Key Points and Methods of Edible Mushroom Strain Preservation Explained in Detail

Strains are a major biological resource and the primary means of production in edible mushroom cultivation. After a superior strain has been selected and bred, its excellent traits must be kept unchanged, or changed as little and as slowly as possible, so that production performance is not reduced and it can be used in production over the long term. Therefore, strain preservation is of great significance in edible mushroom production.

I. Principles of strain preservation

There are many methods of strain preservation, but the principles are much the same. First, select excellent pure strains. Use the spores, spore bodies and vegetative forms of microorganisms; second, according to their physiological and biochemical properties, artificially create conditions such as low temperature, dryness or oxygen deprivation to inhibit the metabolism of the microorganisms, reducing their life activities to an extremely low level or bringing them into a dormant state, thereby prolonging the life of the strain and keeping the strain's original traits, preventing variation. No matter which preservation method is used, the requirements during strain preservation are that the strain does not die, is not contaminated by unwanted microorganisms, and does not degenerate.

II. Methods of strain preservation

1. Low-temperature periodic transfer preservation method. Inoculate the strain to be preserved onto a suitable slant medium, culture at a suitable temperature, and when the mycelium has grown vigorously and covered the slant, take it out and store it in a low-temperature dry place at 3-5°C or in a 4°C refrigerator or freezer, transferring to a fresh tube every 4-6 months, with the specific interval determined by 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 reduces the chance of contamination and also prevents the medium from drying out. Except for the mushroom strain, all other edible mushroom strains can be preserved by this method.

2. Liquid paraffin preservation method. Take chemically pure liquid paraffin (requiring no moisture and no mold) and put it in a triangular flask, plug with cotton and wrap with paper, sterilize for 1 hour at 1 kg/sq cm pressure, then place in a 40°C constant-temperature incubator for several days to evaporate the moisture in it, until the paraffin oil is completely transparent. Transfer the prepared paraffin oil onto a blank slant and culture at 28-30°C for 2-3 days; it can be used only after proving that no contaminating microorganisms grow. 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 low-temperature dry place. The preservation time is more than one year, and 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 it through a 60-mesh sieve to remove coarse particles, then soak it 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 it 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 it and grind it fine, pass it through a 100-mesh sieve, mix the prepared sand and soil at (2-4):1, use a magnet to draw out the iron matter, 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 a sterility test, and use after passing. Take the slant strain that has already formed spores, inject 3-5 milliliters of sterile water under aseptic conditions, 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. Put the inoculated sand-soil tube into a vacuum desiccator containing a desiccant, connect a vacuum pump and draw 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 it into small strips of 4 × 0.8 cm, spread 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. Put the filter paper strips carrying the spores into preservation test tubes, then put 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 @#@245@#@ with no shriveled grains and no impurities, wash them 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 and spread them out to 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 them into test tubes, about 2-3 grams per tube, then clean the test tubes, plug with cotton, sterilize at 1.5 (1.5 kg/sq cm, 2 hours), after passing the sterility test set aside for use, after the test tube substrate cools, inoculate, culture at a suitable temperature, and after the mycelium has covered the substrate, coat and seal the cotton plug with paraffin, and place at low temperature for preservation. Transfer once about every 2 years.

(2) Bran culture preservation method. Take fresh bran, pass it 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 cotton plugs and wrap with paper, autoclave (1.5 kg/sq cm, 30 minutes), after passing the sterility test set aside for use, transfer the vigorous strain growing on the slant medium into the sterile bran culture tubes; during transfer, be careful to stir the bran in the small test tubes evenly so that it is in a loose state, culture at a suitable temperature until the mycelium has covered the bran, place the small bran culture tubes in a desiccator, and preserve at low temperature or a suitable 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 the sterility test set aside for use, inoculate the strain to be preserved into potato glucose liquid medium and culture with shaking at a suitable temperature for 5-7 days. Under aseptic operation, draw a little of the cultured strain and inject it into a test tube of physiological saline that has passed inspection, 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 already cultured, abundantly grown cells or spores in sterilized serum, egg white, or skim milk to make a bacterial suspension, dispense the suspension under aseptic operation into sterilized glass ampoules, about 0.3-0.5 milliliters per tube, then connect with a pressure-resistant rubber tube to a freeze-drying device, place the ampoules in a freezing tank and 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 stored for more than ten years, but the cost is relatively high.

8. Liquid nitrogen ultra-low temperature preservation method. First, make the strain to be preserved into a bacterial suspension for later use; second, prepare ampoules, add 0.8 milliliters of a 10% (volume ratio) glycerol distilled water solution as a cryoprotectant to each bottle, plug with cotton and sterilize (1 kg/sq cm, 5 minutes). After the sterility test, inoculate the strain to be preserved, melt-seal the bottle mouth with a flame, check for air leakage, place the sealed ampoules in a freezer, and lower the temperature slowly at a rate of 1°C per minute, so that the preserved material gradually and evenly freezes, until -35°C; after that the freezing rate does not need to be controlled. After the ampoules are frozen, immediately put them into a liquid nitrogen tank and preserve at -150 to -196°C. This method is used by only a few research institutes.