Tissue Cultures

1. Prepare materials:

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 121^oC for 20 – 30 minutes to ensure complete sterilization.

Let cool down to around 37^oC

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

Do not want to use your own cultures to start here is a video for one of our YouTube friends who also sell cultures too.

1. Prepare materials:

• Special needle (insulated handle)
• Alcohol lamp
• Alcohol
• Cotton (gauze)
• Matches or lighter
• Bottles with PDA
• Laminar flow cabinet (or protected environment)
• UV lamp

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 45^o-degreeangle, 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 #.