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In this article we will discuss about the isolation and identification of fungi.
Isolation of Fungi:
1. Dilution Plate Method.
2. Soil Plate Method.
This method is used to study the ecological distribution of various soil fungi.
Principle:
In this method no fungal spore will be lost as in that of soil dilution and gives a clear picture of the entire population in a particular sample.
Requirements:
1. Soil sample: 0.005 – 0.015 g.
2. Microspatula.
3. Petri plates with fungal media.
4. Sterile distilled water.
5. Incubator.
6. Bunsen flame.
7. Glass marking pencil.
Procedure:
1. Weigh 0.005-0.015 g of soil sample and place it in a sterile Petri dish using a spatula.
2. Crush the soil with spatula adding a drop of sterile water.
3. Pour 8-10 ml of melted (45°C) fungal medium (PDA/Czapek’s) into the plate and rotate the plate gently in order that the soil particles get evenly distributed in the agar medium.
4. Incubate the plates at 37°C for 4—5 days. Pick up the colonies that come up, make pure cultures and identify them.
3. Burried Slide Technique:
Principle:
When slides coated with agar or otherwise are inserted in soil and left for a few days organisms will grow on it.
Requirements:
1. Slides coated with malt agar composition given at the end of this portion or uncoated glass slides.
2. Electric plate (40°-50°C).
3. Knife (sterile).
4. Stain-Rose Bengal.
5. Microscope.
6. Petri dishes.
Procedure:
1. Place sterile slides in sterile Petri dishes.
2. Make a thin coating with molten agar on ¾ th of the slide.
3. Remove the slides, keep them on an electric plate at 40°-50°C until the agar is dried to a thin paper like layer.
4. Make slits in soil with a sterile knife and introduce the slides vertically down.
5. Press the soil around the slides gently so that the soil comes in contact with the slides. Leave the slides for 3-5 days. During this time, dried malt agar absorbs moisture and becomes softened.
6. Remove the slides after 6—8 days, wash them gently, dry and stain with Rose Bengal and observe under the microscope.
Media for Isolation of Fungi:
No single medium can isolate all organisms from a natural ecosystem like soil. Since there are various substrates in soil, the organisms get adapted to it, degrade them and use them as their carbon and energy source.
Hence in order to have a cross section of the soil micro-flora, it is necessary that several synthetic or semisynthetic media should be used. For the isolation of fungi, bacterial growth is suppressed by adding 1-2 mg/litre Strepto- penicillin after autoclaving or 12 mg Rose Bengal before autoclaving.
Some of the commonly used fungal media are:
Potato Dextrose Agar (PDA):
Preparation:
Cook pealed sliced potatoes in 500 ml distilled water for one hour or in a steamer or in an autoclave for 40 minutes. Strain the extract and add to a 500 ml of melted agar along with dextrose. Make up the volume to 1000 ml. Distribute in flasks and tubes and autoclave under 15 lb (121°C) for 15 minutes.
Czapeks Sucrose-nitrate Agar:
Czapek-Dox Agar:
Soil Extract Agar:
Preparation:
Soil extract is prepared by autoclaving 500 g of fertile field soil with 1200-1500 ml of tap water. Autoclave at 121°C for 30 minutes. Cool and filter through cloth of filter paper by adding 0.5 g of CaS04 or CaC03 to get rid of the cloudiness. Add K2HP04 and agar to 1000 ml of this extract.
Sabouraud Dextrose Agar (pH 5.6):
Add 10 µg/ml aureomycin to sterilised cooled (45°C) agar medium.
This medium is mainly used for isolation of human pathogenic fungi mostly dermatophytes.
Identification of Fungi:
Fungi obtained in pure cultures can be identified based on:
1. Morphology of the colony; Waxy, velvetty, fluffy, leathery, cottony, etc.
2. Vegetative thallus; Unicellular, hyphae coenocytic or septate.
3. Asexual spores; Zoospores, sporangiospores, conidia or blastospores.
4. Sexual spores; Oospores, zygospores, ascospores, basidiospores.
5. Reproductive bodies; Ascocarps, basidi- carps, simple conidiophores, synnamata, sporodochia, pycnidia, acervuli
6. Conidial ontogeny and arrangement of conidia; Solitary, in masses, chains, porosporous, arthrosporous, etc.
Mount hyphae, fruit bodies, conidia, etc. in lactophenol after staining them with cotton blue. Using calibrated ocular micrometer measure the various structures and identify the fungus with the aid of taxonomic books and monographs.