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Three Inoculation Methods and Practical Operation Procedures for King Oyster Mushroom

1. Solid Spawn Inoculation

Start inoculation only when substrate temperature drops below 25 °C to lower contamination risks.

• Pre‑operation preparation: Turn on inoculation‑room sterilization devices 4‑5 hours in advance, and activate the air‑purification system 1 hour ahead to keep positive pressure.

• Personnel & tools: Operators wear sterile protective suits, shoes and caps. Disinfect hands with 75% alcohol swabs. Wipe inoculation tools with 75% alcohol, then flame‑sterilize and cool before use.

• Spawn handling: Select and pre‑sterilize spawn on the same working day. Scrape off the aged top mycelium layer (1‑2 cm). Break spawn apart and fill substrate‑bag holes with an inoculation spoon, making sure spawn reaches hole bottoms for fast, even mycelial growth.

• Inoculation dosage: Fully fill pre‑drilled holes and spread a 0.3‑cm‑thick even spawn layer over the substrate surface.

2. Twig Spawn Inoculation

Use twig spawn 10 days after full bag/bottle colonization, to complete mycelium post‑maturation and realize high survival rate and fast colonization. Using spawn right after full colonization will easily cause poor germination and inoculation failure.

Operating steps:

1. Disinfect spawn surface with alcohol swabs; remove bag collar and aged surface mycelium.

2. Take out twig spawn, insert it into pre‑punched holes of sterilized substrate bags.

3. Seal bags with caps.

3. Liquid Spawn Inoculation

3.1 Clean‑room Requirements

The clean‑room system shall include central air filtration, refrigeration and ozone disinfection systems. Functional zones: primary cooling room, rapid cooling room, pre‑inoculation room, inoculation room and spawn‑processing room.

• Workshop entrance: install airlock and disinfection pit.

• Inoculation room: equip with Class‑100 air filtration system and mechanical conveying line.

3.2 Inoculation Process

1. Cool cultivation bottles/bags below 25 °C before inoculation. Run all filtration and disinfection systems per standard startup procedures; personnel shall comply fully with clean‑room disinfection rules when entering and exiting.

2. Transfer crates of cooled bottles/bags onto the special inoculation conveying line. Under high‑efficiency filters, operators use automatic liquid‑spawn machines for inoculation. Set tank pressure at 0.20 MPa, apply 25‑30 ml liquid spawn onto each bottle/bag opening.

3. Maintain the inoculation environment at Class 10 000 cleanliness. For detailed operations, refer to liquid‑spawn inoculation for Flammulina velutipes.

4. After inoculation, move crates off the conveying line and transport them to the incubation room by transfer carts.