Chapter III

METHODOLOGY

This chapter will present discussions of the main method of research

which will be used in the conduct of the study and the procedures in the conduct

of the experiment. It includes the different procedures and data gathering

techniques and statistical treatment and tool which will be used to accomplish the

objectives of the study

Research Design

The study will use experimental design which is showing the treatments

as the independent variable and the bioactivity being the dependent variable.

There are four treatments with three replicates for each. Negative Control will be

the Distilled Water; Treatment 1 will be the P. guajava leaf extract; Treatment 2

will be the C. longa rhizome extract; and Positive control will be the Dithane,

known to cure mildews on plants. Antifungal assay will also be conducted to

determine the zone of inhibition. The research design will also show the

terminologies for each symbol to guide the readers in understanding the design.
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Treatments Antifungal Activity

R1

Antifungal Assay
R2
T1
R3

R1 Zone of Inhibition

T2 R2
Sphaerotheca
Fuliginea
R3
(Powdery
Mildews).
R1

C+ R2

R3 Legend:
Rn- Replicates

R1 T1- P. guajavae leaf extract

T2 C. longa rhizome extract
C- R2 C+- Dithane
(positive control)
R3 C-—-Distiled Water
(negative control)

Research Design
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Preparation of Materials

The materials which will be used by the researchers will be prepared and

sterilized in Orca laboratory in General Santos City. The materials includes 32

pieces petri dishes, two (2) pieces 250 ml beaker, two (2) pieces 1000 ml

Erlenmeyer flask, one (1) piece graduated cylinder, 32 pieces cotton swabs, and

96 pieces filter discs. The researchers will sterilize some of the glasses using

autoclave with distilled water and the others will be placed in a sterilizer oven at

121°C for 30 minutes. In this study, the researchers will use two ethanolic

extracts.

Preparation of Plant Extracts

The P. folium will be collected from Zone 4, Soledad Estate, Brgy. City Heights,

General Santos City. The C. longa rhizomes shall be collected from Aquinoville,

Kaunlaran, 3rd Street, Brgy. San Isidro, General Santos City

In this study, 500 grams of P. guajava leaves and 500 grams of C. longa

rhizomes will be thoroughly washed in running water to remove surface

contaminants before being air-dried for 2-3 days. The dried samples will be cut

into small pieces and soaked in 1000 ml of ethanol before being placed in a

thoroughly washed glass for 72 hours before being submitted to the Orca

Laboratory.
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Preparation of Media Culture

The researcher will use Sabouraud Dextrose Agar (SDA) for Sphaerotheca

Fuliginea (powdery mildew). Such organism will be collected from infected

plants and will be isolated for its culture needed for the experiment.

Preparation of Inoculum

The researcher will use the type of midews known as S. fuliginea. Using

cotton swabs, an amount of S. fuliginea culture will be swabbed and mixed with

100 ml Normal Saline Solution (NSS) for fungus to make the inoculum.

Application Treatment

The researcher will use a sterile cotton applicator in the application of

inoculum. As the agar solidifies, the cotton applicator will be dipped into the

inoculum and will be applied using the streaking plate technique. Half of the petri

dish will be streaked in one direction, the petri dish will then be rotated to

continue streaking in perpendicular to the first application. Filter discs will be

soaked in the different treatments for one hour and will be placed to the culture

media.

Incubation

The petri dishes will be incubated for 24 hours and 48 hours at 37 0C in an

upright position.
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Data Gathering Technique

The data will be gathered after 24 and 48 hours. The researcher will

measure the zone of inhibition of the bacteria and fungi using a Vernier calliper

and will be recorded in a tabulated form.

Statistical Tools

For the quantitative aspect of the study, the One-Way Analysis of

Variance (ANOVA) for Complete-Randomized Design (CDR) with equal

replicates will be used to determine if there is a significant difference in the mean

zone of inhibition of S. fuliginea. The results will be summed up and will be

obtained before subjecting the data for further analysis.