Tissue Culture
Potatoes: 200 gr.
Dextrose: 20 gr.
Agar powder: 20 gr.
Water: 1 liter.
Cotton (gauze)
Note: Visually check potatoes for spots or rot. Buy dextrose and Agar of commercial grade.
2. Wash and cut potatoes into one-centimeter cubes; leave on or remove the skin.
3. Clean small flat bottles (small whiskey bottles as a container can be used).
4. Place potatoes in one liter of water. Simmer for 15 - 20 minutes.
5. Remove potatoes & keep the broth as clear as possible. Add water to broth to reach one liter of liquid PDA
6. Bring water to stove. Add dextrose followed by agar. Slowly stir continuously with regular speed until completely dissolved.
7. Pour liquid PDA in bottle until you reach 5 - 10 mm high.
8. Plug bottle with cotton.
9. Place bottles in autoclave at 121oC for 20 - 30 minutes to ensure complete sterilization. Let cool down to around 37oC.
10. Place bottles in slanted position as to increase surface area of the medium. PDA should come close to the neck but must not touch the cotton plug.
After PDA medium is settled in bottle, transfer all bottles to clean shelf in the clean room.
11. Check for contamination (contamination can be seen when dark spots or lines occur).
Selecting tissue culture
1. Prepare materials:
- Special needle (insulated handle)
- Alcohol lamp
- Alcohol
- Cotton (gauze)
- Matches or lighter
- Bottles with PDA
- Laminar flow cabinet (or protected environment)
2. Select a strong mushroom for culture.
- Healthy.
- Not too mature, not too young.
- Not too humid (at least 2-3 hours after watering)
- With a stiff stalk
- Make sure it is clean and far from any contaminated mushroom.
3. Clean the room, all necessary tools, inside and outside the laminar flow cabinet with alcohol. Transfer PDA bottles and necessary tools into the chamber.
4. Place all cleaned materials inside laminar flow. Turn on UV lamp and laminar flow. After 10-15 minutes, turn off UV lamp but leave laminar flow for the duration of the operation.
5. Clean both hands and bottles with alcohol and insert hands into the cabinet.
6. Hold needle with 2 fingers in a 45o-degree angle, flame needle to disinfect until the needle turns red. Make sure it does not touch any surface after flaming.
7. While needle cools down (15-20 seconds - hold needle not to touch anything or place it on the clean surface of a glass).
8. Using other fingers, tear mushroom lengthwise (DO NOT use knife to cut).
9. With the needle, cut a small piece (2 mm x 2 mm) of fleshy tissue from inside the mushroom (in the middle between the cap and the stalk). Make sure that it is clean and did not touch the outside of the mushroom.
10. Flame around the mouth of the bottle. Using other fingers, remove cotton plug of PDA bottle in front of flame to secure against contamination.
11. Insert the needle in the bottle and inoculate by placing small piece of cut mushroom in the middle of the PDA’s surface. Make sure the piece of mushroom does not touch anything before entering the PDA bottle
12. Close bottle immediately near the flame with cotton plug
Note: the bottom of the bottle should always be lower than the mouth of the bottle and the mouth of the bottle should remain near the flame at all times.
13. Label bottles and indicate: Date, type of mushroom, mother spawn #.
Culture from PDA to PDA
Because of the extremely delicate nature of tissue culture, it is highly recommended that tissue culture be done in only a few bottles of PDA since there is high risk of contamination. Then, several bottles of PDA can be prepared from the extremely pure mycelium.
9. With the needle, cut a small piece (5 mm x 5 mm) of mycelium on PDA Make sure that the PDA not contaminated.
10. Flame around the mouth of the new PDA bottle. Using other fingers, remove cotton plug of PDA bottle in front of flame to secure against contamination.
10. Flame around the mouth of the new PDA bottle. Using other fingers, remove cotton plug of PDA bottle in front of flame to secure against contamination.
11. Insert the needle in the bottle and inoculate by placing small piece of PDA mycelium on the middle of the PDA’s surface. Make sure the mycelium PDA does not touch anything before entering the PDA bottle.
12. Close bottle immediately near the flame with cotton plug
Note: the bottom of the bottle should always be lower than the mouth of the bottle and the mouth of the bottle should remain near the flame at all times.
13. Label bottles and indicate: Date, type of mushroom, mother spawn #.
14. Whether from tissue culture or PDA to PDA, from the time of incubation to full growth mycelium will take about 10 - 15 days. (Depending on species).
15. Keep PDA bottles with mycelium on clean shelf.
Check infection by other fungi in the bottle everyday. Also check growth rate.
16. After mycelium covers whole PDA medium, keep mature mycelium in cool place or in the refrigerator in the vegetables section.
Step 4. 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
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 121o 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
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 121o 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.
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.
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.
