"outline of methodology_thesis_ edit1 by Addie"
Partial Characteriztion of the biodegrading ability of the fungi Xylaria sp. and its four mutants on natural rubber, chicken feathers and polystyrene. Objectives: 1. to determine if Xylaria sp mutants and wildtype can degrade/consume/ assimilate natural rubber as a carbon source 2. to determine if Xylaria sp mutants and wildtype can degrade/consume/ assimilate chicken feathers as a carbon and nitrogen source 3. to determine if Xylaria sp mutants and wildtype can degrade/consume/ assimilate polyurethane as a carbon source 4. to compare the degradation capability of the wild type and the mutants. Outline of methodology I. Preparation of Inoculum Isolate the Xylaria sp. by culturing it in a Potato Dextrose Agar (PDA) medium. Adjust to pH 5 and incubate at 25˚C. After 2-3 days, transfer the fungi into test media. II. Preparation of Pollutants A. Polystyrene - Cut 1x1 cm squares from plastic cups. B. Chicken feather - Obtain fresh feathers from Gallus gallus sp. Wash and sterilize in an autoclave. C. Natural Rubber - Obtain a unused rubber latex glove. Cut 1x1 cm of the gloves. III. Preparation of Test Media* A. For plates: Prepare 2 sets of plates containing 20 ml PDA in triplicate (6 plates per mutant/wild type, per pollutant tested). Add 0.5% glucose in set A and add 0.5% of the liquid pollutant in set B. Adjust to pH 5 by adding small amounts of either 0.1M NaOH or 0.1M HCl. Inoculate the fungi using agar discs from the PDA plate described in I. Agar discs will be obtained by cutting a 2-3 day-old inoculum on the peripheral area of a colony using a 5-8 mm diameter cork borer. The agar discs will be transferred to the PDA plates of sets A and B by using a sterile toothpick. Incubate at 25˚C. Observe the colony growth in set B and compare it always with set A. Set A will be the control group and it would indicate whether the fungi transferred is active. Then measure the colony growth by its diameter. B. For flasks and test tubes: Prepare 2 sets of flasks containing 50 ml Mineral Medium each, in triplicate. Add 0.5% glucose in set A and B. Adjust to pH 5 by adding small amounts of either 0.1M NaOH or 0.1M HCl. Then add the solid pollutant in set B only. Inoculate the fungi by using ………….. When all the glucose has been used up and the fungi had grown into a considerable mass as examined visually, add another MM + 0.5% glucose in set A only, leaving the set B flasks to utilize the solid pollutants as the sole carbon source. The extent of colonization should be carefully examined every day until rate of colony growth can be predicted (growth in mm/day). But if otherwise, continue adding the MMG (mineral medium+0.5%glucose) to both sets of flask until the fungi has grown and thrived. Incubate for 50-80days, with the flasks in a room with more or less 250C in temperature, under shaking conditions, while the test tubes in an incubator with 25 0C in temperature as well. IV. Remove solid pollutants from the culture medium and examine under a scanning electron microscope (SEM). Note: * - this step is intended for each mutant and for the wild type. Since we have 4 mutant strains and a wild type, this step will be repeated five times multiplied with the number of pollutants to be used.