• Introduction
• Layout of a spawn laboratory
• Equipment of spawn laboratory
• Culture media, culture and preservation and storage of culture
• Preparation of Mother Spawn and Planting Spawn
• Qualities of a good spawn and its maintenance
• Transit of Spawn

Introduction

The Spawn comprises mycelium of the mushroom and a supporting medium, which provides nutrition to the fungus during its growth. The yield and quality of spawn is governed by the genetic makeup of the strain, and the technology including the substrates used in spawn production.

Layout of a spawn laboratory

While exact area of the spawn laboratory would depend on several factors, an area 90x30x13’ can easily house a moderate spawn unit. The lab is divided into different work-areas like cooking/autoclaving room, inoculation room, incubation room insulated with air-conditioning, washing area, store office and one cold room heavily insulated for storage of spawn.

Equipment of spawn laboratory

1. Two steam autoclaves (30” dia, 40” h) electrically or steam operated.
2. One boiling vessel,
3. Laminar flow
4. One refrigerator
5. One BOD incubator
6. One pH meter
7. Glassware
8. Chemicals
9. Non-absorbent cotton
10. Polypropylene bags or bottles
11. Air conditioning equipment’s
12. Steel racks
13. Exhaust fans
14. Filters

Culture media, culture and preservation and storage of culture

A. Culture media preparation
The pure culture of cultivated mushroom can be raised on the following media:

1. Wheat extract agar medium : Boil 32-g wheat grains with one litre of distilled water for about 2 hours and filter after 24 h. Add 20g agar to a litre of filtrate and boil. pH of the medium is maintained at 6.5. Fill about 5ml of medium in each test tube. Autoclave test tubes at 15 lb. psi for 20 to 30 minutes.
2. Lambert's agar medium : Add 10 g glucose, 0.5 g magnesium sulphate, 1.9 g potassium dihydrogen phosphate and 20 g agar to one litre of distilled water. The remaining process is the same as for wheat extract agar medium.
3. Malt extract agar medium : Add 20 g malt extract and 20 g agar to one litre of distilled water and boil for 5 minutes. The remaining process is the same as for wheat extract agar medium.
4. Compost extract agar medium : Boil 200 g ready synthetic compost with 2 litres of distilled water for about 2 h and filter after 24 h. Add 35 g agar to 2 litres of filtrate. Adjust pH of medium at 6.5-7.0. Fill about 5 ml medium in each test tube and sterilize at 20 lb. psi for 1 h.
5. Potato dextrose agar medium : Boil 250 g peeled potatoes in one litre of distilled water till these become soft and filter. Add 20 g dextrose and 20 g agar to the filtrate and raise volume of water to one litre. The remaining process is the same as for wheat extract agar medium.

B. Culturing the fungus

1. Tissue culture : Mushrooms used in tissue culture should be atleast 24 to 48 hours old. Any part of the mushroom may be used, although the cap especially the lower portion where the gill plate joins the stem is considered the best tissue for excision. Whenever possible, the mushroom should be at the button stage of development.
   The mushroom is cut lengthwise from cap downwards. With a flamed needle, a small piece of the internal tissue of the broken mushroom is cut and inserted into agar bottle. The inoculated bottles are incubated at 25+1oC preferably in darkness for about 10-15 days.
2. Spore culture : Collect aseptically large sized healthy mushroom with membrane (veil) still intact, surface sterilize the mushroom, mount on a wire stand over a Petridish under glass beaker already sterilized in an oven at 160 o C for about 2 h and cooled. The spores get deposited as spore print. The spores are stored under sterile condition in a refrigerator for future use. These spores can be used for direct inoculation of wheat extract agar medium or Lambert's agar medium.
3. Multispore culture : For raising culture, spore suspension is prepared in sterilized distilled water. One ml of spore suspension containing more than hundred spores is mixed in each test tube containing about 5-7 ml of sterilized wheat extract agar or Lambert's agar liquid medium (45 o C) and slant are prepared. The slants are incubated at 28 o C for spore germination for about 2 weeks. The mycelial threads become visible on slant surface. Then the single spore cultures are raised.

C. Preservation and storage of culture
Proper maintenance of pure cultures of cultivated mushroom is necessary to maintain vigour and productivity. There is no satisfactory way to check and evaluate the qualities of spawn by any rapid on the spot examination. The strains of cultivated mushroom must be suitably preserved and carefully tested from time to time for vigour and productivity.

Conventional methods of culture preservation

1. Periodic transferStock cultures are maintained by periodic transfer on a suitable solid substrate or natural/semi -synthetic agar media.


2. Isolation Pure cultures are raised by using single or mass basidiospore isolation or tissue culture technique from freshly harvested fruit bodies.

Freezing methods of culture preservation
The most effective methods are freeze-drying (Lyophilization), and freezing and storing in liquid nitrogen.

1. Freeze-drying and freezing : It is most economical and effective method of long-term preservation of sporulating fungi. For freeze drying, strains are grown on plates containing suitable agar medium. Three plugs of the advancing edge of the culture are removed with the help of a 5 mm sterile cork borer and transferred in heavy-walled borosillicate glass ampules for freeze-drying and storing in liquid nitrogen.
2. Cryogenic freezing Freeze : drying and freezing methods of preservation cause freezing injury to biological systems. There are compounds that protect living cells and organisms against damage due to freezing and thawing.

Method of Cryogenic Freezing

1. Preparation of Cultures : The culture is raised on agar slants. At optimum production of mycelium, slants are flooded with 10% glycerol or 5% DMSO and gently scraped to obtain a suspension for freezing
2. Filling and Sealing of Ampules : Suspensions of mushroom cultures are filled in heavy walled borosilicate glass ampules and precooled to 5 o C for 30 minutes and then sealed with a semi-automatic sealer.
3. Freezing of cultures : Ampules can be frozen by dipping direct into the liquid nitrogen or by controlled freezing procedure. The ampules, placed onto aluminium cans in boxes, are placed into the chamber of the programmed freezer. The freezing rate is programmed to cool at 1 o C/ minute to -35 o C at which time the temperature is lowered rapidly to below -100 o C. After the ampules have been frozen, they are immediately transferred to storage in liquid nitrogen at -196 o C or liquid nitrogen vapour storage at -150 to180 o C.

Preparation of Mother Spawn and Planting Spawn

The next steps after securing the pure cultures of cultivated mushroom from tissue or spores are preparation of mother spawn, also known as stock or master culture, and planting material (mycelium grown in suitable substrate).

Mother Spawn
The method commonly followed in India is as follows: Ten kg of wheat or Sorghum grains are boiled in 15 litres water for 10-15 minutes and allowed to remain soaked in hot water for 15-25 minutes without boiling. Water is drained off over a wire netting to dry slightly. 120 g gypsum and 30 g lime are mixed with 9 kg of boiled grains. The gypsum prevents the sticking of grains together and lime adjusts the pH.

The grains are filled into half litre milk or glucose bottles upto three-fourth capacity. Bottles are plugged with non-absorbent cotton and sterilized at 20-22 lb. psi (126 o C) for 1 ½ to 2 hours. Sterilized bottles are taken out from the autoclave while still hot and are shaken to avoid clump formation. The bottles are immediately transferred to inoculating room or chamber and allowed to cool down overnight. Next day, bottles are inoculated with two bits of agar medium colonized with the mycelium of pure cultures raised either by tissue or spore by putting the culture bits just opposite to each other in the inner side of glass surface in the middle of the bottle. About 7-10 days after inoculation, bottles are shaken vigorously so that mycelial threads are broken and mixed with grains. Three weeks after incubation, the stock culture becomes ready for further multiplication of spawn. One bottle of stock culture is sufficient to multiply in 30-40 polypropylene bags or bottles. Inoculated bottles are incubated at 25 + 1o C.

Planting Spawn
The technique for raising planting spawn is essentially the same as for stock culture except that instead of glass bottles, polypropylene bags may also be used as the containers for filling grains. Inoculated bottles or polypropylene bags are incubated at 26 +1 o C. In two to three weeks after inoculation, spawn becomes ready for seeding the compost.

Qualities of a good spawn and its maintenance

The spawn should be fast growing in the compost, early cropping after casing, high yielding and should produce better quality of mushroom. In order to maintain the quality, the spawn grower should take care of the following:

1. Select high yielding, early producing and better sporophore quality strain for preparation of spawn.


2. Select unbroken and good quality grain for spawn production.


3. Boiling of grains should be done according to the suggested procedure to maintain about 48-50 percent moisture in the grains.


4. The pH of boiled grains should be adjusted to pH 6.5-7.5 by mixing appropriate quantity of calcium carbonate and gypsum.


5. Prepare spawn from mother culture only. Do not multiply from spawn to spawn.


6. The inoculation should be done in a double chambered closed air-tight inoculation room or under laminar flow.


7. Shake the inoculated bottles throughly and incubate at 24 -26 oC for the growth of mycelium.


8. Sort out and remove the contaminated bottles from spawn room regularly.


9. Store the fully grown spawn at 3-5oC in cold store or refrigerator but for not more than two months.

It is a general experience that spawn prepared on jowar (Sorghum bicolor) or wheat grains gives higher yield over the spawn prepared on bajra, barley, or kodo grains. Both spawn producer and buyer should ensure the following.

1. There should be proper coating of mycelium around grain used as a substrate for spawn production. No loose grains should be seen in the bottle or bags. The grains left over without mycelial coating invite contamination in the compost during spawn-running.


2. The growth of mycelium in the spawn should be silky or strandy type. It should not be cottony because it may lead to stroma formation in casting layer, which interferes with air exchange and absorption of water the casing resulting in lower yields.


3. The growth of spawn should be white. Brown colouration develops as spawn grows. Fresh spawn gives higher yield than the old one.


4. There should be no greenish or blackish spot in the spawn. Such spots indicated ontamination.


5. There should be no slimy liquid in the spawn which indicates bacterial contamination.


6. Old spawn should  not be used because its vigour might have decreased.