Detailed Explanation of Sterilization, Inoculation and Cultivation Methods for Shiitake Mushroom Culture Media
I. Sterilization of Spawn Culture Media
Sterilization of the culture medium is the key to success or failure in spawn production. Unsterilized culture media contain large numbers of microorganisms, which often lead to spawn contamination and cause losses to production. At present, the sterilization method commonly used by mushroom growers is atmospheric steam sterilization.
The atmospheric sterilization stove used for atmospheric steam sterilization is built with bricks. Select an iron pot with a diameter of 110 cm, build a pot cylinder with bricks 100 cm high and 120 cm in diameter, and smooth the inside and outside with cement.
After the culture medium is loaded into the sterilization pot, cover the mouth of the pot cylinder with relatively thick plastic film and burlap pieces, tie it firmly with rope, then heat it over a fierce fire and bring the pot to a boil in the shortest possible time. When the plastic film puffs up into a steamed bun shape, it indicates that the pot has come to a boil. Sterilization timing starts from this point and must continue for 8-10 hours to ensure thorough sterilization. Some mushroom growers are often careless about sterilization, resulting in failure of spawn production due to severe contamination by competing microorganisms. During atmospheric sterilization, the "three preventions" must be achieved. First, prevent a drop in temperature midway; the fire must not be stopped midway. If the temperature in the pot cannot reach 100°C, the purpose of sterilization cannot be achieved within the specified time. Second, prevent burning the pot dry. Before sterilization, enough water must be added to the pot. During sterilization, if the water level in the pot is insufficient, add water promptly through the water injection port. Third, prevent sterilization dead corners. For example, uneven heating at the bottom of the pot, or steam leaking from only one end at the mouth of the pot cylinder, and so on, may all create sterilization dead corners.
II. Inoculation and Cultivation
The sterilized culture medium can be inoculated. Inoculation is the transfer of spawn onto the culture medium. Personnel engaged in inoculation must establish a strict concept of asepsis, keeping the operator and inoculation tools in a strictly aseptic state. Therefore, inoculation tools must be strictly sterilized, the operating environment must be thoroughly disinfected, and the operator's hands and clothing must also be disinfected, strictly following aseptic operating procedures. Inoculation should be carried out inside an inoculation box or inoculation room.
During inoculation, first clear away debris from the inoculation box or inoculation room and spray-disinfect with 2% Lysol. Place the culture medium to be inoculated, the spawn, and the tools to be used on the workbench in the inoculation box or inoculation room, and fumigate the inoculation box or inoculation room with formalin and potassium permanganate for at least half an hour. The dosage is 5 grams of potassium permanganate plus 10 milliliters of formaldehyde per cubic meter of space. Before inoculation, wipe both hands and the outer surface of the mother spawn test tubes or stock spawn bottles with 75% alcohol cotton, then light the alcohol lamp and carry out the operation. After inoculation is completed, extinguish the alcohol lamp and move the inoculated bottles or bags to the cultivation room for cultivation. Generally, one mother spawn tube can be expanded into 4-5 bottles of stock spawn, and one bottle of stock spawn can be expanded into 40-50 bottles or 15-20 bags of cultivation spawn.
The cultivation room should be kept clean and the environmental conditions such as temperature, humidity, air, and light should be well adjusted. The suitable temperature for spawn cultivation is 20-25°C, and the relative humidity of the air is best controlled at about 60%. During the mycelial growth period, dark conditions are required. The cultivation room must always maintain good ventilation conditions. According to this management method, the spawn will fully colonize the bottles or bags in about 40 days. If the spawn that has fully colonized the bottles (bags) is not used immediately, it should be placed in an environment below 0-10°C, where it can be preserved for 1-3 months.