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.

培养基是微生物实验的基础。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、适宜的存储条件和质量控制试验是提供优质培养基的保证。

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分是非常重要的。一般生产商在供应干粉培养基和成品培养基时，其配方和使用说明都会随货发送。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），重要的一点是需遵守其提供的使用说明以保证配制出的培养基质量。如果是成品培养基，随货应有指明有效期和存储条件的分析报告，以及促生长试验、选择性试验所用的微生物。

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.

水是配制培养基常用的溶剂。一般使用纯化水，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 Balance <1251)>.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.

要保证培养基配制的一致性，需要对培养基干粉或培养基组分进行准确称量。应使用经过校准的天平，其称量范围应与所称的重量相符（见1251分析天平称量）。应对称量容器和工具（例如称量勺）进行清洁以保证无异物进入所配培养基。各成分的重量应进行记录。

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. 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.

培养基干粉应在水中完全溶解，然后进行灭菌。如果需要加热溶解，要注意不能过度加热，因为所有的培养基，或多或少，都是对热敏感的。使用合适的设备配制培养基配制，以便控制加热、持续搅拌和混合培养基。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匀。

Preparation of media in poorly cleaned glassware can allow inhibitory substances to enter the media.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 Apparatus <1051> for additional guidance.

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。抑制性物质可能来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. 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.

培养基灭菌应在生产商提供的参数范围内，或用户验证的参数范围内实施。商品化的预制培培养基应随货有所用灭菌方法的资料。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，灭菌周期应进行验证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，因此较大的负载可能需要较长的灭菌时间。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，在培养基灭菌要求和培养基在过热条件下分解中寻找平衡点。在灭菌结束冷却后，不建议将培养基留在灭菌器中存储，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、凝胶强度改变、或pH值超出生产商指定范围、以及促生长和/或具有选择性下降。

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 pH <791> for 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；冷藏的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。

•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（盖不应粘在碟上）

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