Mushroom Culture

preparation
• Successful mushroom production depends upon the proper
maintenance of pure culture spawn capable of providing
higher yields, excellent flavour, palatable texture, colour
and resistance to pests and diseases.

• Maintenance of vigour and genetic characteristics of a strain

in form of a pure culture is very important.

• The isolation, purification and maintenance of mushroom

cultures require technical expertise and aseptic high-tech
laboratory facilities.

• All types of mushroom use dead wood as a source of

nutrient which are made available through decomposition.
• Thus for the growth of mushroom mycelium, the nutritional
requirement of mycelium must be met out by the media or
substrate.

• A media is a substrate containing all the necessary nutrition

for the growth of mushroom mycelium in laboratory.

• A typical media must contain C, H, O, N, P, K, S, Mg, Ca, Cu,

Fe, Mn, Mo, Zn, Co.

• According to chemical composition, media are

(i) Natural Media: Comprise of entirely natural products of

unknown composition.
(ii) Semisynthetic media: Some components of known chemical
composition while others are with unknown composition.

(iii) Synthetic media: Chemically defined media

On the basis of the uses fungal media are

(i) Routine laboratory media: Complex raw material of plant and

animal origin such as malt extract, yeast extract, peptone,
meat extract etc

(ii) Enriched media: Supplemented routine media with some

specific substances

(iii) Selective media: Facilitates the isolation of particular group

of organisms or species from a mixed inoculum.
(iv) Differential media: Supplemented with certain chemicals,
and differentiates between various kinds of organism on the
basis of visible differences. Such type of media are used
more often in bacteriological laboratories.

(v) Assay media: Specifically employed for assay of vitamins,

amino acids, antibiotics, disinfectants etc..

(vi) Biochemical media: Generally used for the differentiation of

microorganisms on the basis of their biochemical activities.
In laboratory, the edible mushroom strains are cultured on
various culture media,

Potato Dextrose Agar

Peeled potato - 200 g Dextrose - 20g
Agar agar - 20 g Distilled water - 1 lit

Malt Extract Agar

Malt Extract - 20 g Dextrose - 20 g
Agar agar - 20 g Distilled water - 1 lit

Oat meal agar

Oat meal flakes - 30 g Agar agar - 20 g
Distilled water - 1 lit
Compost agar
Pasteurized compost - 150 g Agar agar - 20 g
Distilled water - 1 lit

Wheat extract agar

Wheat grain - 32 g Agar agar - 20 g
Distilled water - 1 Lit.

Rice bran decoction medium

Rice bran - 200 g Agar agar - 20 g
Distilled water - 1 Lit

Wheat grain and compost extract are most suitable culture

media for maintaining A. bisporus and A. bitorquis cultures.
Cultures of Volvariella spp. and Pleurotus spp. can be
maintained on PDA or Malt extract agar medium.
• The media is prepared by mixing all
the ingredients and then pH is
adjusted at around 6.5 by using N/10
HCl or NaOH before adding agar.

• The media is pored in to test tubes

almost 40% and closed with cotton
plug.

• The test tubes are sterilized in a

autoclave at 15 p.s.i for 15 minutes.

• The tubes are then placed in a slanting

position to make more surface area
for the mycelial spread.
• It is desirable that cultures are not maintained on the same
type of culture medium in each subculturing.

• Fresh and healthy mushroom fruit body (basidiocarp)

showing all the desirable attributes of that species/strain
should be used to raise mycelial cultures.
 Methods of Culture Preparation
(i) Vegetative mycelium culture (tissue culture)

• Under aseptic conditions using laminar flow, young

basidiocarp is cleaned with sterilized distilled water and
dipped into 0.1% murcuric chloride or 2.5% sodium
hypochlorite solution for 1 min.
• The basidiocarp is air dried and split open longitudinally
from centre and vegetative mycelial bits are cut from the
collar region (junction of pileus and stipe).
• The mycelial bit is transferred to a slant containing suitable
medium under aseptic conditions in a laminar hood and
incubated at 25+1°C in a BOD incubator.

• For Paddy straw and Milky mushrooms the slants are kept at
32°C.
Flow diagram of tissue culture technique in mushrooms
(ii) Multispore culture
• Spore mass is scraped from a fresh spore print or basidiocarp
and suspended in 100 ml of sterilized distilled water in flasks
and shaked to obtain uniform spore suspension.
• A few drops of this suspension are added to lukewarm culture
medium and poured into oven sterilize petriplates.
• The culture medium is allowed to solidify and then petriplates
are incubated at 25±2°C for 3-4 days (32°C for Volvariella
volvacea).
• The growing spores are then transferred to culture tubes and
incubated at 25+1°C in case of Agaricus bisporus and A.
bitorquis and at 32+1C for Volvariella volvacea .
(iii) Single spore culture

• Single spore cultures are procured in same way as that in

multispore cultures.

• For single spore culture isolation, the spore suspension is

serially diluted to obtain 15-20 spores per plate so that
individual germinating spore is marked and could be lifted using
micromanipulator attached with microscope under aseptic
conditions, transferred to culture medium and incubated at
25+2°C for 10-14 days.

• Single spore cultures are mostly self fertile in A. bisporus hence

can be used for selection where as in case of self sterile spores it
can be used for breeding new strains (Pleurotus and A.
bitorquis).
Germinated spores in a petriplate
MUSHROOM SPAWN (SEED)
PRODUCTION
 What is mushroom spawn

 Spawn is used as inoculum or seed for growing mushroom.

 Spawn has been defined as the vegetative mycelium or fungus

growing on a convenient medium.

 Spawn comprises mycelium of the mushroom and a supporting

medium, which gives nutrition to the fungus during its growth.
 SPAWN PREPARATION

 Three steps involved in spawn reparation.

1. Raising pure culture.

2. Substrate preparation.

3. Inoculation and incubation

 SUBSTRATE PREPARATION
 Take wheat, jowar or bajra grains.
 Remove plant debris or soil particles.
 Soak in water for 2 hours
 Boil the grains, till the grains are soft.
• Remove excess water and spread grains on cloth or wire mesh
for 4-8 hours.
• Mix gypsum(Cal.sulphate) 200g and cal.carbonate 50g per 10 kg
of boiled grains.
• Fill in glucose bottle for mother spawn and in PP bags for
commercial spawn.
• Autoclave or sterilize at 22 lb pressure for 2 hrs.
• After cooling bottles or bags can be used for spawn
 INOCULATION AND INCUBATION

 The mother spawn can be prepared by inoculating small bit of

tissue (5mm) from a culture tube.
 Inoculated bottles are incubated at 25or 30 C for 15 days.
 Mother spawn can be used for inoculating spawn prepared in PP
bags.
Directorate of Mushroom Research
(Indian Council of Agricultural Research)
Chambaghat, Solan – 173213 (HP) INDIA

Phone : 01792-230767, 230451

Fax : 01792-231207
Email: directordmr@gmail.com
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