◆Bevel preservation method
•Method: Store the cultured slant strains in a refrigerator at 4-5°C, and regularly transplant them to new slant culture media for further storage. The general shelf life is 3-6 months.
•Note: the temperature of the refrigerator; the evaporation of water in the culture medium (use rubber plugs instead of cotton plugs).
•Advantages: simple and suitable for short-term preservation of bacterial strains in the laboratory.
•Disadvantages: Multiple passages in a short period of time can easily cause bacterial strain mutation and degradation, and increase the chance of bacterial infection.
•Applicable strains: short-term preservation of bacteria, actinomycetes, yeast, filamentous fungi, etc.
◆Puncture preservation method
•Principle: low temperature, lack of oxygen.
•Storage period: 6-12 months in refrigerator at 4-5℃.
•Applicable strains: various aerobic bacteria.
•The function of liquid paraffin: prevent the culture medium from losing water and isolate oxygen.
•Liquid paraffin preservation method: (adding sterilized liquid paraffin to the slant strains) is suitable for strains that cannot use paraffin as a carbon source, such as nitrogen-fixing bacteria, mycobacteria, salmonella, Mucor, Rhizopus, etc.
◆Solid koji preservation method
•Applicable strains: spore-producing fungi.
•In this method, natural agricultural products such as bran, rice, millet or wheat grains are used as a spore-producing medium, allowing the bacteria to produce a large number of dormant bodies (spores) and then preserve them.
•NOTE: Control proper moisture.
•Shelf life: 1-3 years
◆Sand tube drying and preservation method
•Principle: low temperature, oligonutrients.
•Storage period: 1-2 years in refrigerator at 4-5℃.
•Applicable strains: spore-producing filamentous fungi and actinomycetes and spore-producing bacteria.
•Sieved, sterilized and dried sand was used as the preservation medium.
•Silica gel, magnetic beads or porous glass beads can be used, but there are not many microorganisms suitable for these carriers, their shelf life is short, and they are not as practical as sand.
◆Vacuum freeze drying preservation method
•Principle: The cells are quickly frozen at low temperatures to keep the cell structure intact, and then the water is sublimated under vacuum (one of the most effective methods).
•Step:
❶Handling of ampoules:
❷sterilization;
❸Protective agent: Its function is to maintain the vitality of bacterial cells and minimize freezing damage caused by freeze-drying to microorganisms. Skim milk or serum is often used.
❹Freeze-drying (use low-temperature alcohol or dry ice below -15°C to quickly freeze it, and use a vacuum pump to dry it at low temperature).
•Preservation period: seal the ampoule at a low temperature of 4-5°C and store it for 5-10 years.
•Factors affecting the preservation effect: strain, bacterial age and moisture content in the sample.
•Applicable strains: Suitable for various microorganisms.
•Features: long storage period, small variation, wide application range, and ideal storage method.
◆Liquid nitrogen preservation method
•Principle: low temperature.
•Suspension medium: 10% glycerol or 5% dimethyl sulfoxide.
•Mechanism: Glycerol or dimethyl sulfoxide can enter cells and protect cells by reducing strong dehydration.
•Glycerol is generally sterilized by high-pressure steam, while dimethyl sulfoxide is preferably filter sterilized.
•Applicable strains: Suitable for the preservation of all microorganisms, animal and plant cells.
•Storage period: Store in liquid nitrogen (-196℃) for 5-10 years.
•Features: It has the longest storage period, but the storage cost is high. It is only used to preserve strains with high economic value, easy mutation, or strains that cannot be preserved for a long time by other methods.
◆Suspension preservation method
•Principle: Oligotrophic preservation, the bacteria are suspended in a nutrient-free solution.
•Applicable bacterial species: filamentous fungi, yeast-like fungi and intestinal bacteria.
•Storage period: 10℃ or room temperature preservation (18-20℃) for more than 1 year.
◆Cryopreservation
•Method: Add 12ml of bacterial liquid into a small screw-top tube with good sealing performance, seal it and place it directly in a low-temperature refrigerator for preservation.
•Storage period: -20℃ storage period is about 1 year.
◆Precautions for strain preservation
❶The state of the bacteria before preservation
•Dormant bodies such as spores or spores
•Culture time: too short, easy to die; too long, production performance decline
•Culture temperature: Generally, strains cultured at the optimal growth temperature until the spores mature are better preserved.
❷Matrix used for bacterial culture preservation
•The culture medium used for slant preservation should have a smaller proportion of carbon sources and be poorer in nutrients
•When preserving sand and soil tubes, the sand and soil must be thoroughly washed to prevent excessive organic matter from affecting the metabolism of bacteria or producing some toxic substances after sterilization
•The protective agents used in freeze-drying include many substances that are decomposed or denatured by heating, such as reducing sugar and skim milk. Excessive heating can easily form toxic substances
❸Damage to cell structure during operation
•During freeze-drying, slow freezing speed can easily lead to the formation of larger ice crystals in cells, causing damage to cell structures;
•The degree of vacuum drying affects the cell structure. Protective agents are added to reduce the damage to the cell structure caused by freeze-drying.