Secreting a Good Quality Fruiting Culture

(1) Sources of the cultures

(A). Tissue culture. A large healthy mushroom should be chosen either in the later button or egg stage. It should be cleaned with 75% alcohol. The mushroom should be split in half by hand longitudinally and some inside tissue taken from the upper part of the stipe. It should be placed centrally on the surface of the medium with a sterilised needle. The quicker this is done the better. As soon as we transfer the tissue, the test tube should be closed and dated before it is returned to the incubator between 25°C and 34°C depending on the mushroom used. Within two or three days some white, delicate mycelia will be produced from the small piece of the tissue. They grow upwards encircling the inner wall of the test tube. About ten days later the mycelium will grow rapidly and cover the surface of the agar medium. Then it is ready to transfer to spawn substrate to make spawn.

(B). Spore culture. Individual spores properly collected can be transferred singly to a testtube or petri dish and allowed to develop and germinate into mycelium. Some single-spore isolates from homothallic mushrooms, e.g.  Volvariella volvacea  (primary homothallism) or Agaricus bisporus (secondary homothallism) can be used as fruiting culture to make spawn. However if single-spore isolates are from heterothallic mushrooms, e.g.  Lentinula edodes, Pleurotus sajor-caju and Ganoderma lucidum, then they cannot form fruiting cultures and thus cannot make spawn. They have to be mated with a compatible single-spore isolate. After mating they form a dikaryon/fruiting culture. Then they can be used to make spawn.

(C). Pure culture from other laboratories. As an alternative to culturing in the laboratory, as outlined, a test tube culture may be obtained from a research laboratory. The advantage of this is that cultures maintained in reputable culture collections are already tested for their production characteristics and are guaranteed to be pure.

(D). Cultures from another source. Cultures also may be grown from spawn obtained from another source. A piece of the spawn is aseptically transferred to agar slants. However, this is risky because the number of transfers that the spawn culture has undergone is rarely known. If this procedure is followed, it may be advisable to first grow the spawn into fruiting bodies, then make the necessary isolations from the fruiting body.

(2) Culture media  

Mushrooms grow on a variety of culture media and on different agar formulas, both natural and synthetic, depending on the organism to be cultivated and the purpose of the cultivation. Synthetic media are often expensive and time-consuming in preparation hence they are not commonly used for routine purposes in mushroom laboratories.

(A). PDA (potato dextrose agar), is the simplest and the most popular medium for growing mycelia of most cultivated mushrooms. It can be purchased commercially as ready mixed powder which can be used directly to make the medium in the laboratory, with a concentration of 20gm/1 litre of distilled water. 

(B). Alternatively, it can be prepared in the laboratory with the following ingredients: Potato, diced - 200 gm (1/5kg); Dextrose - 20 gm; Powdered agar or agar bars - 20gm; Water - 1 litre. Procedure: Peeled potatoes are washed, weighed, and cut into cubes. They are boiled in a casserole with at least one litre of water until they become soft (around 15 minutes). The potatoes are removed and water is added to the broth to make exactly 1 litre. The broth is
returned to the casserole, and dextrose and the agar added. The solution is heated and stirred occasionally until the agar is melted. The hot solution is then poured into clear flat bottles filling to about 2.5cm from the bottom. When using test tubes for the stock cultures, they are filled with at least 10ml of the liquid agar solution. The bottles or test tubes are plugged with cottonwool. When Petri dishes are available and are used to produce mycelial colonies, the solution can be poured into the dishes to form a layer on the bottom.

(C). A ready-made MEA (malt extract agar) powder is also available commercially. The recommended amount of powder (20gm) is mixed with 1 litre of water, then melted and sterilised. One percent peptone or 0.5% yeast may be added for faster mycelial growth for both PDA and MEA.



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