How to make culture medium? Simple to make
1. Selection of medium formula
same kindmediumTherefore, in addition to the standard method used, which should be strictly formulated according to its provisions, it is generally necessary to collect relevant information as much as possible, compare and check it, and then choose and record its source according to its own purpose of use.
2. Preparation records of the culture medium
Each preparation of the medium should be recorded, including the name of the medium, the formula and its source, and the grades of various ingredientspHThe value, the temperature and time of disinfection, the date of preparation, and the person who prepared it, etc., should be copied, and the original records should be kept for future reference. The copied records should be stored with the prepared medium to prevent confusion.
3. Weigh the components of the medium
The various components of the medium must be accurately weighed and attention should be paid to prevent confusion. It is best to complete the formula at one time without interruption. The formula can be placed on the side. After each component is weighed, it is marked on the side of the preparation, and the medicines to be weighed are taken at one time and placed on the left side. After each weighing is completed, it is moved to the right side. After the complete weighing is completed, an inspection should be carried out.
4. Mixing and melting of various components of the medium
Medium usedchemicalsAll should be chemically pure. The cooking pot used shall not be a copper pot or an iron pot to prevent trace amounts of copper or iron from being mixed into the culture medium, making it difficult for bacteria to grow. It is best to use a stainless steel pot for heating and dissolving. It can be boiled and dissolved in a large beaker or a large flask with a high pressure steam sterilizer or a flowing steam sterilizer. When dissolving in the pot, it can be heated with warm water first and disturbed at any time to prevent coking. If coking is found, the medium cannot be used and should be reprepared. After most of the solid components are dissolved, use a small amount of fire to completely dissolve all the ingredients until boiling. If agar is dissolved, use another part of water to dissolve other ingredients, and then mix the two solutions thoroughly. During the process of heating and melting, the water lost due to evaporation must eventually be replenished.
5. Preliminary adjustment of medium pH
Because the pH of the medium will change during the heating and disinfection process, the pH of the medium should be adjusted initially after the components of the medium are completely dissolved. For example, the pH of the beef infusion can be reduced by about 0.2, while the pH of the intestinal infusion will be significantly increased. Therefore, for this step, the operator should always pay attention to the exploration experience, so as to grasp the final pH of the medium and ensure the quality of the medium. After the pH adjustment, the medium should also be boiled for a few minutes to facilitate the precipitation of the medium sediment.
6. Filtration and clarification of the medium
The liquid medium must be absolutely clear, and the agar medium should also be transparent without significant precipitation. Therefore, filtration or other clarification methods need to be used to achieve this requirement. Generally, the liquid medium can be filtered by filter paper, and the filter paper should be folded into a folding fan or funnel shape to avoid filter paper cracking due to uneven hydraulic pressure.
The agar medium can be filtered hot with a clean white flannel. It can also be filtered with a double-layer gauze with a thin layer of absorbent cotton sandwiched in the middle. When soaking new meat, liver, blood and potatoes, the debris must be filtered off with a flannel first, and then filtered repeatedly with filter paper. If the filtration method cannot meet the clarification requirements, the egg white clarification method must be used. The medium that is about to be cooled to 55-60 ° C is placed in a large triangular flask, and the amount should not exceed 1/2 of the flask capacity. Add 1-2 egg whites to every 1000ml of medium, shake it vigorously for 3-5 minutes, put it in a high-pressure steam sterilizer, heat it at 121 ° C for 20 minutes, take it out and filter it with a flannel while it is hot.
7. Packing of culture medium
The dispensing of culture media should be in appropriate containers such as test tubes and flasks according to the purpose and requirements of use. The dispensing amount shall not exceed 2/3 of the container's capacity. The mouth of the container can be sealed with a tampon covered with moisture-proof paper, and the outside must be wrapped with waterproof paper (the test tubes are generally used with screw caps). It is best to use a semi-automatic or electric quantitative dispenser when dispensing. When dispensing agar bevel media, the dosage amount should be appropriate to form 2/3 of the bottom layer and 1/3 of the bevel. The dispensing container should be cleaned in advance and dry-baked and disinfected to facilitate thorough sterilization of the medium. Each batch of medium should be separately packed with 20ml of medium in a small glass bottle, and sterilized simultaneously with the batch of medium for the purpose of determining the final pH of the batch of medium.
8. Sterilization of culture medium
Generally, the medium can be sterilized by autoclaving at 121 ° C for 15 minutes. In various medium preparation methods, if there are no special regulations, this method can be used for sterilization.
Certain heat-resistant ingredients, such as sugars, should be prepared separately in a concentrate of 20% or more, disinfected by filtration or batch sterilization, and then added to the medium by aseptic operation techniques and quantification. Gelatin media should also be sterilized at a lower temperature. Blood, body fluids, and antibiotics should be drawn and added to the medium cooled to about 50 ° C by aseptic operation techniques.
Agar bevel media should be taken out immediately after sterilization, cooled to 55 ° C -60 ° C, and placed in an appropriate bevel until it solidifies naturally.
9. Quality testing of culture medium
After each batch of medium is prepared, it should be carefully checked. If it is found to be broken, water immersed, abnormal color, tampon contaminated with the medium, etc., it should be picked out and discarded. And determine its final pH.
All media were incubated overnight in a 36 ± 1 ° C incubator, and discarded if bacterial growth was found.
With the relevant standardsstrainInoculate 1 to 2 tubes or bottles of medium for 24 to 48 hours, if aseptic growth or poor growth. The cause should be traced and repeated inoculation. If the results are still the same, the batch of medium should be discarded and cannot be used.
10. Preservation of culture medium
The medium should be stored in a cool and dark place, preferably in a normal refrigerator. The storage time should not exceed one week, and the poured plate medium should not exceed 3 days. Each batch of medium must be accompanied by a subsheet or a clear label for the preparation of the batch of medium.
