(A) the mother species of the separation of culture
The separation of the mother species of edible fungi can be divided into spore isolation method, tissue isolation method, and mycelium isolation in the base, etc..
1. Spore separation method
Spore separation method, is to use the edible fungi sexual spores or asexual spores sprouted into mycelium, cultivated into a variety of methods. This kind of strain has stronger vitality, but there are differences between individual spores, and the natural differentiation phenomenon is more serious, with a large variation, which needs to be tested by mushrooming before it can be applied in production.
(l) Single-spore isolation method: It is a method to take only one tensile spore each time or each test tube and let it germinate into mycelium to obtain pure strains. Mushrooms and straw mushrooms with single-spore isolation of mycelium, have the ability to bear fruit, can be used to isolate the production of pure strains. Single-spore separation production is less used, and the technology is complex, generally use multi-spore separation method.
(2) multi-spore separation method: that is, many spores inoculated on the same medium, so that they sprout, free mating to obtain pure strains of edible fungi a method. Specific methods of operation, there are the following:
①Spore ejection method: choose individual robust, round shape, no pests and diseases, mushroom uniformity, high yield and stable production, adaptability of eight or nine points of maturity of the seed mushrooms, cut most of the stems with aseptic water rinsed a number of times and then use sterilized gauze or skimmed cotton, filter paper to absorb the surface moisture. In the inoculation box or aseptic chamber, hang the gills of the seed mushrooms upside down with a wire under a glass funnel, which is inverted over a Petri dish; the small hole at the upper end is plugged with cotton. The petri dish is placed on an enameled plate covered with gauze and left to stand for 12 to 20 hours, and the spores on the gills will be dispersed inside the petri dish. A layer of powdered spore prints will form (very lavender for flat mushrooms, brown for mushrooms and straw mushrooms, and white for shiitake and enoki mushrooms). Inoculate with a small amount of spores on the outside of agar in a test tube or on a petri dish using an inoculation needle. When the spores germinate and produce colonies, select the colonies with early spore germination and good growth for test-tube culture.
Spores can also be collected with a spore collector. The method is to select the seed mushrooms, according to the above procedure, gently lift the glass bell jar, insert the seed mushroom stalk downward on the wire frame of the spore collector, and place it in the center of the petri dish. Immediately afterward, cover the glass cover and stuff the bell palm around with gauze. And pour a little ascending mercury or sterile water on the gauze. Transfer to a constant temperature box at about 20℃ for incubation.
②The method of applying on the pleats: when separating according to the aseptic operation; you should choose the mature seed mushrooms, use the inoculation needle to insert directly between the pleats, gently wipe the surface of the pleats of the fruiting body of the spores that have not yet been ejected, and then draw a line to inoculate on the culture medium.
3) Hook suspension method: take a few pieces of mature mushroom cap pleats or a small piece of ear piece (black fungus, hairy fungus, white fungus), use sterile stainless steel wire (or wire, cotton thread and other hanging materials) to hang on top of the medium in the triangular vials, and do not make contact with the medium or the surrounding walls of the vials. Set the appropriate temperature culture, transfer can be.
④Application method: according to the aseptic operation of the mature gill or ear piece to take a small piece of melted agar medium or gum arabic, paste, etc. attached to the test tube wall directly above the tube slant medium. After 6 to 12 hours of cultivation, when the spores fall on the slant, immediately transplant the spores together with part of the agar medium into a new test tube culture can be.
Spore separation of the mother species, must be further purified and re-strengthened, when the mother species planted a week or so, mycelium covered with slant, select the mycelium healthy, vigorous growth without aging, no infection of the mother species of weed test tubes, and then turn the tube to expand the general to the cultivation of the species, the transfer of the tube should not be more than five times.
Spore separation of the mother species, must be through the mushroom test, identified as a high-quality strains, before the production of use.
General mushrooms, such as mushrooms, flat mushrooms, cockles, shiitake mushrooms, winter mushrooms and straw mushrooms, etc., can be used to obtain the mother seed by the method of multi-spore separation.
Spore separation of fungus is described as follows:
After obtaining the seed ear according to the aseptic operation, the triangular flask in which the seed ear is hung is cultivated for 12 hours, and there is a "spore print" on the surface of the medium at the bottom of the flask. Take out the seed ear, placed in 20 ℃ -25 ℃ incubator culture 2 ~ 3 days, the surface of the medium will appear milky white transparent paste small colonies, is the yeast-like conidia of the silver ear of the formation of a small number of silver ear mycelium. At this time, transfer to the test tube slant medium, to be full of slant, and then transfer to the nutrient-rich, and the surface is relatively dry medium. After about 30 days, the colony grows white mycelium.
The yeast-like conidia and mycelium of silver fungus can only be beneficial to the germination of silver fungus spores, the colonization of mycelium and the formation of fruiting bodies if they are mixed with the feathery mycelium of the ascomycetes (fragrant ash mycelium) which helps to decompose wood and other fibrous materials and provide nutrients.
In the two kinds of mycelium rendezvous, first selected two kinds of pure mycelium.
Feathery mycelium to be purified selection, generally to select the rapid growth, climbing wall force strong test tube slant or seed bottle, take the apex of the mycelium, turn the tube transfer, set 25 ℃ - 28 ℃ on the culture, repeat the tube a few times can be obtained from the excellent pure species.
The characteristics of silver ear mycelium, mycelium growth is slow, stretcher spores are not easy to sprout. In the two bacteria mixed, first take the 8 ~ IO days of culture of fungus slant, according to the aseptic method of operation in the slant from the fungus about 0.5 cm from the fungus access to a small piece of feathery mycelium. Cultivate at 25℃ for 1 week to get the mixed mother seed of silver fungus.
2. Tissue separation culture method
The use of internal tissues of the substrate, asexual reproduction and obtain the mother of a simple method, that is, tissue separation. The method is easy to operate, mycelium growth and development of fast, varieties of characteristics are easy to save, especially after cross-breeding, excellent strains of bacteria with tissue isolation method can make the genetic characteristics of stability. The following isolation methods are often used.
(l) Separation of the substrate: the seed mushroom should be selected as a large cover thick, short shank, eight or nine minutes of maturity of the best varieties. Cut off the two bases of the mushroom, in the aseptic box with 0.1% mercuric acid water immersion for a few minutes, and then rinse with sterile water and wipe dry or with 75% alcohol cotton balls to wipe the cap and stipe 2 times for surface disinfection. When inoculating, just tear open the seed mushroom and pick a small piece of tissue at the junction of cap and stalk or at the folds; transfer to PDA medium. Cultivate it at 25℃ for 3-5 days, then you can see white fluffy mycelium on the tissue, and get the strain by transferring and expanding the tube. For example, shiitake mushroom, flat mushroom, etc. can be used in this way.
(2) mycorrhizal isolation: Poria, Poria, Leiwan and other fungi of the substrate is not easy to collect. And common is its storage of nutrients in the kernel. Separation with the mycelium, the same can be obtained strains. The method is to wash the surface of the nucleus, sterilized with alcohol or mercury, cut the nucleus, take a small piece of intermediate tissue, about the size of a soybean, inoculated in the PDA medium slant, heat preservation culture. It should be noted that the nucleus is a storage organ, most of which is polysaccharide, containing only a small amount of mycelium, so the picked tissue block should be larger, if the tissue block is too small, it will not be easy to separate out the strains.
(3) mycorrhizal isolation: there is a part of the substrate is not easy to find, there is no nucleus, you can use mycorrhizal isolation. Such as honey ring bacteria, false honey ring bacteria. The method of operation is to use alcohol or mercury to gently wipe the black cortex of the surface of the mycoplasma 2 to 3 times, and then remove the black outer cortex (mycoplasma sheath), draw out the white pith portion of the fungus; with sterile scissors will be a small section of the pith, inoculated in the culture medium, heat preservation and cultivation, that is, to get the bacterial species.
Bacteriophage isolation should be noted: because the bacteriophage is relatively small, the isolate is also relatively small, the isolation is very easy to contaminate the stray bacteria, so it is necessary to operate strictly.
3. base mycelium separation culture method
The use of edible fungi fertility substrate as a separation material, to get a pure strain of a method called base mycelium separation method.
This isolation method is suitable for the substrate that only appears in a particular season and is not easy to harvest because it is born in the morning and dies in the evening. The difference between intra-basal isolation and tissue isolation is that the mycelium in the dried mushroom wood or ear wood is often dormant. Sometimes the growth does not resume immediately after inoculation. Therefore, it is necessary to keep the mycelium for a longer period of time (about 1 month) to determine whether it is viable or not. The mycelium separation method can be divided into the mycelium separation in the material (i.e. mushroom wood or ear wood separation method) and the mycelium separation method in the soil.
(1) mycelium separation in the material: that is, the mushroom wood or ear wood separation method, in order to reduce the infection of stray bacteria, mushroom (ear) wood in the separation of the previous, must be aseptic treatment. Can be mushroom (ear) water surface with alcohol lamp flame gently burned over to burn mold spores, or then 0.1% mercuric acid water immersion spores for a few minutes, and then rinsed with sterile water with aseptic filter paper sucked dry. Care should be taken when cutting the inoculation block. The inoculation block must be cut within the range of mycelial distribution of the fungus. Therefore, the slow-growing species of mycelium should be taken shallowly; the fast-growing species of mycelium can be taken y. At the same time. The range of mycelium distribution should also be determined according to the type of mushroom, wood texture, thickness of mushroom (ear) wood, and length of development time. Then cut and take with a sharp knife. The inoculation block should be as small as possible to reduce the chance of infection by stray bacteria and to improve the purity of the strain. The inoculation block is moved to the medium, it should be put into the greenhouse or warm box at 22℃~26℃ suitable for mycelial growth, so that the mycelium resumes growth.
(2) mycelium separation in the soil: there are many types of edible fungi, many soil-born edible fungi; spores are not easy to germinate. Tissue separation is also not easy to succeed, with the mycelium separation in the soil to obtain pure species of the method, called mycelium separation in the soil.
Mycelium separation in the soil should pay attention to, due to the mycelium in the soil around a variety of soil microorganisms living, so the separation must be as far as possible to avoid the interference of these microorganisms, as far as possible batch to take the tip of the clean mycelium veins, without debris mycelium inoculation, repeatedly rinsed with sterile water, in the medium to add some inhibition of bacterial growth of the drug, such as 40 μg / l of streptomycin or gentamycin. Infected bacteria can be removed from the culture medium if they are found. If infected bacteria are found, the mycelium at the edge of the colony can be picked out and inoculated into the woodchip medium. Since the bacteria do not have the ability to break down lignin, they are not likely to expand easily in the wood-chip medium; they are confined to the inoculation. After the mycelium grows out of the infected area, it can then be expanded and purified.
(3) substrate base separation: from the bottle, bag cultivation or large bed cultivation of substrate base separation of new mycelium method, called substrate base separation, is now bag cultivation of psyllids as an example:
from the ear early, ear rate away from the ear, pests and diseases free cultivation room; select the strongest viability of the ear 5 bags of young ears, moved to the climate of the mild, with the diffuse light of the field place for late cultivation, in order to Enhance the mycelium's vitality. After 7 to 10 days of cultivation, when the diameter of the fruiting body reaches 4 to 5 centimeters, it can be taken back as the mother of separation. Then select the most desirable one, cut off the psyllid fruiting body with a sharp knife, and place it in a refrigerator at 0℃ or in a container with dichlorvos overnight in order to kill the pests in the bottle. Then scrub the ear base and the outside of the bag with 75% alcohol or ascorbic acid to remove impurities, together with the inoculation tools, inoculation medium, etc., and move them into the aseptic room. After sterilization, the old mycorrhizae about 15 mm thick in the upper part of the bag mouth were dug out with an inoculation knife and cultured. When the mouth of the bag reveals white mycelium, use the inoculation needle to pick a piece of half a grain of rice large white mycelium, quickly moved into the center of the parent species test tube medium, gently remove the inoculation needle, plug the cotton plug. In order to be able to obtain a larger number of mother seeds, an inoculation volume of 100-200 test tubes, in order to select from. After isolation, it should be moved into the constant temperature box or greenhouse at 22℃-24℃ for cultivation. Due to more moisture in the medium, mycelium recovery is faster than ear wood separation. After 2-3 days, white mycelium can be seen at the edge of the separated material. It should be observed at least twice a day for purification. Observation and purification methods are the same as those for ear wood separation. Cultivated at the right temperature for 10-15 days, when the inoculation block twisted group appeared red, yellow water droplets, you can expand the original species.
(B) the original and cultivated species of the inoculation culture
Mother species obtained, in order to meet the needs of strain production. Should be selected excellent, high purity of the parent species to further expand the original species.
The original seed culture medium is generally used cottonseed husk, wood chips, grass and other media.
Bottles or bags of original seed culture medium sterilized, can be sent to the inoculation box sterilized, to be bottles of culture medium cold to below 30 ℃, can be inoculated in accordance with aseptic operating procedures.
Species bottles (bags) into the culture room, should be frequently checked, once found stray bacteria contamination, immediately removed. Cultivated into the original species, mycelium must be robust and strong, close to the wall of the bottle without drying, pure color, with a certain aroma, strong vitality, expansion into a cultivated species to eat fast.
Cultivated species is to further expand and cultivate the original species into tertiary species, which has the same inoculation and cultivation as the original species. Generally speaking, the cultivated species of shiitake mushroom, flat mushroom, winter mushroom, monkey head, fungus, silver ear mostly use sawn wood and cotton peel as culture medium. Mushrooms mostly use dung grass as culture material.
Cultivated species require that the material is not dehydrated and dried up. The mycelium is robust and strong, pure color, fragrance, no aging phenomenon, no contamination of stray bacteria, some species permit a small amount of the original base, access to the cultivation material, fast germination, good growth, strong vitality.
Original seeds in the connection with cultivated species, the original bottle mouth of a layer of mycelium should be dug out of use.
(C) the simple cultivation of liquid seed
At present, the production of strains with liquid deep fermentation method, with large production volume, short cycle, the age of the fungus neat, low cost, inoculation is convenient and so on, it is to achieve the goal of the factory production of edible fungi. Now the liquid seed cultivation process is briefly described hereafter for reference.
In short, it is the pure and excellent strains, access to liquid medium (solid medium without coagulant), so that the mycelium propagation to form a large number of small balls, and then mix this medium into wood chips or cottonseed hulls, made of fungus, cultivation, the formation of its substrate. It can also be used to make original seeds and cultivated seeds.
Cultivation of liquid strains, the culture liquid can be loaded into a triangular bottle, about one-fifth of the weight of the empty bottle. Cultivate with a bottle shaker (also known as shaker). Shaking machine has two kinds of rotating and reciprocating, generally use the reciprocating type. Liquid culture medium can be prepared with potato juice (sweetened), malt juice, or cornstarch, soybean cake flour, sugar, inorganic salts, and other preparations. Can be mixed medium, because it is suitable for a variety of edible fungi and medicinal fungi culture, all have good results. Components: soybean cake powder 2%, cornstarch 1%, glucose 3%, yeast 0.5%, potassium dihydrogen phosphate 0.1%, calcium carbonate 0.2%, pH natural, 12 ℃ sterilized for half an hour. Then inoculate the culture.
If the production volume is large, on the basis of triangular bottles, and then expand the seed tank step by step, the fermenter for liquid deep aeration fermentation, producing a large number of strains. However, due to the production requirements of a wide range of equipment, technical, we should also create the conditions; to achieve the modernization of edible mushroom cultivation.
(D) Identification of strain quality
The best way to identify the quality of strains is to make mushroom test. But the time is relatively long. In the acquisition of strains, or in the production of strains, how can we know the growth of mycelium, quickly determine the quality of strains? We introduce several methods:
1. Naked eye observation: excellent strains of mycelium thick white, velvety, thick and dense, neat growth, fast speed, strong flavor.
2. The standard of good strains can be summarized as: "pure, fragrant, positive, strong, moist" five words. Check, open the cork, take out small pieces of mycelium from the middle of the bottle of strains, observe the color, smell the smell, hand pinch the material to check its water content, to see whether it is in line with the above requirements of the standard.
3. Microscopic examination: pick a small amount of mycelium, placed on the microscope to observe its morphology, mycelium branching, segregation, locking joints, the thickness of the cell membrane. Cultivation observation: pick a small piece of mycelium from the strain bottle, inoculate the slant test tube medium, placed in 23 ℃ ~ 25 ℃ constant temperature culture, after five weeks to check the strain viability. If the mycelium grows fast, exuberant and pure, robust and thick, long and neat, it indicates that the strain has strong vitality.
4. Block method: put a piece of white paper on the table, the same age of the strain from the bottle out of a large piece of hand into 2 pieces, and then 2 pieces into 4 pieces, 4 pieces into 8 pieces, and so on. Excellent strains have more whole pieces and less crumbs. Inferior bacteria less whole pieces, more crumbs.
5. Liquid strain identification: when the triangular bottle or fermentation tank culture 3-7 days later, such as bubbles on the liquid surface, resulting in "oil skin", turbidity and other phenomena, indicating that the strain itself with stray bacteria. If the bacterial block floats up, or late growth of a very thin layer of mycelium, it means that the strain is weak. White pieces of mycelium around the fast-growing, thick white, cotton wool-like, indicating that the strain of strong vitality.
(E) strain production process of miscellaneous Yin control
In the production of strains, we must always pay attention to the miscellaneous bacterial contamination, in order to prevent the main, once it occurs, the root cure is not easy. So the whole seed production process must be carried out under sterile conditions. Inoculation box and all separation, inoculation utensils, vessels, should be strictly disinfected and sterilized. Staff hands should be cleaned with disinfectant, and wear disinfected overalls, caps and masks, and move with agility and precision, and try not to talk and walk around to prevent infection by airborne stray bacteria.
When separating the mother seed, select the mushroom early, strong vitality, the first tide of mushrooms is the key, because it is strong vitality, after inoculation quickly occupy the position, no opportunity for invasion of stray bacteria.
The reason for the contaminated strains is multi-faceted, generally due to incomplete sterilization, or due to the inoculation of the aseptic operation is not strict, the bottle cap is not suitable for the cause. So sterilization must be thorough, the best inoculation bud will be placed in the culture medium at about 25 ℃ temperature box to do the effect of checking, after 2 days does not grow bacteria, that is thorough, can be used, the inoculation process must be strictly aseptic operation.
Pollution of stray bacteria another reason may be the separation of the mother seed infected with stray bacteria, due to the seed mushroom surface with bacteria, disinfection is not complete, through the tissue block will be stray bacteria into the culture medium.
In short, there are many opportunities for contamination, pay attention to environmental disinfection, thorough sterilization according to aseptic operating procedures, layer by layer, ring grasp, pay close attention; lift away from the quality of the strain.