The strain is the main biological resource and the primary production data for the production of edible fungi. After an excellent strain has been selected, it must maintain its excellent traits or change as slowly as possible, so as not to reduce the production performance, and can be used in production for a long time. Therefore, the preservation of strains is of great significance in the production of edible fungi.
　　
Because of the different genetic characteristics of various microorganisms, the preservation methods suitable for use are also different. A good and effective preservation method should firstly keep the excellent traits of the original strains unchanged for a long time, and also consider the versatility of the method, the simplicity of operation and the popularity of the equipment. Here are some common methods for preservation of strains:
　　
(1) Inclined cryopreservation method
　　
The strain is inoculated on a suitable slant medium, and after the strain has been completely grown, it is stored in a refrigerator at about 4 ° C, and transferred to a new slant medium at regular intervals (preservation period), after growth. Continue to preserve, so continuous. This method is widely applicable to the short-term preservation of most microbial strains such as bacteria, actinomycetes, yeasts and molds, and the strains that are not suitable for lyophilization. Actinomycetes, molds and spore-forming bacteria can generally be stored for about 6 months, spore-free bacteria can be stored for about 1 month, and yeast can be stored for about 3 months. If the rubber plug is used instead of the cotton plug, and then sealed with paraffin, it is stored in a refrigerator at 4 °C, which not only prevents the water from volatilizing, but also can block the oxygen, and can prevent the tampon from being contaminated by moisture. This improvement can extend the preservation period of the strain.
　　
Due to the low temperature preservation, this method greatly slows down the metabolic propagation rate of microorganisms and reduces the frequency of mutations. At the same time, it also reduces the evaporation of water in the medium, so that it does not dry. The advantages of this method are simple and easy to carry out, easy to promote, and high survival rate. Therefore, most of the commonly used strains in scientific research and production use this preservation method. The disadvantage is that the strain still has a certain degree of metabolic activity, the preservation period is short, the number of passages is large, and the strain is more susceptible to variation and contamination.
　　
(2) Paraffin oil sealing method
　　
In the method, under the aseptic condition, the liquid paraffin which has been sterilized and evaporated has been poured into the slanted surface (or semi-solid puncture culture) of the cultured mature strain, and the paraffin oil layer is raised by 1 cm above the top of the slope to make the culture. It is isolated from the air, sealed with a paraffin seal, and stored vertically in a refrigerator at room temperature or 4 °C. The liquid paraffin used requires high-quality non-toxic, chemically pure specifications. The sterilization conditions are: 150-170 ° C sterilization in the oven for lh; or 121 ° C high-pressure steam sterilization for 60-80 min, and then placed in an oven at 80 ° C to dry Remove moisture.
　　
Since the liquid paraffin blocks the air, the cells are in an oxygen-deficient state, and the water is prevented from volatilizing, so that the culture does not dry, so that the preservation period can be 1 to 2 years, or longer. This method is simple to operate. It is suitable for preserving mold, yeast, actinomycetes, aerobic bacteria, etc. It has a good preservation effect on molds and yeasts and can be stored for several years or even up to 10 years. However, the preservation effect of many anaerobic bacteria is poor, especially for some bacteria that can decompose hydrocarbons.
　　
Some experiments have indicated that this method is suitable for the preservation of Monascus, and the survival rate after storage for 1 to 2 years is 100%. It has also been reported that some of the mycelium mycelium are preserved at 3 to 6 °C with paraffin oil preservation method. It is easy to die, but it is ideal at room temperature, which is worth noting.
　　
(3) Sand pipe preservation method
　　
This is a commonly used method for long-term preservation of strains, suitable for spore-forming actinomycetes, molds and spore-forming bacteria, for some drying-sensitive bacteria such as Neisseria, Vibrio and Pseudomonas and yeast. It does not apply.
　　
The preparation method is that the sand and the soil are separately washed, dried and sieved (the general sand is used for 60 mesh sieve and the soil is used for 120 mesh sieve), and the ratio of sand to soil is (1 to 2): 1 The mixture was placed in a small test tube. The height of the sand was about 1 cm, steam-sterilized at 121 ° C for 1 to 1.5 hours, and intermittently sterilized 3 times. After drying at 50 °C, it is ready for use after inspection. There are also sand or soil for storage. The strains to be preserved should be fully cultured with slant medium, and then 108~1010/ml bacterial suspension or spore suspension can be made into the sand tube with sterile water. Actinomycetes and molds can also directly scrape the spores. The carrier is mixed, and then placed in a desiccator for about 2 to 4 hours, sealed with a flame seal (or sealed with paraffin), placed in a desiccator, and stored in a refrigerator at room temperature or 4 ° C. .
　　
The sand pipe method has the conditions of low temperature, dryness, oxygen barrier and no nutrient, so the preservation period is longer, the effect is better, and the microorganisms are convenient to transfer and economical and simple. It has a longer shelf life than the paraffin oil sealing method, about 1 to 10 years. The Institute of Microbiology, Chinese Academy of Sciences preserves actinomycetes using the sand pipe preservation method.
　　
(4) Bran preservation method
　　
The bran preservation method is also known as the tracing preservation. That is, the bran is used as a carrier to adsorb the spores, and then stored under low temperature and dry conditions. The preparation method is that the bran and the water are mixed at a certain ratio of 1:(0.8-1.5) according to the different requirements of different strains, and the volume is 2/5 of the test tube volume, and after cooling and sterilizing, it is cooled and accessed. The freshly cultured strains are cultured at a suitable temperature until the spores grow. The test tube is placed in a desiccator containing a desiccant such as calcium chloride, and dried at room temperature for several days, and then transferred to a low temperature for storage; after drying, the test tube can also be sealed by flame sealing and then stored, and the effect is better.
　　
This method is applicable to spore-forming molds and certain actinomycetes, and the preservation period is more than one year. Because of the simple operation and economical benefits, the factory is more used. The Institute of Microbiology, Chinese Academy of Sciences uses the bran preservation method to preserve Aspergillus, such as Aspergillus oryzae, Aspergillus niger, Aspergillus awamori, etc., which can be stored for several years to several decades.
　　
(5) Glycerol suspension preservation method
　　
This method is to suspend the strain in glycerin distilled water and store it at low temperature. This method is simpler, but a low-temperature refrigerator is required. If the preservation temperature is -20 °C, the preservation period is about 0.5 to 1 year, and the use of -70 °C, the preservation period can reach 10 years.
　　
The culture solution of the logarithmic phase of the bacteria to be preserved is directly mixed with glycerin steam-sterilized at 121 ° C for 20 min, and the final concentration of glycerol is 10% to 15%, and then distributed in a small centrifuge tube, and placed in a low temperature refrigerator. preservation. Genetically engineered bacteria are often preserved using this method.
　　
(6) Freezing vacuum drying preservation method
　　
Freeze vacuum drying preservation method, also known as freeze-drying preservation method, referred to as freeze-drying method. It is usually prepared by using a protective agent to prepare a cell suspension or spore suspension of the species to be preserved in an ampoule tube, and then rapidly freezing the bacteria-containing sample at a low temperature, and vacuuming under reduced pressure to sublimate the water to dehydrate the sample to form a sample. A completely dry solid mass. It is immediately melted under vacuum conditions, resulting in an oxygen-free vacuum environment, and finally placed at a low temperature to keep the microorganisms in a dormant state and to be preserved for a long time. Commonly used protective agents are skimmed milk, serum, starch, dextran and other high molecular substances.
　　
Because this method has the conditions of low temperature, dry and hypoxic strain preservation, the preservation period is long, generally 5 to 15 years, the survival rate is high, and the mutation rate is low. It is an ideal preservation method widely used at present. . Except for the filamentous fungi that do not produce spores, most other microorganisms such as viruses, bacteria, actinomycetes, yeasts, filamentous fungi, etc. can adopt this preservation method. However, the operation of the method is cumbersome, technical requirements are high, and equipment such as a freeze dryer is required.
　　
When the preserved strain is needed, the ampoule can be opened in a sterile environment, the sterile medium is injected into the ampoule tube, the solid mass is dissolved, the water is shaken, and then inoculated into a slope suitable for the growth of the strain. It can be cultured at a suitable temperature.
　　
(7) Liquid nitrogen ultra-low temperature preservation method
　　
The liquid nitrogen cryopreservation method is abbreviated as liquid nitrogen preservation method or liquid nitrogen method. It is a method of preserving at a liquid nitrogen ultra-low temperature (-196 ° C) using glycerin, dimethyl sulfoxide or the like as a protective agent. The main principle is that the cells of the strains transition from normal temperature to low temperature, and before the temperature is lowered to low temperature, the free water in the cells is exuded out through the cell membrane to prevent the cells from being damaged by free water condensation into ice crystals. When the American ATCC Culture Collection Center adopts this method, the bacterial suspension or the agar block with hyphae is controlled to cool down at a rate of 1 °C/min per minute from 0 °C to -35 °C, and then preserved. In a liquid nitrogen cold box at -150 ° C ~ -196 ° C. If the cooling rate is too fast, the formation of ice crystals will damage the cells because the free water in the cells is too late to ooze out of the cells. According to the research, the speed of cooling is controlled at (1~10) °C/min, and the cell death rate is low; as the speed increases, the mortality rate increases accordingly.
　　
The protective agent for liquid nitrogen cryopreservation generally selects glycerin, dimethyl sulfoxide, dextrin, serum protein, polyethylene nitropentane, Tween 80, etc., but the most commonly used is glycerol (10% to 20%). Different microorganisms should choose different protective agents, and then determine the concentration of the protective agent through experiments. In principle, it is controlled to a concentration that is insufficient to cause microbial death.
　　
This method is simple, efficient and has a storage period of more than 15 years. It is recognized as one of the most effective long-term preservation techniques for bacteria. In addition to a small number of microorganisms sensitive to low temperature damage, the method is applicable to the preservation of various microbial strains, and even algae, protozoa, mycoplasma, etc. can be effectively preserved by this method. Another major advantage of this method is that it can be preserved using microorganisms in a variety of culture formats, either spores or cells, liquid cultures or solid cultures. The disadvantage is that ultra-low temperature liquid nitrogen equipment needs to be purchased, and liquid nitrogen consumption is high, and the operation cost is high.
　　
To use the strain, take the ampoule from the liquid nitrogen tank, and quickly put it into warm water of 35 ~ 40 °C, let it freeze and melt, open the ampoule bottle aseptically, and transfer it to the same medium used before storage. Incubate on a slope. When removing the ampoule from the liquid nitrogen tank, the speed is fast, generally not more than 1 min, in order to prevent other ampoules from heating up and affecting the preservation quality. Always wear special gloves when sampling to prevent accidental explosions and frostbite.
　　
(8) Host preservation method
　　
This method is applicable to obligate living cell parasitic microorganisms (such as viruses, rickettsia, etc.). They can only be parasitic in living animals and plants or other microorganisms, so they can be preserved against the characteristics of the host cells. For example, the plant virus can be mixed with the virus of the young leaves of the plant, frozen or dried. The phage can be expanded by bacterial culture and stored directly in the medium. The animal virus can directly infect a suitable organ or body fluid with a virus, and then sealed in a test tube and stored at a low temperature.
　　
Among the above-mentioned strain preservation methods, the slant surface cryopreservation method, the paraffin oil storage method, and the host preservation method are the easiest, followed by the sand tube preservation method, the bran preservation method, and the glycerol suspension preservation method; The liquid nitrogen cryopreservation method is the most complicated, but its preservation effect is the best. When applied, it can be selected according to actual needs.
　　
In the internationally renowned "American Type Culture Collection Center" (abbreviated ATCC), only the two most effective preservation methods are used, that is, the freeze-vacuum preservation method and the preservation period of the preservation period of generally 5 to 15 years are generally more than 15 years. The liquid nitrogen cryopreservation method is used to minimize the number of passages and avoid the purpose of strain variation and decline. There are three methods for the preservation of strains in China, namely, the low-temperature preservation method of the inclined surface, the cryopreservation method of liquid nitrogen, and the preservation method of freezing vacuum drying.
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