Culture plate of an orchid mycorrhizal fungus.

A plate culture of the fungus Candida albicans. It lives in numerous parts of the body as
normal flora.
Culture of fungi
Saprotrophic fungi can be subcultured on media containing nutrients appropriate to their
growth and development. Several different types of media have been used successfully.
The most commonly used in undergraduate classes consists of a fruit or vegetable, or
their extracts, mixed with sugars and agar, and set in Petri dishes. The organic and
mineral fractions are designed to supply nutrients similar to or commonly found in the
environment of the fungus. A few commonly used materials include:

• Soil [SOIL AGAR]

• Potato [PDA]
• Tomato plus other vegetables [V8 JUICE Agar]
• Malt extract [MA]
• Dung [DUNG AGAR]

These can be more highly defined by replacing the organic component with known
organic materials including:

• Nutrient Dextrose [NDY]

• Sabouraud dextrose [SABOURAUD AGAR]

Most of these media are available from commercial sources. Commercial sources have
a more consistent quality than those prepared from the raw materials each time. 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.

Bacteria are suppressed by adding antibiotics to the agar. As most antibiotics are
denatured by heat, the antibacterial agents are usually added to sterilised molten agar,
that is just above setting temperature (hand warm, but not hot to touch). Penicillin (50
units per ml), Streptomycin (50 unit per ml), Tetracyclin (30 units per ml) are commonly
used this way, either alone or more commonly, in combination. Chlomamphenicol (50mg
per l) can be autoclaved and is added during preparation.

Six phases of mushroom cultivation

Phase Time span Temperature Key points

Regulate water and NH3

1. Phase I content through microbial
6-14 days action.
composting

Add fertilizer / additives

Reduce number of
7-18 days via potentially harmful
2. Phase II composting method, microbes through further
composting or ~2 hours for composting, or apply
pasteurization pasteurization (heat heat sterilization.
sterilization)
Remove unwanted NH3.
3. Spawning and 14-21 days 75°F; to 80°F; must Add starter culture.
growth be above 74°F; for
rapid growth Allow mycelium to grow
through substrate and
Must be below form a colony.
80°F; to 85°F to
avoid damaging Depends on substrate
mycelia dimensions and
 composition.

Finished when mycelium

has propagated through
entire substrate layer
Promote the formation of
rhizomorphs, or
mushroom pins.

Add a top covering or

dressing to the colonized
4. Casing 13-20 days
substrate.

Fertilizing with nitrogen

increases yields.

Induces pinning
Earliest formation of
recognizable mushrooms
from mycelium.
5. Pinning 18-21 days
Adjusting temperature,
humidity and CO2 will
also affect the number of
pins, and mushroom size
Repeated over 7-10
6. Cropping Harvest
day cycles
Culture of Protozoa
The in vitro culture of protozoan parasites involves highly complex procedures, which
are subject to many variables. These parasites have very complex life cycles and,
depending on the life cycle stage, may require different culture parameters. However, in
vitro cultivation is important for many reasons, some of which include: diagnosis, antigen
and antibody production, assessment of parasite immune modulating capabilities, drug
screening, improvements in chemotherapy, differentiation of clinical isolates,
determination of strain differences, vaccine production, development of attenuated
strains, and the continued supply of viable organisms for studying host-parasite
interactions.

There are a number of issues involved in the culture of protozoan parasites that make
these procedures highly complex and subject to many variables, some of which are
known and some of which are still undefined. Certainly protozoan parasites have
complex life cycles. They may have different morphological stages within the life cycle
and may have both cold-blooded and warm-blooded animals as intermediate or
definitive hosts within the life cycle. In vitro culture of organisms at any one of these
stages within the life cycle involves a tremendous number of variables, including parasite
stage, host site, host temperature, host immune responses, parasite species and/or
strain, and parasite-protective mechanisms. To simulate the host environment in an in
vitro culture system can be extremely demanding, assuming one can actually determine
all the relevant variables.
Often the organisms are very fastidious in their growth requirements, and different lots of
media and/or medium components may be toxic. Something as simple as the type of
glass used for the culture container can have tremendous influence over the success or
failure of culture trials. Many culture medium components require filter sterilization and
have relatively short shelf lives. Another requirement for many medium formulations
includes the addition of human or animal sera, which are expensive and highly variable
and may contain factors that are detrimental to parasite growth. Much research has
been devoted to the development of defined medium formulations, although, even with
the elimination of serum, various other components may not have been totally defined.
In some cases, growth factors have been identified and substituted for serum or serum
components.
At the present time, are there any advantages in the in vitro culture of protozoan
parasites, especially when revolutionary developments are going on in the field of
molecular biology? One can argue that many types of research, including the diagnosis
of etiologic agents, can be accomplished even when very small numbers of organisms
are available. However, many areas of research can be undertaken only when large
numbers of parasites with no contaminating bacteria or host materials are available. One
of the great advantages of in vitro culture, especially axenic culture, is that obtaining a
continuous supply of pure organisms without any bacterial and/or fungal contamination
is possible.
In vitro cultivation of parasitic protozoa that cause human disease is invaluable, as it
provides not only information on the development of the parasite but also avenues for
new approaches to the containment and/or eradication of the parasite. In vitro cultivation
is important for a number of reasons, as follows: (i) as an important adjunct to diagnosis;
(ii) to produce antigens used to prepare monoclonal and polyclonal antibodies against
the organisms for use in immunologic tests; (iii) to identify specific proteins that may
enhance the invasive properties of the parasite and in turn the development of
monoclonal antibodies that will help neutralize parasitic invasion; (iv) to assess
functional antibodies and cell-mediated protective systems against the parasites,
assessments that can only be made in a cost-effective manner using in vitro culture; (v)
to screen drugs, in vitro, in order to identify potential therapeutic agents; (vi) to
differentiate susceptible from resistant isolates so that advances in chemotherapy can
be made; (vii) to differentiate clinical isolates using techniques such as isoenzyme
electrophoresis, monoclonal antibody techniques, and/or DNA probe techniques; (viii) to
elucidate isolate and strain differences which will be a useful tool for molecular
epidemiology; and (ix) to produce vaccines, as relatively large numbers of parasites at
specific stages can be produced in culture. (x) In addition, continuous culture over long
periods of time may cause attenuation of strains, and therefore, attenuated strains have
potential in the development of suitable vaccines. (xi) In vitro cultivation also provides a
system to assess vaccine efficacy, since it can only be done by using intact parasites
that can be obtained in large quantities and without the contaminating influences of host
components. Finally, culture can be used (xii) to provide the parasite inoculum used for
experimental animal disease models; (xiii) to study the biochemistry, physiology, and
metabolism of the parasites as well as determine their nutritional requirements; (xiv) to
understand the ultrastructural organization of the parasite; and (xv) to provide a system
suitable for the assay of lymphokines and other cytokines that may block invasion of the
parasite.

ZAMORA, Maria Loreta B.

N2-A