美国药典USP31 71 无菌检查法 双语版
USP36 1117 优良微生物检测规范中英文版
USP36 1117 优良微生物检测规范（中英文1/ 2）2013-08-09 15:30:46| 分类：USP|举报|字号订阅1117 MICROBIOLOGICAL BEST LABORATORY PRACTICES 优良微生物检测规范INTRODUCTION 介绍Good laboratory practices in a microbiology laboratory consist of activities that depend on several principles: aseptic technique, control of media, control of test strains, operation and control of equipment, diligent recording and evaluation of data, and training of the laboratory staff. Because of the inherent risk of variability in microbiology data, reliability and reproducibility are dependent on the use of accepted methods and adherence to good laboratory practices.优良微生物检测规范由一些活动组成，这些活动依赖于几个基本要素：无菌技术、培养基控制、检测用菌株控制、设备操作和控制、完善的记录和数据评估、化验室员工的培训。
由于微生物数据具有天生的不确定性，数据的可靠性和重复性取决于是否使用被接受的方法，以及是否严格遵守化验室规范。
MEDIA PREPARATION AND QUALITY CONTROL 培养基制备和质量控制Media Preparation 培养基制备Culture media are the basis for most microbiological tests. Safeguarding the quality of the media is therefore critical to the success of the microbiology laboratory. Media preparation, proper storage, and quality control testing can ensure a consistent supply of high-quality media.培养基是大多数微生物测试的基础。
[应用]无菌USP71中文版
![[应用]无菌USP71中文版](https://img.taocdn.com/s3/m/7732a7160242a8956aece40a.png)
美国药典USP31 <71>无菌检查法无菌检查法此通那么的各局部已经与欧洲药典和/或日本药典的对应局部做了协调。
不一致的局部用符号〔〕来标明。
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。
只要其性质许可，这些药品将使用供试产品无菌检查法项下的膜过滤法来检测。
如果膜过滤技术是不适合的，那么使用在供试产品无菌检查法项下的培养基直接接种法。
除了具有标记为无菌通道的设备之外，所有的设备均须使用培养基直接接种法进行检测。
在结果的观测与理解项下包含了复验的规定。
由于无菌检查法是一个非常精确的程序，在此过程中程序的无菌状态必须得到确保以实现对结果的正确理解，因此人员经过适当的培训并取得资质是非常重要的。
无菌检查在无菌条件下进行。
为了实现这样的条件，试验环境必须调整到适合进行无菌检查的方式。
为防止污染而采取的特定预防措施应不会对任何试图在检查中发现的微生物产生影响。
通过在工作区域作适当取样并进行适当控制，来定期监测进行此试验的工作条件。
这些药典规定程序自身的设计不能确保一批产品无菌或已经灭菌。
这主要是通过灭菌工艺或者无菌操作程序的验证来完成。
当通过适当的药典方法获得了某物品中微生物污染的证据,这样获得的结果是该物品未能到达无菌检验要求的结论性证据，即便使用替代程序得到了不同的结果也无法否认此结果。
如要获得关于无菌检验的其他信息，见药品的灭菌和无菌保证<1211> 按照下面描述的方法配制实验用培养基；或者使用脱水培养基，只要根据其制造商或者分销商说明进行恢复之后，其能够符合好氧菌、厌氧菌、霉菌生长促进试验的要求即可。
使用经过验证的工艺对培养基进行灭菌操作。
下面的培养基已经被证实适合进行无菌检查。
巯基醋酸盐液体培养基主要用于厌氧菌的培养。
但其也用于检测好氧菌。
大豆酪蛋白消化物培养基适合于培养霉菌和好氧菌。
巯基醋酸盐液体培养基,L-胱氨酸, 氯化钠, 葡萄糖,,酵母提取物(水溶性) , 酪蛋白胰酶消化物,巯基乙酸钠,或者巯基乙酸,刃天青钠溶液(1 比1000)，新配制纯洁水0.5 g 5.0 g 15.0 g 0.5 g 0.3 mL 1.0 mL 1000 mL将L-胱氨酸、氯化钠、葡萄糖、酵母提取物、酪蛋白胰酶消化物与纯洁水混合，并加热至实现溶解。
无菌USP71中文版
美国药典USP31 <71>无菌检查法无菌检查法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。
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。
只要其性质许可，这些药品将使用供试产品无菌检查法项下的膜过滤法来检测。
如果膜过滤技术是不适合的，则使用在供试产品无菌检查法项下的培养基直接接种法。
除了具有标记为无菌通道的设备之外，所有的设备均须使用培养基直接接种法进行检测。
在结果的观测与理解项下包含了复验的规定。
由于无菌检查法是一个非常精确的程序，在此过程中程序的无菌状态必须得到确保以实现对结果的正确理解，因此人员经过适当的培训并取得资质是非常重要的。
无菌检查在无菌条件下进行。
为了实现这样的条件，试验环境必须调整到适合进行无菌检查的方式。
为避免污染而采取的特定预防措施应不会对任何试图在检查中发现的微生物产生影响。
通过在工作区域作适当取样并进行适当控制，来定期监测进行此试验的工作条件。
这些药典规定程序自身的设计不能确保一批产品无菌或已经灭菌。
这主要是通过灭菌工艺或者无菌操作程序的验证来完成。
当通过适当的药典方法获得了某物品中微生物污染的证据,这样获得的结果是该物品未能达到无菌检验要求的结论性证据，即便使用替代程序得到了不同的结果也无法否定此结果。
如要获得关于无菌检验的其他信息，见药品的灭菌和无菌保证<1211> 按照下面描述的方法配制实验用培养基；或者使用脱水培养基，只要根据其制造商或者分销商说明进行恢复之后，其能够符合好氧菌、厌氧菌、霉菌生长促进试验的要求即可。
使用经过验证的工艺对培养基进行灭菌操作。
下面的培养基已经被证实适合进行无菌检查。
巯基醋酸盐液体培养基主要用于厌氧菌的培养。
但其也用于检测好氧菌。
大豆酪蛋白消化物培养基适合于培养霉菌和好氧菌。
巯基醋酸盐液体培养基,L-胱氨酸, 氯化钠, 葡萄糖, 琼脂，呈颗粒状(水分含量不超过15%) 0.75 g 0.5 g 2.5 g 5.5/5.0 g L-胱氨酸,酵母提取物(水溶性) , 酪蛋白胰酶消化物,巯基乙酸钠,或者巯基乙酸,刃天青钠溶液(1 比1000)，新配制纯净水0.5 g 5.0 g 15.0 g 0.5 g 0.3 mL 1.0 mL 1000 mL将L-胱氨酸、氯化钠、葡萄糖、酵母提取物、酪蛋白胰酶消化物与纯净水混合，并加热至实现溶解。
美国药典USP31翻译版
Many Pharmacopeial articles either are hydrates or contain water in adsorbed form. As a result, the determination of the water content is important in demonstrating compliance with the Pharmacopeial standards. Generally one of the methods given below is called for in the individual monograph, depending upon the nature of the article. In rare cases, a choice is allowed between two methods. When the article contains water of hydration, the Method I (Titrimetric), the Method II (Azeotropic), or the Method III (Gravimetric) is employed, as directed in the individual monograph, and the requirement is given under the heading Water.很多药典物品要么是水合物，要么含有处于吸附状态的水。
因此，测定水分含量对于证实与药典标准的符合性是很重要的。
通常，在具体的各论中会根据该物品的性质，要求使用下面若干方法中的一个。
偶尔，会允许在2个方法中任选一个。
当该物品含有水合状态的水，按照具体各论中的规定，使用方法I（滴定测量法）、方法II（恒沸测量法）、或方法III（重量分析法），这个要求在标题水分项下给出。
美国药典USP31-71-无菌检查法-双语版
美国药典USP31-71-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。
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。
71无菌检查法
＜71＞无菌检查（USP29-NF24）◆这个章节的大部分内容都和欧洲药典或日本药典相应检测部分保持一致。
有一些不一致的地方已用特殊标记（◆◆）注明。
◆ 下述步骤被用于检查药典中规定要求无菌的是否满足其专论中相应的无菌要求。
供试品的性状允许时，可以采用供试品的无菌检查中的薄膜过滤法来检测。
如果薄膜过滤方法不能使用，则采用供试品的无菌检查中的直接接种法进行检测。
所有的器具，除了标明的无菌器皿外，都采用直接接种法进行检测。
重新检测的规定条款见结果的观测和分析。
因为无菌检测是一项非常严格的操作过程（必须保证无菌操作才能得到一个准确的检测结果），所以对操作人员进行相关的培训和鉴定是非常重要的。
无菌检查即在无菌状态下实行。
为了达到这个状态，检测的环境必须适合无菌操作。
要防止检验过程中暴露的微生物受污染。
要对无菌检验的操作环境进行适当地取样检测和合适的控制来监控。
不仅仅通过这些药典中的步骤就能确保一批产品无菌或已经灭菌。
确认在无菌状态下操作或按无菌操作步骤操作则是首先需要的。
当用药典中合适的药典方法检测产品中出现了微生物污染，其结果宣判该无菌检测所需方法无效，甚至是更换了操作步骤得到了不同的结果。
◆额外的无菌检测信息，参见＜1211＞灭菌和确保无菌概略。
◆培养基按以下处方制备培养基，或使用按该处方生产的符合规定的脱水培养基，其能满足需气菌，厌气菌，霉菌的增殖培养。
培养基采用有效的程序灭菌。
下面培养基适合用于无菌检查。
硫乙醇酸盐流体培养基主要用于厌气菌的培养。
还用于需气菌的培养。
大豆粉-酪蛋白消化物培养基适合霉菌和需气菌的培养。
硫乙醇酸盐流体培养基取L-光胺酸，氯化钠，葡萄糖，酵母浸出物和酪蛋白胰酶消化物混合，加热直到培养基被活化。
加入巯基乙酸钠或巯基乙酸酯，如有必要，加1N 氢氧化钠，调节PH 值使灭菌后为7.1±0.2。
如需过滤，加热培养基不需要煮沸，热时滤过滤纸。
加入刃天青钠溶液，混合，马上分装至合适容器中，这样在培养期间最多培养上层基颜色发生变化。
美国药典无菌检查法31中文版
美国药典USP31 71 无菌检查法中文版美国药典USP31-NF26无菌检查法《71》.doc71 STERILITY TESTS 无菌检查法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。
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。
只要其性质许可，这些药品将使用供试产品无菌检查法项下的膜过滤法来检测。
如果膜过滤技术是不适合的，则使用在供试产品无菌检查法项下的培养基直接接种法。
除了具有标记为无菌通道的设备之外，所有的设备均须使用培养基直接接种法进行检测。
在结果的观测与理解项下包含了复验的规定。
由于无菌检查法是一个非常精确的程序，在此过程中程序的无菌状态必须得到确保以实现对结果的正确理解，因此人员经过适当的培训并取得资质是非常重要的。
无菌检查在无菌条件下进行。
为了实现这样的条件，试验环境必须调整到适合进行无菌检查的方式。
为避免污染而采取的特定预防措施应不会对任何试图在检查中发现的微生物产生影响。
通过在工作区域作适当取样并进行适当控制，来定期监测进行此试验的工作条件。
这些药典规定程序自身的设计不能确保一批产品无菌或已经灭菌。
这主要是通过灭菌工艺或者无菌操作程序的验证来完成。
当通过适当的药典方法获得了某物品中微生物污染的证据，这样获得的结果是该物品未能达到无菌检验要求的结论性证据，即便使用替代程序得到了不同的结果也无法否定此结果。
如要获得关于无菌检验的其他信息，见药品的灭菌和无菌保证<1211>按照下面描述的方法配制实验用培养基；或者使用脱水培养基，只要根据其制造商或者分销商说明进行恢复之后，其能够符合好氧菌、厌氧菌、霉菌生长促进试验的要求即可。
使用经过验证的工艺对培养基进行灭菌操作。
下面的培养基已经被证实适合进行无菌检查。
巯基醋酸盐液体培养基主要用于厌氧菌的培养。
但其也用于检测好氧菌。
大豆酪蛋白消化物培养基适合于培养霉菌和好氧菌。
- 1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
- 2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
- 3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。
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。
只要其性质许可，这些药品将使用供试产品无菌检查法项下的膜过滤法来检测。
如果膜过滤技术是不适合的，则使用在供试产品无菌检查法项下的培养基直接接种法。
除了具有标记为无菌通道的设备之外，所有的设备均须使用培养基直接接种法进行检测。
在结果的观测与理解项下包含了复验的规定。
Because sterility testing is a very exacting procedure, where asepsis of the procedure must be ensured for a correct interpretation of results, it is important that personnel be properly trained and qualified. The test for sterility is carried out under aseptic conditions. In order to achieve such conditions, the test environment 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 by appropriate sampling of the working area and by carrying out appropriate controls.由于无菌检查法是一个非常精确的程序，在此过程中程序的无菌状态必须得到确保以实现对结果的正确理解，因此人员经过适当的培训并取得资质是非常重要的。
无菌检查在无菌条件下进行。
为了实现这样的条件，试验环境必须调整到适合进行无菌检查的方式。
为避免污染而采取的特定预防措施应不会对任何试图在检查中发现的微生物产生影响。
通过在工作区域作适当取样并进行适当控制，来定期监测进行此试验的工作条件。
These 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 procedures.这些药典规定程序自身的设计不能确保一批产品无菌或已经灭菌。
这主要是通过灭菌工艺或者无菌操作程序的验证来完成。
When evidence of microbial contamination in the article is obtained by the appropriate Pharmacopeial method, the result so obtained is conclusive evidence of failure of the article to meet the requirements of the test for sterility, even if a different result is obtained by an alternative procedure. For additional information on sterility testing, see Sterilization and Sterility Assurance of Compendial Articles 1211 .当通过适当的药典方法获得了某物品中微生物污染的证据，这样获得的结果是该物品未能达到无菌检验要求的结论性证据，即便使用替代程序得到了不同的结果也无法否定此结果。
如要获得关于无菌检验的其他信息，见药品的灭菌和无菌保证<1211>MEDIA 培养基Prepare media for the tests as described below, or dehydrated formulations may be used provided that, when reconstituted as directed by the manufacturer or distributor, they meet the requirements of the Growth Promotion Test of Aerobes, Anaerobes, and Fungi. Media are sterilized using a validated process.按照下面描述的方法配制实验用培养基；或者使用脱水培养基，只要根据其制造商或者分销商说明进行恢复之后，其能够符合好氧菌、厌氧菌、霉菌生长促进试验的要求即可。
使用经过验证的工艺对培养基进行灭菌操作。
The following culture media have been found to be suitable for the test for sterility. Fluid Thioglycollate Medium is primarily intended for the culture of anaerobic bacteria. However, it will also detect aerobic bacteria. Soybean–Casein Digest Medium is suitable for the culture of both fungi and aerobic bacteria.下面的培养基已经被证实适合进行无菌检查。
巯基醋酸盐液体培养基主要用于厌氧菌的培养。
但其也用于检测好氧菌。
大豆酪蛋白消化物培养基适合于培养霉菌和好氧菌。
Fluid Thioglycollate Medium 巯基醋酸盐液体培养基Mix the L-cystine, sodium chloride, dextrose, yeast extract, and pancreatic digest of casein with the purified water, and heat until solution is effected. Dissolve the sodium thioglycollate or thioglycolic acid in the solution and, if necessary, add 1 N sodium hydroxide so that, after sterilization, the solution will have a pH of 7.1 ± 0.2. If filtration is necessary, heat the solution again without boiling, and filter while hot through moistened filter paper. Add the resazurin sodium solution, mix, and place the medium in suitable vessels that provide a ratio of surface to depth of medium such that not more than the upper half of the medium has undergone 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 between 2 and 25 in a sterile, airtight container. If more than the upper one-third of the medium has acquired a pink color, the medium may be restored once by heating the containers 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.将L-胱氨酸、氯化钠、葡萄糖、酵母提取物、酪蛋白胰酶消化物与纯净水混合，并加热至实现溶解。