液制备同培养基的灵敏度检查中的金黄色葡萄球菌），加菌量小于100cf u，供试品用量同供试品无菌检查每份培养基接种的样品量。
阳性对照管培养48～72小时应生长良好。
阴性对照：供试品无菌检查时，应取相应溶剂和稀释液同法操作，作为阴性对照。
阴性对照不得有菌生长。
无菌试验过程中，若需使用表面活性剂、灭活剂、中和剂等试剂，应证明其有效性，且对微生物生长及存活无影响。
无菌检查法包括薄膜过滤法和直接接种法。
只要供试品性状允许，应采用薄膜过滤法。
进行供试品无菌检查时，所采用的检查方法和检验条件应与验证的方法相同。
操作时，用适宜的消毒液对供试品容器表面进行彻底消毒。
如果容器内有一定的真空度，可用适宜的无菌器材（如带有除菌过滤器的针头），向供试品容器内导入无菌空气，再按无菌操作开启容器取出内容物。
供试品处理及接种培养基除另有规定外，按下列方法进行。
1.薄膜过滤法薄膜过滤法应优先采用封闭式薄膜过滤器，也可使用一般薄膜过滤器。
无菌检查用的滤膜孔径应不大于0.45um，直径约为50mm。
根据供试品及其溶剂的特性选择滤膜材质。
滤器及滤膜使用前应采用适宜的方法灭菌。
使用时，应保证滤膜在过滤前后的完整性。
水溶性供试液过滤前先将少量的冲洗液过滤以润湿滤膜。
油类供试品，其滤膜和过滤器在使用前应充分干燥。
为发挥滤膜的最大过滤效率，应注意保持供试品溶液及冲洗液覆盖整个滤膜表面。
供试液经薄膜过滤后，若需要用冲洗液冲洗滤膜，每张滤膜每次冲洗量为100ml，且总冲洗量不宜过大，以避免滤膜上的微生物受损伤。