Introduction to microbial culture medium configuration and sterilization methods and procedures

The microbial culture medium is a mixed nutrient for the growth, reproduction and metabolism of microorganisms. Because microorganisms have different types of nutrients, the requirements for nutrients are also different, and the purpose of the research is different. Therefore, there are many kinds of culture media, and the raw materials used are also different, but from the perspective of nutrition, the microbial culture medium is generally Contains carbon sources, nitrogen sources, inorganic salts, auxins, and water necessary for microorganisms. In addition, the medium should also have a suitable pH, a certain buffering capacity, a certain redox potential and a suitable osmotic pressure. In order to achieve specific requirements for research, media preparation and sterilization are required. How should microbial culture media be formulated and sterilized?

1. Preparation solution

Add a part of the required amount of water to the container, and according to the formula of the medium, weigh the various raw materials, add them in order to dissolve them, and finally make up the required water. For the peptone, meat paste and other substances, it needs to be heated and dissolved, and evaporated by heating. The moisture should be added to the water after all the raw materials are dissolved.

When preparing the solid medium, first boil the prepared liquid medium, then add the weighed agar, continue heating until it is completely melted, and stir constantly to avoid burning the agar bottom.

2. Adjust pH

The pH of the test medium is tested with a pH test paper (or pH potentiometer, hydrogen ion concentration colorimeter, BLBIO-2020 pH controller). If it is not met, it can be adjusted with 10% HCl or 10% NaOH until it is adjusted. The pH required for the formulation.

3. Filter

Filter the culture medium with filter paper, gauze or cotton. When filtering with gauze, it is best to fold into six layers. When filtering with filter paper, the filter paper can be folded into a tile shape and filtered on a funnel.

4. Packing

The filtered medium should be dispensed. If you want to make a slant medium, you must dispense the medium in a test tube. If you want to make a plate medium or a liquid, semi-solid medium, you must divide the medium into a cone. Inside the bottle.

When dispensing, hold the spring clip in one hand to allow the medium to flow out, and hold the test tube or conical flask in the other hand and pick up the medium in turn. When disassembling, be careful not to allow the medium to adhere to the nozzle or the bottle mouth to avoid contamination of the bacteria by soaking the cotton plug.

The amount of the medium to be placed in the test tube depends on the size of the test tube and the conical flask and the need. Generally, when the slant medium is prepared, each test tube of 15 × 150 mm is about 3 to 4 ml (1/4 to 1/1). 3 test tube height), such as the preparation of deep medium, about 12 to 15 ml of each tube of 20 × 220 mm. The medium filled in each conical flask is generally half of its volume.

5. Add tampon

After the dispensing is completed, the tampon is required to block the nozzle or the mouth of the bottle. The main purpose of the plug is to filter the air and avoid pollution. The tampon should be made of ordinary fresh and dry cotton. Do not use absorbent cotton to avoid the tampon being unusable due to the absorbent cotton. When making the tampon, spread the cotton to the appropriate thickness according to the size of the tampon, grab the size of the palm of your hand, place it in the round hole formed by the thumb and index finger of the left hand, insert the middle index finger into the middle of the cotton, and the index finger of the left hand and the thumb are slightly When gripped, a long stick-shaped tampon will be formed. After the tampon is made, it should be quickly inserted into the nozzle or the mouth of the bottle. The tampon should be tightly attached to the inner wall without gaps to prevent the microbes in the air from invading along the wrinkles. Do not over-tighten the tampon. After plugging, it is suitable to use a portable cotton plug, tube or bottle. 2/3 of the tampon should be inside the tube or inside the bottle, and a little cotton is exposed at the upper end for easy extraction. Test tubes and conical flasks with tampon should be covered with thick paper and bundled for preparation.

6. Making slant medium and plate medium

After the medium is sterilized, for example, a slant medium and a plate medium are prepared, and the sputum medium is not solidified.

(1) Make a slant medium. On the experimental bench, put a wooden strip with a length of about 0.5 to 1 meter and a thickness of about 1 cm. The head of the test tube is placed on the wooden strip, so that the medium in the tube is naturally inclined, and after solidification, it becomes a slant medium.

(2) Make a plate medium. Place the conical flask and petri dish containing the culture medium just off the experimental table, ignite the alcohol lamp, hold the bottom of the conical flask with your right hand, pull the tampon from the left hand, and slightly open the bottle on the alcohol lamp. Add the burning, open the Petri dish cover with your left hand, and pour the culture medium into the Petri dish with your right hand. Pour 10 ml into each dish to cover the bottom of the dish. Place the medium and leave it for about 15 minutes. After solidification, a stack of 5 culture dishes is placed, inverted, placed in an incubator, and inspected 24 hours later. For example, the bacteria at the end of the medium can be used to culture microorganisms.

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