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APPENDIX-I

Composition of Media: The following culture media were used in the course

of the investigations:

Czapek Dox Agar (CzDA): It consisted of the following composition

1) Sucrose -30g

2) Dipotassium hydrogen phosphate (K2HPO4)-1.0g

3) Magnesium sulphate (MgSO47H2O)-0.5g

4) Potassium chloride (KCl)-0.5g

5) Ferrous sulphate (FeSO47H2O)-0.01g

6) Agar-15g

7) Distilled water-1000ml

Czapek Dox broth: It consisted of the following composition

1) Sucrose-30g

2) Dipotassium hydrogen phosphate (K2HPO4)-1.0g

3) Magnesium sulphate (MgSO47H2O)-0.5g

4) Potassium chloride (KCl)-0.5g

5) Ferrous sulphate (FeSO47H2O)-0.01g

6) Distilled water-1000ml

Potato Dextrose Agar (PDA): It consisted of the following composition:

1) Peeled Potato- 200g

2) Dextrose-20g

3) Agar Agar- 20g

4) Distilled water- 1000ml

Peeled and sliced 200g of fresh potato tubers were boiled in 1 litre of water for

40 mins. Strained through 4 layers of cheesecloth and discarded the potato

slices. Added to the potato broth 20g glucose and 20 g agar (with stirring), then

enough water to bring the volume back to one litre. Autoclaved and allowed to

cool, and poured on to the plate.

Potato Dextrose Broth: It consisted of the following composition:

1) Peeled Potato-200g

2) Dextrose-20g

3) Distilled water- 1000ml

Peeled and sliced 200g of fresh potato tubers were boiled in 1 litre of water for

40 mins. Strained through 4 layers of cheesecloth and discarded the potato

slices. Added to the potato broth 20g glucose, then enough water to bring the

volume back to one litre. Autoclaved and allowed to cool, and poured on to the

plate.

Oatmeal agar: It consisted of the following composition:

1) Oatmeal-60g

2) Agar-12.50g

3) Distilled water-1000ml

Oatmeal broth: It consisted of the following composition:

1) Oatmeal-60g

3) Distilled water-1000ml

Nutrient agar: It consisted of the following composition:

Peptone-5.0g

Beef extract-3.0g

Yeast extact-1.5g

Sodium chloride-5.0g

Agar agar-15.0g

Nutrient broth: It consisted of the following composition:

Peptone-5.0g

Beef extract-3.0g

Yeast extact-1.5g

Sodium chloride-5.0g

The ingredients of each of the above medium were boiled in distilled water till

the ingredients dissolved. After dissolving the ingredients the media were

distributed in 500ml flask and plugged the mouth of the flask with sterile

cotton and autoclaved at 15 pounds pressure for 15 minutes. Then, about 25ml

of the melted medium was poured in one petridish, and kept them at room

temperature for three days to ensure that there is no contamination. When there

was no contamination, then the media were ready for use. The antibiotics

penicillin, 20 units and streptomycin, 30 microgram milligrams/milliliter of

medium were added, when cooled to about 450C (Tilak, 1987). The pH of all

the media prepared and used was corrected to 6.0±1 by using either 0.1N

NaOH solution or 0.1N HCl solution.

0.01% Chloramphenocol/Streptomycine was used in cultural studies to avoid

bacterial contamination.

Stock cultures and pure culture of fungal isolates were maintained in slants and

plates at 40C±1 in refrigerators. Subcultures were made at regular intervals or

whenever required.

Appendix-II

Stains-Lacto-phenol: It consisted of the following composition:

Lactic acid-100ml

Phenol-100g

Glycerine-100ml

Distilled water-100ml

The phenol was dissolved in water without heat to prevent oxidation, then

added glycerine and lactic acid.

Cotton Blue: It consisted of the following composition:

Saturated solution of Cotton Blue in alcohol (95%-100%)

Glycerine-10ml

Distilled water-80ml

The material was covered with lactophenol for 1 to 2 minutes. Then warmed

gently over a flame and added a drop of Cotton blue. Sometimes Cotton blue

may be introduced before warming. The excess stain was drained off and

examined.