outline of methodology_thesis_ edit3 by addie

							Janine Erica P. Dayao                                  Mary Bernadette V. Egloso

Partial Characteriztion of the potential biodegrading ability of the fungi
Xylaria sp. and its four mutants, [mutant 1, mutant 2, mutant 3, mutant
4] on natural rubber, chicken feathers, and polystyrene

Objectives:
  1. to determine if Xylaria sp. mutants and wild type can degrade/consume/
      assimilate natural rubber as a carbon source
  2. to determine if Xylaria sp. mutants and wild type can degrade/consume/
      assimilate chicken feathers as a carbon and nitrogen source
  3. to determine if Xylaria sp. mutants and wild type can degrade/consume/
      assimilate polystyrene as a carbon source
  4. to compare the biodegrading ability of the wild type to each mutant


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. Petroleum

          B. Polystyrene
                    Cut 1x1 cm strips from polystyrene food containers
          C. Chicken feather
                    Obtain fresh feathers from Gallus gallus sp. Sterilize by
             putting inside SM plastic bag and autoclave for 20 mins. at 15psi.
          D. Rubber
                  - Obtain rubber latex 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. Set A
             is to verify if the fungi inoculated to both set-ups are active. 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 250C 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.


For Set B:

*RUBBER
      Natural latex gloves will be cut into pieces with masses of 0.25 g Mineral
medium then autoclave subsequently. Inoculate the fungi after the medium has
cooled. Then incubate at 25 ˚C at pH 5. Or cultivate the fungi on a latex
overlay agar plate technique wherein a layer of concentrated natural rubber is
added on top of the mineral medium or Potato dextrose agar at a concentration
of 0.02% (wt/vol).
*CHICKEN FEATHERS

The basic medium used for isolation and fermentation of the feather-degrading
microorganisms contained the following constituents (g/L): NaCl (0.5), KH2PO4
(0.7), K2HPO4 (1.4), MgSO4 (0.1) and feathers (10), pH 7.2. Cultivation was
done using 500 ml Erlenmeyer flasks containing 100 ml medium. Feather agar
medium containing the basic medium and 20 g/L of agar was used for
screening the microorganisms in plates. For the medium used for screening
mutants, 10 g casein was used instead of feathers. Luria-Bertani (LB) medium
(peptone 1% (w/v), yeast extract 0.3% (w/v), NaCl 0.5% (w/v), pH
7.2) was used for inoculum preparation and isolate maintenance.
       The fungi will be inoculated on the feather medium-containing flasks and
will be shaken at 25 ˚C.


*POLYSTYRENE
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. Ten pieces of polystyrene strips cut into 1x1 cm
from polystyrene food containers are added to the set A flasks only, and were
then inoculated with a loopful of Xylaria sp. mycelium. Set B without
polystyrene strips were used as control. All the sets are incubated for 50-
80days, in a room with more or less 250C in temperature.

*PETROLEUM