Medium commonly used in microbiology laboratories
mediumIt can be prepared by prescription or use a prescribed dehydrated medium.
In the preparation of media, dehydrated media or individual formula components that meet the requirements of quality should be selected for preparation. Dehydrated media should be accompanied by prescription and instructions for use. When formulating, it should be operated according to the requirements on the instructions for use to ensure that the quality of the media meets the requirements. No agglomerated or color-altered dehydrated media should be used. Dehydrated media or individual formula components should be stored under appropriate conditions, such as low temperature, dry and protected from light. All containers should be sealed, especially those containing dehydrated media. In addition to prescription and instructions for use, commercialized finished media should also indicate the valid period, storage conditions, quality control bacteria for suitability inspection tests and uses.
In order to ensure the stability and reliability of the quality of the medium, each dehydrated medium or each formulation component should be accurately weighed and require a certain degree of precision. The most commonly used solvent for preparing the medium is purified water. In special cases, deionized water and distilled water may be required. The weight of each weighing object and the amount of water used should be recorded. The container and supporting utensils used for preparing the medium should be clean, and the glassware can be rinsed with purified water to eliminate the residue of detergent and foreign substances. For heat-sensitive media such as sugar fermentation medium, the sub-packaging container should generally be sterilized in advance to ensure the sterility of the medium.
The dehydrated medium should be completely dissolved in water and then packaged and sterilized. If heating is required to assist dissolution during preparation, care should be taken not to overheat to avoid darkening the color of the medium. If other components need to be added, they should be fully mixed after addition.
Media should be sterilized according to parameters provided by the manufacturer or verified by the user. Commercial finished media must be accompanied by information on the sterilization method used. Medium sterilization generally uses moist heat sterilization technology, and special media can be sterilized by membrane filtration.
The use of inappropriate heating and sterilization conditions in the medium may cause color changes, reduced transparency, agar coagulation power or changes in pH. Therefore, the medium should be sterilized by a verified sterilization procedure. The method and conditions of medium sterilization should be verified by sterility test and growth promotion test. In addition, the steam circulation system of the autoclave should also be verified to ensure normal heat distribution under certain loading methods. An autoclave with a slow rise in temperature may cause the medium to overheat, and excessive sterilization may destroy the growth-promoting quality of the vast majority of bacterial and fungal media. The volume and loading method of the medium in the sterilizer will also affect the rate of heating. Therefore, a comprehensive verification of the sterilization procedure should be carried out according to the characteristics of the sterilized medium.,
The pH of each batch of media should be determined after sterilization (measured by cooling to room temperature 25 ° C). If the pH range is listed in the medium prescription, the pH range should not exceed the specified ± 0.2 unless it is verified that the pH range of the medium is allowed to vary widely.
Media made into plates or sub-packaged in test tubes should be inspected as follows: containers and lids should not be broken, the amount should be the same, the formation of bubbles should be avoided as much as possible, cracks or ripples should not occur on the surface of solid media, crystallization should not be formed at refrigerated temperatures, and microorganisms should not be contaminated. The batch quantity, valid period and sterility inspection of the medium should be checked and recorded.
Storage of culture medium
Self-prepared media should be labeled with information such as name, batch number, preparation date, etc., and stored under verified conditions. Commercial finished media should be labeled with the name, batch number, production date, expiration date and relevant characteristics of the medium. Manufacturers and users should store according to the requirements of the medium instruction manual. The storage and transportation conditions should be used to minimize the loss of moisture of the finished medium and provide mechanical protection.
After the medium is sterilized, if it is stored in an autoclave, the quality may be affected, and this storage method is generally not recommended.agar mediumDo not store at or below 0 ° C, as freezing may damage the gel properties.
The medium should be stored away from light. If it is to be stored for a long time, it should be placed in a closed container to prevent water loss. The agar plate is best used now. If it is stored in a refrigerator, it should generally not exceed one week, and it should be tightly packed. If the storage is extended, the storage period needs to be verified and determined.
The re-melting of the solid medium after sterilization is only allowed once to avoid the quality of the medium being degraded or microbial contamination due to excessive heating. The re-melting of the medium generally uses water bath heating or circulation steam. If using a microwave oven, excessive heating of the medium and evaporation of water should be avoided, and more attention should be paid to safety. The melted medium should be placed in a water bath at 45~ 50 ° C for no more than 8 hours. When pouring the medium, the water on the outer surface of the medium container should be dried to avoid water droplets from the outer wall of the container entering the medium and causing pollution.
Used culture media (including failed culture media) should be disposed of in accordance with national regulations on the disposal of contaminated waste.
Quality control test of culture medium
The laboratory should establish a quality control program for the experimental medium (that is, there must be a medium quality control sop) to ensure that the quality of the medium used meets the needs of relevant testing.
The quality of finished media prepared in the laboratory or commercialized depends on its preparation process, and media prepared by inappropriate methods will affect the growth or recovery of microorganisms, thus affecting the reliability of test results.
The prepared medium should be subject to regular quality control tests. The routine monitoring items for the media prepared in the laboratory are pH, suitability check tests, and regular stability checks to determine the valid period. The quality of the medium should be determined according to the suitability check test during the valid period. The length of the valid period will depend on the stability of the composition of the medium under certain storage conditions (including container characteristics and tightness).
In the laboratory, if the medium is prepared using a proven formulation and sterilization procedure and the process is controlled, the suitability test of the same batch of dehydrated media can be carried out only once. The strains tested can be selected from the relevant appendices according to the purpose of the medium, or the contaminating strains commonly found in the production environment and products can be added. If the quality control test of the medium does not meet the regulations, the cause of the non-conformity should be found to prevent the problem from recurring. Any medium that does not meet the requirements should not be used.
Media used for environmental monitoring must be particularly protected. Media used for critical area monitoring should preferably be double-packed and end point sterilized. If end point sterilized media cannot be used, 100% pre-culture should be carried out before use to prevent foreign contaminants from being introduced into the environment and to avoid false positive results.
In fact, in our microbiology laboratory, the quality control of culture media has always been a problem that many laboratories are very troubled by. I believe it is very helpful for everyone to study GB 4789.28-2013 "Quality Requirements for Media and Reagents".
