Mushroom Culture

Isolation techniques

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.
1. Spore Culture
a) Spore Print :

• In order to get a spore print or collection of spores , 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 .The surface nearby should be thoroughly sterilized. 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 exactly like radial symmetry of the gills .The spore print on the paper can be preserved for a longer time by cutting and folding it into two halves.

Fig. 2.17 Healthy fruit body kept covered for getting spore print
Fig. 2.18 Spore print collected after 24 -72 hours

b) Spore transfer and germination:

• In order to get a pure culture , 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. Minimum, three agar dishes should be inoculated for each spore print and the culture developed after its incubation at appropriate temperature is known as multispore culture.

2.Tissue Culture

• 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 and 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 ± I C for 6-12 days and observed at different intervals for the mycelial growth. All Petri plates / glass tubes showing contaminations should be discarded and only the ones with pure growth should be retained for further use after ascertaining the purity and true to type nature of the culture.

Fig. 2.19 & 2.20 Healthy fruit body picked for tissue isolation in an Air flow chamber


Fig. 2.21 Agar slants and pure culture of a mushroom in test tubes
Fig. 2.22 Pure cultures of a mushroom in agar medium in Petri dishes.

Fig. 2.23 The inoculated tubes and Petri dishes kept for incubation in B.O.D incubator
Fig. 2.24 Pure cultures in tubes and Petri dishes stored in refrigerator

Sub-culturing:
The pure culture of edible mushroom, once established either through spore culture or tissue culture technique , is maintained properly in cool atmosphere or a refrigerator. Sub-culturing is done from time to time by aseptically transferring a small piece of growing pure culture along with the culture medium on the test tube slants containing same or other suitable medium.

The pure culture of a mushroom can be used for preparing master cultures for large scale spawn production on commercial scale . It will be discussed in the next lesson in detail.