Procedure

1. Select well grown, disease free button mushroom early in the morning and keep it on a clean paper for 2-3 hr, to get certain amount of moisture present in the mushroom to get evaporated.
2. Clean the culture room/ laminar flow chamber with antiseptic solution.
3. Keep the sterilized PDA slants, razor blades, forceps etc. inside the chamber and put on he UV light.
4. After 20 minutes put off the UV light and start working after 5 minutes.
5. Sterilize all the instruments to be used by exposing to Bunsen burner.
6. Take in the mushroom and split open the mushroom longitudinally into two halves.
7. Using a blade cut a small piece of tissue from the centre of the spilt mushroom at the junction of pileus and stipe.
8. Remove the cotton plug of the agar slant and the tissue is aseptically placed inside the slant by using a sterilized forceps and closes it immediately.
9. After transferring tissues from the mushroom , the tube are arranged in a wire basket and kept in a clean room at room temperature for the growth of the fungus
10. Observe the tube at periodical intervals and remove the contaminated ones. The tubes will be ready for further use within another ten days. The base spawn is used for preparation of mother spawns.
    

Precautions to be observed:

• Wash the hands with antiseptic lotion before start working inside the chamber. If possible, it is better to use hand gloves while operation.
• It is better that the maximum of two persons may work inside the room at a time. Avoid unnecessary talking while working inside the room.
• While separating the tissue from the centre of the mushroom it should not touch the bottom or sides of the mushroom.

Mother Spawn:
Mother spawn is nothing but the mushroom fungus grown on a grain based medium. Among the several substrate materials tested by TNAU, Coimbatore, sorghum grains are the best substrate for excellent growth of the fungus. Well-filled, disease- free sorghum grains are used as substrate for growing the spawn materials. The various steps involving in preparation of mother spawn are listed below here under.

Procedure

1. Wash the sorghum grains in water thoroughly to remove chaffy and damaged grains.
2. Cook the grains in an autoclave / vessel for 30 minutes just to soften them.
3. Take out the cooked grains and spread evenly over a Hessian cloth on a platform to remove the excess water.
4. Mix Calcium carbonate (CaCO3) thoroughly with the cooked, dried grains @ 20 g / kg.
5. Fill the grains in polypropylene bags up to ¾ th height (approximately 300-330 g/bag), insert a PVC ring , bold the edges of the bag down and plug the mouth tightly with non-absorbent cotton wool.
6. Cover the cotton plug with a piece of waste paper and tie tightly around the neck with a jute thread.
7. Arrange the bags inside an autoclave and sterilize under 20 lbs. pressure for 2 hours.
8. Take out the bags after cooling and keep them inside the culture room and put on the UV light.
9. After 20 minutes put off the UV light and start working in the culture room. Cut the fungal culture into two equal halves using a inoculation needle and transfer one half portion to a bag. Similarly, transfer another half portion of the culture to an another bag.
10. Incubate the inoculated bags in a clean room under room temperature for 10 days for further use to prepare bed spawn.

Bed spawn:
 The method of preparation of bed spawn was same as that of mother spawn. The cooking, filling and sterilization were similar to that of mother spawn. After sterilization , the bags are taken for inoculation.

Precautions to be observed:

• Avoid over cooking of sorghum grains on the floor. Always dry over hessian cloth spread on a raised platform.
• Don’t dry the cooked grains on the floor. Always dry over hessian cloth spread on a raised platform
• Use only recommended dose of CaCo3 for mixing with the cooked grains. Mixing over dose reduces the fungal growth in the inoculated bags.
• Avoid further sub culturing of the second-generation bed spawns. This leads to lose of virulence of the spawn lead to reduced yield and repeated sub culturing lead to complete lose of virulence wherein the fungal growth may be noted in the beds but no buttoning is completely arrested.

Procedure:

1. The sterilized bags are placed inside the culture room and put on the UV light.
2. After 20 minutes put off the UV light and take in the well-grown mother spawn.
3. Transfer spawn from the mother spawn to sterilized bags @ 10 g per bag.
4. After inoculation the spawn bags are kept in a clean room for fungal growth. (This is first generation of bed spawn).
5. Use the bed spawn after 10 days of inoculation for bed preparation.
6. Sub culture the first generation bed spawn as mother spawn to produce one more generation, which is second generation bed spawn.
   

Updated on Dec 2013