Bacterial culture technology in rabbit farm laboratory (what about fungal infection in rabbit farm)

Isolation and culture of bacteria is a very important step in laboratory diagnosis of rabbit f

Bacterial culture technology in rabbit farm laboratory (what about fungal infection in rabbit farm)

Isolation and culture of bacteria is a very important step in laboratory diagnosis of rabbit farm. Through the isolation and pure culture of bacteria, we can make preliminary identification according to the morphology, size, structure, color, smell and hemolysis of colonies, and then make identification or research through further staining and microscopic examination or special culture, so as to provide basis for the diagnosis of pathogens.

1. medium preparation is a very important material in the process of bacteriological examination, which contains nutrients necessary for bacterial growth. Only after the bacteria are cultured in the culture medium can they be accurately examined and reliable results can be obtained.

(1) Preparation of meat water:

① Material: 500g lean beef, 1000 ml distilled water (or ordinary water).

② Method: Remove fat and tendons from lean beef, cut it into pieces or mince it, soak it in water at the ratio of 1kg beef and 2000ml water for one night, boil it for one hour, preliminarily filter the meat residue with gauze, squeeze out the meat water, filter it with filter paper, make up the raw water, put it in a flask, and sterilize it with 120℃ autoclave for 20 minutes to make meat water.

Another method of making meat water is: take 0.5- 1.0g of beef paste, add distilled water 100ml, heat to dissolve, filter with filter paper, and sterilize as above.

③ Use: the basis for making various culture media.

(2) Nutritional broth:

① materials: meat water 1000ml, peptone 10g and sodium chloride 5 g.

② Methods: Peptone and sodium chloride were added into the meat water, dissolved by slight heating, the pH value was adjusted to 7.4-7.6 with sodium hydroxide solution, and then heated to make the solution uniform. Filtering, packaging, and autoclaving.

③ Usage: It can be used for general bacterial growth needs, checking bacterial growth performance, and can also be used as the basis for making solid culture medium.

(3) nutrient agar:

① Materials: Nutrition agar powder 40g, distilled water 1000ml.

② Method: Add agar powder into distilled water, stir and soak for 65,438 0 hours, heat until it is completely dissolved, and autoclaved for later use.

Purpose: Pure culture of isolated bacteria and as the basis of other special culture media.

(4) blood agar medium:

① Materials: defibrinated blood 5- 10 ml, nutrient agar 100 ml.

Methods: Prepare a sterile triangular bottle filled with glass beads, aseptically collect blood, inject it into the triangular bottle, and keep shaking until the blood fibers are separated, which usually takes 10 minutes.

After the autoclaved nutrient agar is melted, when it is cooled to 55-60℃, defibrinated blood is added. Pay attention to aseptic operation. After adding blood, gently shake it to avoid bubbles, and immediately pour it into a sterile dish or sub-package it in a sterile test tube for later use.

③ Use: To observe the hemolysis of bacteria. Some bacteria need this culture medium for their first separation.

(5) Anaerobic liver slice broth:

① Materials: nutrient broth, animal liver block or meat residue, liquid paraffin.

② Methods: A small amount of minced meat, meat residue or animal liver was used as broth, which was divided into test tubes, and 5 ml of nutrient broth was added, and 0.5- 1.0 ml of liquid paraffin was added to the upper layer, and 120℃ was sterilized for 20 minutes.

③ Use: Cultivation of anaerobic bacteria.

(6) Mai Kangkai agar:

① Materials: Mai Kangkai agar powder 38g, distilled water 1000ml.

Methods: Mai Kangkai agar powder was added into distilled water, stirred and soaked for 1h, heated until it was completely dissolved, and autoclaved for later use.

③ Use: It is often used for the isolation and culture of Escherichia coli.

2. Bacterial inoculation method

(1) plate streaking inoculation method: plate streaking inoculation method is the most commonly used method in bacterial isolation and culture, which can make bacteria grow dispersedly and form a single colony. Hold the Petri dish with your left hand, and uncover the thumb, forefinger and middle finger at an angle of no more than 20 degrees. Hold the inoculation ring in your right hand, and the inoculation ring forms an angle of about 45 degrees with the agar surface. Smear a little diseased material on the edge of the culture medium, flame sterilize the inoculation ring, touch the diseased material area on the edge of the culture medium after cooling, and draw a horizontal line to one side, so that you can draw a plot continuously, flame sterilize the inoculation ring, after cooling, change the angle of the flat plate, draw a plot from the first plot to the outside, and then draw the flat plate in the same way, paying attention to crossing the previous plot only at the initial position.

(2) Agar slant inoculation method: This method is mostly used for the preservation of strains and can also be used for the separation of bacteria. Hold the slant medium in the left hand and the inoculation ring in the right hand. After flame sterilization and cooling, pick out the required colonies, hold the cotton plug or rubber plug with the little finger of the right hand, take it out after slight rotation, pay attention to avoid pollution, immediately sterilize the nozzle and plug with flame, put the inoculation ring into the test tube, draw a curve from the bottom of the inclined plane to the nozzle, and then sterilize the nozzle and plug with flame and cover it tightly for culture.

(3) Puncture inoculation method: This method is suitable for inoculation of semi-solid, solid culture medium and micro fermentation tube, and is used for biochemical and kinetic experiments of bacteria. Puncture along the center of the culture tube until it is about 0.5 cm away from the bottom of the tube, and then exit the inoculation needle along the puncture line.

(4) liquid culture medium inoculation method: the method is basically the same as the slant inoculation method. The inoculation ring rubs under the liquid level of the culture tube wall, then vibrates several times in the liquid to plug the plug. Pay attention to the sterilization operation of plugs and nozzles.

3. Bacterial culture method

According to the growth characteristics, cultivation purposes and requirements of bacteria, the cultivation methods of bacteria are different. Mainly divided into the following categories:

(1) Aerobic culture method: general culture method. Bacteria were inoculated into the culture medium and cultured in an incubator at 37℃. Suitable for the separation and culture of aerobic bacteria and facultative anaerobic bacteria.

(2) Carbon dioxide culture method: Some bacteria can grow well in a certain carbon dioxide environment.

① Candle jar method: put the inoculated culture medium into a dryer, light a candle, seal it, and put it in an incubator together with the container for culture.

(2) Chemical method: according to the ratio of 0.4 g sodium bicarbonate and 3.5 ml hydrochloric acid in each liter container, put it into two small containers, put it into a dryer, and contact it after sealing to produce carbon dioxide.

③ Carbon dioxide incubator method: the culture medium is cultured in a special carbon dioxide incubator, and the concentration of carbon dioxide can be adjusted as needed.

(3) Anaerobic culture method:

① Ventilation method: put the culture medium into a dryer, vacuum the dryer, then fill the mixed gas of nitrogen, hydrogen and carbon dioxide, and put it in an incubator for culture.

② pyrogallic acid method: pyrogallic acid forms pyrogallic acid salt in alkaline solution, which can absorb oxygen in the air, thus creating an anaerobic environment suitable for the growth of anaerobic bacteria. Culture method: Remove the cover of the Petri dish, turn it over, put pyrogallic acid 1g, then cover it with a layer of absorbent cotton, drop 1ml of IO% sodium hydroxide on the absorbent cotton, then cover the turned cover, quickly seal it with wax, and put it in an incubator at 37℃ for 2-4 days, and observe the results. If the inoculated culture medium is a test tube, take a large test tube, put 2g pyrogallic acid at the bottom, add glass beads above 10, put the culture medium test tube on the glass beads, then drop 2ml 10% sodium hydroxide solution along the wall of the test tube, plug the mouth of the test tube with a rubber stopper, and put it in an incubator at 37℃ for culture.

③ Inoculating anaerobic liver slices in broth: Because liver slices or meat residue contain glutathione, oxidation reaction can occur, thus reducing the oxidation potential energy in the environment and facilitating the separation of anaerobic bacteria. The inoculation method is to take diseased materials with an inoculation ring hook, inoculate them into the culture medium, and cultivate them in an incubator at 37℃ for 2-4 days, and observe the results.