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。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 manufacturer's 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 (See Weighing 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. See Cleaning 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 conditions—for commercially prepared or internally prepared media—may result in a difference in color change, loss of clarity, altered gel strength, or pH drift from the manufacturer's 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121℃15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. See pH791for 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

—Crystal formation 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）

—Lot number 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 manufacturer's 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 manufacturer's 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。[NOTE—Failed 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 manufacturer's 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 (see Sterility 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. [NOTE—Growth-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 –30℃ or 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，或以冻干形式存贮，菌种可以无限期地存贮。这些冷冻存贮的菌种可以用作每周或每月的工作菌种。未使用的部分应该弃去以最大程度降低活性损失的风险和存贮污染的风险。

The number of transfers of working control cultures should be tracked to prevent excessive subculturing that increases the risk of phenotypic alteration or mutation. The number of transfers allowable for specific compendial tests may be specified in that test. One passage is defined as the transfer of organisms from a viable culture to a fresh medium with growth of the microorganisms. Any form of subculturing is considered to be a transfer/passage.

工作控制菌种的传代应可以追溯，以防止过度传代，从而增加表型变异性或异化的风险。特定的药典检测所允许传代的次数必须在该检测中指明。一种途径可以界定为从活菌传代至新鲜培养基，在其中微生物可以生长。任何形式的接种都应被作为是传代/通道。LABORATORY EQUIPMENT 实验室设备

Most equipment (incubators, water baths, and autoclaves) is subject to standard validation practices of incoming qualification, operational qualification, and performance qualification. Additionally, periodic calibration (generally annually) is commonly required. New equipment, critical to the operation of the laboratory, should be qualified according to a protocol approved by the quality assurance unit (QAU). In addition, regular cleaning and sanitization of equipment such as incubators, refrigerators, and water baths should be performed to minimize the potential for contamination in the laboratory. Door seals of incubators and refrigerators should be cleaned and checked for state of repair.

主要设备（培养箱、水浴锅、灭菌器）需要进行标准验证，包括进厂确认、操作确认和性能确认。另外，一般还需要对其进行周期性校正（一般每年校正）。化验室操作的关键新设备应根据质量部门批准的方案进行确认。此外，对设备，如培养箱、冰箱和水浴锅，进行定期清洁和灭菌都是需要的，这样可以最大程度降低化验室中污染的风险。培养箱和冰箱的门封应进行清洁并对维修状态进行检查。

Instruments (pH meters and spectrophotometers) used in a microbiology laboratory should be calibrated on a regular schedule and tested to verify performance on a routine basis. The frequency of calibration and performance verification will vary based on the type of instrument and the importance of that equipment to the generation of data in the laboratory.

在微生物化验室使用的仪器（pH计和光度计）应按计划进行定期校正，并测试以按照常规要求确认其性能。校正频率和性能确认可以根据仪器的类型和仪器重要性而制订不同的周期。

Equipment that is difficult to sanitize (such as refrigerators and incubators) should be dedicated to aseptic operations (such as storage of media for testing and incubation of sterility test samples) and live culture operations to minimize the potential for inadvertent contamination of the tests.

难以灭菌的设备（例如冰箱和培养箱），如果用于无菌操作（例如测试用培养基存贮和无菌测试样品的培养）和活菌操作，则分开专用，以最大程度降低可能无意的污染。Autoclaves are central to the operation of the laboratory and must have proper validation in place to demonstrate adequate sterilization for a variety of operations. Autoclave resources must be available (and validated) to sterilize waste media (if performed in that laboratory) as well as the media prepared in that laboratory. The choice of one or several autoclaves is not driven by a need to separate aseptic and live operations (everything in the properly maintained autoclave is sterile after the cycle) but rather driven by resource considerations (see below).

灭菌器是化验室操作的核心设备，必须经过适当验证以证明其不同操作的灭菌保障。灭菌器资源（经过验证）应支持废培养基灭菌（如果在化验室灭菌），在化验室制备的培养基灭菌。是选择用一台还是多台灭菌器不需要根据将无菌操作和活菌操作分开的原因（所在经过适当维护的灭菌器灭菌的物质均是无菌的），而是应根据化验室要保证提供操作所需的充分资源（参见下文）来考虑。

LABORATORY LAYOUT AND OPERATIONS 实验室平面设计和操作

Laboratory layout and design should carefully consider the requirements of good microbiological practices and laboratory safety. It is essential that

cross-contamination of microbial cultures be minimized to the greatest extent possible, and it is also important that microbiological samples be handled in an environment that makes contamination highly unlikely.

化验室平面设计应全面考虑优良微生物规程和化验室安全要求。对此基本的要求是应尽可能降低微生物培养基的交叉污染，同样重要的是样品操作的环境应保证无污染。

In general, a laboratory should be divided into clean or aseptic areas and live culture areas. Areas in which environmental or sterile product samples are handled and incubated should be maintained completely free of live cultures, if possible. If complete separation of live and clean culture zones cannot be accomplished, then other barriers and aseptic practices should be employed to reduce the likelihood of accidental contamination. These barriers include protective clothing, sanitization and disinfection procedures, and biological safety cabinets designated for clean or aseptic operations only. Procedures for handling spills or mishaps with live cultures should be in place, and all relevant technical personnel should be trained regarding these methods.

一般来说，微生物检验室应分为洁净区域或无菌区域、活菌区域。如有可能，环境监控样品或无菌产品样品操作和培养的区域应保持无活菌。如果活菌和无菌培养区域无法完全隔离，则应采用物理隔断和灭菌措施以降低可能的意外污染。这些物理隔断包括防护服、灭菌和消毒程序，仅供洁净或无菌操作所用的生物安全柜。应对操作中活菌洒出或灾祸制订书面程序，所有相关的技术人员均应接受此相关培训。

Some samples will demonstrate microbial growth and require further laboratory analysis to identify the contaminants. When growth is detected, the sample should be taken from the clean section of the laboratory to the live culture section without undue delay. Subculturing, staining, microbial identification, or other investigational operations should be undertaken in the live culture section of the laboratory. If possible, any sample found to contain growing colonies should not be opened in the clean zone of the laboratory. Careful segregation of contaminated samples and materials will reduce false-positive results.

有些样品可能会发现有微生物生长，需要进一步的实验分析以确定污染物。如果发现有微生物生长，样品应立即从洁净区域移至活菌区域。次培养、染色、微生物鉴别，或其它调查操作应在活菌区域进行。如有可能，所有发现有菌株生长的样品均不可化验室洁净区域打开。对受污染样品和物料小心进行隔离可以减少假阳性结果。

Staff engaged in sampling activities should not enter or work in the live culture handling section of a laboratory unless special precautions are taken, including wearing protective clothing and gloves and careful sanitizing of hands upon exiting.

Ideally, staff assigned to sampling activities, particularly those in support of aseptic processing, should not work in the vicinity of live culture laboratory operations.

取样人员不应进入或在活菌操作区域工作，除非采取了特殊的预防措施，包括穿着防护服和手套、退出时进行仔细的手消毒。理想情况下，取样人员尤其是那些无菌工艺的取样人员，不应在活菌操作附近工作。

It is important to consider that microbial contamination of samples, which leads to false-positive results, is always possible unless careful aseptic precautions are taken. Facilities should be designed so that raw material and excipient sampling can be done under controlled conditions, including proper gowning and sterilized sampling equipment. It may not always be possible to sample utility systems, such as water systems, under full aseptic conditions; however, it should be noted that when samples are not taken aseptically, their reliability is inevitably compromised.

考虑样品被污染的可能性是非要重要的，这种情况可能会导致假阳性结果，除非采取了严格的无菌预防措施，否则总会存在这种可能性。设施的设计应使原料和辅料取样操作在控制环境下进行，包括进行适当的更衣和对取样工具进行灭菌。对公用系统进行无菌条件下取样有时可能无法实现，例如水系统，此时如果不是在无菌条件下取样应注意其可靠性不可避免地会降低。

Environmental sampling methods should require minimal aseptic handling in loading and unloading sampling instruments. Whenever possible, sampling equipment should be loaded with its microbiological recovery media in the environment that is to be sampled.

环境取样方法应尽可能减少取样工具的无菌操作。只要可能，取样工具应在将要取样的环境里载入其微生物回收培养基。

All testing in laboratories used for critical testing procedures, such as sterility testing of final dosage forms, bulk product, seed cultures for biological production, or cell cultures used in biological production, should be performed under controlled conditions. Isolator technology is also appropriate for critical, sterile microbiological testing. Isolators have been shown to have lower levels of environmental contamination than manned clean rooms, and therefore, are generally less likely to produce false-positive results. Proper validation of isolators is critical both to ensure environmental integrity and to prevent the possibility of false-negative results as a

result of chemical disinfection of materials brought into or used within isolators

(see Sterility Testing—Validation of Isolator Systems1208).

在化验室进行的所有关键检测，例如制剂无菌测试、原料药、生物制品接种、或生物制品细胞培养，均应在受控环境下进行。分离技术也适用于关键的无菌微生物测试。隔离器经证明比手工清洁房间具有更低的环境污染水平，因此一般不可能产生假阳性结果。对隔离器进行适当的验证很关键，可以保证环境的完整性，以及防止由化学消毒物质引起的可能的假阳性结果。（参见无菌测试----隔离系统验证1208）

SAMPLE HANDLING 样品制备

Viable microorganisms in most microbiology samples, particularly water, environmental monitoring and bioburden samples, are sensitive to handling and storage conditions. Critical parameters in these conditions include product (or sample) composition, container composition, time of storage, and temperature of storage. Therefore, it is important to minimize the amount of time between the sampling event and the initiation of testing and to control, as much as possible, the conditions of storage. If the sample is to be transported to a distant location for testing, then the conditions of transport (time, temperature, etc.) should be qualified as suitable for that test and sample. Guidance for water testing in this regard can be found in Water for Pharmaceutical Purposes 1231 . Product mixing before sampling may need to be evaluated and applied in order to ensure microbial dispersement and representation in the sample aliquot.

大多数微生物样品中的活菌，尤其是水样、环境监控样品和生物负载样品，对处置过程和存贮条件非常敏感。这些环境下的关键参数包括产品（或样品）成分、容器成份、存贮时长、存贮温度。因此，尽量缩短取样和检验开始的时间间隔非常重要，以及尽可能控制存贮环境。如果样品需要送到一个较远的检测场所，则传送条件（时间、温度等）应针对所做的检测和样品进行确认以保证其合适。有关水检测的指南参见1231制药用水。在取样前即进行混合的产品可能需要进行评估，以保证微生物水平及样品代表性。All microbiological samples should be taken using aseptic techniques, including those taken in support of nonsterile products. If possible, all microbiological samples should be taken under full aseptic conditions in specialized sampling areas. The areas should be as close to the point of use as possible to minimize contamination during transit.

所有用于微生物检测的样品均应在无菌环境下取样，包括用于非无菌制剂的样品。如有可能，所有微生物检测用样品应在特定的无菌条件的取样区域取样，该区域应与使用时的环境尽可能接近，以最大程度降低转移过程中的污染。

Samples submitted to the microbiology laboratory should be accompanied by documentation detailing source of the sample, date the sample was taken, date of sample submission, person or department responsible for the submission, and any potentially hazardous materials associated with the sample. The testing department should acknowledge receipt of the sample and reconcile the identity and number of samples as part of this sample documentation.

样品传递至微生物化验室时应附有详细的记录，包括样品来源、取样日期、样品传递日期、传递人员或部门、样品可能会含有的危险物质。检验部分应告知样品收到，并核对编号和样品数量，在取样记录中注明。

MICROBIOLOGICAL MEDIA INCUBATION TIMES 微生物培养基培养时间Incubation times for microbiological tests of less than 3 days' duration should be expressed in hours: e.g., ―Incubate at 30 to 35 for 18 to 72 hours‖. Tests longer than 72 hours' duration should be expressed in days: e.g., ―Incubate at 30 to 35 for 3 to 5 days‖. For incubation times expressed in hours, incubate for the minimum specified time, and exercise good microbiological judgment when exceeding the incubation time. For incubation times expressed in days, incubations started in the morning or afternoon should generally be concluded at that same time of day.

微生物测试的培养时间如果少于3天，则应表述为小时，例如“在30-35℃培养18-72小时”。如果大于72小时，则应表述为天数，例如“在30-35℃培养3-5天”。如果培养时间以小时计，则培养时间应为指定的最短时间，如果超出指定的培养时间，则需要应用好的微生物判断。如果培养时间以天计，则上午或下午开始进行培养一般会在结束天的同一时间进行判定。

TRAINING OF PERSONNEL 人员培训

Each person engaged in each phase of pharmaceutical manufacture should have the education, training, and experience to do his or her job. The demands of microbiological testing require that the core educational background of the staff, supervisors, and managers be in microbiology or a closely related biological science.

They should be assigned responsibilities in keeping with their level of skill and experience.

参与药品生产各阶段活动的所有人员均需要进行教育、培训，具备其所操作岗位的经验。微生物检测岗位要求操作人员、主管和经理的基本教育背景为微生物学或相关生物学科。公司应根据人员的技能水平和经验赋予不同的责任。

A coherent system of standard operating procedures (SOPs) is necessary to run the microbiology laboratory. These procedures serve two purposes in a training program. Firstly, these SOPs describe the methodology that the microbiologist will follow to obtain accurate and reproducible results, and so serve as the basis for training. Secondly, by tracking the procedures in which a particular microbiologist has demonstrated proficiency, the procedure number or title also serves to identify what training the microbiologist has received specific to his or her job function.

要使微生物室正常运行，需要有一套条理分明的标准操作规程（SOP）。这些程序对于培训来说有两个目的，一是这些SOP描述了微生物化验员需要遵守以获得准确和可重复的结果的方法，从而作为培训的基础教材，二是在SOP中由指定的微生物化验员进行了专业描述，该SOP的编号和题目可以用以识别微生物化验员接受了其岗位所对应的培训。

Training curricula should be established for each laboratory staff member specific to his or her job function.

对各化验室人员，应根据其岗位要求制订相应的培训课程。

He or she should not independently conduct a microbial test until qualified to run the test. Training records should be current, documenting the microbiologist's training in the current revision to the particular SOP.

化验室人员在未获得资格前，不得独立进行微生物测试。培训记录应保持更新，记录微生物化验员根据特定SOP现行版本所进行的培训。

Periodic performance assessment is a wise investment in data quality. This performance testing should provide evidence of competency in core activities of the microbiology laboratory such as hygiene, plating, aseptic technique, documentation, and others as suggested by the microbiologist's job function.

对化验员进行周期性的绩效评估是对数据品质的明智投资。这种行为测试应提供微生物核心活动合格的证据，如卫生、铺碟、无菌操作、记录和其它微生物化验员工作内容的活动。

Microbiologists with supervisory or managerial responsibilities should have appropriate education and inhouse training in supervisory skills, laboratory safety, scheduling, budgeting, investigational skills, technical report writing, relevant SOPs, and other critical aspects of the company's processes as suggested in their role of directing a laboratory function.

微生物室具有监督或管理职责的人员应具有适当的教育背景，以及公司内部培训，内容包括监管技巧、化验室安全、制订计划、制订预算、调查技巧、技术报告书写、相关SOP、公司程序所要求其岗位应具体的领导化验室的职责的其它关键方面。Competency may be demonstrated by specific course work, relevant experience, and routinely engaging in relevant continuing education. Achieving certification through an accredited body is also a desirable credential. Further, it is expected that laboratory supervisors and managers have a demonstrated level of competence in microbiology at least as high as those they supervise. Expertise in microbiology can be achieved by a variety of routes in addition to academic course work and accreditation. Each company is expected to evaluate the credentials of those responsible for designing, implementing, and operating the microbiology program. Companies can thus ensure that those responsible for the program understand the basic principles of microbiology, can interpret guidelines and regulations based on good science, and have access to individuals with theoretical and practical knowledge in microbiology to provide assistance in areas in which the persons responsible for the program may not have adequate knowledge and understanding. It should be noted that microbiology is a scientifically based discipline that deals with biological principles substantially different from those of analytical chemistry and engineering disciplines.

人员是否合格可以通过特定工作、相关经验和日常从事相关持续教育进行证明。获得相关机构的证书也是一种理想的方法。另外，最好化验室主管和经理能具备其所监管的微生物工作的至少同样水平的能力。除通过专业课程和专业机构证书外，也可以通过不同的方法达到微生物专业水平。公司应评估负责微生物程序设计、实施和操作的负责人员的证书，从而保证这些人员具备基本的微生物原理，可以对指南和法规基于科学理论进

行解释，并知晓哪些人具备理论和实践微生物知识，能在负责某程序的人不具备充分知识和理解时提供相关的帮助。需要注意的是，微生物学是一门科学严谨的学问，是与生物原则相关的学科，根本区别于分析化学和工程学原理。

Many times it is difficult for individuals without specific microbiological training to make the transition.

一个人如果没有特定的微生物培训知识，要进行知识传递是非常困难的。LABORATORY RESOURCES 化验室资源

The laboratory management is responsible for ensuring that the laboratory has sufficient resources to meet the existing testing requirements. This requires some proficiency in budget management and in determining appropriate measures of laboratory performance. A measure of laboratory performance is the number of investigations performed on tests conducted by the laboratory, but this measure alone is not sufficient. In addition to tracking investigations, the period of time between sample submission and initiation of testing should be tracked, as well as the period of time between end of test and report release (or test closure). Significant delays in these measures are also indications of an under-resourced laboratory staff.

实验室管理人员应保证实验室具有充足的资源，以满足现有的检测要求。要做到这点，需要有充足的预算以及对化验室表现做出适当的评估。化验室表现的评估标准之一是化验室所做检测的调查数量，但仅采用该标准是不够的。除了追踪调查之外，还应追踪样品传送间隔和检测开始时间，以及检测结束到出具报告（或检测关闭）的时间间隔。这些行为的延迟也说明化验室人员未满足资源要求。

The laboratory management should have sufficient budget to meet testing requirements. Particular measures of budgetary requirements will be specific to the given laboratory, but budgetary considerations related directly to the need of the laboratory for sufficient resources must be addressed to ensure reliable testing results.

化验室管理应具备充足的预算，以满足检测的要求，预算应细化针对各特定的化验室，与化验室资源是否充分直接相关的预算要求则必须进行说明，以保证获得可靠的检测结果。

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