USP36 1117 优良微生物检测规范中英文版.doc

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。保证培养基的质量因而成为微生物实验室成功的关键。培养基的制备、合适的存贮和质量控制检测可以保证持续高质量培养基供应。It is important to choose the correct media or components in making media based on the use of accepted sources or references for formulas. The manufacturers formula and instructions for preparation routinely accompany dehydrated media and ready-made media. Because different media types may have different preparation requirements (e.g., heating, additives, and pH adjustment), it is important to follow these instructions to ensure preparation of acceptable media quality. A certificate of analysis describing expiration dating and recommended storage conditions accompanies ready-made media, as well as the quality control organisms used in growth-promotion and selectivity testing of that media.在制备培养基过程中使用已接受的来源或配方标准，选择正确的培养基或成份是非常重要的。一般生产商在供应培养基干粉和配制好的培养基时，其配方和配制指示都会随货发送。由于不同的培养基类型可能有不同的配制要求（例如，加热、添加剂，和pH值调节），重要的一点是需要遵守其提供的配制指示以保证配制出的培养基质量。如果是已制备好的培养基碟/瓶，随货会收到指明有效期和推荐的存贮条件的分析报告，以及促生产试验用微生物种类，和该培养基的选择性试验用微生物种类。Water is the universal diluent for microbiological media. Purified Water is most often used for media preparation, but in certain cases the use of deionized or distilled water may be appropriate. Water of lesser quality should not be used for microbiological media preparation. The volume of the water used should be recorded.水是通用的微生物培养基稀释剂。纯化水常用于培养基制备，但在某些情况下，也可以使用去离子水或蒸馏水。更低品质的水不应用于微生物培养基制备。水的用量应记录。Consistent preparation of media requires accurate weighing of dehydrated media or media constituents. A calibrated balance with the appropriate weight range for the ingredients should be used (SeeWeighing on an Analytical Balance1251). Clean weighing containers and tools (such as spatulas) should be used to prevent foreign substances from entering the formulation. The weight of the components should be recorded.要保持培养基配制的一致性，需要对培养基干粉或培养基组份进行准确称量。应使用在所需称量范围内经过校正的天平（见1251分析天平称量）。应对称重容器和工具（例如料勺）进行清洁以保证无异物进入所配物料。各成份的重量应进行记录。Dehydrated media should be thoroughly dissolved in water before dispensing and sterilization. If heating is necessary to help dissolve the media, care should be taken not to overheat media, because all culture media, to a greater or lesser extent, are heat-sensitive. Equipment used in the preparation of media should be appropriate to allow for controlled heating, constant agitation, and mixing of the media.培养基干粉应在水中完全溶解，然后进行配制和灭菌。如果需要加热助溶，要注意不能过热，因为所有的培养基，或多或少，都是对热敏感的。用于培养基配制的设备应适当，以便控制加热、持续搅拌和培养基混合。Darkening of media (Maillard-type reaction or nonenzymatic browning) is a general indication of overheating. When adding required supplements to media, adequate mixing of the medium after adding the supplement should be performed.培养基变黑（美拉德类型反应或非酶褐变）一般说明过热。在向培养基中加入所需要的补充成分时，在加入后需要进行充分搅拌混合。Preparation of media in poorly cleaned glassware can allow inhibitory substances to enter the media.在清洁不彻底的玻璃器皿中配制培养基会使得抑制性物质带入培养基。Inhibitory substances can come from detergent residue after cleaning glassware or from prior materials used in the glassware. Be sure that the cleaning process removes debris and foreign matter, and that the detergent is thoroughly rinsed out with Purified Water. SeeCleaning Glass Apparatus1051for additional guidance.抑制性物质可能来自于玻璃器皿中的清洁剂残留，或来自于玻璃器皿中上次所盛装的物料。要保证清洗程序可以去除残渣和外来物质，并且清洁剂可以被纯化水彻底冲洗掉。参见1051玻璃容器的清洁。Sterilization of media should be performed within the parameters provided by the manufacturer or validated by the user. Commercially prepared media should provide documentation of the sterilization method used.培养基灭菌应在生产商提供的参数范围，或用户验证的参数范围内实施。商业制备好的培养基应随货有所用的灭菌方法。Autoclaving by moist heat is the preferred sterilization technique, except in instances when boiling is required in order to avoid deterioration of heat-labile components of the media. Sterilization by filtration may also be appropriate for some formulations.湿热灭菌是较好的灭菌技术，除非需要煮沸以避免培养基中不耐热成分被破坏。有些配方可能适用过滤灭菌。The effects of the sterilization method and conditions on the media should be validated by sterility and growth-promotion testing of the media. In addition, if sterilized by moist heat, the autoclave cycle should be validated to ensure proper heat distribution for selected loads and volumes. Typically, manufacturers recommend using an autoclave cycle of 121 for 15 minutes using a validated autoclave. These conditions apply to time at temperature of the media. As container size and the load configuration of the autoclave will influence the rate of heating, longer cycles may be required for larger loads. However, the sterilization time will be dependent on the media volume and autoclave load. Sterilization cycles in which the autoclave is slow to come up to temperature may result in overheating of the media. Therefore, care must be taken to validate a sterilization cycle, balancing the need for sterile media against the tendency of the media to degrade under excessive heating. Storage of the media in the autoclave after the liquid cycle is completed is not recommended after cooling, as it may damage the media. Improper heating or sterilizing conditionsfor commercially prepared or internally prepared mediamay result in a difference in color change, loss of clarity, altered gel strength, or pH drift from the manufacturers recommended range, as well as reduced growth-promotion activity and/or selectivity.灭菌方法和条件对培养基的影响应经过无菌验证和培养基促生长试验确认。另外，如果采用湿热灭菌，灭菌周期应进行验主下以保证在所选的负载和体积下的热分布。生产商一般会推荐采用12115分钟作为灭菌条件，培养基灭菌即采用此条件。由于容器尺寸和灭菌器的负载参数会影响加热速度，如果负载较大时，应延长灭菌时间。当然，灭菌时间还是取决于培养基体积和灭菌负载。升温较慢的灭菌条件可能会导致培养基过热，因此需要特别注意灭菌周期的验证，在培养期灭菌要求和培养基在过热条件下分解中寻求平衡点。在灭菌结束冷却后，不建议将培养基留在灭菌器中存贮，因为可能会对培养基造成损坏。不适当的加热或灭菌条件-对于商业制备的培养基或公司自制的培养基-可能会导致颜色变化差异、澄清度差、胶强度改变、或pH值超出生产商指定范围、以及促生产试验表示出活性下降和/或具有选择性。The pH of each batch of medium should be confirmed after it has cooled to room temperature (20 25 ) by aseptically withdrawing a sample for testing. Refrigerated purchased media should be allowed to warm up to ambient room temperature if it is to be checked for pH confirmation. A flat pH probe is recommended for agar surfaces, and an immersion probe is recommended for liquids. SeepH791for guidance with pH measurement and instrument calibration. The pH of media should be in a range of 0.2 of the value indicated by the manufacturer, unless a wider range is acceptable by the validated method.每个批次培养基在冷却到室温后（20-25），应采用无菌方式取样检测pH值；冷冻的采购来的培养基应在升温至室温后检测pH值。建议对培养基平面采用扁平的pH探头，液体培养基则采用浸入式探头。pH值测量和仪器校正指南见pH791.除非有验证过的方法可以接受一个更宽的范围，否则培养基的pH值应在生产商指示的pH值0.2之内。Prepared media should be checked by appropriate inspection of plates and tubes for the following:培养基制备好后，应对碟或管进行以下检查Cracked containers or lids容器或盖裂开Unequal filling of containers容器填充不匀Dehydration resulting in cracks or dimpled surfaces on solid medium脱水导致固体培养基裂开或凸陷Hemolysis溶血Excessive darkening or color change太黑或颜色变化Crystalformation from possible freezing可能因为冷冻引起的结晶Excessive number of bubbles过量气泡Microbial contamination微生物污染Status of redox indicators (if appropriate)氧化还原指示剂状态（适用时）Lotnumber and expiration date checked and recorded检查批号和有效期并记录Sterility of the media培养基灭菌Cleanliness of plates (lid should not stick to dish)培养基用碟清洁（盖不应该粘在碟上）Media Storage培养基存贮It is prudent to consider how the manufacturer or supplier transports and stores media before distribution to the end user. Manufacturers of media should use transport and storage conditions that minimize the loss of moisture, control the temperature, prevent microbial contamination, and provide mechanical protection to the prepared media.培养基的生产商或供应商在培养基销售给最终用户前的运输和存贮方式是需要慎重考虑的。培养基生产商所采用的运输和贮存条件应最大程度降低水份损失、保证温度的控制、防止微生物污染，提供对已制备培养基的物理保护。Media should be labeled properly with batch or lot numbers, preparation and expiration dates, and media identification. Media should be stored according to the manufacturers instructions. Media prepared in house should be stored under validated conditions. Do not store agar at or below 0, as freezing could damage the gel structure. Protect stored media from exposure to light and excessive temperature. Before prolonged storage, agar plates should be placed into a sealed package or container to retard moisture loss.培养基应标识批号，制备时间和有效期，以及培养基识别信息。培养基应根据生产商指示进行存贮。公司自己制备的培养基应在经过验证条件下存贮，不要将培养基存贮等于或低于0条件下，因为结冻可能会对胶质结构造成损伤。存贮期间培养基应避光，避免超温。如果要延长存贮时间，培养基碟应放在密封的包装或容器中以减少水分损失。Remelting of an original container of solid media should be performed only once to avoid media whose quality is compromised by overheating or potential contamination. It is recommended that remelting be performed in a heated water bath or by using free-flowing steam. The use of microwave ovens and heating plates is common, but care should be taken to avoid damaging media by overheating and to avoid the potential injury to laboratory personnel from glass breakage and burns. The molten agar medium should be held in a monitored water bath at a temperature of 45 to 50 for not more than 8 hours.固体培养基只能解冻一次，以避免培养基由于过热或潜在污染造成质量问题。建议在热水浴中或使用自由流动蒸汽中解冻培养基。通常会使用微波炉和加热盘，但需要注意避免因为过热造成培养基损伤，避免因玻璃仪器破裂和燃烧引起化验室人员受伤。熔融的培养基应在40-45水浴中不超过8小时。Caution should be taken when pouring the media from a container immersed in a water bath to prevent water from the bath commingling with the poured sterile media. Wiping the exterior of the container dry before pouring may be advisable.将培养基从浸在水浴中的容器中倒出时要特别注意，避免水浴用水混入倒出的无菌培养基中。最好在将容器从水浴中取出后、倾倒前，将容器外表面擦干。Disposal of used cultured media (as well as expired media) should follow local biological hazard safety procedures.使用过的培养基处理（和过期的培养基）需要按照微生物危险控制程序处理。Quality Control Testing质量控制检测Although growth media can be prepared in a laboratory from individual components, many laboratories, for ease of use, use dehydrated media or purchase commercially prepared media in plastic plates or glass containers. Manufacturers of media attempt to standardize raw materials from biological sources, but must constantly deal with unavoidable differences in raw materials obtained from natural sources, and therefore, lot-to-lot variability of media must be considered. In addition, the performance of media prepared in a laboratory or by a manufacturer is highly dependent on preparation and storage conditions.虽然生产用培养基可以采用各组份配制而成，但许多化验室为了方便，采用培养基干粉或采购商业制备的装在塑料培养皿或玻璃容器中的培养基。培养基生产商会尽量采用同一生物来源的原料，但这些原料不可避免会有差异，因此需要考虑到培养基批次之间的差异。另外化验室制备的或者生产商制备的培养基的性能很大程度度上取决于培养条件和存贮条件。Improper media preparation can cause unsatisfactory conditions for microbial growth or recovery and unreliable results.培养基制备不当会导致微生物生长或回收条件不理想，从而导致不可靠的结果。Therefore, quality control tests should be performed on all prepared media, including media associated with swabs or media in strips and other nontraditional formats. Tests routinely performed on in-house prepared media should include pH, growth promotion, inhibition, and indicative properties (as appropriate), and periodic stability checks to confirm the expiration dating.因此，需要对制备的所有培养基进行质量控制检测，包括擦拭结合培养基、条状培养基和其它非传统方式。一般公司自制培养基检测应包括pH值、促生长试验、抑制试验和指示性试验（如需要），并进行定期稳定性检查以确认有效期。When in-house prepared microbiological media are properly prepared and sterilized using a validated method, the growth-promotion testing may be limited to each incoming lot of dehydrated media, unless otherwise instructed by the relevant compendial method. If the media preparation procedure was not validated, then every batch of media should be subjected to growth-promotion testing. Test organisms may be selected from the appropriate compendial test chapter. In addition, microorganisms used in growth-promotion testing may be based on the manufacturers recommendation for a particular medium, or may include representative environmental isolates (but these latter are not to be construed as compendial requirements).如果公司采用经过验证的方法进行培养基制备和灭菌，则促生产试验可以是只针对购入的每个培养基干粉批次，除非相关药典方法另有要求。如果培养基制备程序未经验证，则所制备的每个批次培养基均需要进行促生长试验。检验用菌种可以从相应的药典检测章节中选择。另外，对于特殊的培养基，用于促生长试验的菌株可以采用生产商推荐的品种，或包括有代表性的环境隔离物（但后者不构成药典要求的内容）。Expiration dates on media should have supporting growth-promotion testing to indicate that the performance of the media still meets acceptance criteria up to and including the expiration date. The length of shelf life of a batch of media will depend on the stability of the ingredients and formulation under specified conditions, as well as the type of container and closure.培养基的有效期应能支持促生长试验，在培养基有效期内及到到有效期时，该培养基应仍符合可接受标准。一个批次培养基货架期长度取决于配料成份的稳定性、在特定条件下的配方，以及容器和密封的形式。When a batch of media does not meet the requirements of growth-promotion testing, an investigation should be initiated to identify the cause. This investigation should include a corrective action plan to prevent the recurrence of the problem. Any batch of media that fails growth-promotion testing is unsuitable for use.如果培养基的促生长试验失败，则应启动调查程序寻找原因。该调查应包括纠正措施以防止相同问题重复发生。所有促生产试验失败的培养基不得用于检测。NOTEFailed growth-promotion test results may not be used to negate positive test results. 注：促生产试验失败可能会导致阳性检测为阴性。Some reagents are used for diagnostic purposes to help support identification of microbial organisms, e.g., Gram stain and oxidase test reagents. These may have attributes that can be quality control tested similar to microbiological media. Select the correct quality control standard microorganisms, following the manufacturers instructions, and perform the testing before unknown sample diagnostic testing. All relevant diagnostic reagents should be subjected to incoming quality confirmation before use.有些诊断试剂用于帮助鉴别微生物种类，例如革兰氏染色和氧化酶检测试剂。这些试剂具有与微生物培养基类似的进行质量控制测试的特性。选择正确的质量控制标准微生物，按照生产商指示，在未知样品诊断检测前进行测试。所有相关的诊断试剂均应进行进厂质量确认才可使用。Special care should be taken with media that is used in sterility tests (seeSterility Tests71for requirements) and in environmental monitoring studies. Media used for environmental monitoring of critical areas should preferably be double-wrapped and terminally sterilized. If terminal sterilization is not performed, media should be subjected to 100% pre-incubation and inspection before use within a critical area. NOTEGrowth-promotion testing for this media must be performed after the preincubation stage. This will prevent extraneous contamination from being carried into controlled environments and will prevent false-positive results. A raised agar level for surface contact plates should be verified.用于无菌检测（见71无菌检测）和环境监控研究的培养基需要特别注意。用于关键区域环境监控的培养基最好用双层包装，并采用最终灭菌方式。如果无法进行最终灭菌，则在使用前应进行100%预培养并检查【注：该培养基的促生长试验必须在预培养阶段后进行】。这样能防止由于携入受控制环境中所产生的额外污染，防止假阳性结果。接触碟培养基厚度应进行确认。MAINTENANCE OF MICROBIOLOGICAL CULTURES微生物培养维护Biological specimens can be the most delicate standards to handle because their viability and characteristics are dependent on adequate handling and storage. Standardizing the handling and storage of cultures by the user laboratory should be done in a way that will minimize the opportunity for contamination or alteration of growth characteristics. The careful and consistent treatment of stock cultures is critically important to the consistency of microbiological test results. Cultures for use in compendial tests should be acquired from a national culture collection or a qualified secondary supplier.生物种属可能是最精致的分类标准，因为其活性和属性取决于充分的处理和存贮。在使用菌种的化验室，其菌种处理和存贮应采用标准化程序以最大程度降低污染或生长特性变异。对库存菌种进行小心的处理对于保证微生物检验结果的一致性是至关重要的。在药典检测中使用的菌种应从国家菌种保藏中心或有资质的二级供应商处获取。They can be acquired frozen, freeze-dried, on slants, or in ready-to-use forms. Confirmation of the purity of the culture and the identity of the culture should be performed before its use in quality control testing.菌种可以是冰冻、冻干、斜面或即用即配形式。在将其用于质量控制检测前应对培养物的纯度进行确认和鉴别。Ready-to-use cultures should be subjected to incoming testing for purity and identity before use. The confirmation of identity for commonly used laboratory strains should ideally be done at the level of genus and species.临用现配菌种应在进厂时检测其纯度和鉴别方可使用。化验室常用菌株的鉴别最好在其属和种的层次进行。Preparation and resuscitation of cultures should follow the instructions of the supplier or a validated, established method. The “Seed-Lot” technique is recommended for storage of stock cultures.菌种的制备和接种应遵守供应商的指示，或遵守经过验证的已有的方法。建议对菌种保藏采用“种子批”技术。The original sample from the national culture collection or a qualified secondary supplier is resuscitated and grown in an appropriate medium. Aliquots of this stock culture (the first transfer or passage) are suspended in a cryoprotective medium, transferred to vials, and frozen at 30or below, until use. If stored at 70, or in lyophilized form, strains may be kept indefinitely. These frozen stocks can then be used to inoculate monthly or weekly working cultures. Once opened, do not refreeze unused cell suspensions after culturing a working suspension. The unused portion should be discarded to minimize the risk of loss of viability and contamination of the stock.从国家菌种保藏中心或有资质的二级供应商获得的原始样品在适当的培养基中进行复苏和生产。库存菌种（第一次传递或通道）的分装为低温防护培养基形成混悬液，转移至小瓶，在-30下冷冻直至使用。如果在-70下存贮，或以冻干形式存