According to the preliminary clinical diagnosis and bacterial species and the different gas needs of bacteria to be examined, it can be divided into aerobic culture method, carbon dioxide culture method and anaerobic culture method. This experiment is derived from the laboratory instruction of Mudanjiang Medical College undergraduate 5-year testing program.
Operation method
bacterial culture experiment Move A. Aerobic culture method: This method is the most commonly used culture method in clinical bacteriological room, suitable for general aerobic and parthenogenetic anaerobic bacteria culture. The common temperature of aerobic culture is 35-37℃, which is suitable for the growth of most pathogenic bacteria. In addition, there are 4 ℃ culture, such as Listeria monocytogenes in addition to 37 ℃ in the growth. Listeria monocytogenes can also grow at 4 ℃, while other Gram-positive bacteria can not, so it can be used to identify. Listeria monocytogenes has power when cultured at 25℃, while Campylobacter fetus can grow at 25℃. For more product details, please visit Aladdin Scientific website.
Carbon dioxide culture method: some bacteria such as Neisseria meningitidis, Neisseria gonorrhoeae and Brucella, when first isolated from clinical specimens, need to have 5% to 10% CO2. environment, these bacteria to grow well. The usual methods of supplying COZ are:
1, M carbon dioxide incubator: a special box, that is, to adjust the content of CO2, but also to adjust the required temperature (generally 35-37 ℃), CO2 is a cylinder regularly filled with 99.99% content of CO2, and then the cylinder and the CO2 incubator CO2 delivery tube connected to the vacuum gauge on the cylinder to indicate the amount of CO2 output, this method can be used for large-scale laboratory applications.
2、Candle cylinder method: Inoculate the plate or tube culture medium, put it into a desiccator, and put in a lighted candle. The edge of the lid of the desiccator coated with petroleum jelly, cover the lid, the candle after a few minutes of self-extinguishing, at this time the CO2 content of the desiccator accounted for about 5-10%, and then put the culture of the desiccator in the 37 ℃ incubator culture.
3, chemical method: commonly used sodium bicarbonate hydrochloric acid method. According to the volume of each liter of sodium bicarbonate 0.4 g, 0.35 ml of concentrated hydrochloric acid ratio, respectively, into the container, the container into the desiccator, to be placed in the culture, tighten the lid of the desiccator, tilt the container so that concentrated hydrochloric acid and sodium bicarbonate contact to generate CO2.
Third, anaerobic culture method: this method is applicable to the culture of specialized anaerobic bacteria. There are chemical methods, biological methods and comprehensive methods. The commonly used anaerobic methods are:
l, Butcher's meat medium method: prepared from horse meat or beef dregs with appropriate amount of broth, applicable to all anaerobic bacteria, especially the culture of Clostridium difficile.
2, pyrogallic gallic acid: in a clean glass plate placed on a piece of sterilized sandcloth, add a small amount of pyrogallic gallic acid and KOH (NaOH) on the sandcloth. Immediately cover the plate culture medium of the inoculated specimen, and seal the plate around with paraffin to prevent oxygen from entering. After the chemical reaction of the above drugs, oxygen can be absorbed, creating an anaerobic environment.
This method is simple and suitable for general laboratories, but the chemical reaction produces pyrogallic gallic acid, which has a certain toxic effect on bacteria, so the isolation rate is not high.
3, anaerobic tank method: the use of cans can be sealed to physical or chemical methods to cause anaerobic environment. Commonly used pumping method and cold catalyst method. The former draw out the air in the tank, filled with nitrogen, so repeated three times, and finally filled with 10% H2, 10% CO2, 80% N2 gas mixture, and palladium for the catalyst, catalytic O2 and H2 synthesis of water, to achieve anaerobic environment. Inside the tank, there is a melamine indicator liquid, which is blue when there is oxygen, and is reduced to colorless when there is no oxygen. The latter in the anaerobic tank placed in the gas bag, the bag is equipped with potassium borohydride, sodium bicarbonate and citrate made of tablets, when used to cut open the upper corner, add 10 ml of water, and immediately cover the jar lid, twisting the fixed card, at this time by the gas bag produced by the H2, the target particles in the tank beforehand catalyzed by the canister with the same tank of the combination of O2 into water, 10-30s that is, the lid of the canister and the wall of the canister appeared on the droplets, the tank anaerobic conditions indicated by the liquid melamine indicator. The anaerobic condition in the tank is indicated by liquid melamine indicator. This method is effective and suitable for large and medium-sized laboratories.
4、Gas bag method: Inoculate the plate culture medium, put it into the special plastic gas bag, and seal the mouth of the bag, the anaerobic principle is the same as the anaerobic cold catalyst method. This method is simple, suitable for bedside inoculation and grass-roots laboratories.