University of Sulaimani 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: 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.

The accuracy of this technique is low when only one plate is counted. 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.

(2) Selective techniques Next lab.