Peptone 1g

0.5g sodium chloride

Agar 2g

Tap water 100 ml

Sterilization at PH 7.0~7.21.05 kg/cm2 for 22 min.

Specific preparation steps

1.

Beef paste, peptone and NaCl are accurately weighed in turn according to the proportion of culture medium formula, and put into a beaker. Beef sauce is generally a glass rod, weighed in a small beaker or watch glasses, melted with hot water and poured into the beaker. It can also be placed on weighing paper and directly put into water after weighing. At this time, with a little heating, the beef sauce will be separated from the weighing paper, and then the paper will be taken out immediately. Peptone absorbs moisture easily, so it should be weighed quickly. In addition, when weighing drugs, be careful not to mix drugs. Use a horn spoon for one medicine, or after weighing one medicine, wash it and dry it, and then weigh another medicine. Don't put the wrong cap on the bottle.

thaw

In a beaker, you can first add less water than you need, stir it with a glass rod, and then heat it on an asbestos net to dissolve it. After the drug is completely dissolved, add water to the required total volume. If a solid culture medium is prepared, the weighed agar is put into the dissolved medicine and then heated to melt it. In the process of agar melting, it is necessary to keep stirring to prevent the beaker from breaking at the bottom of agar paste. Finally, make up the lost water.

Adjust pH value

Before adjusting the pH value, measure the original pH value of the culture medium with precision pH test paper. If the pH is acidic, add 1mol/L NaOH into the culture medium drop by drop with a dropper, while stirring, and measure the pH value with a pH test paper at any time until the pH reaches 7.6. Otherwise, use 1mol/L HCl for adjustment. Be careful not to adjust the pH too high to avoid callback, otherwise it will affect the ion concentration in the culture medium.

For some microorganisms that need more accurate pH value, the pH value can be adjusted with an acidity meter (please refer to the relevant instructions for usage).

filter

Filter with filter paper or multi-layer gauze while it is hot, so as to facilitate observation of the results. If there is no special requirement, this step can generally be omitted (filtering is not needed in this experiment). Many don't need to be filtered.

Step 5: Repackage.

According to the experimental requirements, the prepared culture medium can be divided into test tubes or triangular bottles. See figure V- 1 for the subpackage equipment.

Be careful not to touch the culture medium on the nozzle or bottle mouth during packaging, so as not to pollute the tampon and cause pollution.

(1) The suitable height of liquid packaging is about 1/4 of the height of test tube.

(2) Pack the solids in separate test tubes, the loading capacity shall not exceed 1/5 tube height, and sterilize the test tubes to make inclined planes, as shown in Figure V-2. The quantity of repackaged triangular bottles should not exceed half of the volume of triangular bottles.

(3) Generally, the height of semi-solid packaging test tube should be 1/3, and it should be placed vertically after sterilization.

6.gasser

After repackaging the culture medium, put a cotton plug on the test tube mouth or triangular bottle mouth to prevent external microorganisms from entering the culture medium to cause pollution and ensure good ventilation performance (the method of making cotton plug is attached to the back of this experiment).

bind up

After plugging, tie all test tubes with hemp rope, then wrap a layer of kraft paper around the cotton plug to prevent the condensed water from wetting the cotton plug during sterilization, and then tie them with hemp rope. Mark the name, group and date of the media with a marker. The triangular cork is wrapped in kraft paper and tied with hemp rope in the form of slipknot, which is easy to untie when used. Similarly, the name, group and date of the culture medium are indicated by marks.

disinfect

The above culture medium was autoclaved at1.05 kg/cm2 (15 lb/in2) and 12 1.3℃ for 20 minutes. If it cannot be disinfected in time due to special circumstances, it should be put into the refrigerator for temporary storage.

9. Leave an inclined plane

The sterilized test tube culture medium is cooled to about 50℃, and the cotton plug end of the test tube is placed on the glass rod, and the length of the inclined plane is not more than half of the total length of the test tube.

10. Sterility inspection

Put the sterilized culture medium in a greenhouse at 37℃ for 24-48 hours, and check whether the sterilization is complete.

1. Living material: Bacillus thuringiensis.

2. Culture medium: beef paste peptone agar culture medium (Appendix III, 1).

3. Equipment: 90ml sterile water, 9ml sterile water, sterile tray, lml sterile straw, balance, sample weighing bottle, marker, glass scraper, etc.

Fourth, experimental methods.

1. Preparation of sample diluent: accurately weigh 10g of the sample to be tested, put it in a 250ml triangular bottle filled with 90ml sterile water and small glass beads, shake it with hands or a shaker for 20min to disperse microbial cells, and let it stand for 20-30s to obtain 10- 1 diluent; Suck 1 0-1diluent lml with a sterile pipette of1,transfer it into a test tube filled with 9ml sterile water, suck it for three times, and mix the bacterial liquid evenly to obtain 10-2 diluent; Another sterile pipette sucks 10-2 diluent 1ml, moves it into a test tube filled with 9ml sterile water, and sucks it for three times to obtain l0-3 diluent; And so on, by continuous dilution, 10-4, 10-5, 10-6, 10-7, 10-8, 10-9, etc. (Figure 22-9

Fig. 22- 1 plate counting method for sample dilution and diluent sampling and culture

When counting with dilution plate, the dilution of the bacteria to be detected should be determined according to the sample. When the number of bacteria to be detected in the sample is large, the dilution should be high, and vice versa. Usually, the dilutions of 10-7, 10-8 and 10-9 are used to determine the number of bacteria, and the dilutions of 10-4, 10-5 and 10-6 are used to determine the number of bacteria in soil.

2. Plate inoculation culture There are two methods of plate inoculation culture: mixed plate culture and smear plate culture.

(1) The numbers of sterile plates in mixed plate culture method are 10-7, 10-8 and 10-9, and each number has three replicates. According to the requirements of aseptic operation, suck the diluent 10-9 1 ml with a sterile pipette and put it in the serial number 1ml. Suck 10-8 diluent per lml in the same way and put it in the three plates numbered 10-8, and then suck 10-7 diluent per lml and put it in the three plates numbered 10-7 (the pipette does not need to be replaced when the concentration changes from low to high). Then pour the bacterial culture medium that has been melted and cooled to 45-50℃ into 9 plates respectively (Figure 22-2), gently rotate the plates to mix the bacterial liquid and the culture medium evenly, invert it after cold suspicion, and cultivate it at a suitable temperature. You can count the colonies until they grow up.

(2) Smear counting method The smear counting method is basically the same as the mixing method, except that the culture medium is melted and poured into a sterile plate while it is hot, and numbered after solidification, and then 0. 1ml bacterial liquid is sucked by a sterile pipette and inoculated on agar plates with different dilution numbers (each number is set to three replicates). Then spread the bacterial liquid evenly on the flat plate with a sterile spatula (Figure 22-3), and use a sterile spatula for each dilution. When changing the dilution, it is necessary to burn and disinfect the spatula. When smearing from low concentration to high concentration, the spatula may not be replaced. Lay the coated plate flat on the table for 20-30 minutes, so that the bacterial liquid can penetrate into the culture medium, then turn the plate upside down, keep the temperature for culture until the colonies grow, and then count.