Isolation techniques for getting pure cultures and their maintenance:
There are two methods to have a mushroom culture –
- The Spore Culture and
- Tissue Culture technique.
- Spore Culture Technique:
a) Spore Print :
– The cap from a healthy, disease free mushroom is removed , surface cleaned with a swab of cotton dipped in alcohol and placed on a clean sterilized white paper or on clean glass plate or on surface of the clean glass slides .
– To prevent air flow , place a glass jar or clean glass or cup over the cap surface.
– Spores will fall on the white paper or slide surface within 24- 48 hours.
b) Spore transfer and germination:
– The scalpel is sterilized by keeping it on a burning flame for 8- 10 seconds till it becomes hot red.
– Cool it by dipping in a sterilized medium.
– Scrap some spores from the spore print taken on a paper or glass slide and transfer them by gently streaking on the agar medium aseptically.
– Three agar dishes should be inoculated for each spore print and the culture developed after
its incubation at appropriate temperature is known as multi-spore culture.
- Tissue Culture Technique:
– A small bit from the pileal region is cut with the help of a sterilized blade or scalpel,
– Washed several times in sterilized distilled water
– Dried in a clean tissue paper before inoculating aseptically on a Petri plate or tube containing suitable culture medium.
– The inoculated Petri plates are incubated at 25 ± 1 °C for 6-12 days and observed at different intervals for the mycelial growth.
– All Petri plates / glass tubes showing contamination should be discarded and only the ones with pure growth should be retained for further use.