(1) Sorghum grain : should be free from pest and diseases. Avoid red and black coloured sorghum grains
(2) Glucose bottles: cleaned with soap water, rinsed with fresh water and sun dried. Which is available in hospitals and bottle sellers.
(3) Non absorbent cotton : This is used to plug the mouth of the bottles
(4) Paper : Size of 10 cm x 10 cm used to cover the mouth of the bottle over the cotton plug.
(5) Jute thread Size of 30 cm used to tie around the neck of the bottle.
(6) Vessel: The vessel should be a non corrosive with zinc coating.
(7) Hessian cloth/Khada cloth : This is used to dry the off cooked sorghum grains.
(8) Calcium carbonate : to adjust the pH of the grains and to remove excess moisture.
(9) Base Spawn ( Culture tube containing desired fungus) - Get it from reliable source like University. (IIHR, Bangalore and ANGRAU, Hyderabad ).
(a) Put the sorghum grains in water to remove chaffy and damaged grains
(b) Half cook the sorghum grains for 30 minutes in a vessel.
(c) Test the half cooked grains by gently pressing the grains between the thumb and index finger or chew a grains by incisors. Grains should slightly break but should not be sticky.
(d) Remove the half cooked grain, drain out water and spread cover a hessian cloth evenly to allow excess water to evaporate. (Approximate time required is 30 minutes)
(e) Mix thoroughly 20 g of calcium carbonate for every kg of grains
(f) Fill the grains in glucose drip bottles / PP covers up to ¾th height (300 g / bottle)
(g) Tightly plug the mouth of the bottles with non - absorbent cotton
(h) Cover the cotton plug with the paper and tie it around the neck of the bottle using jute thread.
(i) Keep all the bottles in an autoclave or pressure cooker.
(j) Sterilize the bottles with grains at 20 P.S.I. for 2 hours
(k) After cooling, the bottles are ready for inoculation.
METHOD OF INOCULATION
(a) Keep required number of sterilized bottles containing grains in culture room/LFC.
(b) Transfer with the help of sterilized inoculation needle, a bit of fungal growth (base spawn) into each bottle.
(c) Incubate the spawn bottles at room temperature (30±2oC).
(d) Observe the growing mycelium. In about 15 days complete growth of white mycelium covering the entire bottle could be seen. Allow the spawn to mature for 4 to 5 days more. Now the spawn is ready for use. Use the mother spawn with in 25-35days.
POINTS TO BE NOTED
(a) While cooking avoid full boiling of sorghum grain. Half cooking is sufficient.
(b) Mixing of 20 g of CaCo3/kg dry weight of sorghum seeds helps to remove the excess moisture from seeds as well as maintain the pH of the seeds in neutral
(c) Immediately after two hours of steam sterilization. Remove the sorghum grains bottle from the pressure cooker. Otherwise the grains in bottom bottles will reach its full cooked stage.
Bed Spawn (Commercial Spawn)
(1) Base grain (Sorghum/paddy/paddy husk//husk) taken in glucose bottles/PP covers, sterilized as in above case.
(2) Mother spawn bottle
(3) Hooked 5mm Iron road
(a) From a single mother spawn bottle 30 bed spawn bottles could be prepared.
(b) Two persons are required to transfer the inoculum from the mother spawn bottles to bed spawn bottles. This should be done in the laminar flow chamber or inoculation room.
(c) One person should hold the mother spawn bottle in his left hand and open the plug with right hand by keeping the mouth of the bottle near the flame.
(d) With the help of a hooked sterilized 5mm iron rod, stir the mother spawn to get individual grains with the fungal growth.
(e) Second person, now, can open the sterilized bottle with grains in the same way holding the cotton plug in his right hand.
(f) Now the first person holding mother spawn bottle should transfer about 10 g of sorghum grains with the fungus to the grain containing in new bottles.
(g) Heat the mouth of the inoculated bottle and quickly close with the same cotton plug. All the above steps should be done near the flame to avoid contamination.
(h) Incubate the bed spawn bottle at room temperature. Within 15 days milky white growth could be seen.
(i) 25 days old bed spawn is ideal for bed preparation.
(j) When bed spawn stored under normal room temperature condition, can be used upto a period of 35 days from the date of inoculation.
(k) The temperature below 150C and above 300C is not favorable for spawn growth. Under refrigeration at 50C it can be stored for 2 months.
Submitted by rajudesu on Mon, 08/08/2011 - 22:59