METHOD FOR LONG-TERM PRESERVATION OF OBLIGATE ANAEROBIC BACTERIA BY DRYING FROM FROZEN STATE Russian patent published in 2024 - IPC C12N1/04 

FIELD: biotechnology; microbiology.

SUBSTANCE: disclosed is a method for long-term preservation of obligate anaerobic bacteria by drying from a frozen state. Method uses surface cultivation of strains of obligate anaerobic bacteria on a dense nutrient medium corresponding to their physiological needs, in anaerobic conditions in an atmosphere of a three-component gas mixture (N₂ – 80%, CO₂ – 10%, H₂ – 10%). Biomass of the grown colonies of obligate anaerobic bacteria in the exponential growth phase is removed from the surface of the dense medium with a dummy and mixed with a protective stabilizing medium, in form of defatted milk or saccharose-gelatinous medium in amount of 20% of the volume of the bacterial suspension, in ratio of 2 cups of a monolayer of a pure bacterial culture, so that the initial number of cells is 10⁹–10¹² cells in 3 ml of a protective stabilizing medium, poured into vials for lyophilization under anaerobic conditions in an atmosphere of a three-component gas mixture of 80% N₂, 10% CO₂, 10% H₂, then the bottles are closed with cotton stoppers and pre-frozen in a protective stabilizing medium for 16 hours at a temperature of minus 70 °C by moving for 5 minutes from anaerobic conditions into a low-temperature freezer. After 16 hours, bottles with bacterial suspension of obligate anaerobic bacteria are placed in lyophilic drying, which is pre-cooled to minus 50 °C, then the freeze drying chamber is evacuated to 20,000 Pa (0.02 bar) for 60 minutes, followed by heating the freeze drying shelves to minus 15 °C at rate of 30 deg/hour and continued heating of shelves to 30 °C at rate of 3 deg/h and held at this temperature for 48-72 hours. Prior to disconnection and removal from lyophilic drying of bottles with dried bacterial cells of obligate anaerobic microorganisms, lyophilic drying chamber is filled with gaseous nitrogen through the union on the cover, 15 minutes later, the bottles with the lyophilised obligate anaerobic bacteria are placed in an anaerobic atmosphere from a three-component gas mixture of 80% N₂, 10% CO₂, 10% H₂, the cotton plugs are replaced with rubber plugs and sealed with a metal cap.

EFFECT: method enables to unify the process of preparing viable bacterial cultures for lyophilization, unify protective media and lyophilisation process, minimize losses of bacterial obligate anaerobic microorganisms during lyophilisation process to 0.01–0.02%.

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