Is it possible to produce high-quality fungal strains? This is a key criterion for evaluating whether specialty food households are truly producing specialty foods. More importantly, self-cultivating strains can significantly reduce the cost of purchasing seeds and improve economic efficiency. There are generally two approaches for obtaining the strains used in food and medicinal fungi: one is using tissue or spore separation techniques for self-cultivation, and the other is introducing species from relevant research institutions.
First, strain isolation.
There are two main methods for isolating strains of food and medicinal fungi: tissue separation and spore isolation. These methods are commonly used by scientific research units. The isolated strains must be tested for mushroom production and can only be used after successful application.
(1) Tissue Separation: This method involves asexual reproduction or cloning. Theoretically, the resulting bacterial strain does not undergo genetic recombination or mutations, making it ideal for production.
To begin, select uncontaminated and fully matured mushroom strains (6-point ripening), place them in a sterile paper bag, and take them back to the inoculation room. Place them in an inoculation box along with the parent culture medium and other supplies. Perform internal disinfection. In the aseptic environment of the inoculation box, first disinfect your hands and tools with 75% alcohol, then disinfect the mushroom surface with 0.1% mercury. Use a sterile scalpel to cut open the junction between the cap and the stem, and then cut strips at that point. Use an inoculation hook to take a small piece of truffle meat, quickly insert it into the middle of the slant of the medium, and plug the tampon. After culturing at 25°C for 3-5 days, white, sparse, and delicate hyphae will grow around the tissue blocks. After 15-18 days, the mycelium will develop, and test tubes are selected. They must pass the mushroom test before being used for production.
Figure 6-1: Tissue Isolation
(b) Spore Isolation: This method involves sexual reproduction. The resulting strains may have better traits than the parent or worse. There are two methods for spore separation: single spore isolation and polysporum isolation. Single spore isolation is mainly used for genetic breeding, while polysporum isolation is often used for multiple species. The spore collection process is as follows:
The hook collecting method uses mature fruit bodies (spotted spores) and sterilizes them in a sterile room. Then, hang the bacteria folds (toadstools) or bacteria holes (porous fungi) downward in an aseptic container. Allow the spores to naturally shoot out at 22-26°C for 5-10 hours. White spores will appear on the bottom of the container. Take sterile water, collect a small amount of spores, make a spore suspension, use a sterile syringe or dropper to transfer 1-2 drops onto the slant of the test tube, and incubate at 25°C for germination. Choose clean slopes for expansion and rigorous mushrooming tests, which are preferred for large-scale production.
Figure 6-2: Collecting spores with hooked mushrooms
1. Hook 2. Mushroom 3. Medium
The tube collection method involves taking a small piece of fruit body under aseptic conditions, rubbing a small amount of sterile agar or paste, and adhering it to the opposite glass surface of the test tube culture medium. After placing 20 tampons, leave them at approximately 25°C. Spores may be ejected onto the slant medium, and the bacterial mass on the test tube wall is removed. After further cultivation, multiple spores germinating into mycelia are obtained.
Figure 6-3: Collecting spores on test tubes
Second, mother strain production.
In the production of edible and medicinal fungi, strain preparation is usually divided into two levels: the parent strain and the original strain. The parent strain is also called the first-level seed. Because the mycelium grows on the slope of the test tube, it is also known as a test tube seed or a slant seed. To ensure quality and prevent degradation, the number of transfers should not exceed three times. As the number of transfers increases, the yield tends to decrease. For long-term preservation, rejuvenation testing is required after mushrooming, and it can then be used for large-scale production or sales. Producers typically introduce the mother strain and use it to prepare the original strain after propagation.
(A) Parent Culture Medium: The masterbatch medium is commonly made with agar as a solidifying agent. It is extracted from seaweed, cauliflower, or other red algae and is transparent, odorless, and appears as vermicelli or powder. The main components of agar include galactose sulfate, such as D-glucuronic acid, L-galactose, D-galactose, 3,6-anhydro-L-galactose, and pyruvic acid. Dry agar typically contains 16% water, 4.4% ash, 1.15% calcium oxide, 0.77% magnesium oxide, and 0.4% nitrogen. Agar has a stable chemical structure and is not easily decomposed or utilized by the mycelium. It acts only as a coagulant in the medium. It melts into a liquid at 96°C and solidifies again when cooled below 45°C. Since the solid medium prepared with agar is clear and transparent, it is easy to observe the morphology of the hyphae, making it an essential material for conventional slant area plate culture media. The amount varies depending on the season, purpose, and quality of the agar itself. It is generally 1.5%-1.8% in winter and 2% in summer; 2.5% when isolating strains and 1.5%-2% during production. If the amount is too high, the medium becomes hard, retains more water, and is less likely to dry, but the cost is higher. Even the purest agar still contains trace amounts of nitrogenous compounds and residual inorganic salts, so it is best not to use agar in experiments requiring exact nutrient requirements. For production, a nutrient-rich medium is needed to support robust mycelium growth, so various enriched media are used. Commonly used mother culture media include:
1. Potato-glucose-agar medium (PDA): 200g peeled potato, 20g glucose, 20g agar, and 1000ml water. This medium has fewer nutrients and the mycelium does not grow long, often used for isolation, purification, and preservation.
2. 200g peeled potato, 20g glucose, 2.5g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 3g peptone, 120mg vitamin B, 20g agar, and 1000ml water.
3. 200g peeled potatoes, 20g glucose, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 20g agar, 100g chestnut wood, and 1000ml water.
4. 50g sorghum powder, 10g glucose, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 20g agar, and 1000ml water.
5. 200g oyster mushrooms, 20g glucose, 2.5g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 3g peptone, 20g agar, and 1000ml water.
(B) Preparation of Test Tube Slopes
1. Prepare the medium: Choose high-quality potatoes, peel them (remove eyes), cut into thin slices, weigh 200g, add 1000ml of water to a small aluminum pan, boil for 20-30 minutes, filter the broth through a 3-layer gauze, and replenish with hot water to 1000ml. Add agar and continue heating, stirring with a glass rod until the agar melts. Add glucose and replenish to 1000ml.
2. Allocate test tubes: Usually used as containers for the parent culture medium, common specifications are 18mm x 18mm and 20mm x 20mm. The medium capacity is 10-15ml, which is 1/4 to 1/5 of the test tube's length. The medium must not adhere to the inner wall of the test tube. Wipe clean.
3. Make a tampon: Take the appropriate amount of cotton to make a tampon. The tampon should be smooth and sturdy. The total length of the tampon is 3-4 cm. A standard tampon should have a large plug and not be easily deformed. The portion inserted into the test tube should account for 2/3 of the total length of the plug, and the exposed portion should be 1/3 (not less than 1 cm). The size and elasticity of the tampon should match the test tube port. The tampon inserted into the test tube should be close to the tube wall without leaving gaps. Overtightness hinders air circulation and is inconvenient to operate; too loose cannot achieve the purpose of preventing contamination, and the tampon may fall out. The tightness should allow the tampon to be lifted without the test tube coming off, and there should be a slight sound when removing it.
4. Sterilization: Combine 6-8 tubes, cover them with kraft paper or double-layer newspapers, fasten with rubber bands or cords to prevent the tampon from getting wet. Stand upright in a pressure cooker for sterilization. When heating and sterilizing, drain the air in the pan. When the temperature rises to 121°C (pressure 1.0 kg/cm²), stop heating for 30-40 minutes. Wait until the pointer returns to zero, first open 1/5 of the lid. Use waste heat to dry the tampon, wait until there is no direct steam, then open the lid and remove the test tube.
5. Slope the hot surface and place the end of the test tube on the wooden strip to make a certain angle of slope. Generally, the length of the inclined surface should be 1/2 of the full length of the test tube. When the temperature is low, cover the insulation to prevent excessive condensation, and wait until it is completely solidified.
6. Check the sterilization effect: Randomly select 5 test tubes and incubate at 25°C for 3-5 days. Check if there are any bacteria on the slope. If any bacteria are found, it means the sterilization is incomplete, and it should be discarded or re-sterilized. No germs can appear for inoculation.
(3) Inoculation and Cultivation
1. Sterilize the inoculation equipment, culture medium, and strains before inoculation. Place them in the inoculation box (or inoculation room). Then, fumigate with 5g potassium permanganate and 10ml 40% formaldehyde solution per cubic meter for 30 minutes. If there is a UV lamp, inoculate after 30 minutes of exposure.
2. When inoculating the test tube, do not leave the aseptic area next to the flame of the alcohol lamp. Incinerate the inoculation hook and wait for it to cool. Cut the grasses on the slope horizontally and vertically into small pieces of large grains (Fig. 6-4). Insert the seed block appropriately into the center of the inclined surface of the test tube. After pulling out the inoculation hook, bake the mouth of the test tube and quickly plug the tampon after overheating. With repeated inoculations, generally, 30-50 test tubes can be transferred for each parent.
3. Cultivate the inoculated tubes in a 25°C incubator or culture room. In constant temperature culture, Grifola frondosa requires 12-15 days of culture to allow the hyphae to grow over the test tube slope. During the cultivation process, check at any time, pick out test tubes with mixed bacteria or abnormal phenomena to ensure the quality of the mother seed.
Figure 6-4: Transfer method of female parent (Chen Shiyu)
Third, original strain production.
The original strain is the species (secondary species) in which the parent strain is transferred to a culture bottle or bag containing the culture medium for expansion. The original strain preparation procedure is as follows:
(I) Formulas and spices
1. Hardwood has 78% of wood chips, 20% of bran, 1% of brown sugar (or white sugar), and 1% of gypsum. The water content is about 60% and the pH value is 5.5-6.5.
2. 90% cottonseed hull, 8% bran, 1% brown sugar, and 1% plaster. The water content is about 60% and the pH value is 5.5-6.5.
3. 42% cotton seed hull, 40% wood chips, 16% bran, 1% brown sugar, and 1% plaster. The water content is about 60% and the pH value is 5.5-6.5.
4. 99% wheat and 1% plaster. The wheat kernels are soaked in 1% lime water for 12-24 hours (depending on the temperature), boiled for 15 minutes with heat, and the wheat kernels are not suitable for flowering. The wheat grains are then slightly dried and mixed into the gypsum immediately. Or infusion bottles, each bottle of 300 grams of wet wheat, a thin layer of cottonseed husk sealing material at the shoulder to reduce the contamination of bacteria.
During the batching process, the following points must be noted:
(1) Sawdust shall be sifted through 2-3 mesh, and remove hard objects such as wooden blocks, wooden strips, wood chips, and mildew masses. Ingredients shall be prepared according to the production plan. Mix well with the machine or by hand. Dissolve brown sugar in water and make it uniform with water. Mix in the material. To achieve the main and accessories uniform, dry and wet uniform, suitable pH, no raw material group.
(2) Doing a good job in environmental sanitation. The best place to mix materials is concrete. It must be flushed with water in advance to minimize pollution.
(3) Flexibly control the moisture content. It is advisable to add more water to dry materials and fine materials, and to add less water to wet materials and coarse materials. It is advisable to add less water to the cement site and add more water to dry ground to infiltrate the water. The specific amount of water should be filled with good materials for half an hour. Do not drop drip.
(4) Operation should be rapid at high temperatures (around 28°C). If the mixture is sterilized for too long, the material will become acid. Light influences germs, while weight does not cause bacteria. Therefore, we must reasonably arrange the production volume so as to ensure no more than 4 hours from the seasoning to the sterilization in the high-temperature period, and not overnight from the seasoning to the sterilization in the low-temperature period.
(2) Bacteria bottles used for bottling and sealing should be brushed in advance and stored dry after use. Wood chips or cottonseed husk culture materials are bottled, and the material can be bottled and pressed to the bottleneck. The material surface must be below the shoulder of the bottle. Then, in the middle of the material, a hole with a diameter of 1.5-2 cm should be drilled to clean the inside and outside of the bottle mouth.
Take a complete, slightly larger than the palm of the cotton rolled tampon, tampon slightly thicker than the bottle, a little harder to screw into the bottle is appropriate. Insert 2/3 of the bottle, exposed l/3, and the tampon's head is flush with the bottom of the bottle neck. Too tight, affecting ventilation and slow bacterial growth. Too loose not only tampon is easy to fall off, but also does not achieve the purpose of filtering bacteria, causing bacteria infection. After plugging the tampon, cover it with kraft paper or double-layered newspaper and fasten it with a rubber band.
(iii) Sterilization using autoclave or atmospheric pressure sterilization. Autoclave pressure is usually 1.5-2 kg/cm² and sterilization time is 2-2.5 hours. Pressure sterilization takes 8-10 hours.
(4) Before inoculation, the bottle temperature must be reduced to about 30°C to prevent high temperature inoculation of heat-destroying bacteria. The cooling chamber is cooled in the cooling chamber, and no cooling chamber can be directly cooled in the inoculation chamber. Each mother can transfer 3-4 bottles of original species. Before inoculation, first clean the inoculation room (box), spray 2% to clean the air, and then move all the sterilized strainer bottles and utensils into the room, then strictly follow the vaccination room (box) disinfection. Inoculation must be performed strictly according to aseptic procedures.
The specific method of operation is as follows: The vaccinator holds the mother test tube, rubs the tube twice with an alcohol swab, and then pulls the tampon off. The test tube mouth is aligned with the flame of the alcohol lamp, and the tube is burnt with a flame. The burned vaccine is used. Quickly insert the glass tube into the tube to cool it, remove the 1 cm long piece of mycelium from the front of the bevel, and divide the remaining slope into 3-4 sections. Another person is above the flame of the alcohol lamp, and the cultivar picks up the bacteria. Unplug the original bottle tampon, the vaccinator will remove the bacteria seed block, quickly access the inoculation hole of the original species, the tampon plugged after the flame. Such a test tube can receive 3-4 bottles of original species. After each test tube is inoculated, the inoculation cartridge should be re-sterilized to prevent cross-infection. After the seeds are picked up, the countertops are immediately cleaned and various residues such as test tubes, spilled culture medium, and used cotton for disinfection are cleared out and a second round of inoculation is performed according to the method described above.
(e) To cultivate the original species culture, note the following:
1. Clean the culture room a few days in advance and clean the cement walls and floors, and strictly disinfect them before use. The culturing room is required to keep the air dry, clean, and avoid strong light, leaving open and closed vents, and no dead space for air circulation.
2. Move the inoculated seed bottle into the culturing chamber, keep the temperature at 25°C, and maintain the air humidity 55-65%. Generally 3-5 days mycelium can eat material, 7-10 days mycelium can cover. After the cover of the mycelium, strengthen the ventilation and keep the indoor air fresh. Normally, 25-85 days of hyphae can be grown to fill the bottle.
3. During the training period, it must be checked at any time and it should be promptly taken to find contaminated bacteria. Different kinds of bacteria in the original species breeding sites are different, the pollution causes are also different: If the tampon is damp, the air humidity is too high, the bottle mouth is easy to breed Trichoderma fluorescens. If bacteria and other molds, such as green mold and mucor, occur inside and below the bottle, the sterilization may not be thorough. If random bacteria are found around the inoculation block, it may be due to inexperienced inoculation or tampon loosening. For example, germs that grow in the vicinity of several bottles may be miscellaneous bacteria or vaccination tools. Regardless of the situation, it must be removed as soon as possible after the discovery of the harmful bacteria, in order to prevent the spread of bacterial bacteria in the bacteria bottles. Bottles that are initially contaminated can be completely sterilized and re-inoculated without delay.
In addition to bacteria, sometimes the seed does not germinate. The reason is: First, the inoculation tool burns after being burned, the seed blocks were excavated without cooling, and the mycelium was burned to death or the flame was burned when the mycelium passed the flame. The second is that the parent plant shrinks and ages and loses its germination power. The third is that the culture temperature is not suitable, the strainer bottle is not cooled after being sterilized, and the bacteria species are heated and die.
Although germ seed germination sometimes, but do not eat material, the main reasons are: bacterial seed blocks and culture materials are not closely integrated; culture material is dry; the pH of the culture material is not suitable; the culture material is added with an excess of anti-bacterial drugs such as carbendazim and so on. Therefore, when inoculating, strains should be tightly combined with the culture material; adhere to the bottle with the ingredients, and sterilize in time to prevent acidification of the culture materials; when mixing wood chips, be sure to prevent mixing with pine wood chips; do not add or excessively add bacteria when mixing ingredients. Antibacterial drugs such as Ling, to prevent the growth of mycelium is inhibited; to maintain indoor air humidity 55-65%, away from heat, so that the bottle heat evenly.
(vi) The main criteria for good original species of the original species are:
1. The hyphae are white and free of bacteria, and the tampon paper lids are free of mildew and the mycelium is full of bottles. After the mycelium grows over the bottle, the brown droplets are secreted on the surface, and a few primordia are formed and can be considered normal.
2. Open the stopper with the unique aroma of edible medicinal bacteria, no musty and sour smell.
3. Randomly excavate a strain of bacteria from the original type of bottle into pieces that are flexible and not loose. The pieces were grafted onto a new medium, and the strains germinated normally at 25°C.
The performance of poor quality seeds is the appearance of sclerotial markings (antagonistic lines) on the surface of mycelium, thin hyphae, atrophy of detachment, yellowing and aging, and the original species cannot be used for production.
Fourth, cultivation of seed production.
The preparation, inoculation, and culture methods of the cultivars are basically the same as those of the original species. The main differences are as follows: First, the original species is inoculated with the test tube parent species, and the cultivars are propagated with the original species; second, the cultivars, in addition to the strainer bottle and can bottle, the container used can also be used as a culture material container with polypropylene plastic bags of various specifications.
V. Preservation of strains.
The preservation of strains is a basic work of the food and medicinal fungi industry, and it is a necessary means to ensure the genetic stability of wild germplasm resources and the relative stable genetic performance of existing production species. There are many methods for the preservation of strains. Some of them have good preservation effects but they have high investment or complicated operations. In the production practice, there is a need for a simple method of less equipment investment, ensuring that the bacteria species do not die, no pollution, and maintaining the best possible seed quality, such as the method of combining low-temperature preservation of the slope with long-term preservation of the natural matrix.
In general, long-term preservation of the mother species, short-term preservation of the original species. It is necessary to ensure that the traits and vigor of the good strains do not mutate, die, and are not contaminated to ensure their purity. Therefore, the preservation method should have the advantages of easy material extraction, convenient operation, insusceptible degradation of bacterial species, and long-term preservation of non-polluting bacteria. The commonly used strains are stored as follows:
(a) The advantage of the cryogenic preservation of the slope is that it is easy to preserve and takes up less space. The specific approach is: After the mycelium is covered with slant, it is stored at 0-5°C. After a certain period of time (2-3 months), change the tube once. The PDA culture medium cannot be used for long-term preservation of the tube, otherwise the species may be easily degraded, and the ability of the medicinal and medicinal bacteria to degrade lignocellulose is weakened. Therefore, after a certain period of time (approximately 1 year), the bacteria species must be transferred to the woodchip medium for rejuvenation, and then the robust and non-polluting hyphae are selected and then returned to the PDA medium for preservation. In the long-term preservation process, it is necessary to prevent the tampon from infesting with bacteria. The strain test tube mouth is preferably sealed with a wax seal to prevent excessive evaporation of water in the culture medium. Increasing the amount of agar (2.5-3%) can slow the evaporation of water. It is also necessary to prevent the detachment of the label on the tube of the bacteria species and the resulting germination.
The cryogenic cryopreservation method is simple and easy to observe, and the viability and purity of the preserved strains can be observed at any time. Once it is mixed, the naked eye can find it in time. Use this method to pay attention to the following points:
1. The lean and rich media used the author's practice in the preservation of bacteria for many years and found that the culture media of the preserved strains cannot be too nutritious, otherwise the hyphae grow too prone to aging and produce more harmful metabolites. The bacteria died. Therefore, potato-glucose-agar slant (PDA), which is less nutritious, is used. Although the growth of edible and medicinal bacteria in the PDA medium is delicate, they are not easy to age and have a long life span. However, the mycelial growth on the PDA medium with rich (such as peptone, yeast extract, bran extract, etc.) Easy to age, life is short. Therefore, the Grifola frondosa inoculum is conserved in the rich and rich culture medium, which is beneficial to the maintenance and rejuvenation of its superior species.
2. Frequently changing medium If the same strain is used for a long period of time, the growth rate of the mycelium will be slow, and the mycelia will appear weak, broken and sparse. This is because the hyphae always grow on the same semi-synthetic substrate. On the other hand, unlike some natural growth environments, certain enzymes and active substances are not activated for a long period of time, and passivation or loss of vitality. By replacing the medium, especially the replacement of natural nutrients, carbon, nitrogen, and mineral elements can be changed to restore hypha vitality.
3. The age of the bacteria and the preservation temperature must be suitable for the mycelial culture of the bacteria to be preserved. The age of the bacteria is related to the length of the preservation period. Such as fungus, Agaricus blazei mycelium covered with slant and then cultured for 2-3 days, so that the mycelium is more dense, thick, is conducive to bacteria resistant to low temperature, refrigerator temperature set at 4-8 °C, can extend the strains Vitality; while Pleurotus ostreatus, Flammulina velutipes and other species can be placed in the refrigerator when the mycelium is just overgrown or will be covered with an inclined surface. The hyphae of these species are themselves resistant to low temperatures and can grow slowly under low temperature conditions, and the refrigerator is preserved. The best temperature is 0-4 °C, which can delay the fruiting body of the mushroom.
4. Sealed with porous rubber plugs to extend the storage period of tampon sealing medium is prone to loss of water and shrinkage; and use of non-porous rubber plug sealing caused by hypoxia, hyphae will die due to lack of oxygen or physiological reactions (hypha or matrix production pigment). If drilling holes in rubber stoppers with a hole diameter of 1.5 mm, then seal the holes with cotton to coordinate the conflict between water retention and ventilation. After 9-12 months of storage, the strains will still survive 80% or more.
(b) Natural matrix preservation This method is based on the characteristics of edible fungi, using natural substrates to preserve their species. Wood rot may be cultured using wood chips as the main material, and grass rot may be cultured as main material. The nutrition of the natural substrate is comprehensive, and most of the nutrients are sustained-release nutrients, and it is not easy to produce excess nutrients or hunger; secondly, the medium has good physicochemical properties, and can absorb or buffer harmful substances that are excreted by mycelium, and the balance between moisture and ventilation is balanced. Into the matrix growth, not exposed in the air, hyphae low respiratory intensity, strong vitality.
1. Raw materials and formulas When preparing the sawdust medium, the hardwood chips are the best, and the proportion of coarse and fine wood chips should be appropriate. The coarse wood chips should have a particle size of about 2 mm. The fine wood chips should be sawdust, and the ratio of rough and fine should be 1 : 2, add 20% of bran and 1% sugar of dry wood chips, water content is 60%. Such a substrate has good air permeability, nutrient-rich and long time for mycelial decomposition and utilization, and mycelium that grows up to the inside of the matrix is more tolerant than hyphae that are exposed outside the substrate and exposed to the air. Proper proportions of different particle sizes of the matrix can also harmonize the conflict between gas and water. Too much coarse particles, "overhead mycelium", excessive fine particles, poor air permeability, and slow mycelial growth. The application method is as follows:
78% of sawdust, 20% of bran, 1% of gypsum, 1% of sucrose, and 65% of water, and put it into a thicker tube. The loading amount is 1/3-1/2 of the tube and 1.5 kg/cm². Bacteria 1.5 hours. After cooling, access the strains that need to be preserved and culture at 28°C. When the mycelium grows on the sawdust medium, remove it and replace it with a sterile rubber plug. Store it in the refrigerator at 0-5°C for 1-2 years.
2. The container and the amount of filling use a glass bottle with a capacity of 250 ml, which is clean and clean, the loading capacity is generally not more than 3/5 of the glass bottle capacity, the filler can not be overfilled, the remaining particles of the bottle wall must be wiped clean, otherwise the preserved bacteria Species can easily cause pollution.
3. If tampon is used for sterilization during sterilization and refrigerated sterilization, the surface of the substrate is likely to lose water, and the survival rate of the inoculation is low. Even if the mycelium is revived and the growth is very slow, it is better to use a polypropylene membrane seal and change it after inoculation. Cotton vines were cultured until the mycelium became full and then replaced with a holed sterile stopper.
After the strains deposited by the above methods were stored for 3 years, the survival rate reached 90%, and the verified yield and fruit body morphology characteristics of the mushroom had no significant changes. Therefore, the natural matrix preservation method has the characteristics of long shelf life and relatively stable genetic properties of the bacterial species.
(c) Paraffin and oxygen sealing: The liquid paraffin is divided into conical flasks, the volume of which is l/3 of the bottle space, and the tampon is plugged, sterilized at 121°C for 2 hours, and then placed in a 40°C incubator to make it watertight. Evaporate, or put in a desiccator for several days to remove water, paraffin wax is transparent. Under sterile conditions, use a sterile pipette to load the beveled test tube with the hyphae so that the liquid level is about 1 cm above the top of the bevel. Sterile rubber stopper and store it vertically. This method allows bacteria to survive for more than 3 years, but it is better to transplant it once every 1-2 years. It is not necessary to pour out paraffin during transplantation. Take a piece of hyphae with the inoculation hook. Due to paraffin transfer, the growth of mycelium is weak, and it needs to be transferred again and again for 1-2 times to rejuvenate.
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