Different types of media preparation processes
Different types ofmediumThe preparation procedures are also different, and can be mainly divided into 8 steps: batching, melting, pH correction, clarification and filtration, sub-packaging, sterilization, and verification.
1. Ingredients: Weigh various ingredients accurately according to the medium prescription, first add a small amount of distilled water to the triangular flask, and then add various ingredients to prevent peptone from adhering to the bottom of the bottle, and then rinse the bottle wall with the remaining water.
2. Melting: Mix various ingredients in water, preferably with circulating steam for half an hour. If it melts on an electric furnace, it should be stirred at any time. If there are agar ingredients, more attention should be paid to preventing spillage. After melting, make up for the lost moisture.
3. Correct pH: pH determination, take 3 test tubes of the same diameter as the standard tube (usually Fahrenheit test tubes), add 5ml of the medium to be measured in the first and third tubes, and add 0.2g/L of phenol red 0.25ml in the first tube as the measuring tube, mix well. pH correction, if the measurement tube peracid or peralkali can be corrected with 0.1mol/L sodium hydroxide or 0.1mol/L hydrochloric acid solution until the color is the same as the standard tube, add alkali or acid to be accurate and slow, every 1 drop should be fully mixed, and the second drop (sometimes only half a drop) should be added after colorimetry to accurately record the amount added. Calculation, set 5ml medium to correct pH to 7.4 requires 0.1mol/L sodium hydroxide 0.15ml, existing medium 4990ml, the amount of sodium hydroxide to be added can be calculated according to the following method: 5:4990 = 0.15: X = 0.15 × 4990/5 = 149.7 (ml), if this 0.1mol/L sodium hydroxide is replaced by 1mol/L sodium hydroxide, 14.9ml is required.
4. Filtration clarification: After the medium is prepared, there is generally sediment or turbidity, which needs to be filtered into clear and transparent before use. The commonly used filtration methods are as follows:
The liquid medium must be clear in order to observe the growth of bacteria. Filter paper is commonly used. Before heating, add chicken protein diluted with water (1000ml medium with 1 chicken protein) and heat at 100 ° C for 60 to 70 ° C for 40 to 60 minutes, so that its insoluble substances adhere to the coagulated protein and precipitate, and then siphon out the supernatant or filter it with filter paper. Solid medium such as agar medium, after heating and melting, it needs to be filtered with flannel or two layers of gauze in absorbent cotton while hot; it can also be used
Natural precipitation method, coming soonagar mediumIn an aluminum pot or a wide-mouth enamel container, melt with high-pressure (103.43kPa) steam for 15 minutes, let it stand in a pressure cooker overnight, pour out the agar the next day, cut off the bottom sediment with a knife, and then melt to collect clear agar medium.
5. Sub-packing: Sub-packing the medium in triangular flasks and test tubes of different capacities as needed. The sub-packing amount should not exceed 2/3 of the container to avoid spillage during sterilization. The sub-packing amount of agar inclined surface is 1/5 of the capacity of the test tube. After sterilization, it must be placed into an inclined surface while it is hot. The length of the inclined surface is about 2/3 of the length of the test tube. The sub-packing amount of semi-solid medium is about 1/3 of the length of the test tube. After sterilization, it should stand upright while it is hot and solidify after cooling. The liquid medium should be sub-packed in the test tube, which is about 1/3 of the length of the test tube. Agar plate: After sterilization (or heating and melting), the culture medium is cooled to about 50 ° C, and it is poured into the sterilized flat plate with aseptic procedures. The flat plate with an inner diameter of 9cm is poured with about 13~ 15ml of the culture medium, and the bottom of the flat plate is gently shaken. Medical | Education Network collects and organizes, so that the culture medium is tiled on the bottom of the flat plate. After solidification, it is ready. When pouring, do not open all the lids of the plate to avoid dust and Egyptian bacteria in the air falling into it. The newly made flat plate culture medium (referred to as the flat plate) has a lot of moisture on the surface, which is not conducive to the separation of bacteria. Usually, the flat plate should be placed upside down in the 37 ° C incubator for about 30 minutes until the flat plate is dry. Use.
6. Sterilization: Different ingredients and properties of culture media can be sterilized by different methods. Autoclave Sterilization: The temperature and time of autoclave sterilization vary with the type and quantity. Generally, a small amount of high-pressure (103.43kPa) can be sterilized for 15 minutes. When the dosage is large, it can be sterilized by high-pressure (103.43kPa) for 30 minutes, and the sugar-containing medium can be sterilized by high-pressure (55.16kPa) for 15 minutes. To avoid the destruction of sugar.
Verification: After each batch is made, it must be verified before it can be used. During the inspection, the medium is incubated in a 37 ° C incubator for 24 hours to prove sterile. At the same time, the growth and reproduction and biochemical reactions on this medium are checked with known strains. Only those who meet the requirements can use it.
8. Storage: The prepared medium should not be stored for too long, but should be done with a small amount of diligence. Each batch should be marked with the name, sub-packaging amount, production date, etc., and placed in the refrigerator at 4 ° C for later use.
