MULTIPLYING SPAWN ON SORGHUM SEEDS

1. Prepare materials:

• Sorghum seeds
• Bottles (flask type)
• Cotton (gauze)
• Paper squares 7 cm x 7 cm
• Rubber bands
• Alcohol lamp
• Alcohol bottle

Note: Various types of grains can be used: Sorghum, millet, wheat

Grains must:

• Have been recently harvested
• Contain few broken kernels
• Little contamination
• No fungi, no insects
• No more than 12% humidity

2. Soak sorghum for one night; 2 liters of water per 1 kg of grain.

• Wash and strain sorghum seeds to remove all water.

3. Steam sorghum seeds for 30-45 minutes to soften grains and cook about 25%.
4. Drain water and spread sorghum seeds to cool down and decrease moisture.
5. Fill ¾ of bottle with sorghum seeds.
6. Carefully prepare cotton plug
7. Tightly plug mouth of bottle with cotton and leave out for ventilation.
8. Transfer all prepared bottles to the sterilization chamber.

Close chamber. Fire-up burner or stove to heat chamber. Make sure to release all air from the chamber before starting. Keep pressure in the chamber at 15 lb./sq.inch. or 121^o Celsius for 30 minutes for small chambers and 45 minutes for medium chambers.

Let bottles cool down.

9. Transfer bottles to a clean and cool place.
10. Bottles must be cleaned and well prepared.

Prepare the well verified PDA bottles

11. Clean laminar flow chamber using alcohol.
12. Transfer PDA, sorghum seed bottles, paper and rubber bands in laminar flow chamber. Light UV lamp for 10 – 15 minutes before starting. Place needle in alcohol.

Turn off UV. Clean both hands with alcohol and insert hands into the chamber.

13. Using 2 fingers, take out needle, pass through fire as to burn alcohol, and disinfect needle. Make sure the needle turns red.
14. After the needle cooled down to normal state, use needle to cut small square (5mm x 5mm) of PDA with mycelium (white color).
15. Close bottle immediately. Remain near flame at all times.
16. Using other hand flame around the mouth and shoulder of the sorghum seed bottle.

Using other fingers, open spawn bottle near flame to avoid contamination.

17. Insert needle and inoculate sorghum seeds with PDA mycelium by placing small square piece in the middle of the bottle. Make sure the PDA mycelium does not touch anything before entering the sorghum seeds bottle.

Note: The mouth of the bottle should be near the flame. The mouth should remain higher than the bottom part at all times. Do not touch mouth of bottle with piece of PDA.

18. Close bottle immediately.
19. Place square paper over cotton and tie with plastic neck or rubber band.
20. Label inoculated sorghum bottles writing: Date, Spawn no., ref., and inoculation time.

Note: It takes about 10 – 15 days to get full-grown sorghum grain mycelium, depending on the species.

21. Keep mature sorghum seeds in a cool place or in the vegetable compartment of the refrigerator. Check for infection regularly.
22. Remove contaminated bottles.

Transfer contaminated bottles to cleaning site. Clean bottles as normal glassware.

23. Observe and collect data. Take notes to draw conclusions.

Note: A loss of about 3% is to be expected.