HANDS ON TRAINING

(HOT)

EAM 421(0+10)
MUSHROOM CULTIVATION
TECHNOLOGY

TOPIC:
Mother Culture Preparation
of Mushroom
MUSHROOM

Mushroom is a fleshy fruiting body of

some fungi arising from a group of
mycelium buried in substratum.
Most of the mushrooms belong to the
Sub-Division: Basidiomycotina and a
few belong to Ascomycotina of King-
dom-Fungi.

For cultivation of mushroom primarily

we need a Pure culture.
 • The pure cultures are raised on a
convenient culture medium which
are generally in solid state. Pure
culture is essential for the
preparation of Mother culture .
• Isolation of pure culture is done
by two methods
1. Spore culture
• Spore print
• Spore Transfer and Germination
2. Tissue culture
PURE CULTURE
DIFFERENT CULTURE MEDIA

Different culture media used for culturing pure cultures of different

mushrooms are :
• Potato-dextrose agar media(PDA)
• Potato – dextrose Yeast agar media(PDYA)
• Malt extract agar media(MEA)
• Compost extract agar media(CEA)
• Malt peptone grain agar media(MPGA)
SPORE CHARACTERS OF DIFFERENT MUSHROOMS

Agaricus bisporus: The Button Mushroom

• Cap is convex to broadly convex
• Spore print is brown
Pleurotus spp : The oyster mushroom
• The cap is tongue shaped, maturing to
a shell shaped form
• Spores are whitish to lilac grey
• Mycelium is whitish, fast growing
Volvariella spp : The paddy straw mush-
room
• Cap is egg shaped
• Spores are pinkish to pinkish brown in
mass
Auricularia spp : Jew’s ear mushroom
• Cap is ear or shell shaped
• Spores are white, cream or yellowish
Lentinulla spp : Shiitake mushroom
• Cap is umbrella shaped
• Spores are white in color
SPORE CULTURE METHOD

1. 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 .
SPORE CULTURE cont…

2. 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.
 TISSUE CULTURE METHOD

• A small bit from the pileal region is cut with

the help of a sterilized blade or scalpel
• It is 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.
Tissue culture method for mushroom
PURE CULTURES OF DIFFERENT MUSHROOMS

Oyster Button
mushroom mushroom

Shiitake
mushroom
 SUB-CULTURING

• The pure culture of edible mushroom, once

established either through spore culture or
tissue culture technique, should be 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
commercial spawn production.
 • Mother culture is the seed of mushroom
that is directly prepared from the pure
culture. It is usually grown on a grain
medium.
• Here the vegetative mycelial network
of a mushroom developed after the
germination of one or more than one
fungal spore grown on a convenient
medium.
• Among the various base mediums,
sorghum grains are considered to be the
MOTHER CULTURE best for mother culture production
MOTHER CULTURE PREPARATION

For the prepreparation of Mother culture, following materials are

required :
• Suitable medium – wheat/sorghum/rye
• Pure culture
• Gypsum
• Calcium carbonate
• Polythene covers
• Non absorbent cotton

* The whole procedure is carried out in aseptic conditions in Laminar air flow *
PROCEDURE

1. Weigh 1 kg of sorghum seeds and soak the sorghum grains in

clean water to remove chaffy and damaged grains.
2. Add 500ml of water and cook the grains in a vessel for
30 minutes.
3. Take out the cooked grains and dry them by spreading evenly
on the platform to remove excess water.
4. Add 2% CaSO4 and CaCO3 to the grains and mix the chemicals
well.
5. Pack the seeds into bags
weighing 400 to 450g and
tie the bags by placing non
absorbent cotton.
6. Arrange the bags inside an autoclave and sterilize under 20-lbs.
pressure for 2 hours. 
Allow the bags to cool down. Then place the bags in laminar air
flow and start working. These bags are inoculated with the bits
of agar medium colonized with the mycelium of pure culture.
These are incubated at 25±1°C.
After 7 days of inoculation, bags are shaken vigorously so that
mycelial threads are broken and become well mixed with the
grains. 2 weeks after inoculation, Mother culture is ready which
can be further made use for the preparation of commercial
spawns.
LIST OF DIFFERENT SUBSTRATES USED

Mushroom species Substrate Colonization period

Agaricus Cereal grain 20-25 days

Auricularia Saw dust/ grain 18-20 days

Lentinula Saw dust / grain 20-25 days

Volvariella Used tea leaves/ paddy 10-12 days

straw +saw dust/ cereal
grains

Pleurotus Cereal grains 10-15 days

PRECAUTIONS TO BE OB-
SERVED
• Avoid overcooking of grains as it may lead to
splitting of grains.

• Don’t dry the cooked grains on the floor. Always

dry over hessian cloth spread on a raised platform
or on a wire mesh tray .

• Use only recommended dose of CaCO3 for

mixing with the cooked grains.

• Avoid further sub culturing of the second

generation spawn. This leads to loss of
vigour of the spawn which again leads to
reduced yield.
CHARACTERS OF GOOD
SPAWN
• There should be proper coating of the mycelium
around every grain used as substrate for spawn. 
• The growth of the mycelium in the spawn bag
should not be cottony or fluffy type but it should
be strandy.
•  The growth of fresh spawn is more or less white.
Brown coloration develops as spawn grows. 
• There should not be any slimy growth in the
spawn bottles which is an indication of bacterial
contamination.
•  There should not be any greenish or blackish
spot in the spawn bottles. Such type of spots indi-
cates that the spawn is contaminated with
moulds.
MUSHROOM REPOSITORIES

Mushroom repositories/Banks play a vital role in supply of pure and authentic

cultures to most of the mushroom spawning producing units. One can
obtain pure cultures for making cultures from any of the National /
International repositories. Some of them are :
National
• National Research center for Mushroom, Chambaghat, Solan, HP.
• Division of Mycology and Plant pathology, IARI, Pusa, New Delhi
• Indian Institute of Horticultural Research, Bangalore, karnataka.
• Institute of Microbial Technology(IMTECH),Sector 39D, Chandigarh
International
• International Mycological Institute, Kew, Surrey, UK.
• Dutch Mushroom Experimental Station, The Netherlands .
THANK
YOU
PRESENTED BY :
MONICA RG
ALB8137
IV th year B sec