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In this article we will discuss about the isolation of specific groups of fungi: 1. Acrasiales 2. Chytridiales 3. Saprolegniales and Pythiales 4. Ascomycotina 5. Yeasts.
1. Acrasiales:
The food used by this group of organisms is mainly bacteria.
Requirements:
a. Soil sample.
b. Escherichia coli cultures.
c. Nutrient agar plates.
d. 25 ml specimen bottles with cover.
e. Sterile distilled water.
f. Incubator.
g. Slants with E. coli.
h. Inoculating loop/needle.
Procedure:
1. Inoculate Escherichia coli cultures on agar plates and incubate at 37°C for 24 hours.
2. Skim the cells from the agar plate and suspend them in sterile water.
3. Place soil samples in 25 ml specimen bottles and sprinkle them with bacterial suspension so as to moisten the soil—not inundating it.
4. Cover the bottles loosely with caps.
5. Incubate them at 20°-23°C.
If Acrasiales members are present in soil, pseudoplasmodia will develop along with fruit bodies.
6. Transfer them to slants with E. coli and use for further studies.
2. Chytridiales:
Chytrids can be isolated from soil and pond waters, using baits like cellophane, bleached grass leaves, onion scales, etc.
Requirements:
1. Soil/pond water.
2. Baits cellophane, bleached grass leaves etc.
3. Microscope.
4. Petri plates.
Procedure:
1. Suspend 3 g of soil in water and place in Petri dishes or place pond water in Petri dishes.
2. Introduce baits and incubate under room temperature.
3. Zoospores present in soil or pond water settle on the baits and develop their thalli.
4. Mount the baits on slides and observe under the microscope.
3. Saprolegniales and Pythiales:
Members of these orders can be isolated on hemp seeds from soil or pond water.
Requirements:
1. Soil/pond water.
2. Sterilised hemp seeds.
3. Petri plates.
4. Microscope.
Procedure:
1. Add soil in Petri dish and add sterile distilled water. Allow the water to clear. Take pond water also in Petri dishes.
2. Sprinkle autoclaved hemp seeds (5-10) in each Petri dish.
3. Incubate at room temperature near a window.
After two days different forms like Pythium, Phytophthora, Achlya Saprolegnia, etc. appear on hemp seeds. After mounting them and identifying them they can be grown on corn meal agar.
Corn meal Agar:
Preparation:
Cook corn meal for one hour at 60°C in 500 ml distilled water. Filter through cheese cloth and add filtrate to melted agar in 500 ml distilled water and make up the volume to 1000 ml.
4. Ascomycotina:
Ascospores are normally dormant in soil and to break their dormancy soil is steamed and used for isolation of this group of fungi.
Requirements:
a. Steamed soil.
b. Water blanks.
c. Pipettes.
d. Petri plates.
e. Beakers.
Procedure:
1. Take 125 g of soil each in different beakers and steam at different intervals of time like 2, 4, 6, 8 and 10 minutes (Different intervals are used since according to soil type, its structure and moisture content may have an effect on the selective action of steam).
2. Use this soil for dilutions after removing 1 cm soil from the surface and plate on agar media.
5. Yeasts:
i. Dilution plate:
Colonies that come up can be sub-cultured on medium containing:
Glucose (W/V) 4%
Peptone (W/V) 1 %
pH 4
or on potato dextrose agar with 0.003% Rose Bengal.
ii. Enrichment technique:
Requirements:
1. Tap water.
2. Corn steep liquor.
3. Soil sample.
4. Aureomycin.
5. Water blanks.
6. Petri dishes.
7. Microscope.
Procedure:
1. Add 250 ml tap water and 2.5 ml corn Steep liquor in 1 lit flask, stopper and autoclave.
2. After cooling each flask is inoculated with 1 got soil. Aureomycin at a concentration of 100 µg/ml is added.
3. Shake the flasks well to disperse soil particles and incubate under room temperature near window.
4. Yeasts grow as a thin film. Mount them and observe.
5. When yeast population comes to 2-3 cells per high power field, make dilutions.
6. Add 1 ml of the final dilution to each Petri plate.
7. Pour melted cooled (45°C) nutrient agar with aureomycin (50 µg/ml) into the plates, rotate, allow to solidify and incubate.
8. Subculture the yeast colonies on nutrient agar slants mount and observe.