Separation and cultivation of microorganisms

Separation and cultivation of microorganisms

一、Separation and cultivation

Microorganisms exist in a mixed state in nature. To obtain the desired strains, they must be separated from them. If it is contaminated accidentally when it is preserved, it must be purified. There are many methods for the separation and purification of microorganisms, but the basic principle is similar. That is, the sample to be separated is diluted to a certain extent, and the cells (or spores) of the microorganism are kept in a dispersed state as much as possible, and then they are grown into pure single colony. However, the above work is inseparable from the process of inoculation, which is to transfer a microorganism to another sterilized medium.

Microbiological inoculation classification material and tool:

Constant temperature incubator, inoculation ring, glass rod, pipette, alcohol lamp, culture medium, E. coli, Bacillus subtilis, Staphylococcus aureus, yeast, etc.

Operation method of inoculation:

1. Slant inoculation (connect Staphylococcus aureus)

Before operating, wipe your hands with 75% alcohol, and ignite the alcohol lamp after the alcohol volatilizes. Hold the strain tube and the inclined surface between the left thumb and the other four fingers, so that the inclined surface and the side with the strain are facing upward and in a horizontal position. First, rotate the strain and the tampon of the slope to make it easy to pull out when inoculating. Hold the inoculation ring in your left hand (like holding a pen), sterilize the ring end on the flame first, and then sterilize it by extending into the rest of the test tube. Use the ring finger, pinky finger and palm of your right hand to pull out the germ tube and the cotton plug or test tube cap of the inclined test tube at the same time, and then slowly sterilize the test tube mouth (do not burn too hot). Extend the burned inoculation ring into the seed tube, contact the inoculation ring on the inner wall of the test tube or the medium without moss, let it cool down sufficiently, then gently scrape a little moss and then pull it out of the tube Vaccination ring. Quickly extend the inoculated ring with bacteria into another test tube to be connected. Make a "Z" shape from the bottom of the slope up and down densely. Sometimes it is also possible to use an inoculation needle to pull a line in the center of the medium for oblique inoculation, so as to observe the growth characteristics of the bacteria. After the inoculation is completed, the inoculation loop is withdrawn and the cotton plug is inserted. Sterilize the vaccination ring. Put down the inoculation loop and tighten the cotton plug.

2. Liquid inoculation

Connect the inclined medium to the liquid medium. This method is used to observe the growth characteristics of the bacteria and the measurement of the biochemical reaction. The operation method is the same as before, but the test tube mouth is inclined upward, so as to prevent the culture fluid from flowing out and connecting the bacteria, inoculate Rub the ring and the inner wall of the tube a few times to wash the bacteria on the ring. After inoculation, a cotton plug is inserted, and the test tube is gently tapped in the palm of the hand to fully disperse the bacteria. Inoculate the liquid medium from the liquid medium. When the strain is liquid, use a sterile pipette or dropper in addition to the inoculation ring. When inoculating, just pull out the cotton plug next to the flame, pass the mouth of the tube through the flame, use a sterile pipette to suck the bacterial solution into the culture solution, and shake it evenly.

3. Plate inoculation

Score and spread the bacteria on the plate. Cross-line inoculation See separate cross-line method. Spreading and inoculation Inject the bacterial solution into the plate with a sterile pipette, and evenly coat the surface of the plate with a sterilized glass rod.

4. Vaccination

Inoculate the bacteria into the solid deep culture medium. This method is used to inoculate anaerobic bacteria or observe physiological performance when identifying bacteria. The operation method is the same as above, but the inoculation needle used should be straight. Puncture the inoculation needle from the center of the culture medium until it is close to the bottom of the tube, but do not penetrate, and then slowly pull out the original puncture route.

二、Separation operation method:

1. Dilution separation method

Disperse the separated sample to the minimum by continuous dilution, then draw a certain amount into the plate and mix it with the agar medium that is suitable for melting, so that the dispersed bacteria are fixed in place to form a single colony. Escherichia coli or yeast is prepared with sterile water as a bacterial suspension. Take several sterile test tubes, each containing 9ml of sterile water. Pipette 1ml of the prepared bacterial suspension and place it in the first test tube containing 9ml of sterile water, so that it is diluted 10 times, which is 10-2. Draw 1ml from the first test tube (10-2) and inject it into the second test tube containing sterile water, so that it is diluted 100 times, which is 10-2. Operate in the same way until it is diluted to 10-5-10-6. Accurately draw 0.2ml of the bacterial solution of each dilution at 10-5-10-6 and add it to the numbered empty sterile dish. Repeat the same dilution to make three dishes. Pour the agar medium that has been melted and cooled to 45 into each of the above-mentioned plates, gently rotate to mix the medium and the bacterial suspension thoroughly, after solidification, put it in a 37 or 38 degree incubator and incubate for 24-48 hours, observe The growth and distribution of colonies on the plate.

2. Flat-bed scribe separation method

Plate streaking separation method is a method to separate microorganisms by dividing the inoculation ring on the surface of the plate medium by zoning. The principle is to dilute the microbial sample "from point to line" on the surface of the solid medium several times to achieve the purpose of separation. Pour the plate Dissolve the beef extract peptone agar medium, pour the plate, and let it stand horizontally until it sets. Burn the inoculation ring on the flame of alcohol lamp, and wait for cooling, take a mixed inoculation liquid of Staphylococcus aureus and Escherichia coli. Hold the agar plate in the left hand and lift the lid slightly, while approaching the flame. Hold the inoculation ring in the right and extend into the dish. Make an scribe line in an area on the plate. The inoculation ring is 30-40° from the surface of the plate. Gently touch the angle, and use your wrist force to slide lightly on the surface. Do not scratch or embed the surface of the tablet into the base. Burn the inoculation cup to kill the remaining bacterial liquid on the inoculation ring. After cooling, extend the inoculation ring into the dish. After making a little contact with the line crossed in the first area, turn 90°. The two areas continue to be crossed. After marking, inoculate the inoculation cup, after cooling, use the same method to draw lines in other areas. After all the lines are marked, use a special crayon on the bottom of the dish to indicate the strain, date, group and name. Place the entire Petri dish upside down and place it in a constant temperature incubator. 37 After 24-48 hours of cultivation, take out and observe. Pay attention to the characteristics of the colony switch, size, color, edge, surface structure, transparency, etc.

三、Precautions：

1. The inoculation room should be kept clean, scrub the countertops and walls with pulverized phenol soap liquid, and fumigate regularly with lactic acid or formaldehyde. Before each use, it should be sterilized by UV lamp. Regularly check the sterility of the inoculation room. Before entering the vaccination room, personal hygiene should be done first, and work shoes, hats, work clothes, and masks should be replaced in the buffer room. Work clothes, work shoes and masks are only allowed to be used in the vaccination room. It is not allowed to go to other places, and it should be replaced and disinfected regularly. The inoculated test tubes, triangles and bottles should be marked with the name and date of the culture medium and strain. All items moved into the inoculation room must be wiped clean with 70% alcohol in the buffer room. Before inoculation, sterilize both hands with 70% alcohol or Xinjieer, do not leave the flame of the alcohol lamp during the operation; the tampon is not placed randomly; the inoculation tools need to be flame sterilized before and after use.

2. The incubator should be cleaned and disinfected frequently.