CN112080396A - Stropharia rugosoannulata liquid strain incubator and culture method - Google Patents
Stropharia rugosoannulata liquid strain incubator and culture method Download PDFInfo
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Abstract
The invention discloses a stropharia rugoso-annulata liquid strain incubator which is characterized by comprising a liquid stock culture tank (1), a temperature-control magnetic rotary disc (2) and a combined stirrer (3), wherein the liquid stock culture tank (1) is arranged above the temperature-control magnetic rotary disc (2), stock culture solution containing liquid stock seeds is loaded in the liquid stock culture tank (1), and the combined stirrer (3) is placed in the stock culture solution; the rotation of the temperature control magnetic turntable (2) drives the combined stirrer (3) to rotate, thus realizing the non-contact stirring and heating of the stock culture solution. Compared with the solid stock culture, the culture device disclosed by the invention can shorten the stock culture time by 40-60 days and shorten the spawn running time of the cultivated species by 30-50 days.
Description
Technical Field
The invention belongs to the field of mushroom cultivation, and particularly relates to a liquid strain incubator and a culture method for stropharia rugoso-annulata.
Background
Stropharia rugoso-annulata (Stropharia rugoso-annulata) also named as red tricholoma matsutake and corious versicolor, the pileus is wine red in color and crisp and tender in meat quality, is one of ten mushrooms in the international mushroom trading market, can consume various agricultural and forestry wastes, is simple in cultivation mode, can be cultivated in open field or facilities by adopting raw materials, and is suitable for solving the problem of agricultural straw recycling caused by prohibition of straw burning in China.
At present, the second-stage strains (stock strains) of the stropharia rugoso-annulata are divided into solid strains and liquid strains, the solid strains grow slowly, the difference between the activity of the upper parts of the strains and the activity of the lower parts of the strains is large, the inoculation operation is slow, and the growth speed of hyphae is inconsistent after the third-stage strains are inoculated; the liquid strain production method generally adopts the technologies of a shaking table and a fermentation tank, the efficiency of producing the secondary liquid strain through the shaking table is low, and the fermentation tank is high in cost and inconvenient to transport. The culture time of the third-class stropharia rugoso-annulata seeds is long, the growth speed of hypha is about 2mm/d, the room temperature is controlled to be about 25 ℃ when the strains are prepared in summer, the hypha is filled in the bag for about 85 days, and the culture cost is far higher than the material cost and the labor cost.
Disclosure of Invention
The invention provides a liquid strain incubator and a culture method for stropharia rugoso-annulata, aiming at the problems in the background technology. Can shorten the culture time of the strains, reduce the cost and can quickly transport the liquid strains.
The invention discloses a stropharia rugoso-annulata liquid strain incubator which comprises a liquid stock culture tank, a temperature-control magnetic rotary disc and a combined stirrer, wherein the liquid stock culture tank is arranged above the temperature-control magnetic rotary disc, stock culture solution containing liquid mother seeds is loaded in the liquid stock culture tank, and the combined stirrer is placed in the stock culture solution; the rotation of the temperature control magnetic turntable drives the combined stirrer to rotate, so that the non-contact stirring and heating of the stock culture solution are realized.
Preferably, the top of the liquid stock culture tank is provided with a strain injection opening and a liquid feeding opening, the strain injection opening is provided with a rubber cover to realize opening and closing, and the liquid feeding opening is provided with a rubber plug to realize opening and closing; the bottom of the liquid stock culture tank is provided with a liquid taking faucet which is sealed by a sealing film in the stock culture solution preparation process; the liquid taking faucet is connected with the rubber tube in the inoculation process, the outlet end of the rubber tube is a glass conical opening, glass beads are arranged in the rubber tube, and the diameter of the glass beads is slightly larger than that of the rubber tube; the bottom of the liquid stock culture tank is provided with an annular support leg; a graduated scale and a handle are arranged on the body of the liquid stock culture tank; the top center downside of cultivateing the jar sets up the recess, cultivates the central upside of bottom of jar and corresponds and set up the recess, and two recesses are used for the both ends of spacing combination stirring.
Preferably, a magnetic disc is arranged at the top of the temperature control magnetic rotary disc and is controlled by a motor to rotate; the body of the temperature control magnetic turntable is provided with a power switch, a temperature adjusting knob, a rotating speed adjusting knob, a display screen and a power line.
Preferably, the stirrer comprises a magnetic rotor and a plastic rotor, and the magnetic rotor and the plastic rotor are connected through a bearing.
Preferably, the plastic rotor is longitudinally movable in an upper portion of the bearing, and the magnetic rotor is fixed in a lower portion of the bearing.
Preferably, the magnetic rotor is a hexagonal cross rotor, and the plastic rotor is an olive-shaped cross rotor without edges and corners.
The invention also discloses a method for culturing the liquid strain of stropharia rugoso-annulata, which comprises the following steps of
Liquid mother culture solution: 4-6 parts of wood dust powder, 4-6 parts of glucose, 0.5-1.5 parts of soybean meal and 992 parts of water 986-;
stock culture solution: 9-11 parts of corn flour, 3-5 parts of sawdust powder, 9-11 parts of glucose, 2-4 parts of soybean meal and 977 parts of water 969-containing material;
cultivating seed nutrients: 38-42 parts of 3mm sawdust and 38-42 parts of 8mm sawdust; 7-9 parts of rice husk, 4-6 parts of soybean meal, 5-7 parts of corn flour, 0.5-1.5 parts of light calcium carbonate and 892-908 parts of water;
the method comprises the following steps:
s1, preparing a liquid mother culture solution, subpackaging the liquid mother culture solution into a plurality of conical flasks, plugging the flask openings with silica gel plugs with air holes, placing the flasks into an autoclave, and cooling to room temperature after sterilization is finished; obtaining a liquid mother culture solution;
s2, taking the test tube mother seeds stored in a refrigerator, picking hypha blocks by using an inoculation needle, inoculating into a liquid mother seed culture solution, and standing for 24 hours; putting into a shaking table, rotating at 190rpm, and culturing for 48 h; standing for 24 h; continuously putting the mixture into a shaking table, rotating at the speed of 190rpm, and culturing for 192 h; obtaining a liquid mother seed;
s3, taking out the cultured liquid mother seeds, crushing hyphae in a homogenizer in a superclean workbench, and injecting all the liquid mother seeds into stock culture solution from a rubber stopper by using a sterilized needle cylinder, wherein the stock culture solution is loaded in a liquid stock culture tank;
s4, standing the liquid stock culture tank for 12 hours, then placing the liquid stock culture tank on a temperature-controlled magnetic turntable, starting the temperature-controlled magnetic turntable, keeping the temperature at 25 ℃, and increasing the rotating speed to enable the liquid in the tank to slowly flow;
s5, preparing a culture material according to a culture material formula, filling the culture material into a polypropylene fungus bag, sealing the bag with a special lantern ring for edible fungi, inserting a mesh inoculating rod into the lantern ring by using a power cone, covering a lantern ring cover, putting the lantern ring cover into a sterilization pot, sterilizing for 2 hours at 121 ℃ by adopting high-pressure sterilization, putting the fungus bag into a cooling chamber fumigated and sterilized by using sodium isophoronic urate after the sterilization is finished, and cooling to room temperature;
s6, taking down a sealing film on a liquid taking faucet in a super-clean workbench, burning the liquid taking faucet on the outer flame of an alcohol lamp, connecting a sterilized rubber tube to the liquid taking faucet after the liquid taking faucet is cooled, opening a lantern ring cover of a cultivated strain, pinching glass beads on the rubber tube, squeezing 5ml to 10ml of liquid strain into a grid inoculation rod, enabling the liquid strain to flow to the bottom along the grid inoculation rod, covering the lantern ring cover, and putting the inoculated strain bag in an environment at 25 ℃ for cultivation for 45 days.
Preferably, in S3, the stock culture solution is obtained by:
s3-1, preparing stock culture solution;
s3-2, filling the stock culture solution into a liquid stock culture tank (1) through a liquid filling port (1-7), plugging the liquid filling port (1-7) by a silicon rubber plug (1-8), keeping the liquid stock culture tank (1) in a closed state, and putting the liquid stock culture tank into an autoclave for sterilization at 121 ℃ for 20 min;
s3-3, sterilizing the liquid stock culture tank, and cooling to room temperature in an aseptic environment.
The invention has the advantages of
The device is simple, low in cost, light and movable, the culture area is not limited, the rotor in the fermentation bottle is driven by magnetic force to enable the culture solution to rotate at a proper rotating speed, and the method can accelerate the growth speed of the strains, increase the biomass of hyphae and enable the strains to grow to a usable state in a short time. The device can uniformly spawn in the spawn running room, and the spawn running is transported to an inoculation place after the spawn running is finished.
The application also provides a brand-new liquid mother culture solution, stock culture solution, culture seed nutrient formula and a corresponding cultivation method. The device of the invention is used in cooperation to accelerate the growth speed of strains to the maximum extent, shorten the hypha cultivation time and improve the hypha biomass.
Drawings
FIG. 1 is a schematic view of a liquid stock culture tank according to the present invention
FIG. 2 is a schematic view of the structure of the temperature-controlled magnetic turntable of the present invention
FIG. 3 is a schematic view of the structure of the stirrer of the present invention
FIG. 4 is a schematic view of the structure of the power-assisted cone and the inoculation rod of the present invention
FIG. 5 is a schematic view showing the use of the inoculation rod of the present invention
Detailed Description
The invention is further illustrated by the following examples, without limiting the scope of the invention:
with reference to fig. 1-5, a stropharia rugoso-annulata liquid strain incubator comprises a liquid stock culture tank (1), a temperature-controlled magnetic rotary disc (2) and a combined stirrer (3), wherein the liquid stock culture tank (1) is arranged above the temperature-controlled magnetic rotary disc (2), stock culture solution containing liquid stock seeds is loaded in the liquid stock culture tank (1), and the combined stirrer (3) is placed in the stock culture solution; the rotation of the temperature control magnetic turntable (2) drives the combined stirrer (3) to rotate, thus realizing the non-contact stirring and heating of the stock culture solution.
Preferably, a strain injection port (1-1) and a liquid feeding port (1-7) are arranged at the top of the liquid stock culture tank (1), a rubber cover (1-2) is arranged on the strain injection port (1-1) to realize opening and closing, and a rubber plug (1-8) is arranged on the liquid feeding port (1-7) to realize opening and closing; a liquid taking faucet (1-3) is arranged at the bottom of the liquid stock culture tank (1), and the liquid taking faucet (1-3) is sealed by a sealing film in the stock culture solution preparation process; the liquid taking faucet (1-3) is connected with the rubber tube (1-4) in the inoculation process, the outlet end of the rubber tube (1-4) is a glass conical opening (1-5), glass beads (1-6) are arranged in the rubber tube (1-4), and the diameter of the glass beads (1-6) is slightly larger than that of the rubber tube (1-4); the bottom of the liquid stock culture tank (1) is provided with an annular support leg (1-9); a graduated scale (1-10) and a handle (1-11) are arranged on the body of the liquid stock culture tank (1); the bottom center upside of the culture tank (1) is correspondingly provided with a groove, and the two grooves are used for limiting the two ends of the combined stirrer (3).
Preferably, a magnetic disc (2-1) is arranged at the top of the temperature control magnetic rotary disc (2), and the magnetic disc (2-1) is controlled by a motor to rotate; the body of the temperature control magnetic turntable (2) is provided with a power switch (2-2), a temperature adjusting knob (2-3), a rotating speed adjusting knob (2-4), a display screen (2-5) and a power line (2-6).
Preferably, the stirrer (3) comprises a magnetic rotor (3-1) and a plastic rotor (3-2), and the magnetic rotor (3-1) and the plastic rotor (3-2) are connected through a bearing (3-3).
Preferably, the plastic rotor (3-2) is longitudinally movable at the upper part of the bearing (3-3), and the magnetic rotor (3-1) is fixed at the lower part of the bearing (3-3).
Preferably, the magnetic rotor (3-1) is a hexagonal cross rotor, and the plastic rotor (3-2) is a non-angular cross rotor and is an olive-shaped cross rotor.
The following describes how to use the culture apparatus of the present invention to culture liquid strains of Stropharia rugosoannulata, with reference to specific examples, the specific dimensions of the culture apparatus used in the examples are described as follows:
150ml of conical flask; the diameter of the liquid stock culture tank (1) is 300mm, the height is 450mm, and the total volume is 30L; the width of the annular supporting leg (1-9) is 9cm, and the height is 3 cm; the width of the liquid adding port (1-7) is 8 cm; the width of the strain injection port (1-1) is 26 mm; the outer diameter of the temperature control magnetic turntable (2) is 30cm, and the total height is 120 mm; the diameter of the magnetic disc (2-1) is 100mm, and the height is 25 mm; the controllable range of the rotating speed is 0-500 rpm, and the controllable range of the temperature is 0-35 ℃; the polypropylene fungus bag (4-1) is 15mm multiplied by 28 mm; 35mm edible mushroom special lantern ring (4-2); the length of the grid inoculation rod (4-5) is 150mm, the outer diameter is 21mm, the inner diameter is 20mm, and the outer part is smooth; the length of the power-assisted cone (4-3) is 350mm, and the outer diameter is 19.5 mm.
Example 1: in parts by weight
Liquid mother culture solution: 4 parts of wood chip powder, 4 parts of glucose, 0.5 part of soybean meal and 992 parts of water;
stock culture solution: 9 parts of corn flour, 3 parts of sawdust powder, 9 parts of glucose, 2 parts of soybean meal and 977 parts of water;
cultivating seed nutrients: 38 parts of 3mm sawdust and 38 parts of 8mm sawdust; 7 parts of rice husk, 4 parts of soybean meal, 5 parts of corn flour, 0.5 part of light calcium carbonate and 908 parts of water.
Example 2: in parts by weight
Liquid mother culture solution: 6 parts of wood dust, 6 parts of glucose, 1.5 parts of soybean meal and 986 parts of water;
stock culture solution: 11 parts of corn flour, 5 parts of sawdust powder, 11 parts of glucose, 4 parts of soybean meal and 969 parts of water;
cultivating seed nutrients: 42 parts of 3mm sawdust and 42 parts of 8mm sawdust; 9 parts of rice husk, 6 parts of soybean meal, 7 parts of corn flour, 1.5 parts of light calcium carbonate and 892 parts of water.
Example 3: in parts by weight
Liquid mother culture solution: 5 parts of wood dust, 5 parts of glucose, 1 part of soybean meal and 990 parts of water;
stock culture solution: 10 parts of corn flour, 4 parts of sawdust powder, 10 parts of glucose, 3 parts of soybean meal and 972 parts of water;
cultivating seed nutrients: 40 parts of 3mm miscellaneous wood chips and 40 parts of 8mm miscellaneous wood chips; 8 parts of rice husk, 5 parts of soybean meal, 6 parts of corn flour, 1 part of light calcium carbonate and 900 parts of water.
Based on the formulas in the above 3 examples, the culture of the stropharia rugoso-annulata liquid spawn is carried out according to the following steps:
s1, preparing a liquid mother seed culture solution, subpackaging into a plurality of conical flasks, filling 70ml of the culture solution into each flask, plugging the flask opening with a silica gel plug with an air hole, placing into an autoclave, and cooling to room temperature after sterilization is finished; obtaining a liquid mother culture solution; s2, taking the test tube mother seeds stored in a refrigerator, picking hypha blocks by using an inoculation needle, inoculating into a liquid mother seed culture solution, and standing for 24 hours; putting into a shaking table, rotating at 190rpm, and culturing for 48 h; standing for 24 h; continuously putting the mixture into a shaking table, rotating at the speed of 190rpm, and culturing for 192 h; obtaining a liquid mother seed;
s3, taking out the cultured liquid mother seeds, crushing hyphae in a homogenizer in a superclean workbench, injecting all the liquid mother seeds into stock culture solution from a rubber stopper by using a sterilized needle cylinder, and injecting the stock culture solution into a liquid stock culture tank (1) from the rubber stopper (1-2) by using the needle cylinder;
s4, standing the liquid stock culture tank (1) for 12h, then placing the liquid stock culture tank on a temperature-controlled magnetic turntable (2), starting the temperature-controlled magnetic turntable (2), keeping the temperature at 25 ℃ and increasing the rotating speed to enable the liquid in the tank to slowly flow;
s5, preparing compost according to a culture seed nutrient formula, filling the compost into a polypropylene fungus bag (4-1), sealing the bag with a special lantern ring (4-2) for edible fungi, inserting a grid inoculation rod (4-5) into the lantern ring (4-2) by using a power cone (4-3), covering a lantern ring cover (4-6), putting the bag into a sterilization pot, sterilizing the bag at 121 ℃ for 2 hours by adopting high pressure sterilization, putting the bag into a cooling chamber fumigated and sterilized by sodium isochlorodiphenic urate after the sterilization is finished, and cooling the bag to room temperature;
s6, taking down a sealing film on a liquid taking faucet (1-3) in a super-clean workbench, burning the liquid taking faucet (1-3) on outer flame of an alcohol lamp, connecting a sterilized rubber tube (1-4) to the liquid taking faucet (1-3) after the liquid taking faucet (1-3) is cooled, opening a collar cover (4-6) of a cultivated strain, pinching glass beads (1-6) on the rubber tube (1-4), extruding 5ml-10ml of liquid strain into a grid inoculation rod (4-5), enabling the liquid strain to flow to the bottom along the grid inoculation rod (4-5), covering the collar cover (4-6), and putting the inoculated strain bag in an environment at 25 ℃ for cultivation for 45 days.
In the S3, the stock culture solution is obtained by the following steps:
s3-1, preparing stock culture solution;
s3-2, filling stock culture solution into liquid stock culture tanks (1) through liquid filling ports (1-7), filling 15L of culture solution into each tank, plugging the liquid filling ports (1-7) with silicon rubber plugs (1-8), keeping the liquid stock culture tanks (1) in a closed state, and sterilizing in an autoclave at 121 ℃ for 20 min;
s3-3, sterilizing the liquid stock culture tank (1), and then putting the sterilized liquid stock culture tank into a sterile environment to cool to room temperature.
The practical operation is carried out according to the three embodiments, and the strain properties and the cultivation time are obtained as follows:
by adopting the formula and the device disclosed by the application to culture hyphae, the strain culture time can be obviously shortened, and the cost is reduced.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
Claims (8)
1. A stropharia rugoso-annulata liquid strain incubator is characterized by comprising a liquid stock culture tank (1), a temperature-control magnetic rotary disc (2) and a combined stirrer (3), wherein the liquid stock culture tank (1) is arranged above the temperature-control magnetic rotary disc (2), stock culture solution containing liquid stock culture is loaded in the liquid stock culture tank (1), and the combined stirrer (3) is placed in the stock culture solution; the rotation of the temperature control magnetic turntable (2) drives the combined stirrer (3) to rotate, thus realizing the non-contact stirring and heating of the stock culture solution.
2. The stropharia rugoso-annulata liquid strain incubator according to claim 1, characterized in that a strain injection port (1-1) and a liquid filling port (1-7) are arranged at the top of a liquid stock culture tank (1), a rubber cover (1-2) is arranged on the strain injection port (1-1) to realize opening and closing, and a rubber plug (1-8) is arranged on the liquid filling port (1-7) to realize opening and closing; a liquid taking faucet (1-3) is arranged at the bottom of the liquid stock culture tank (1), and the liquid taking faucet (1-3) is sealed by a sealing film in the stock culture solution preparation process; the liquid taking faucet (1-3) is connected with the rubber tube (1-4) in the inoculation process, the outlet end of the rubber tube (1-4) is a glass conical opening (1-5), glass beads (1-6) are arranged in the rubber tube (1-4), and the diameter of the glass beads (1-6) is slightly larger than that of the rubber tube (1-4); the bottom of the liquid stock culture tank (1) is provided with an annular support leg (1-9); a graduated scale (1-10) and a handle (1-11) are arranged on the body of the liquid stock culture tank (1); the bottom center upside of the culture tank (1) is correspondingly provided with a groove, and the two grooves are used for limiting the two ends of the combined stirrer (3).
3. The liquid spawn incubator of Stropharia rugosoannulata according to claim 1, characterized in that the magnetic disc (2-1) is arranged on the top of the temperature-controlled magnetic rotary disc (2), and the magnetic disc (2-1) is controlled by a motor to rotate; the body of the temperature control magnetic turntable (2) is provided with a power switch (2-2), a temperature adjusting knob (2-3), a rotating speed adjusting knob (2-4), a display screen (2-5) and a power line (2-6).
4. The liquid spawn incubator of Stropharia rugosoannulata according to claim 1, characterized in that said stirrer (3) comprises a magnetic rotor (3-1) and a plastic rotor (3-2), the magnetic rotor (3-1) and the plastic rotor (3-2) are connected by a bearing (3-3).
5. The liquid spawn incubator of Stropharia rugosoannulata according to claim 4, characterized in that said plastic rotor (3-2) is longitudinally movable on the upper part of the bearing (3-3), and the magnetic rotor (3-1) is fixed on the lower part of the bearing (3-3).
6. The liquid spawn incubator of Stropharia rugoso-annulata according to claim 4, characterized in that said magnetic rotor (3-1) is a hexagonal cross rotor, said plastic rotor (3-2) is a non-angular, olive-shaped cross rotor.
7. A method for culturing liquid strains of stropharia rugoso-annulata is characterized in that the liquid strains are cultured according to the parts by weight
Liquid mother culture solution: 4-6 parts of wood dust powder, 4-6 parts of glucose, 0.5-1.5 parts of soybean meal and 992 parts of water 986-;
stock culture solution: 9-11 parts of corn flour, 3-5 parts of sawdust powder, 9-11 parts of glucose, 2-4 parts of soybean meal and 977 parts of water 969-containing material;
cultivating seed nutrients: 38-42 parts of 3mm sawdust and 38-42 parts of 8mm sawdust; 7-9 parts of rice husk, 4-6 parts of soybean meal, 5-7 parts of corn flour, 0.5-1.5 parts of light calcium carbonate and 892-908 parts of water;
the method comprises the following steps:
s1, preparing a liquid mother culture solution, subpackaging the liquid mother culture solution into a plurality of conical flasks, plugging the flask openings with silica gel plugs with air holes, placing the flasks into an autoclave, and cooling to room temperature after sterilization is finished; obtaining a liquid mother culture solution;
s2, taking the test tube mother seeds stored in a refrigerator, picking hypha blocks by using an inoculation needle, inoculating into a liquid mother seed culture solution, and standing for 24 hours; putting into a shaking table, rotating at 190rpm, and culturing for 48 h; standing for 24 h; continuously putting the mixture into a shaking table, rotating at the speed of 190rpm, and culturing for 192 h; obtaining a liquid mother seed;
s3, taking out the cultured liquid mother seeds, crushing hyphae in a homogenizer in a superclean workbench, and injecting all the liquid mother seeds into stock culture solution from a rubber stopper by using a sterilized syringe, wherein the stock culture solution is loaded in a liquid stock culture tank (1);
s4, standing the liquid stock culture tank (1) for 12h, then placing the liquid stock culture tank on a temperature-controlled magnetic turntable (2), starting the temperature-controlled magnetic turntable (2), keeping the temperature at 25 ℃ and increasing the rotating speed to enable the liquid in the tank to slowly flow;
s5, preparing compost according to a culture seed nutrient formula, filling the compost into a polypropylene fungus bag (4-1), sealing the bag with a special lantern ring (4-2) for edible fungi, inserting a grid inoculation rod (4-5) into the lantern ring (4-2) by using a power cone (4-3), covering a lantern ring cover (4-6), putting the bag into a sterilization pot, sterilizing the bag at 121 ℃ for 2 hours by adopting high pressure sterilization, putting the bag into a cooling chamber fumigated and sterilized by sodium isochlorodiphenic urate after the sterilization is finished, and cooling the bag to room temperature;
s6, taking down a sealing film on a liquid taking faucet (1-3) in a super-clean workbench, burning the liquid taking faucet (1-3) on outer flame of an alcohol lamp, connecting a sterilized rubber tube (1-4) to the liquid taking faucet (1-3) after the liquid taking faucet (1-3) is cooled, opening a collar cover (4-6) of a cultivated strain, pinching glass beads (1-6) on the rubber tube (1-4), extruding 5ml-10ml of liquid strain into a grid inoculation rod (4-5), enabling the liquid strain to flow to the bottom along the grid inoculation rod (4-5), covering the collar cover (4-6), and putting the inoculated strain bag in an environment at 25 ℃ for cultivation for 45 days.
8. The method for culturing liquid spawn of Stropharia rugosoannulata according to claim 7, wherein in S3, the stock culture solution is obtained by:
s3-1, preparing stock culture solution;
s3-2, filling the stock culture solution into a liquid stock culture tank (1) through a liquid filling port (1-7), plugging the liquid filling port (1-7) by a silicon rubber plug (1-8), keeping the liquid stock culture tank (1) in a closed state, and putting the liquid stock culture tank into an autoclave for sterilization at 121 ℃ for 20 min;
s3-3, sterilizing the liquid stock culture tank (1), and then putting the sterilized liquid stock culture tank into a sterile environment to cool to room temperature.
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