Detailed Explanation of the Technical Regulations for the Production of Edible Mushroom Spawn
Edible mushroom spawn refers to the pure mycelium of edible mushrooms cultivated artificially, usually divided into mother spawn, stock spawn and planting spawn. Mother spawn is pure mycelium that has been rigorously screened and confirmed to be of value in production. Mother spawn is cultured on test-tube slant medium, also known as tube spawn or first-level spawn. The spawn obtained by further propagating and transferring the mother spawn onto sawdust or grain medium is called stock spawn or second-level spawn, and the culture containers are mostly cans or spawn bottles. The spawn further propagated from the stock spawn is called planting spawn or third-level spawn, which can be used directly for production cultivation.
I. Mother spawn preparation method
1. Prepare the mother spawn slant medium. The most commonly used is PDA medium, and the preparation method is: take several disease-free potatoes, wash and peel them, cut into thin slices, weigh 200 grams, add 1000 milliliters of water and boil, boil until the potatoes are soft but not falling apart, filter the resulting liquid through 6 layers of gauze, top up the filtrate with clean water to 1000 milliliters, then add 20 grams each of agar and glucose, continue heating until dissolved, and while hot dispense into test tubes, about 8 milliliters per tube. Use test tubes of the 180 x 10 millimeter specification, and scrub them clean before use. After dispensing, plug with cotton plugs; bundle every 10 tubes into a group, place them upright in a small wire cage, sterilize with an autoclave or a simple steam pot, then while hot arrange them into slants, and once the medium solidifies it is ready for inoculation.
2. Prepare mother spawn by tissue isolation method. Select fruiting bodies that grow robustly, are neat in appearance, large in cap and thick in flesh, free of pests and diseases, and possess the characteristics of the variety, and harvest them when they are 70 to 80 percent mature to serve as isolation material. Cut off the stipe, wipe the surface of the mushroom body with a 75% alcohol cotton ball, and move it to an inoculation box. The inoculation box should be fumigated with formaldehyde in advance. Wipe the fruiting body material once more with an alcohol cotton ball, tear it open longitudinally from the cap, and use a sterilized blade or small tweezers to take material from the junction of the cap and the cup, cutting a piece of tissue the size of a wheat grain; use an inoculation needle to move it to the middle of the test-tube slant medium, one piece per tube. During inoculation, the mouth of the test tube must always be above the flame of an alcohol lamp, and the inoculation needle must also be heated red on the flame and allowed to cool at the tube mouth before taking inoculation material. Dozens of tubes can be inoculated each time, then send them to a constant-temperature incubator or culture room for culturing. Keep the temperature at 23-26 degrees C and the relative air humidity at about 70%. After 3 days of culturing, white fluffy mycelium will be seen growing around the tissue pieces, and in about a week it will cover the test tube. If green, red, yellow, black or milky white colonies appear on the slant medium, it means contamination by foreign microorganisms and they should be discarded promptly. Strictly select the spawn that has covered the slant, choosing those that grow fast and have dense, white, thick mycelium, and transfer them to tubes once more to obtain mother spawn.
3. Tube-transfer culturing of mother spawn. Tube transfer is carried out in an inoculation box, and several slant media are placed in the inoculation box in advance, with routine disinfection of the inoculation box. Operators wash their hands, wear a white lab coat and a white cap, covering their hair, and wipe their hands and the tube walls with 75% alcohol cotton. Hold the mother spawn test tube and the medium test tube in the left hand, hold the inoculation needle in the right hand, open the cotton plug of the test tube above the flame of an alcohol lamp, and burn the inoculation needle and the tube mouth on the flame at the same time, keeping the tube mouth always next to the flame. Let the burned inoculation needle cool at the tube mouth wall, then use the needle tip to cut out a small piece of medium covered with mycelium about the size of a wheat grain, quickly take it out from the tube mouth, inoculate it into the middle of the medium slant, then burn the tube mouth on the flame again, quickly plug the cotton plug, and inoculate the next tube. After all inoculation is completed, send them to a constant-temperature incubator or culture room.
II. Stock spawn preparation method
1. Preparation of stock spawn medium. Formula: broadleaf tree sawdust 78%, wheat bran 20%, sucrose and gypsum powder 1% each, pH value 6.5, water content 60%. Corn kernels or wheat kernels can also be used to make stock spawn medium, but the cost is high. According to the production quantity, weigh the materials in the formula proportions, mix them thoroughly, and make the water content appropriate, that is, when the material is squeezed in the hand, water beads appear between the fingers but do not drip; then fill bottles up to the shoulder of the bottle, use a finger-thick wooden stick to make a round hole in the center to increase aeration, tie the bottle mouth with polypropylene film, and sterilize promptly, either with an autoclave or a simple steam pot.
2. Inoculation and culturing. Routinely disinfect the inoculation room and inoculation box. Bring the sterilized stock spawn medium cans or spawn bottles into the inoculation box, 200 bottles at a time, and fumigate once more with formaldehyde. Operators must strictly carry out aseptic technique. Wipe the outer wall of the mother spawn test tube with alcohol cotton, bring it into the inoculation box, open the cotton plug over the flame, hold an inoculation spatula or large tweezers in the right hand, heat them red on the flame and let them cool, then pick out a piece of mycelium the size of a bean (with some medium attached) from the mother spawn tube, open the film of the stock spawn medium bottle, quickly put the mother spawn mycelium into the center of the stock spawn medium so that the mycelium surface closely touches the medium surface in the bottle, immediately cover the film, and then inoculate the next bottle in turn. One mother spawn tube can inoculate 15-20 bottles of stock spawn. Promptly label the inoculated spawn bottles, noting the edible mushroom variety, level (which level of spawn), inoculation date, inoculator, etc. Promptly send them to the culture room for culturing. Keep the temperature at 23-26 degrees C and the relative air humidity at about 70%. The room should be shaded or only weakly scattered light is needed. After about 30 days of culturing, when the mycelium has covered the bottle, it can be used to propagate planting spawn. If not used for the time being, store it short-term under dry, low-temperature and light-proof conditions.
III. Planting spawn preparation method
Planting spawn is the spawn used directly for production cultivation, and its preparation method is basically the same as that of stock spawn. One bottle of stock spawn can propagate about more than 100 bottles of planting spawn. The medium is the same as that for stock spawn. After sterilization, inoculate in the inoculation box, and under the control of the alcohol lamp flame, take out a piece of mycelium the size of a fingernail from the stock spawn bottle, put it into the center of the planting spawn medium, cover the film, and then inoculate the next bottle; after all inoculation is completed, send them to the culture room and place them on shelves for culturing. Culture in the dark for 30 days, and once the mycelium has covered the bottle, it can be used for cultivation.