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Freeze drying machine for microbial strains: liquid nitrogen cryopreservation technology

Time: 2020-09-11 10:04 Source: unknown Author: Bander liquid nitrogen tank Click: second
The vacuum freeze-drying machine for microbial strains is a kind of equipment for drying materials at low temperature under the condition of vacuum. It is mainly used for the drying of microbial preparations and bioactive substances. The bacteria preservation room is used for vacuum freeze-drying preservation of microorganisms. Freeze drying is one of the best methods for long-term preservation of bacteria. Because of its convenience for mass production, long-term preservation, high vitality and simple storage and transportation equipment, it has become a widely used preservation method and has been widely used to preserve most bacteria, fungi and some viruses. liquid nitrogen tank

1. Preparation of ampoule tube or frozen storage tube
Use round bottom borosilicate glass products ampoule tube, or screw mouth plastic frozen tube. Note that the glass tube must not have cracks. Clean the frozen storage tube or ampoule tube and sterilize it at 121 ℃ for 15-20 minutes.

2. Preparation of protective agent
The types of protective agents should be selected according to the types of microorganisms. When preparing protective agent, attention should be paid to its concentration. Generally, 10-20% glycerol is used.

3. Preparation of microbial preservation
It has an effect on the survival rate of microorganisms at different stages of maturation. It should be operated under aseptic condition.

4. The following methods can be used for the preparation of strains:
Scrape the spores or bacteria on the slant of the culture, mix them with the protective agent, and then add them into the cryopreservation tube;
Inoculate the liquid culture medium, mix the bacterial suspension and protective agent after oscillatory culture, and then put them in the cryopreservation tube;
The culture material was cultured on a plate to form bacteria. Some small pieces of uniform size (about 5-10 mm in diameter) were cut from the plate with a sterile perforator. The bacteria blocks on the edge of the colony were taken from the fungus and mixed with the protective agent, and then added into the frozen storage tube;
Put 1.2-2 ml agar medium into the small ampoule tube, inoculate the strain, and add protective agent after 2-10 days of culture.

5. Pre freezing
Generally, the freezing speed should be controlled at 1 ℃ per minute to freeze the sample to - 35 ℃.
At present, there are three commonly used methods of temperature control
——The method of programmed temperature reduction, which uses the electronic computer program to control the cooling device, can steadily and continuously reduce the temperature and control the cooling rate well.
——Section cooling method: the bacteria in the refrigerator of different temperature levels or liquid nitrogen tank It can be cooled in sections or suspended in ice mist. Generally, the ampoule tube or plastic tube is put into the refrigerator from - 20 ℃ to - 40 ℃ for 1-2 hours, and then it is taken out and put into the refrigerator liquid nitrogen tank Freezing is fast. In this way, the freezing rate decreases by about 1-1.5 ℃ per minute.
——For low temperature resistant microorganisms, it can be directly put into gas phase or liquid phase nitrogen.

6. Preservation
Put the ampoule tube or plastic frozen tube in the liquid nitrogen tank for storage. Generally, the temperature in the gas phase is - 150 ℃ and the temperature in the liquid phase is - 196 ℃.
Preservation cycle
Generally more than 10 years.

7. Recovery methods
Take out the ampoule tube or plastic frozen storage tube from the liquid nitrogen tank, put it in the water bath at 38 ℃ - 40 ℃ for rapid recovery and shake it properly. Until the internal ice dissolves completely, it usually takes about 50 seconds to 100 seconds. Open the ampoule tube or plastic cryopreservation tube and transfer the contents to the appropriate medium for culture.

8. Scope of application
All kinds of microorganisms.
Precautions for liquid nitrogen storage
——Take care of the safety belt and gloves to prevent frostbite
——The screw cap must be tightened for plastic frozen storage pipe
——When transporting liquid nitrogen, special containers must be used. Never store or transport liquid nitrogen in closed containers. Do not use thermos to store liquid nitrogen
——Pay attention to the indoor ventilation of liquid nitrogen container to prevent excessive nitrogen from suffocating people
——If liquid nitrogen seeps into the tube, the volume expansion of liquid nitrogen will be about 680 times when it is taken out of the liquid nitrogen container, and the explosion force is very large. Therefore, special care should be taken liquid nitrogen tank
——Pay attention to observe the residual amount of liquid nitrogen in liquid nitrogen container and replenish liquid nitrogen regularly.

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