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. With the needle, cut a small piece (5 mm x 5 mm) of mycelium on PDA Make sure that the PDA not contaminated.
1) Flame around the mouth of the new PDA bottle. Using other fingers, remove the cotton plug of PDA bottle in front of the flame to secure against contamination.
2) Insert the needle in the bottle and inoculate by placing a small piece of PDA mycelium in the middle of the PDA’s surface. Make sure the mycelium PDA does not touch anything before entering the PDA bottle.
3) Close bottle immediately near the flame with a 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.
4) Label bottles and indicate: Date, type of mushroom, and mother spawn #.
5)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).
6)Keep PDA bottles with mycelium on a clean shelf.
Check infection by other fungi in the bottle every day. Also, check the growth rate.
7) After mycelium covers the whole PDA medium, keep mature mycelium in a cool place or in the refrigerator in the vegetable section.
8) Check for contamination. Separate contaminated bottles. Transfer contaminated bottles to clean.
9) Keep detailed notes of observations.