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.
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.
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.
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.
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.
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.
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.
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.
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.
Prepared media should be checked by appropriate inspection of plates and tubes for the following:
•Cracked containers or lids 容器或盖子裂开
•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凹陷
•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）