In nature, edible fungi do not exist alone, but live with many bacteria, radioactive bacteria, molds and so on. The so-called strain separation is to separate these mixed bacteria that coexist with edible fungi and obtain pure and excellent strains through culture. Denominator species, original species and cultivated species of the strain. \x0d\ (1) separation and culture of parent species \ x0d \ x0d \ The separation of parent species of edible fungi can be divided into spore separation, tissue separation and substrate mycelium separation. \x0d\ 1。 Spore separation method \x0d\ Spore separation method is a method of using sexual spores or asexual spores of edible fungi to germinate into hyphae and culture them into strains. The strain has strong viability, but there are differences among spores, and the natural differentiation phenomenon is serious and the variation is great. It needs to be tested before it can be used in production. \x0d\(l) Single spore isolation method: It is a method to take only one basidiospore at a time or in each test tube and let it germinate into mycelium to obtain pure strains. Agaricus bisporus and Volvariella volvacea mycelium separated by single spore have the ability to produce mushrooms, so this method can be used to separate and produce pure strains. Single spore separation is rarely used in production, and the technology is complex. Generally, multi-spore separation method is used. \x0d\(2) Multi-spore separation method: It is a method of inoculating many spores on the same culture medium, allowing them to germinate and mate freely, and obtaining pure strains of edible fungi. The specific operation methods are as follows: \x0d\① Mushroom spore ejection method: select 89% mature mushrooms with strong individuals, round flowers, no pests and diseases, uniform fruiting, high and stable yield and strong adaptability, cut off most of them, rinse the stalk with sterile water several times, and then use sterile gauze or absorbent cotton and filter paper to absorb the surface moisture. In the inoculation box or aseptic room, the mushroom pleats are hung upside down under the glass funnel with iron wires, and the funnel is covered on the Petri dish upside down; The small hole at the upper end is plugged with cotton. Put the Petri dish on an enamel plate covered with gauze and let it stand 12 ~ 20 hours, and the spores on the bacterial folds will be scattered in the Petri dish. Form a layer of powdery spore print (Pleurotus ostreatus is extremely lavender, mushrooms and straw mushrooms are brown, and mushrooms and Flammulina velutipes are white). Dip a small amount of spores with an inoculation needle and draw agar on a test tube or Petri dish for inoculation. When spores germinate and form colonies, colonies with early spore germination and good growth are selected for in vitro culture. \x0d\ You can also collect spores with a spore collector. The method is: after selecting the seed mushroom, according to the above procedure, gently open the glass bell jar, insert the seed mushroom handle down on the steel wire frame of the spore collector and put it in the center of the Petri dish. Then cover the glass cover and plug the bell palm with gauze. Pour a little mercuric chloride or sterile water on the gauze. Culture in an incubator at about 20℃ ... \x0d\② Folding smear method: when separating according to aseptic operation; Mature seed mushrooms should be selected, and the inoculation needle should be directly inserted between the folds, and the spores on the surface of the folds that have not been ejected by the sub-body should be gently wiped, and then crossed and inoculated on the culture medium. \x0d\③ Hook-hanging method: Take several folds or a small ear piece (Auricularia auricula, Auricularia auricula, Auricularia auricula) of a mature mushroom lid and hang it on the culture medium in a triangular flask with sterile stainless steel wire (or other hangers such as iron wire and cotton thread) so as not to touch the culture medium or the surrounding bottle wall. Culture and transfer at suitable temperature. \x0d\④ Attachment method: Take a small piece of mature bacterial fold or ear piece according to aseptic operation, and attach it to the test tube wall directly above the pipeline inclined culture medium with dissolved agar medium or Arabic gum and paste. After 6 ~ 12 hours of culture, when the spores landed on the inclined plane, the spores and part of agar medium were immediately transplanted into new test tubes for culture. \x0d\ The mother seed obtained by spore separation must be further purified and rejuvenated. When the mother seed is planted for about a week and the hyphae are covered with inclined planes, select the mother seed test tube with strong hyphae, vigorous growth, no aging and no infection of miscellaneous bacteria, and then transfer the tube to expand propagation. In general, it is not advisable to transfer cultured seeds in vitro for more than 5 times. \x0d\ The mother seed obtained by spore separation must pass the fruiting test and be identified as a high-quality strain before it can be used for production. \x0d\ General fungi, such as mushrooms, Pleurotus ostreatus, Pleurotus ostreatus, Lentinus edodes, Lentinus edodes, Volvariella volvacea, etc., can be obtained by multi-spore separation. \x0d\ Isolation of tremella fuciformis spores is briefly described as follows: \x0d\ After obtaining tremella fuciformis by aseptic operation, the surface of the culture medium at the bottom of the bottle has "spore print" after the triangular flask containing tremella fuciformis is cultured 12 hours. When Auricularia auricula is taken out and cultured in an incubator at 20℃-25℃ for 2 ~ 3 days, a milky white transparent paste colony will appear on the surface of the culture medium, which is a small amount of tremella mycelium formed by yeast conidia of tremella auricula. At this time, it is transferred to the slant culture medium of the test tube, and then transferred to the nutrient-rich dry culture medium after the slant is full. After about 30 days, the colony grows white hyphae. \x0d\ Only when yeast conidia and hyphae of Tremella fuciformis are mixed with ascomycetes of pinnate hyphae, the latter can help to decompose fibrous substances such as wood and provide nutrition, which is beneficial to the germination of tremella fuciformis spores, the colonization of hyphae and the formation of fruiting bodies. \x0d\ When two kinds of hyphae meet, first choose two kinds of pure hyphae. \x0d\ Feather hyphae should be purified and propagated. Generally, it is necessary to select a test tube inclined plane or seed bottle with rapid growth and strong wall climbing ability, take the top mycelium, transfer it to a test tube, culture it at 25℃-28℃, and repeat the test tube rotation several times to obtain excellent purebred. \x0d\ Tremella fuciformis mycelium is characterized by slow growth of mycelium and difficult germination of basidiospores. When the two strains are mixed, firstly, the slant of Tremella fuciformis mycelium cultured for 8 ~ 10 days is taken, and a small piece of feathered mycelium is inoculated on the slant about 0.5 cm away from Tremella fuciformis mycelium according to the aseptic operation method. Cultured at 25℃ for 65438 0 weeks, the mixed mother seed of Tremella fuciformis was obtained. \x0d\ 2。 Tissue isolation culture method \ x0d \ A simple method for asexual reproduction by using the internal tissues of fruiting bodies to obtain mother seeds, namely tissue isolation method. This method is simple in operation, fast in mycelium growth and development, and easy to preserve variety characteristics. Especially after cross breeding, the genetic characteristics of excellent strains can be stabilized by tissue separation. The following separation methods are often used. \ x0d \( 1) Separation of fruiting bodies: The cultivation of mushrooms should choose fine varieties with thick covering, short stalk and maturity of 89%. Cut off the two bases of mushrooms, soak them in 0. 1% mercuric chloride water for a few minutes in a sterile box, then rinse them with sterile water and dry them, or wipe the cap handle twice with 75% alcohol cotton balls for surface disinfection. When inoculating, just tear off the seed mushroom and pick a small piece of tissue at the joint between the bud cover and the handle or at the mushroom fold; Transplanted into PDA medium. Cultured at about 25℃ for 3-5 days, white fluffy hyphae can be seen on the tissue, and the strain can be obtained by expanding the tube. Such as mushrooms, oyster mushrooms, etc. Can be used in this method. (2) Sclerotinia separation: The fruiting bodies of Poria cocos, Polyporus umbellatus and Wan Lei are difficult to collect. It is common that it stores vegetative sclerotia. Bacteria can also be obtained by sclerotia separation. The method is to clean the surface of sclerotia, disinfect it with alcohol or mercuric chloride, cut the sclerotia, take a small piece of intermediate tissue, about the size of soybean, inoculate it on the slant of PDA culture medium, and keep warm for culture. It should be noted that sclerotia is a storage organ, most of which is polysaccharide and contains only a small amount of hyphae, so the selected tissue block is large, and if the tissue block is too small, it is difficult to isolate the strain. \x0d\(3) Isolation of bacteriocin: Some fruiting bodies are not easy to find and have no sclerotia, so they can be separated by bacteriocin. Such as Armillaria mellea and Armillaria pseudomellea. The operation method is as follows: firstly, gently wipe the black cortex on the surface of bacteriocin with alcohol or mercuric chloride for 2-3 times, and then remove the black outer cortex (bacterial sheath) to extract white bacterial slurry; Cut off the bacterial marrow with sterile scissors for a short time, inoculate it on the culture medium, and keep the temperature for culture to obtain the strain. \x0d\ Attention should be paid to the separation of bacteriocin: Because bacteriocin is relatively small and the separator is relatively small, it is easy to pollute miscellaneous bacteria during separation, so strict operation is required. \x0d\ 3。 Matrix mycelium separation and culture method \ x0d \ A method of obtaining pure strains by using the matrix of edible fungi as separation material is called matrix mycelium separation method. \x0d\ This separation method is suitable for fruiting bodies that only appear in a specific season, and it is not easy to harvest in a flash. The difference between intrabasal separation method and tissue separation method is that the hyphae in dried mushroom wood or auricularia auricula are often dormant. Sometimes the growth will not resume immediately after inoculation. Therefore, it takes a long time to preserve (1 month or so) to determine whether the mycelium can survive. Matrix mycelium separation method can be divided into: mycelium separation method in wood (that is, mushroom wood or auricularia wood separation method) and mycelium separation method in soil. \x0d\( 1) mycelium separation method: that is, mushroom wood or auricularia wood separation method. In order to reduce the infection of miscellaneous bacteria, mushrooms (auricularia auricula) must be sterilized before separation. The surface of mushroom (ear) water can be lightly burned with alcohol lamp flame to make mold spores burn, or the spores can be soaked in 0. 1% mercuric chloride water for a few minutes, then washed with sterile water and sucked dry with sterile filter paper. Pay attention when cutting the inoculation block. The inoculation block must be cut within the mycelium distribution range of the strain. Therefore, varieties with slow mycelium growth should be taken lightly; Fast-growing species can be obtained deeply. Meanwhile. The distribution range of mycelium should also be determined according to the type of mushroom, wood grain, the thickness of mushroom (ear) and the length of development time. Then cut it with a sharp knife. Inoculation block should be as small as possible to reduce the chance of infection by miscellaneous bacteria and extract the purity of strains. When the inoculation block is moved to the culture medium, it should be placed in a greenhouse or incubator at 22℃ ~ 26℃ suitable for mycelium growth to restore mycelium growth. \x0d\(2) Isolation of hyphae in soil: There are many kinds of edible fungi, most of which are native edible fungi; Spores do not germinate easily. Tissue separation is not easy to succeed, and the method of obtaining purebred hyphae in soil is called hyphae separation in soil. \x0d\ Pay attention to the separation of hyphae in the soil. Because there are various soil microorganisms living around the mycelium in the soil, it is necessary to avoid the interference of these microorganisms as much as possible when separating. When inoculating hyphae, use the method with clean tip and no impurities as far as possible, rinse repeatedly with sterile water, and add some drugs to inhibit the growth of bacteria, such as streptomycin or chlortetracycline at 40 μ g/L. If infected bacteria are found, hyphae at the edge of colonies can be picked out and inoculated into sawdust culture medium. Because bacteria have no ability to decompose lignin, it is not easy to expand in sawdust culture medium; Only at the inoculation site. After the mycelium grows out of the infected area, it can be expanded and purified. \x0d\(3) Separation of fruiting body base: The method of separating new hyphae from fruiting body base cultivated in bottle, bag or big bed is called separation of fruiting body base. Taking bag cultivation of tremella fuciformis as an example, it shows that x0d in the cultivation room has early ear emergence, high ear emergence rate and no plant diseases and insect pests. Five bags of young panicles with the strongest viability were selected and moved to fields with mild climate and scattered light for post-culture to enhance the viability of mycelium. After 7 ~ 10 days of culture, when the diameter of fruiting body reaches 4 ~ 5 cm, it can be recovered as a separated mother seed. Then choose the best one, cut off the tremella fruiting body with a sharp knife, and put it in a refrigerator at 0℃ or a container filled with dichlorvos overnight to kill the pests in the bottle. Then use 75% alcohol or mercuric chloride to scrub the impurities outside the ear base and ear bag, and move them into the sterile room together with the inoculation tool and inoculation medium. After sterilization, the old mycorrhiza with a thickness of about 65438±0.5mm above the bag mouth was dug out with an inoculation knife and cultured. When the white hyphae are exposed at the mouth of the bag, use the inoculation needle to pick out a piece of half a grain of white fungus, quickly move it into the center of the mother seed test-tube culture medium, gently remove the inoculation needle and plug it with cotton. In order to get more mother seeds, there should be 100-200 test tubes to choose from at a time. After separation, it should be transferred to a constant temperature box or greenhouse at 22℃-24℃ for culture. Because there is a lot of water in the culture medium, mycelium recovery is faster than auricularia auricula separation. After 2-3 days, white hyphae can be seen at the edge of the isolate. Observe at least twice a day for purification. The method of observation and purification is the same as that of auricularia auricula separation. After 10- 15 days of suitable temperature culture, when red and yellow water drops appear in the clump of inoculation block, the original seed can expand. \x0d\\x0d\ (II) Inoculation and culture of original seeds and cultivated seeds \x0d\\x0d\ After the mother seeds are obtained, in order to meet the needs of strain production. Excellent and high-purity mother seeds should be selected and further expanded into original seeds. \x0d\ The original seed culture medium generally adopts cottonseed hull, sawdust, grass and other media. \x0d\ Bottled or bagged seed culture medium can be sent to sterilization inoculation box after sterilization. When the culture medium in the bottle is cooled to below 30℃, it can be inoculated according to aseptic operation procedures. \x0d\ Strain bottles (bags) should be checked frequently after being put into the culture room, and if there is any contamination by miscellaneous bacteria, they should be taken out immediately. The mycelium of the original seed must be thick, close to the bottle wall and not shrink, with pure color, certain fragrance and strong vitality, and it can be eaten quickly when it is expanded into a cultivated seed. \x0d\ Cultivated species is to further expand and cultivate the original species into tertiary species, which is the same as the inoculation culture of the original species. Generally, the cultivated species such as Lentinus edodes, Pleurotus ostreatus, Lentinus edodes, Hericium erinaceus, Auricularia auricula, Tremella fuciformis, etc. mostly use sawn wood and cotton skin as the culture medium. Mushrooms often use dung grass as culture material. \x0d\ Cultivated species require that the wood block cannot be dehydrated and shrunk. The mycelium is robust, pure in color, fragrant, non-aging, and free from mixed bacteria pollution. Some species allow a small amount of primordium. After inoculation with cultivation materials, hyphae grow fast, grow well and have strong vitality. \x0d\ When the original seed is grafted with the cultivated seed, a layer of mycelium at the bottle mouth of the original seed should be removed. \x0d\\x0d\ (3) Simple culture of liquid strains \x0d\\x0d\ At present, submerged fermentation is used to produce strains, which has the characteristics of large yield, short cycle, neat age, low cost and convenient inoculation, and is the goal of industrial production of edible fungi. The culture process of liquid strains is briefly described as follows for reference. \x0d\ In short, pure and excellent strains are inoculated into liquid culture medium (solid culture medium without coagulant) to make hyphae propagate to form a large number of small fungus balls, and then this culture medium is mixed with sawdust or cottonseed hulls to make fungus blocks and cultured to form fruiting bodies. It can also be used to make original seeds and cultivated seeds. \x0d\ For cultivating liquid strains, the culture solution can be put into a triangular bottle, accounting for about one fifth of the weight of the empty bottle. Cultivate with a shaker (also called a shaker). There are two kinds of bottle shaker: rotary and reciprocating, and reciprocating is generally used. The liquid culture medium can be made of potato juice (with sugar) and wort, or corn flour, bean cake powder, sugar and inorganic salt. Can be mixed with culture medium, and is suitable for the cultivation of various edible fungi and medicinal fungi with good effect. Ingredients: bean cake powder 2%, corn flour 1%, glucose 3%, yeast powder 0.5%, potassium dihydrogen phosphate 0. 1%, calcium carbonate 0.2%, natural pH, 12℃ for half an hour. Then inoculated and cultured. \x0d\ If the production capacity is large, the seed tank will be gradually expanded on the basis of triangular flask, and liquid submerged aeration fermentation will be carried out in the fermentor to produce a large number of strains. However, due to the variety of equipment and strong technology needed in production, conditions should also be created; Realize the modernization of edible fungi cultivation. \x0d\\x0d\ (4) Identification of strain quality \x0d\\x0d\ The best identification method of strain quality is mushroom test. But it will take a long time. When purchasing strains, or in strain production, how can we know the mycelium growth and quickly judge the strain quality? We introduce several methods: \x0d\ 1. Visually, the hyphae of excellent strains are thick and white, fluffy, thick and dense, grow neatly, fast and fragrant. \x0d\2。 The standard of excellent strains can be summarized as "pure, fragrant, upright, strong and moist". When checking, open the cork, take out a small piece of mycelium from the middle of the strain bottle, observe the color and smell, pinch the material block by hand, and check its water content to see if it meets the above requirements and standards. \x0d\3。 Microscopic examination: select a small amount of hyphae and observe their morphology, hyphae branching, separation, locking and cell membrane thickness under the microscope. Culture observation: a small piece of mycelium was selected from the strain bottle, inoculated on the inclined test tube culture medium, and cultured at a constant temperature of 23℃ ~ 25℃. After five weeks, the viability of the strain was examined. If the mycelium grows fast, vigorous and pure, firm and dense, long and neat, it shows that the strain has strong vitality. \x0d\4。 Blocking method: put a piece of white paper on the table, take out a large piece of bacteria of the same age from the bottle, divide it into two pieces by hand, then divide the two pieces into four pieces, and divide the four pieces into eight pieces in turn. There are more excellent strains and less crumbs in one piece. Less inferior bacteria, more crumbs. \x0d\5。 Identification of liquid strains: cultured in triangular flask or fermentor for 3-7 days, if bubbles appear on the liquid surface, "oil skin" and turbidity appear, indicating that the strains are contaminated by miscellaneous bacteria. If the bacterial mass floats or slowly grows a thin mycelium layer, it shows that the viability of the strain is weak. The mycelium around the white block grows fast, thick and white, like cotton wool, which shows that the strain has strong vitality. \x0d\\x0d\ (5) Prevention and control of miscellaneous bacteria in the production of strains \x0d\\x0d\ When producing strains, we should always pay attention to the pollution of miscellaneous bacteria, so as to nip in the bud and prevent it from being eradicated. Therefore, the whole seed production process must be carried out under aseptic conditions. The inoculation box and all instruments used for separation and inoculation should be strictly disinfected and sterilized. Workers should wash their hands with disinfectant, wear sterilized work clothes, hats and masks, move quickly and accurately, and try not to talk or walk around to prevent the infection of miscellaneous bacteria in the air. \x0d\ When separating the mother species, it is the key to select mushrooms with early fruiting and strong vitality. Because of its strong vitality, it quickly occupied the position after inoculation, and there was no opportunity for foreign bacteria to invade. \x0d\ Contaminated strains are caused by various reasons, such as incomplete sterilization, lax aseptic operation during inoculation and inappropriate bottle caps. Therefore, sterilization must be thorough. After inoculation, it is best to check the effect of the culture medium in an incubator of about 25℃. After 2 days, there is no miscellaneous bacteria, which means that it is thorough and can be used. Strict aseptic operation must be carried out during inoculation. Another reason for the contamination of \x0d\ mixed bacteria may be that the mixed bacteria are infected when the mother species is separated. Because there are bacteria on the surface of the mushroom, disinfection is not complete, and the mixed bacteria are brought into the culture medium through the tissue block. \x0d\ In a word, there are many pollution opportunities, so we should pay attention to environmental disinfection, thoroughly disinfect according to aseptic operation procedures, check at different levels and pay close attention; Extract the quality of the strain.