CULTURE MEDIA,

CULTIVATION,
STERILE
TECHNIQUE
 FUNGI ARE HETEROTROPHS!
biotroph
saprotroph

dead cells

living cells
Important ecological roles as decomposers, mutualists, parasites
Fungus cells are totipotent.

Fungi differentiate into a variety of structures including spores which

enable continuation over space and time. The mechanisms for formation of
each type of structure is largely determined by the perception of the
environment by the thallus. Water and organic nutrients are particularly
important for fungi, thus we would expect explosions of fungi in humid,
energy rich habitats. However, a few fungi also tolerate extreme
environments.

Growth of most fungi is indeterminate. The diversity of stages of any

one mycelium means that fungi, at any one time, may have hyphae in
extension, productive, fruiting and senescing phases. The concept of a
single response of a body, such as an animal, does not apply to the
filamentous fungi.
Environmental Factors:

Oxygen is used in respiration in most organisms. The fungi include species that
are obligately aerobic or obligately anaerobic (eg rumen fungi). However many
fungi are in between, with the capacity to function facultatively in aerobic and
anaerobic conditions.
CO2 -The presence of carbon dioxide is also required for some fungi.

Water availability has a major influence on the function of fungi. Most fungi require
very high water availability (relative humidity), and rapidly dry out or senesce in dry
conditions.

pH - fungi can tolerate a wide range of pH, though most media used to culture
fungi are acidic.

Temperature - Fungi can normally tolerate the range of temperature of the

environment from which they are taken. Their response to temperature is quite
varied, however. Active growth will usually be associated with a limited range of
temperatures. Those fungi that grow between 15 and 35 C are called mesophilic,
and above thermophilic. Those that grow at freezing are called psychrophilic.

Light has an important influence on fungal growth in specific cases. The effect of
UV radiation on spore and sporocarp formation, and phototropic release are clear
examples of light being important. UV radiation also reduces viability of spores
especially in air. Overall, light does not play a major part in metabolism and growth
of fungi.
 As Heterotrophs Fungi typically require:

Carbon Source - Glucose (dextrose) is the most widely utilizable carbon source, Fructose and
mannose are the next most commonly utilized sugars by fungi. More complex sources in sugar
alcohols, starches, cellulose, hemicellulose, and other complex carbohydrates.

Nitrogen sources include peptone, yeast extract, malt extract, amino acids, ammonium and
nitrate compounds.

*Macro l nutrients: M, K, P, Mg, S, Ca. *Micronutrients: Fe, Cu, Mn, Zn, Mo. (*minerals)

Fungi have natural deficiencies for vitamins that are satisfied at mM to nM concentrations. The
most common naturally occurring vitamin deficiencies are two B vitamins thiamin and biotin.
Other organic nutrients such as glucose are often contaminated with vitamins sufficient to
supply the growth requirements of fungi.
Culture Medium - Potato dextrose (D-glucose or grape sugar )agar

Percentage of U.S. Recommended Daily

Calories ..................110
Allowances for a Medium Potato
Protein ................... 3 grams
Protein .................. 6 %
Carbohydrate ......... 23 grams
Vitamin C ............. 50 %
Fat .......................... 0 grams
Thiamin ................ 8 %
Dietary Fiber .......... 2710 mg
Riboflavin ............ 2 %
Sodium ................... 10 mg
Niacin .................. 10 %
Potassium ............... 750 mg
Iron ....................... 8 %
Vitamin B6 ........... 15 %
Folacin (folic acid). 8 %
Phosphorus ............ 8 %
Magnesium ............ 8 %
Zinc ....................... 2 %
Copper ... ............... 8 %
Pantothenic acid .... 4 %
Iodine .................... 15 %
 Growth Media

Liquid medium Solid medium

Malt Extract from malted grains

Agar-agar is a natural gelling substance from the cell walls of red

algae, of, like Gelidium and Gracialaria.
Most culture media fit into one of three categories: (1) synthetic, (2) semi-
synthetic, and (3) natural.

Synthetic media are composed of ingredients of known chemical composition

and concentration. These media are useful in physiological or descriptive studies
when it is necessary to duplicate exactly a previous batch of medium or to record
the effects of the deletion or addition of a particular substance.

Semi-synthetic media resemble synthetic media in containing a known set of

ingredients, but differ in that at least some of the ingredients are of unknown or
variable composition. We know that the yeast extract contains thiamine and other
vitamins, but we do not know the exact amounts or what else might be present.
The result is a medium of quite predictable composition but one not completely
known chemically. Semi-synthetic media are widely used in routine work and offer
something of a compromise between synthetic and natural media.

Natural media are so called because they are partly or completely composed of
natural materials. A slice of potato is a natural culture medium, as is a piece of
meat or bread. Natural media are often very good and allow sporulation in fungi
that may otherwise remain sterile. Their major disadvantage is that they may differ
considerably from batch to batch and thus not yield reliable experimental results.
Nevertheless, natural media are widely used in laboratory work .
 SYNTHETIC

Czapek's Solution Agar

Sucrose 30 g
NaNO3 3.0 g
K2HPO4 1.0 g
MgSO4.7H2O 0.5 g
KCl 0.5 g
FeSO4.7H2O 0.01 g
Agar 15 g
Distilled water 1000 ml

Czapek's Solution Agar is a synthetic

medium widely used in mycological
laboratories, particularly for the
identification of species of Aspergillus and
Penicillium. Many moulds produce very
characteristic colonies on it and may also
exude pigmented substances. Aerial
growth is often suppressed and sporulation
may be enhanced.
 Semi-Synthetic Medium

Potato Dextrose Agar

Thinly sliced, peeled white potatoes 500 g
Glucose 20 g
Agar 15 g
Distilled water 1000 ml

Heat potatoes at 60°C for 1 hour and filter through

cheesecloth. Make up volume to 1000 ml and add
other ingredients. Cook 1 hour and then sterilize.

Potato Dextrose Agar, or PDA, as it is usually

called, is an old formula used by plant pathologists
and many mycologists for general laboratory use.
Natural Media
 To transfer the culture we do the following:

1.Take an inoculating needle, usually a thin needle or wire at the end of a long pencil-like handle, and heat it in
an alcohol or gas flame until it glows bright red (Figure 10A).

2.Allow the needle to cool for about 15 seconds. (A hot needle will kill the mold that is to be transferred).

3.Open the Petri dish containing the culture just wide enough to allow entry of the needle.

4.With the heat-sterilized needle, cut out a small portion of the colony margin. Hyphal tip transfers work best as
they are usually the most active parts of the culture; in addition, transfers from the heavily sporulating central
portions will result in spores being spread into the air. Especially in medical work, hyphal tip transfers are
essential. The excised colony margin should be only about 1 mm square (Figure 10B).

5.Transfer the square of colony margin to the sterile plate, making sure that the lid is opened only wide enough
to admit the needle and make the transfer. Place the block at the center, withdraw the needle and flame it until it is
red hot, to kill all adhering spores and hyphae (Figure 10C,D).

6.Close the lid; label the plate with a marking pen, including name of culture and date. We usually wrap a thin
strip of paraffin film around the sides of the plate to cover the opening, but this is not absolutely necessary; just a
couple of pieces of masking tape to hold the lid down will do.

7.Leave the culture to grow in a protected place that has as little air movement as possible.

Figure 10. Steps in sterile technique. Inoculating needle is heated in an alcohol flame (A). Small piece of colony is
removed from Petri dish (B) and transferred to a new dish of agar (C), yielding a plate containing a piece of the old
culture at its center (D).
 Obligate Symbiotic Fungi

In vivo culture with host plant –

During the 1980s, the improvement of technologies related to the

cultivation of excised roots allowed the successful cultivation of
some AM fungi strains.

In vitro culture on excised roots

 Malt agar

Spawn on grain!

plugs
 I. Growth parameters for mushroom cultivation

1. Moisture – humidity and substrate

2. Air Exchange - CO2
3. Temperature
4. Lighting

II. Three main stages in Mushroom growth and

reproduction
1. Spawn Run – mycelium growing in substrate
2. Primordia Formation - initiation of mushrooms
3. Fruitbody Development – ending in cropping
 Creating Conditions for Mushroom Fruiting

High humidity. Most species like 80 to 95% humidity.

Ideal temperature for fruiting — varies with species and strain.

Oyster and shiitake have cold and warm weather strain

Good air exchange — ventilation or fan, low CO2 levels

Enough light. Indirect sunlight for most species. The button mushroom,
Agaricus bisporus an exception — prefers darkness Enoki, low light, high C02

http://www.mykoweb.com/articles/cultivation.html
Under high CO2 levels or with
less frequent ventilation,
mushrooms produce
long stipes with small caps,
while they produce short stipes
with broad caps under low CO2
levels or frequent ventilation.