How do I mix the media evenly?

Generally, as long as it is a drug with good solubility, stirring it evenly in the process of preparing the medium can ensure that the drug can be evenly dissolved in the medium. Even for drugs that are not easily dissolved, the solubility of the drug will increase as the temperature increases during the autoclaving process, and after sterilization, gently shake the Erlenmeyer flask to further ensure that the drug is mixed in the medium.

Prepare 50 ml of complete medium in a ratio of 50 1050.

After adding serum, antibiotics and other substances to the basal medium, it is called complete medium, also called (serum) cell culture medium. The basal medium can only maintain cell survival, in order to make the cells grow and reproduce, it is necessary to add natural medium, commonly used is bovine serum, because bovine serum contains various growth factors that promote cell proliferation and a variety of other substances that are conducive to cell survival.

Culture medium

Due to the different raw materials prepared, the use requirements are different, and the storage and storage aspects are also slightly different. After being heated and moisture-absorbed, the general medium is easy to be contaminated or decomposed by bacteria, so the general medium must be stored in a moisture-proof, dark and cool place. For some media that need to be strictly sterilized (such as tissue culture media), they must be stored in the refrigerator for a long time.

Since the liquid medium is not easy to store for a long time, it is modified into powder.

1. Prepare the solution.

Add a part of the required amount of water, weigh various raw materials according to the formula of the medium, add them in turn to dissolve, and finally make up the required water. For peptone, meat paste and other substances, it is necessary to heat and dissolve, and the water evaporated during the heating process should be supplemented with water after all the raw materials are dissolved.

When preparing the solid medium, first boil the liquid medium that has been prepared above, then add the weighed agar, and continue to heat until it is completely melted. And stir constantly so that the bottom of the agar paste does not burn.

2. Adjust the pH value.

Test the pH of the medium with pH test paper (or pH potentiometer or hydrogen ion concentration colorimeter), if it does not meet the needs, it can be adjusted with 10% HCl or 10% NaOH until the pH value required by the formulation is adjusted.

3. Filtration. Filter the prepared medium while it is hot with filter paper, gauze, or cotton. When filtering with gauze, it is best to fold it into six layers, and when filtering with filter paper, the filter paper can be folded into a tile prismatic shape and spread on the funnel for filtration.

4. Packaging. Filtered media should be aliquoted. If you want to make bevel media, the medium must be aliquoted in test tubes. If you want to make plate media or liquid, semi-solid media, the medium must be aliquoted in Erlenmeyer flasks.

When dispensing, pinch and loosen the spring clip with one hand to let the medium flow out, and hold several test tubes or Erlenmeyer flasks with the other hand to pick up the medium in turn. When dispensing, be careful not to let the medium adhere to the mouth of the tube or bottle, so as not to soak the tampon and cause bacterial contamination.

The amount of media to be loaded into the tube depends on the size and needs of the tube and Erlenmeyer flask. In general, when making bevel medium, each 15 150 mm tube should be filled with about 3 4 ml (1 4 1 3 tube height), and if deep medium was made, each 20 220 mm tube should be filled with about 12 15 ml. The medium contained in each Erlenmeyer flask is generally half of its volume.

5. Add tampons.

After the dispensing is completed, the nozzle or bottle mouth needs to be plugged with a tampon. The main purpose of tampons is to filter the air and avoid contamination. The tampon should be made of ordinary fresh and dried cotton, and do not use absorbent cotton, so as not to make the tampon unusable due to the absorption of absorbent cotton.

When making tampons, spread the cotton to an appropriate thickness according to the size of the tampon, take a piece the size of the palm of the hand, spread it in the round hole formed by the left thumb and index finger, insert the middle of the cotton with the index finger of the right hand, and hold the index finger of the left hand slightly at the same time, which will form a long rod-shaped tampon.

After the tampon is made, it should be quickly inserted into the nozzle or bottle mouth, and the tampon should be close to the inner wall without leaving a gap to prevent the invasion of microorganisms in the air along the folds. The tampon should not be too tight or too loose, and after the plug is done, it is appropriate to keep the hand-held tampon, tube and bottle from falling. The 2 3 of the tampon should be in the tube or bottle, with a little cotton exposed at the upper end for easy pulling.

Tubes and Erlenmeyer flasks with tampons should be covered with thick paper and tied with rope and prepared for sterilization.

6. Make inclined medium and plate medium.

After the medium is sterilized, if the inclined medium and plate medium are made, it should be carried out while the medium is not solidified.

(Summary of personal original creation).

Although there are many types of culture media, the preparation process and steps are basically the same. The approximate preparation procedure is as follows:

1.Accurately weighed according to the composition, suspended in a flask filled with distilled water, heated and dissolved;

2.adjust the pH of the medium;

3.filtration clarification; The prepared medium will produce precipitation or turbidity, which needs to be filtered and clarified, which is conducive to the observation of bacterial growth.

4.Aliquoting: The prepared medium is dispensed according to the needs of actual use.

In the test tube, it can be made into agar bevel, semi-solid medium, agar layer, liquid medium, etc.; Basal medium can be stored in large bottles for later use. Some media need to be aseptically aliquoted after sterilization, such as agar plates.

5.Sterilization: The prepared medium must be sterilized before use. Different sterilization methods are adopted according to the composition and nature of the medium, and the commonly used sterilization methods include high-pressure steam sterilization, flow steam batch sterilization and serum coagulator sterilization.

6.Quality inspection All made media must pass the quality inspection before they can be used. At a minimum, sterility testing and potency testing should be performed.

7.Storage The prepared medium should be marked with the name and date of preparation, and stored in a cold and dark place or 4 refrigerator. The plate should be turned upside down. The medium should not be stored for too long.

Complete medium, also known as total nutrient medium, is a medium with complete nutrients and appropriate proportions.

Different uses, the formula of complete medium is also different, such as for cell culture, plant tissue culture, microbial culture, etc., each has different complete medium formulations.

Below is a complete medium formulation for microbial culture.

Peptone yeast extract glucose sodium chloride 5g, agar added to 1000ml of distilled water, boiled and dissolved, adjusted pH, and then sterilized at 121 for 15min, taken out, cooled slightly, and poured into a petri dish with heat.