Most microorganisms can be stored under cryopreservation, and cryopreservation is the most widely used microbial preservation method. Microbial species that require complex nutrients, such as those that cannot be maintained by other storage methods (such as plant pathogenic fungi), can usually be preserved by ultra-low temperature methods (ATCC 1983; Halliday, Baker 1985). In this method, cells frozen in small tubes or ampoules are frozen at a slower freezing rate (1 ° C / min) until -150 ° C, and these tubes are stored in liquid nitrogen at -150 ~ -196 ° C.
An ultra-low temperature cryoprotectant is different from the cryogen used in the lyophilization process. ATCC commonly uses a mixture of glycerol (10%), dimethyl sulfoxide (5%) and culture medium to preserve most cell lines. These chemicals enter the cell to avoid freezing damage to the inner membrane. Care must be taken when resuscitating cells stored in cryocontainers. When the small tube is heated, the formed ice crystals will kill the cells, and this accident can be avoided by proper operation. As long as the sample is quickly thawed, the loss of vitality can be reduced by quickly placing the sealed tube into water at 37 ° C until all the ice has melted, then opening the nozzle and moving the contents into the medium.
Micro-preserved microorganisms must always be stored in a very low temperature environment, so liquid nitrogen tanks are required. Always pay attention to the replenishment of liquid nitrogen during long-term storage. This type of storage requires more funding than lyophilization, including the labor and liquid nitrogen necessary to maintain storage temperatures.
Materials for cryopreservation of material viruses include: heat-shrinkable plastic tubes, liquid nitrogen tanks, protective gloves and masks, permanent markers, gas burners, and vacuum flasks filled with liquid nitrogen.
Method (1) Cut a heat-shrinkable plastic tube with a length of 2 cm beyond the length of the cryotube at both ends.
(2) Ice bath with clarified virus suspension (supernatant medium for tissue culture, or cell lysate in tissue culture medium), and 0.2 ml ice bath supernatant with a sterile pipette The suspension is dispensed into the freezing tube and the lid is tightened.
(3) Place the frozen tube with the virus in the middle of the heat-shrinkable plastic tube and insert the correct label.
(4) Carefully heat the heat shrinkable plastic tube with a blower and wrap it around the freezing tube. Be careful not to heat the shrink plastic tube with too high a temperature.
(5) Carefully heat both ends of the heat-shrinkable plastic tube and clamp the tube's port to a complete seal with a large tweezers.
(6) Quickly freeze the sealed cryotube in a vacuum flask filled with liquid nitrogen (requires a mask and gloves when handling).
(7) Place the frozen cryotube into the grid in the liquid nitrogen tank, and record the detailed storage position, experiment number and storage date of the sample.
(8) When needed, quickly take out the required sample from the liquid nitrogen tank, and after thawing in a 37 ° C water bath, cut the heat shrinkable plastic tube at the siliconized gasket of the freezing tube with a scalpel blade. It is not necessary to remove the heat shrinkable plastic tube when unscrewing the lid of the freezing tube.
(2) Ice bath with clarified virus suspension (supernatant medium for tissue culture, or cell lysate in tissue culture medium), and 0.2 ml ice bath supernatant with a sterile pipette The suspension is dispensed into the freezing tube and the lid is tightened.
(3) Place the frozen tube with the virus in the middle of the heat-shrinkable plastic tube and insert the correct label.
(4) Carefully heat the heat shrinkable plastic tube with a blower and wrap it around the freezing tube. Be careful not to heat the shrink plastic tube with too high a temperature.
(5) Carefully heat both ends of the heat-shrinkable plastic tube and clamp the tube's port to a complete seal with a large tweezers.
(6) Quickly freeze the sealed cryotube in a vacuum flask filled with liquid nitrogen (requires a mask and gloves when handling).
(7) Place the frozen cryotube into the grid in the liquid nitrogen tank, and record the detailed storage position, experiment number and storage date of the sample.
(8) When needed, quickly take out the required sample from the liquid nitrogen tank, and after thawing in a 37 ° C water bath, cut the heat shrinkable plastic tube at the siliconized gasket of the freezing tube with a scalpel blade. It is not necessary to remove the heat shrinkable plastic tube when unscrewing the lid of the freezing tube.
Other related biological reagents:
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Oropharyngeal Airway,Disposable Orapharyngeal Airway,Oral Airway,Opa Oropharyngeal Airway
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