Freeze-drying and liquid nitrogen preservation strain technology
It is recommended to restore the cultured freeze-dried strains by the following methods.
-, the bottle opening
1. Wipe the ampoule with a cotton wool soaked in 70% alcohol.
2. Heat the top of the ampoule with a flame.
3. Drip sterile water to the top of the heated ampoule to crack the glass.
4. Use a knives or tweezers to knock the top of the cracked ampoule.
Second, the strain recovery culture
1. Using a sterile pipette, draw 0.3-0.5 ml of the appropriate liquid medium, drip into the ampoule tube, and gently shake to freeze the cells in suspension.
2. Take 0.1 to 0.2 ml of the bacterial suspension, transplant it on a suitable agar slant/plate medium, and fill the remaining bacterial solution into a suitable liquid medium, and then incubate at the recommended temperature.
3. If it is not activated, or if there is any doubt after activation, please inform the deposit center within 1 month after receiving the strain. After the verification is correct, it will be reissued free of charge.
Third, matters needing attention
1. Before the strain is activated, store the ampoule in a 6 to 10 °C environment.
2. The cultivation of anaerobic bacteria, if not specified, from the opening to the completion of the inoculation, is required to be filled with oxygen-free gas to maintain the anaerobic state.
3. After some freeze-dried and preserved certain strains, the delay period is longer, and it is necessary to carry out two successive subcultures to grow normally. In this case, repeat step 2 (2).
Liquid nitrogen cryopreservation program
1. Container selection: 2 ml plastic ampoules with screw caps are used.
2. Check the ampoules for leaks: soak them in a 0.05% aqueous solution of methylene blue at 4 °C for 30-45 minutes, rinse well, discard the ampoules containing the blue dye, and use the remaining ampoules.
3. Print the label (laser printing).
4. Sterilization of the container: first rinse the ampoules with distilled water, then sterilize them in distilled water at 121 ° C for 15 minutes in a sterilizing pot, dry and put the printed labels into the ampoules, tighten the screw cap slightly, then Sterilize at 121 ° C for 30 minutes.
5. Preservation bacteria age: at the stage of maximum growth or late in the logarithmic growth phase.
6. Protective agent: 5-10% glycerol or dimethyl sulfoxide is used as a protective agent.
1) Preparation of glycerin: Sterilization at 121 ° C for 15 minutes, storage at 2-8 ° C. Glycerol is typically stored in an aqueous solution of twice the final desired concentration and then mixed with an equivalent amount of cell suspension. If the cell suspension is not used, it is stored in an aqueous solution of the final desired concentration.
2) Dimethyl sulfoxide: Filtered and sterilized using a 0.22 um polytetrafluoroethylene filter membrane. The filter membrane was previously rinsed with methanol and dimethyl sulfoxide. Sterile dimethyl sulfoxide was stored in a 10-15 ml volume at 2-8 ° C protected from light.
7. Packing: Under sterile conditions, the cell suspension (5 cm in diameter from the plate) is dispensed into an ampule, and a protective agent is injected to tighten the screw cap.
8. Fix the ampule on the aluminum clip and stick the label. For ease of access, only one strain is placed on one aluminum clip.
9. Pre-cool the temperature control system to 4 ° C, then put the aluminum clip into it. The temperature was lowered to -40 ° C at 1 ° C per minute, and then cooled to -90 ° C at 10 ° C per minute. After cooling, the aluminum clips are transferred to a reservoir in liquid nitrogen for storage.
10. Reactivation of strains: The strains preserved by liquid nitrogen were taken out and quickly thawed in a 37 ° C water bath. It usually takes 2 to 2.5 minutes, and after complete thawing, it is transferred to a suitable medium for timely cultivation.
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