Is it possible to make qualified strains? It is a criterion to evaluate whether specialty food households produce specialty foods. What's more important is that self-cultivating strains can greatly reduce the cost of buying seed and further increase economic efficiency. There are generally two approaches for the source of strains of food and drug bacteria: one is to use tissue or spore separation technology for self-cultivation, and the other is to introduce species from relevant research departments.
First, the separation of bacteria
There are two methods for the separation of strains of food and drug bacteria: tissue separation and spore isolation. These methods are generally adopted by scientific research units. The isolated strains must be tested for the mushroom products and can only be used after successful application.
(1) Tissue separation This method is asexual reproduction or cloning technique. Theoretically, the obtained bacterial strain does not undergo genetic recombination or other mutations and is therefore often used in production.
Select uncontaminated and mushroom-like mushroom strains (6-point ripening), pick them up, place them in a sterile paper bag, and take them back to the inoculation room. Place them in the inoculum box together with the parent tube culture medium and other supplies. Internal disinfection. In the inoculation box aseptically, first disinfect the hands and tools with 75% alcohol, then disinfect the mushroom surface with 0.1% mercury, and use a sterile scalpel to slit the middle of the joint between the cap and the stipe. Open, cut into strips at the junction of the cap and stipe (Figure 6-1), use inoculum hook to take the small truffle meat, quickly access the middle of the slope of the medium, plug the tampon, at 25 °C After culturing for 3-5 days, white sparse and delicate hyphae grow around the tissue blocks. The mycelium can grow over 15-18 days and test tubes are selected. It must be used for production after passing the mushroom test.
Figure 6-1 Tissue Isolation
(b) Spore isolation This method is sexual reproduction. The obtained strains have undergone genetic recombination and other variations. The production traits may be better than the mothers, and may also be inferior to the mothers. The spores are separated by two methods: single spore separation and polysporum separation. The single spore separation is mainly used for genetic breeding research, and the polysporum separation is mostly used for the production of multiple species. Spore separation techniques are as follows:
The hook collecting method adopts mature fruit body (spotted spores) bacteria caps, and after sterilization in the sterile room, suspending the bacteria folds (toadstool) or bacteria holes (porous bacteria) downwards in the aseptic container Collect the spores (Figure 6-2) and allow them to naturally shoot spores at 22-26°C for 5-10 hours. White spores appear on the bottom of the container. Take sterile water, stick a small amount of spores to make a spore suspension, use a sterile syringe or dropper to take the spore suspension, dripping 1-2 drops on the inclined surface of the test tube, and incubate the spores at 25°C to germinate. Choose no pollution. The slopes are expanded and subjected to rigorous mushrooming experiments, which are preferred for production.
Figure 6-2 Collecting spores with hooked mushrooms
1. Hook 2. Mushroom 3. Medium
The tube collection method takes a small piece of fruit body tissue under aseptic conditions, rubs a small amount of sterile agar or paste, adheres to the opposite glass surface of the bevel of the test tube culture medium (Figure 6-3), and adds 20 tampons after When left to stand at approximately °C, spores in the bacilli may be ejected onto the slant medium, and then the bacterial mass on the wall of the test tube is removed. After further culturing, multiple spores germinating mycelia are obtained.
Figure 6-3 Collecting spores on test tubes
Second, the mother species production
In the production of strains of edible and medicinal bacteria, strain preparation is usually divided into two grades, namely the parent species and the original species. The female parent is also called a first-class seed. Because the mycelium grows on the slope of the test tube, it is also called a test tube seed or a slant seed. In order to ensure quality and prevent degradation, the mother breeder's transfer expansion does not exceed 3 times, the number of transfer expansions increases, and the output tends to decrease. For long-term preservation of the strains, rejuvenation must be tested after mushrooming, can be used for large-scale production or sales. General producers introduce mother species and are used to prepare the original species after propagation.
(A) The parent culture medium masterbatch medium is commonly used as a solidifying agent for agar, which is also known as agar or frozen powder. It is extracted from seaweed cauliflower or other red algae, and is transparent, odorless, vermicelli or powdery. The main chemical components of agar are galactose sulfate, including 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 and utilized by the mycelium. It only acts as a coagulant in the medium. It melts to a liquid at 96°C, and re-solidifies when cooled to less than 45°C. Since the solid medium prepared with agar was clear and transparent, it was easy to observe the morphology of the hyphae, so it became an indispensable material for the conventional slant area plate culture medium. The amount varies depending on the season of use, the purpose, and the quality of the agar itself. It is generally 1.5%-1.8% in winter and 2% in summer; 2.5% when strains are isolated and 1.5%-2% during production. The amount is too much, the medium is hard, the water retention is good, and it is not easy to dry, but the cost is high. It must be pointed out that even the most pure agar still contains trace amounts of nitrogenous compounds and residual inorganic salts, so it is best not to use agar in experiments where nutrient requirements are exact. For production, the nutrient medium is required to be rich in nutrients and the mycelium is robust, so various enriched media are used. The commonly used mother culture medium is as follows:
1. Potato-glucose-agar medium (PDA): 200 g of peeled potato, 20 g of glucose, 20 g of agar, and 1000 ml of water. This medium has less nutrients and the mycelium is not long and it is often used to isolate, purify, and preserve bacteria.
2. 200 g of peeled potato, 20 g of glucose, 2.5 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 3 g of peptone, 120 mg of vitamin B, 20 g of agar, and 1000 ml of water.
3. 200 grams of peeled potatoes, 20 grams of glucose, 2 grams of potassium dihydrogen phosphate,
Magnesium sulfate 0.5 g, agar 20 g, chestnut wood 100 g, water 1000 ml.
4. 50 grams of sorghum powder, 10 grams of glucose, 2 grams of potassium dihydrogen phosphate, sulfuric acid
Magnesium 0.5 g, agar 20 g, water 1000 ml.
5. 200 grams of oyster mushrooms, 20 grams of glucose, 2.5 grams of potassium dihydrogen phosphate, 0.5 grams of magnesium sulfate, 3 grams of peptone, 20 grams of agar, and 1000 ml of water.
(B) Preparation of Test Tube Bevels
1. Prepare medium Choose high-quality potato, peel (digging away eyes), cut into thin slices, weigh 200g, add 1000ml of water into a small aluminum pan and boil for 20-30 minutes, then remove it with a 3-layer gauze The filtrate was replenished with 1000 ml of hot water and poured back into a small aluminum pan. Add agar to continue heating and stir with a glass rod until the agar melts. Add glucose and replenish to 1000 ml.
2. Aliquots of test tubes are usually used as a container for the parent culture medium, commonly used specifications
For 2 types of 18mm 18mm and 20mm 20mm, the medium capacity is 10-15ml, which is 1/4 to 1/5 of the length of the test tube. 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. The standard tampon should have a large plug and not be easily deformed. The portion of the tube that is inserted into the tube should account for 2/3 of the total length of the plug, and the portion exposed outside the tube should be 1/3 (not less than 1 cm). The size and elasticity of the tampon should be matched with the test tube port. The tampon inserted into the test tube should be close to the tube wall without leaving gaps. Overtightness hinders the circulation of air and it is also inconvenient to operate; too loose can not achieve the purpose of bacteria, and tampon easily fall into or out of the test tube. The tightness is such that the tampon is lifted and the test tube does not come off, and it is appropriate to dial out the tampon with a slight sound.
4. Sterilization Combine 6-8 tubes into one, cover them with kraft paper or double-layer newspapers, and fasten them 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. Pendulum 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. Sterilization effect check 5 test tubes are randomly selected and incubated at 25°C for 3-5 days. Check whether there are any bacteria on the slope. If any bacteria are found, it means the sterilization is not complete, and it should be discarded or re-sterilized. No germs can appear for inoculation.
(3) Inoculation and cultivation
l. Sterilization Inoculation equipment, culture medium, and strains are placed in the inoculation box (or inoculation room) before inoculation. Then, the amount of potassium permanganate 5 g, 40% formaldehyde solution 10 ml per cubic meter Fumigate for 30 minutes. If there is a UV lamp, inoculate after 30 minutes of exposure.
2. When inoculating the inoculation 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, and cut the grasses on the slope horizontally and vertically into small pieces of large grains (Fig. 6-4). Depth is appropriate with a little medium, and then the seed block is quickly placed in the center of the inclined surface of the test tube to be taken. After the inoculation hook is pulled out, the mouth of the test tube is baked, and the tampon is quickly plugged after overheating. With repeated inoculations, generally, 30-50 test tubes can be transferred for each parent.
3. Cultivation Put the inoculated tube into 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 slant. In the course of cultivation, it is necessary to check at any time, picking 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, the original species production
The original species is the species (secondary species) in which the parent species is transferred to a culture bottle or bag containing the culture medium for expansion. The original species 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 bottle 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 bacteria 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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