Methodology Collection of Refuse Core Samples: 1.

All the samples will be collected at the Legazpi City Sanitary Landfill is a category 2 disposal facility, located in Barangay Banquerohan, 22 km from the city proper. At present, it has an area of 21 hectares (21,000 square meters). 2. Samples will be collected at four points in the landfill. Mainly in the North, South, West and East sections. 3. 8 core samples will be collected at the 4 points at depths of 5cm and 10 cm of refuse by using a 5cm x 5cm improvised wooden corer. 4. The 8 core samples will be stored at separate sterilized plastic containers at room temperature. Preparation of Refuse Samples: 1. First, practice aseptic technique on the work area, on the laboratory equipment and materials and on the laboratory experimenter all throughout the different procedures of the experiment. 2. For each core sample, suspend the sample in 200mL of distilled water in a 500mL beaker. Stir for 2 minutes with a glass rod to evenly distribute particles. 3. After stirring, filter the solution at least 2 times through a funnel with filter paper in another 500mL beaker so that insoluble particulates will be absent from the sample to be diluted. But for the second filtration, let it filter into a sterilized 250mL Erlenmeyer Flask with a cotton plug to prevent contamination. Preparation of the Potato Dextrose Agar Medium: 1. Prepare sterile agar medium by following the instruction on the container and using the appropriate ratio of the agar and solvent in a beaker then transfer 250 mL portions in an Erlenmeyer flask, autoclaved for 15 min at 121C and 15 psi. 2. Prepare medium well in advance and let solidify before remelting and tempering. Do not re-melt solidified medium more than once or under pressure. 3. To inhibit bacterial growth, amend agar medium with either antibiotics or sterile 10% tartaric acid solution (to be done after agar has been tempered and immediately before pouring plates) as follows: a. Antibiotics. Chiortetracycline-HCl, at agar medium concentration of 40 ppm, is recommended. Other antibiotics may be used (e.g., chloramphenicol, streptomycin) but should always be used at the same concentration as chlortetracycline-HCl and in addition to it. Prepare stock solutions by dissolving 1g of antibiotic in 100 mL of sterile distilled water. Store stock solutions in dark at

4-8C. Shelf life should exceed 1 month. Equilibrate stock solutions to room temperature immediately before use. If agar medium is in 250 ml aliquots, add 1 ml of 100 ml stock solution to obtain 40 ppm concentration. If medium aliquots are greater or less, adjustments will be necessary. b. Tartaric acid solution. A 10% solution may be used to adjust agar medium to pH 3 5 0.1. Titrate to determine amount of solution needed to adjust pH to 3.5. Type and aliquot volume of medium will affect amount of solution needed. After adding solution to medium, verify pH by letting a portion of medium solidify and checking with pH meter. Do this for every new lot of medium prepared. Preparing the Serial Dilutions: 1. Dilute 1mL of inoculums (culture) from the filtered sample to a test tube with 9mL of distilled water (diluent). This will be the 10(-1) dilution factor of a ten-fold serial dilution. 2. Repeat until you have reached a 10(-4) dilution factor. 3. Repeat steps 1-2 for the other filtered samples. Preparing the Spread Plates: 1. After autoclaving, cool the agar to between 45C and 50C prior to pouring the plates to minimize the amount of condensation that forms. 2. Pour 15-25 mL tempered agar medium containing either antibiotic(s) or tartaric acid solution into the Petri plates for each sample dilution. 3. Freshly prepared plates do not work as well as dry plates as it takes longer for the inoculum to absorb into the agar. Plates may be dried by keeping them at room temperature for roughly 24 hours. Plates will dry faster in lower humidity so placing them in a laminar flow hood will speed the drying process. 4. Once dried, plates may be used or refrigerated in closed bags or containers until required. Spreading the Samples: 1. Pipette 0.1-0.2mL of the dilution sample on the center of the agar plate. 2. A reusable glass or metal spreader should be flame sterilized by dipping in alcohol (such as 70% isopropyl or ethanol), shaking off the excess alcohol, and igniting the residue .The spreader is then allowed to cool. 3. The spreader is placed in contact with the inoculum on the surface of the plate and positioned to allow the inoculum to run evenly along the length of the spreader. Even pressure is applied to the spreader and the plate is spun or by hand.( Avoid spreading the inoculum all the way to the edge of the agar. )

4. Avoid disturbing plates for 10 to 20 minutes after spreading to permit the absorption of the inocula. 5. Repeat these steps for all the plates for each dilution. 6. Incubate plates in dark at 20-25C for 3-7 days. Do not stack plates higher than 3 and do not invert. Let plates remain undisturbed until time for counting. Identifying fungi and Labeling Fungi: 1. After 3-7 days of incubating the samples, examine the appearance of the fungal colonies of each plate with the unaided eye. 2. Make smears by using a sterilized wire loop to collect the fungal specimens in varying colonies on the surface of each plate. 3. Place a small drop of distilled water on the center of the glass slide and smear the sample and place a cover slip. 4. Observe under the microscope the morphology of individual fungi and fungal colonies. 5. Compare and contrast the morphological characteristics of individual fungi and fungal colonies and label them.