Practical Mycology
3rd Class
Lab. 3 : 26 .10. 2014
University of Sulaimani
School of Science
Biology Department
ISOLATION OF
FUNGI
Isolation of Fungi

Isolation techniques are numerous and often complex
but can be quite effective in yielding just the desired
fungus, while excluding all the others. Isolation
techniques can be divided into two broad categories:-
(1) Direct methods.
(2) Selective methods.

Both methods can be further divided into a number
of subtypes.
ISOLATION OF FUNGI
1- Direct isolation techniques:
A)Direct transfer
 Putting a piece of the habitat or substrate under a
dissecting microscope. An inoculating needle is
used to transfer some of the spores into a sterile
plate of culture medium.
 Adhering or moistening the needle with a tiny piece
of agar or with a slight amount of glycerine, so that
spores will stick the needle and transferred to the
culture media.

The direct transfer technique is also used to obtain
pure cultures of mushrooms and other large fungi.
The fruiting body is broken open, without touching
the newly exposed flesh, and some of the tissue is
transferred to a sterile culture medium.
B) Moist chambers
1- The container is filled with dry moss and then
water is added. Peat moss is like a sponge in that it
can absorb and hold a tremendous amount of water.
2- Place a single or double layer of filter-paper or
paper towel over the moss layer so that the specimen
does not come in contact with it.
3- Add the specimen, moisten it slightly (unless it is
already damp) and place it on top of the filter-paper.
4- Cover the dish and leave it in a place where the
temperature is reasonably constant.
5- Within a few days fungi will begin to appear on the
specimen.
6- Examine the material with a magnification of at
least 15-20X.
7- Follow the direct transfer procedure to obtain a
pure culture. We can use water agar instead of moist
chamber.
Moist chambers can be used for all kinds of
materials, such as dung, wood, leaves, old stems,
bark, seeds, fruits, old fungi, dead insects, and
numerous other things.
This technique is useful to discover the cause of a
particular decay, diseased plants, and
manufactured products.
C) Direct plating

Often it is most convenient to place materials that
are of interest directly on a nutrient agar medium.

It is a simple technique, requiring the placing of
small bits of the substance on the surface of the.

After a few days of incubation fungal colonies
appear on the surface, and can be transferred into
pure culture.

This technique is commonly used in soil studies,
requiring only a pinch of soil, evenly dispersed
over the surface of the agar.
D) Dilution plating
1- In this technique 1 gram (dry weight) of the
material to be studied is ground up (if necessary) and
dispersed in 9 ml of sterile water.
2- One milliliter (1 ml) of this solution is transferred to
a second tube containing 9 ml of sterile water,
resulting in a 0.01 dilution of the spore mass in the
original material.
3- The process is repeated to yield dilutions of 0.001,
0.0001, and 0.00001 or even further if necessary.
4- A 1-ml portion from each dilution is pipetted to a
separate Petri dish, the solution can be put on the
surface of solidified medium and spread evenly
throughout.
5- After a few days' incubation, colonies will appear
in varying densities.
6- The number of spores present in the original
sample can be calculated roughly by selecting the
plates showing 40-100 colonies and writing down the
colony count.
Colonies per (gram or ml) of original sample =
𝐜𝐨𝐥𝐨𝐧𝐲 𝐜𝐨𝐮𝐧𝐭
𝑫.𝑭×𝒗𝒐𝒍𝒖𝒎𝒆 𝒖𝒔𝒆𝒅
The accuracy of this technique is low when only one
plate is counted.
1- Improper dispersion of spores during dilution.
2- Failure to break up spore masses.
3- Mutual inhibition of growth by certain fungi.
E) Airborne fungi

Certain fungi are more likely to get their spores into the
air than others. Airborne fungal spores can serve as an
infective agent of plant disease and may also be allergenic.

the simplest technique for airborne fungi involve the
horizontal or vertical placement of Petri dishes containing
nutrient agar.

So that they trap any spores that fall on or blow into
them.

Usually 30 minutes to 3 hours out of doors is required for
a good sample using this technique.
F) Baits

Many fungi have quite specific nutrient requirements
and are specialized to use materials that other fungi
use with difficulty or not at all.

We can take advantage of this for the isolation of
fungi by presenting a particular substance to the
environment for colonization.

Kinds of baits might be pieces of wood, insects,
plastics, hair, or anything else one can name.

The bait can be submerged in a particular habitat in
nature or in a moist chamber.

To isolate dermatophytes, for example, it is customary
to place hair on moist soil in a moist chamber and
examine it.
Homework
Everyone must prepare a moist chamber at home and
bring it to lab. next week.
Next lab.
(2) Selective techniques