Spawn is the mushroom seed that is grown on grains to produce the mycelium used for mushroom cultivation. High quality spawn is essential for successful mushroom production. Spawn is produced through processes that include preparing sterile culture media, isolating pure mushroom cultures, growing the cultures on grains to produce the mycelium, and storing the spawn under proper conditions. Quality spawn exhibits full mycelial coverage of grains, fast growth in compost, early and high crop yields, and produces mushrooms of good quality.

1. SPAWN PRODUCTION TECHNOLOGY
Spawn is seed of the mushroom crop. It plays an important role in mushroom industry
because the failure or success of mushroom production depends upon the availability and quality
of spawn. Spawn, the mushroom seed is comparable to vegetative seed in crop plants. It is
merely the vegetative mycelium from a selected mushroom grown in a convenient medium. The
success of mushroom cultivation and its yield depends to a large extent on the purity and quality
of the spawn used. 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 spawn laboratory
The spawn laboratory is divided into different working-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.
Equipments required for spawn laboratory:
1. Autoclave
2. Boiling vessel
3. Laminar flow
4. Refrigerator
5. BOD incubator
6. Glassware
7. Chemicals
8. Non-absorbent cotton
9. Polypropylene bags or glass bottles
10. Air conditioning equipments
11. Steel racks
The preliminary requirement for making spawn is to make a pure culture of the desired
mushroom species on a suitable medium.

2. Culture Media Preparation
The medium on which the pure mushroom mycelium is grown is called culture medium.
These media vary widely in form and composition, depending on the organism to be cultivated.
Some of the common media used are:
Potato Dextrose Agar medium
Composition
Potato = 250g
Dextrose = 20g
Agar = 20g
Distilled water = 1000 ml
Two hundred and fifty grams of peeled potatoes are boiled in one litre of distilled water
till they become soft. This is then filtered through a cheese cloth. The liquid is then collected in a
graduated cylinder. The volume of decoction is made up to 1000 ml by adding fresh distilled
water. 20g of Dextrose and 20g of Agar powder is then added and boiled by stirring with glass
rod. The medium is then transferred to test tubes and plugged with non-absorbent cotton.
Malt Extract Agar medium
Composition
Malt extract = 20g
Agar = 20 g
Distlled water = 1000 ml
Dissolve 50g of commercially available malt extract Agar in 1000 ml of distilled water.
Boil it and pour it in test tubes and plug with non absorbent cotton. pH of media should range
between 6.5 to 7.0
Sterilization
The media should be sterilized immediately after its preparation. Sterilization is done in
autoclave at 15 lb. pressure, which gives a temperature of 121o
C for 15-20 minutes. After
sterilization, tubes are kept in an inclined position for 8-10 hrs to get the media in slant shape.

3. Isolation of Pure Culture
Initial mushroom cultures can be obtained by any of the following two ways:
1. Tissue culture: For tissue culture, a healthy mushroom with intact veil on the lower side of
the mushroom cap (pileus) is selected from cropping trays or bags. Lower portion of the stem
is cut with a sharp knife and washed with running tap water to remove traces of soil or
compost adhering to it. This is then dipped in 0.1 percent solution of HgCl2 for 30-60
seconds for surface sterilization. Small pieces of tissue from junction of stipe and pileus is
taken out with a sterilized inoculating needle and transferred aseptically to Potato Dextrose
Agar slants. The inoculated tubes are incubated at 25±1o
C for 10-15 days, till the surface of
medium is fully covered with mycelial growth of the desired fungus.
2. Spore Print: For raising mono or multi spore culture, spores are collected under sterile
conditions from a large sized healthy mushroom with intact veil. The mushroom after surface
disinfection process as described in tissue culture is mounted on a wire stand over a petridish
containing a sterilized filter paper and covered with a sterilized glass beaker. On opening of
veil, the spores are discharged and deposited on the filter paper in petridish. This spore mass
is known as spore print. This spore mass can be kept under sterilized conditions in a
refrigerator for further use. These spores may be used for direct inoculation on potato
dextrose medium.
Spore culture: For raising culture, spore suspension is prepared in sterilized distilled water.
One ml. of spore suspension is mixed in each test tube containing about 5-7 ml of sterilized
wheat extract agar or Lambert's agar liquid medium (450
C) and slant are prepared. The slants are
incubated at 280
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: Proper maintenance of pure cultures of cultivated
mushrooms is necessary to maintain vigour and productivity. There is no satisfactory way to
check and evaluate the quality of spawn by any rapid or 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

4. 1. Periodic transfer: Stock 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: Drying and freezing methods of preservation may 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: Suspension of mushroom cultures are filled in heavy
walled borosilicate glass ampules and precooled to 50
C for 30 minutes and then sealed
with a semi-automatic sealer.
3. Freezing of cultures: Ampules can be frozen by dipping directly 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 10
C per minute to -350
C and the temperature is lowered rapidly to below
-1000
C. After the ampules have been frozen, they are immediately transferred to storage
in liquid nitrogen at -1960
C or liquid nitrogen vapour storage at -150 to -1800
C.

5. Preparation of Mother Spawn: The next step after securing the pure cultures of cultivated
mushroom from tissue or spores is preparation of mother spawn, also known as stock or master
culture and planting material (mycelium grown in suitable substrate). Commonly, wheat grains
are used for spawn preparation. Wheat grains are cleaned and washed. Ten Kg of wheat grains
are boiled in 15 litre hot water for 10-15 minutes and allowed to remain soaked in same hot
water for 15-25 minutes. Water is then drained off over a wire netting and dried under shade to
remove surface water. On the same day 2.0% CaSo4 to prevent sticking and 0.5% CaCo3 to
maintain the pH is mixed to the grains .
The grains are filled into half litre glass bottles upto three-fourth capacity. Bottles are
plugged with non-absorbent cotton and sterilized at 20-22 lb. psi (1210
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, the bottles are inoculated with bits of agar medium colonized
with the mycelium of pure cultures raised either by tissue culture or spore print 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.
Preparation of commercial spawn: For commercial spawn production, the substrate and
technique remains the same. Instead of glass bottles polypropylene bags are used for commercial
spawn. One bottle of stock culture is sufficient to multiply in 8-10 polypropylene bags.
Inoculated polypropylene bags are incubated at 25 ±10
C.
Qualities of good spawn:
The spawn should be fast growing in the compost, yield 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 points:
1. Select high yielding, early producing and better sporophore quality strain for spawn
preparation.
2. Select unbroken and good quality grains for spawn production.

6. 3. Boiling of grains should be done according to the suggested procedure to maintain
about 48-50 per cent 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 calcium sulphate.
5. Prepare mother spawn from pure culture only.
6. The inoculation should be done in a double chambered closed air-tight inoculation
room or under laminar flow.
7. Sort out and remove the contaminated spawn bottles from spawn room regularly.
8. Store the fully grown spawn at 3-50
C in cold store or refrigerator but for not more than
two months. However, care should be taken not to store the spawn of Calocybe indica
at low temperature.
Spawn producer and consumer should ensure the following points:
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 casing layer, which interferes with air
exchange and absorption of water.
3. The growth of spawn should be white. Old spawn changes to different colours. Fresh
spawn gives higher yield than the old one.
4. There should be no greenish or blackish spots in the spawn. Such spots indicate
contamination.
5. There should be no slimy liquid in the spawn which indicates bacterial contamination.
Transit of spawn:
Care must be taken during transportation of spawn. Following precautions must be ensured
during transit of spawn:
1. Don’t expose spawn bottles to temperature higher than 350
C
2. Always use fresh spawn and its storage should be avoided. However spawn stored at 40
C
can be used up to three months.