||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
- Two steam autoclaves (30 dia, 40 h)
electrically or steam operated.
bags or bottles
Culture media, culture and preservation and
storage of culture
||A. Culture media preparation
culture of cultivated mushroom can be raised on the following media:
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.
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.
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.
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.
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
Culturing the fungus
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.
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.
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.
Preservation and storage of culture
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
Stock cultures are maintained by periodic transfer on a suitable solid substrate
or natural/semi -synthetic agar media.
Pure cultures are raised by using single or mass basidiospore isolation or tissue
culture technique from freshly harvested fruit bodies.
methods of culture preservation
effective methods are freeze-drying (Lyophilization), and freezing and storing in liquid
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.
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.
of Cryogenic Freezing
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
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.
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.
II. Preparation of Mother Spawn and Planting
||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).
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.
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.
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
high yielding, early producing and better sporophore quality strain for preparation of
unbroken and good quality grain for spawn production.
of grains should be done according to the suggested procedure to maintain about 48-50 per
cent moisture in the grains.
of boiled grains should be adjusted to pH 6.5-7.5 by mixing appropriate quantity of
calcium carbonate and gypsum.
spawn from mother culture only. Do not multiply from spawn to spawn.
inoculation should be done in a double chambered closed air-tight inoculation room or
under laminar flow.
the inoculated bottles throughly and incubate at 24 -26 oC for the growth of mycelium.
and remove the contaminated bottles from spawn room regularly.
the fully grown spawn at 3-5oC in cold store or refrigerator but for not more than two
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.
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.
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.
growth of spawn should be white. Brown colouration develops as spawn grows. Fresh spawn
gives higher yield than the old one.
should be no greenish or blackish spot in the spawn. Such spots indicated ontamination.
should be no slimy liquid in the spawn which indicates bacterial contamination.
spawn should not be used because its vigour might have decreased.
Transit of Spawn
||During transportation, spawn should be exposed
to temperatures higher than 35 oC. The spawn bottles or bags can be packed in thermocol
boxes containing ice. Alternatively, spawn can be transported during night. Immediately
after reaching the destination, the spawn bags/bottles should be take out of packing from
thermocol boxes for spawning. Spawn should be used fresh. The spawn can be stored at 5-10
oC for one month, if not used. Volvariella spawn should never be refrigerated. Once the
container is opened, spawn should be used in its entirely.
of contamination in a spawn making plant is the grain used to prepare the substrate.
Modern equipment and facilities for sterilization and maintenance of sterile conditions
reduce fungal and bacterial contamination to 0.1 per cent of the number of spawn units.
Quality control in spawn making ordinarily consists of inspection to eliminate spawn units
visibly contaminated or exhibiting unacceptable differences in appearance, growth colour