The cultivation of Agaricus blazei three-grade strains typically relies on wheat kernels as the primary raw material. However, in actual production, high contamination rates and abnormal growth have been observed. These issues are often attributed to non-standardized production practices. To address this, detailed guidelines and operational techniques have been developed to assist farmers in improving their strain cultivation processes.
1. **Raw Material Preparation**: Cow manure is first sifted and mixed with water at a ratio of 1:1.2. The mixture is then fermented and turned daily for about 20 days until it turns brown and emits a normal fermented odor. After drying, it is ready for use. Wheat grains must be soaked thoroughly, drained, and dried in a shaded, well-ventilated area. Once the outer layer is dry, they should be lightly misted to maintain moisture. After 24 hours, germination begins. When the sprouts reach 1–2 cm in length, the grains are ready for use.
If the wheat has been worm-eaten, it can still be used, but the grains with holes must be washed multiple times. Soaking them in lime water (pH 8–9) for 6 hours ensures that the grains are fully cooked. After soaking, they should be rinsed with cold water until no white core remains.
2. **Basic Formula**: The standard mix includes 100 kg of dried wheat grains, 30 kg of cow dung powder, 1 kg of gypsum, and 1.5 kg of calcium carbonate.
3. **Substrate Mixing**: Wheat grains are mixed with cow dung powder in a 1:0.2 ratio, ensuring full coverage of the grains. Other additives are also incorporated during mixing. Any leftover cow dung powder is stored for future use.
4. **Bottling Process**: Small-scale operations often use 500 ml infusion bottles instead of standard ones to reduce costs. A thin layer of cow dung is placed at the bottom of each bottle, followed by the wheat grains. The grains should be filled to about 1 cm below the bottle's neck. Then, 1–2 pieces of cow dung powder are added, and the mixture is compacted using an "L"-shaped tool before being sealed with a tampon.
5. **Sterilization and Cooling**: Autoclaving is performed at 0.15 MPa for 2–2.5 hours. Once the pressure drops to zero, the bottles are moved to a sterilized cooling room. The room should be cleaned and disinfected in advance, and the bottles are inoculated once cooled to 30°C.
6. **Inoculation Procedure**: Before inoculation, the room is sprayed with Saibai 09 solution. The original culture is wiped with 75% alcohol, and after 10 minutes, the inoculation process begins. Each 500 ml bottle can be inoculated with more than 40 cultures.
7. **Cultivation Environment**: The culture room should be cleaned and sprayed with a disinfectant solution. After two days of confinement, lime powder is applied to the floor to transfer the inoculated strains. The temperature should be maintained around 25°C, not exceeding 28°C. If farmers lack temperature control, they can cool the bottles by placing them in groundwater or using plastic membranes over bricks. Water should be changed frequently to keep temperatures below 25°C. Spraying disinfectants every few days helps prevent contamination. High-efficiency cypermethrin can be used to eliminate pests like flies and mosquitoes.
8. **Contamination Control**: On the third day post-inoculation, any contaminated bottles should be removed immediately. For Streptomyces contamination, the bottles are carefully extracted and disposed of. General bacterial contamination can be treated by spraying and reusing the bottles after drying. Clostridium contamination requires burning or deep burial. Empty bottles should be scalded with hot water and washed before reuse.
9. **Temporary Storage**: After 35–45 days, the strains will begin to produce spores. Unused strains should be stored at 2–4°C to maintain quality.
10. **Quality Assessment**: Healthy Agaricus bisporus mycelium is thick, white, and shows strong, uniform growth. The "2796" strain, commonly used, has a semi-aerobic structure with consistent hyphae. Unhealthy signs include yellowing, clumping, discoloration, or the presence of pests and odors. If the mycelium is weak, sparse, or shows uneven growth, it may indicate improper soaking, excessive heat, or aging. Such strains should not be used for planting.
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