Original document can be found at http://classes.plantpath.wsu.edu/plp521/Word documents/421media.doc
Introduction
A wide range of media are used for
growing fungi. Most mycologists develop preferences for certain
types of media based on experience and peculiarities of the type of
fungi that are routinely grown. Media will affect colony morphology
and color, whether particular structures are formed or not, and may
affect whether the fungus will even grow in culture. For example,
some fungi lack the necessary enzymes to utilize different carbon
sources. All fungi require several specific elements for growth and
reproduction. The requirements for growth are generally less
stringent than for sporulation, so it is often necessary to try
several types of media when attempting to identify a fungus in
culture. Most fungi thrive on Potato Dextrose Agar (PDA), but this
can be too rich for many fungi, so that excessive mycelial growth is
obtained at the expense of sporulation. I have found that most of
the fungi isolated from soil, or from substrates in the soil, i.e.,
plant debris, grow well on Corn Meal Agar (CMA), a relatively weak
medium compared to PDA. Similarly, wood-inhabiting fungi and
dematiaceous (dark pigmented) fungi often sporulate better on CMA or
Oat Agar, both of which have less easily digestible carbohydrate than
PDA. Cellulose-destroying fungi and spoilage fungi retain their
ability to produce cellulase when grown on a weak medium such as
Water Agar (WA) or Potato Carrot Agar (PCA) with a piece of sterile
filter paper, wheat straw or lupin stem placed on the agar surface.
The introduction of pieces of tissue, such as filter paper, wheat
straw, rice, grains, leaves or dung, often produces good sporulation
dependent on the organism grown.
Constituents of Media
Media generally contain a source of
carbon, nitrogen and vitamins. Glucose (dextrose) is the most widely
utilizable carbon source, and hence is the most commonly used in
growth media. Fructose and mannose are the next most commonly
utilized sugars by fungi and are found in media from natural sources.
Sucrose (table sugar) may be used in some media. Nitrogen sources
include peptone, yeast extract, malt extract, amino acids, ammonium
and nitrate compounds. Casamino Acids, a Difco product, is
acid-hydrolized casein, a mixture of amino acids. It is a good
general source of nitrogen but is vitamin-free. Bacto-Peptone,
another Difco product, contains nitrogen and a high peptone and amino
acid content. Salts, including Fe, Zn and Mn, are often added to
‘defined’ media, but are usually not added to the common media
used for routine culture. Fungi have natural deficiencies for
vitamins that are satisfied at mM
to nM concentrations. The most common naturally occurring vitamin
deficiencies are thiamin and biotin. Deficiency of both is quite
common among the Ascomycota. Other organic nutrients such as glucose
are often contaminated with vitamins sufficient to supply the growth
requirements of fungi.
Isolation Media--Water Agar,
Antibiotic Agar and Acidified Cornmeal Agar
Most fungi are difficult to isolate
from infected tissues, infested soil or decaying organic material due
to the rapid and often antagonistic development of associated fungi,
bacteria and actinomycetes with faster growth rates. Successful
isolation of many of these fungi can be achieved by the used of
selective media that either slow down the growth or inhibit the
growth of these antagonistic organisms. One approach is to surface
sterilize the material to eliminate surface contaminants, then place
the material on Water Agar. Most fungi and bacteria will grow on WA,
but at such a slow rate that it is relatively easy to isolate the
target fungus. Selective exclusion can be accomplished by several
approaches. One is by selective inhibition, i.e., the use of
antimicrobial chemicals, primarily antibiotics such as streptomycin
sulfate and penicillin. We have Antibiotic Agar (AA) in the lab for
this purpose. Antibiotic Agar is a good agar to use when isolating
from material that cannot be surface sterilized and hence will have a
heavy load of bacteria. Another approach is to create an
unfavorable environment in the isolation medium such as high pH in
which the fungus will grow but the bacteria will be inhibited.
Acidified cornmeal agar (ACMA) works well for the isolation of fungi
from various types of plant material. It is best to use
surface-sterilized material in conjunction with ACMA.
Growth Media
We will use mostly ‘natural’ media
based on materials such as cornmeal, carrots, hay, potatoes, oatmeal,
soil, etc. ‘Semi-synthetic’ media, containing both natural
ingredients and defined components include Malt Extract Agar, Malt
Agar. We probably will not be using ‘Synthetic’ or ‘defined’
media which contain precise amounts of a carbon source, vitamins and
minerals.
As previously noted, Cornmeal Agar is a
good medium for the culture of dematiaceous fungi and fungi isolated
from soil, soil substrates and wood. Malt Extract Agar (MEA) is also
frequently used for culturing fungi from soil and wood.
Water molds, such as those obtained
from baiting with hemp seed and snake skin, can be grown on Cornmeal
Agar, Water Agar or Emerson’s YpSs agar. See ‘Zoosporic Fungi in
Teaching and Research’ pages 125-127 for a full description of
methods.
Ascomycota isolated from fruiting
bodies forming on dung, wood or soil can be grown on Malt Agar,
Potato Carrot Agar and Potato Dextrose Agar. The first step is to
attempt to get the ascospores to germinate by streaking them out or
getting forcible discharge onto a selective or isolation medium.
Look for spore germination and transfer a small piece of agar with
the germinating spore(s) to one of these richer media.
Basidiomycota growing on wood can be
grown on Malt Extract Agar. Cultures derived from mushrooms can be
grown on Potato Dextrose Agar, Potato Sucrose Agar and Malt Extract
Agar. Potato Dextrose Yeast Extract Agar (PDYA) also is a
recommended medium for growing cultures of Agaricus,
Pleurotus, Lentinus, Stropharia, Flammulina,
and some of the Psilocybe species.
Summary of media and common use
Water Agar (WA)--use for
isolating fungi from surface-sterilized substrates.
Antibiotic Agar (AA)--use for
isolating fungi from substrates not readily surface-sterilized, or to
clean up a culture contaminated with bacteria.
Acidified Cornmeal Agar (ACMA)--use
for isolating fungi from substrates that are likely to be
contaminated with bacteria. Not a substitute for AA, but the acidity
inhibits bacteria and the medium supports the growth of a wide range
of fungi.
Cornmeal Agar (CMA)--use for
growing a wide range of fungi, particularly members of the Fungi
imperfecti; provides a good balance of mycelial growth and
sporulation.
Potato Carrot Agar (PCA)--considered
a relatively weak medium somewhat comparable to CMA, good for some
Fungi imperfecti.
Malt agar (MA)--lacks peptone,
and is useful for culturing many Ascomycota; sporulation in some
species is inhibited by peptone.
Malt Extract Agar (MEA)--a good
growth medium for soil fungi, fungi isolated from wood,
basidiomycetes, etc. An all-purpose type of medium.
Potato Dextrose Agar (PDA)--a
relatively rich medium for growing a wide range of fungi.
Potato Dextrose-Yeast Extract Agar
(PDYA)---good for growing cultures derived from mushrooms.
REFERENCES
Descriptions of methods for isolating
and growing all groups of fungi can be found in the following
reference, available in room 326:
Stevens, R. B., editor. 1981.
Mycology Guidebook. University of Washington Press,
Seattle.
Descriptions of methods for isolating
and growing mushrooms can be found in the following references, also
available in room 326 or from the instructor:
Stamets, P. and J. S. Chilton. 1983.
The Mushroom Cultivator. A Practical Guide to Growing
Mushrooms at Home. Agarikon Press, Olympia, WA.
Stamets, P. 1993. Growing
Gourmet and Medicinal Mushrooms. Ten Speed Press and
Mycomedia, Olympia, WA.
Watling, R. 1977. How To
Identify Mushrooms to Genus V: Cultural and Developmental Features.
Mad River Press, Eureka, CA.
Techniques for isolating and growing
all types of zoosporic fungi can be found in:
Fuller, M. S. and A. Jaworski. 1987.
Zoosporic Fungi in Teaching and Research. Southeastern
Publishing Corporation, Athens, GA.
Techniques for isolating and growing
members of the Protoctista and Chytridiomycota can be found in Margulis, L., J. O. Corliss, M.
Melkonian and D. J. Chapman. 1990. Handbook of Protoctista.
Jones and Bartlett Publishers, Boston.
