The details of the method for isolation of fungi from intestinal contents frog
Two frogs were captured from institute’s garden and kept in washed and dried containers with air ventilation and 50 ml of distilled water. After 24 hours frogs were released from the container and water containing excreta of the frogs was filtered through Whatman No.1 filter paper. Filtered excreta were used for the isolation of organism by following agar overlay technique (Drechsler 1952) which is as follows:
Fine excreta particles moistened with distilled water were placed above (onto the lid), already poured and solidified MGYP media. Plates were incubated at 28° ±2°C for one week. After 48 hours of incubation, colonies developing from germinating conidia which were forcibly discharged on to MGYP media, readily developed bacteria free colonies which were aseptically picked up and transferred on to the fresh plates containing the same media.
- The procedure for morphological analysis
Young colonies pre grown for 48 hrs on MGYP medium were transferred to sterile water agar and incubated for a further period of 48 h under condition of nutritional starvation. Following this the fungus was stained with aceto-orcein and measurements were taken microscopically. (Waingankar et al. 2008)
- Table of fungal isolates
Table for screening of fungal isolates for extracellular chitinase production
Medium containing 1% colloidal chitin and 0.2% yeast extract powder was prepared for plate assays. With the help of a cork–borer, an agar punch from the leading edge of culture grown on MGYP for 48 hrs was cut and placed in the centre of the colloidal chitin plate. Plates were incubated at 28°±2ºC for 3-4 days. After incubation, plates were observed against the light for the zones of clearance.
Table: Screening of fungal isolates for extracellular chitinase production
Sr. No. / NFCCI Accession No. / Name of the Isolate / Source / Zone of Clearance(mm)
1 / NFCCI 1235 / Conidiobolus coronatus a / Plant Litter / 8
2 / NFCCI 582 / Conidiobolus macrosporus / Plant Litter / 3
3 / NFCCI 720 / Conidiobolus sp.1 / Plant Litter / 3
4 / NFCCI 719 / Conidiobolus couchii / Plant Litter / 4.5
5 / NFCCI 723 / Conidiobolus globuliferous / Plant Litter / 2
6 / NFCCI 1229 / Conidiobolus eurymitus / Plant Litter / 3
7 / NFCCI 718 / Conidiobolus coronatus b / Plant Litter / 4
8 / NFCCI 722 / Conidiobolus sp.2 / Plant Litter / +ve
9 / NFCCI 724 / Conidiobolus sp.3 / Soil / 2
10 / NFCCI 1922 / Basidiobolus haptosporus a / Frog excreta / 4
11 / NFCCI 726 / Basidiobolus haptosporus var. minor / Plant Litter / 3
12 / NFCCI 725 / Basidiobolus sp. / Soil / 2
13 / NFCCI 1923 / Basidiobolus haptosporus b / Frog excreta / 4