What are the methods for preserving edible mushroom strains?
I. Principles of strain preservation
There are many methods for strain preservation, but the principles are largely the same. First, select excellent pure strains. Utilize the spores, endospores and vegetative bodies of microorganisms; second, according to their physiological and biochemical characteristics, artificially create conditions such as low temperature, dryness or oxygen deprivation to inhibit the metabolism of microorganisms, reducing their life activities to an extremely low level or bringing them into a dormant state, thereby extending the life of the strain and keeping the strain's original traits, preventing variation. No matter which preservation method is used, during the strain preservation process the requirements are: no death, no contamination by unwanted microorganisms, and no degeneration.
II. Methods of strain preservation
1. Low-temperature periodic subculture preservation method. Inoculate the strain to be preserved onto a suitable slant medium, culture at an appropriate temperature, and when the mycelium robustly covers the slant, take it out and store it in a dry place at 3-5°C low temperature or in a 4°C refrigerator or freezer, subculturing to a fresh tube once every 4-6 months; the specifics should be determined according to the characteristics of the strain. During preservation, note that the ambient temperature must not be too high, to prevent mold from entering the tube through the cotton plug. Therefore, if a cotton plug is used, it can be wrapped with clean sulfuric acid paper or kraft paper, which can reduce the chance of contamination and also prevent the medium from drying out. Except for the straw mushroom strain, all other edible mushroom strains can be preserved by this method.
2. Liquid paraffin preservation method. Take chemically pure liquid paraffin (required to be free of water and not moldy), put it in an Erlenmeyer flask, add a cotton plug and wrap with paper, sterilize at 1 kg/sq cm pressure for 1 hour, then place in a 40°C constant-temperature incubator for several days to evaporate the water in it, until the paraffin oil is completely transparent. Transfer the treated paraffin oil onto a blank slant, culture at 28-30°C for 2-3 days, and use only after proving there is no growth of unwanted microorganisms. Then, using aseptic technique, inject the liquid paraffin into the slant test tube to be preserved. The injection amount should be 1-1.5 cm above the slant medium, plug with a rubber stopper, seal the mouth with solid paraffin, and store upright in a dry place at low temperature. The preservation time is more than one year; at low temperature, the preservation time can be extended further.
3. Sand-soil tube preservation method. Take river sand, soak and wash it with water several times, pass through a 60-mesh sieve to remove coarse particles, then soak in 10% hydrochloric acid for 2-4 hours to remove organic matter, then rinse with water until the pH of the running water reaches neutral, and dry for later use. At the same time, take poor soil or garden soil, soak it in water to make it neutral, after settling discard the supernatant, dry and grind it fine, pass through a 100-mesh sieve, mix the treated sand and soil at (2-4):1, use a magnet to draw out the iron, then dispense into small test tubes or ampoules, 0.5-2 grams per tube, plug with cotton, wrap with paper and sterilize (1.5 kg/sq cm, 1 hour), then dry-heat sterilize (160°C, 2-3 hours) 1-2 times, carry out sterility testing, and use after passing. Take the slant strain that has already formed spores, under aseptic conditions inject 3-5 milliliters of sterile water, scrape the lawn to make a bacterial suspension, then use a sterile pipette to draw the bacterial liquid and drip it into the sand-soil tube until the sand-soil is soaked through. Place the inoculated sand-soil tube into a vacuum desiccator containing a desiccant, connect a vacuum pump and pump air for several hours, until the sand-soil is dry. The vacuum drying operation must be completed within 48 hours after the spores are inoculated, to avoid the spores germinating. Seal the prepared sand-soil tube with paraffin; it can be preserved for 2-10 years at low temperature.
4. Filter paper strip preservation method. Take white (for collecting dark-colored spores) or black (for collecting white spores) filter paper, cut into small strips of 4 x 0.8 cm, lay them flat in a Petri dish, wrap with paper and sterilize (1 kg/sq cm, 30 minutes). Use the hook suspension method to collect spores, letting the spores fall onto the filter paper strips. Place the filter paper strips carrying the spores into preservation test tubes, then place the preservation test tubes into a desiccator for 1-2 days to remove the moisture from the filter paper, making the moisture content of the filter paper about 2%, then preserve at low temperature. 5. Natural substrate preservation method
(1) Wheat grain preservation method: take wheat without shriveled grains and without impurities, wash it clean, soak for 12-15 hours, add water and boil for 15 minutes, continue hot soaking for 15 minutes, so that the wheat grains swell but do not break, drain the water, spread out and air-dry, so that the moisture content of the wheat grains is about 25%. Mix calcium carbonate and gypsum into the cooked wheat grains (ratio of wheat grains, calcium carbonate, gypsum is 10 kg:133 g:33 g), after mixing evenly put into test tubes, about 2-3 grams per tube, then clean the test tubes, plug with cotton, sterilize 1.5 (1.5 kg/sq cm, 2 hours), after passing sterility testing set aside for use, after the test tube substrate cools, inoculate, culture at an appropriate temperature, and after the mycelium covers the substrate, coat and seal the cotton plug with paraffin, and place at low temperature for preservation. Subculture once about every 2 years.
(2) Bran culture preservation method. Take fresh bran, pass through a 60-mesh sieve to remove coarse particles. Mix the bran and tap water at 1:1, put into small test tubes, filling each tube to about 1/3 of its height, add a cotton plug, wrap with paper, autoclave (1.5 kg/sq cm, 30 minutes), after passing sterility testing set aside for use, transfer the robust strain growing on the slant medium into the sterile bran culture tubes; during transfer, be careful to pound and mix the bran in the small test tubes evenly, in a loose state, culture at an appropriate temperature until the mycelium covers the bran, place the bran culture small tubes in a desiccator, and preserve at low temperature or at an appropriate temperature.
6. Physiological saline preservation method. Take 0.7-0.9 grams of pure sodium chloride, put it into 100% milliliters of distilled water, stir evenly and dispense into test tubes, 5-10 milliliters per tube, sterilize (1 kg/sq cm, 30 minutes), after passing sterility testing set aside for use, inoculate the strain to be preserved into potato dextrose liquid medium and culture with shaking at an appropriate temperature for 5-7 days. Using aseptic technique, draw a little of the cultured strain and inject it into a test tube of physiological saline that has passed testing, plug with a sterile rubber stopper, coat and seal with paraffin, and preserve at room temperature or low temperature.
7. Freeze vacuum drying method. Suspend the cultured, abundantly grown cells or spores in sterilized serum, egg white, or skim milk to make a bacterial suspension, dispense the suspension by aseptic technique into sterilized glass ampoules, about 0.3-0.5 milliliters per tube, then connect with the freeze-drying apparatus using a pressure-resistant rubber tube, place the ampoules in a freezing bath to freeze rapidly at -30°C to -40°C, and vacuum-dry in the frozen state, and seal the ampoules by melting under vacuum, store at 20°C, generally can be preserved for more than ten years, but the cost is relatively high.
8. Liquid nitrogen ultra-low temperature preservation method. First, prepare the strain to be preserved into a bacterial suspension for later use; second, prepare ampoules, add 0.8 milliliters of cryoprotectant 10% (volume ratio) glycerol distilled water solution to each bottle, plug with cotton and sterilize (1 kg/sq cm, 5 minutes). After sterility testing, inoculate the strain to be preserved, melt-seal the bottle mouth with flame, check for air leakage, place the sealed ampoules in a freezer, slowly lower the temperature at a rate of 1°C per minute, so that the preserved material gradually and evenly freezes, until -35°C, after which the freezing rate no longer needs to be controlled; after the ampoules are frozen, immediately put them into a liquid nitrogen tank, and preserve at -150 to -196°C.