Rs Chapter 3.docx

CHAPTER 3 METHODOLOGY

The methods and procedures that are to be used in conducting this study are presented in this chapter. It includes the research design, procedure, materials used, and statistical analysis of the data collected about the antibacterial activity of the leaf extract of A. carambola (Star Fruit).

Research Design The study observes the experimental type of research. Figure 1 illustrates the process in the conduct of the study. It shows the variables of the antibacterial property of the leaf extract of A. carambola (Star Fruit) that is to be subjected to test. The researchers engage three treatments in the conduct of the study. There are three replicates in each treatment. The following treatments are: T 1 will be using distilled water as negative control; T2 will be using Amoxicillin (antibiotic drug as the positive control and T3 will be using the leaf extract of A. carambola (Star Fruit) as the experimental treatment. There are the three samples made in each treatment, serving as the replicates. The dependent variable was the antibacterial property of the leaf extract of A. carambola (Star Fruit) on the growth of the bacteria. While on the other hand the independent variable was the leaf extract of A. carambola (Star Fruit).

Ethanolic extract of A. carambola (Star Fruit)

Bioactivity

R1 T1

R2

Antibacterial Activity

R3

Zone of inhibition

R1 T2

R2 R3

R1 T3

R2

Legend: T1- Distilled water (negative control) T2- Amoxicillin (positive control) T3- Leaf extract of A. carambola (Star Fruit) Rn- Replicates

R3

Figure1. Research Design

Preparation of Materials These are the materials that are to be used in antibacterial assay: 5g of Nutrient Agar for B. subtilis, 5g of Eosine Methylene Blue Agar (EMB Agar) for E. coli, 450mL distilled water, rotary evaporator, masking tape, scissors, cotton swab, forceps, 20 pieces of petri dish, 54 pieces of filter discs, hotplate, incubator, ruler in millimeters, vials for plant extract of A. carambola (Star Fruit), E. coli and B. subtilis and 500 mg of Amoxicillin.

General Procedure These are the steps that the researchers need to conduct the experiment: A. Asking for Permission Prepare a request letter to use the facilities of the Science Laboratory in Notre Dame of Dadiangas University to start the conduct of the experiment and will submit it to the laboratory in charge. Then, the A. carambola (Star Fruit) as their specimen will be collected in Alabel, Sarangani Province.

B. Collection of A. carambola leaves (Star Fruit) and Ethanolic Extraction Three hundred grams of A. carambola (Star Fruit) leaves are to be collected for the antibacterial assay. These leaves will be washed with tap water. After washing the leaves, it will be air-dried, chopped into pieces, and then soak in 500 mL of ethanol. After 24 hours the extract will be removed by filtration and subject to rotary evaporator.

C. Preparation of Treatment Each treatment will be placed in a single petri dish with labels. For T1, distilled water is to be prepared also for diluting 500 ml Amoxicillin in 5ml of distilled water and which serves as T2, and the ethanolic extract of A. carambola (Star Fruit) which serves as T3. Then, 18 pieces of filter disk will be soaked in each treatment for an hour

D. Preparation of Culture Media 5g of Nutrient Agar, Eosine Methylene Blue Agar each and 210ml of distilled water will be used in preparing the culture media for the two bacteria subjects. Eighteen (18) petri dishes are needed, and will undergo sterilization process.

E. Filter Disk Diffusion Method B. subtilis, E. coli and are the test subjects that can be obtain in the Science Laboratory of Notre Dame of Dadiangas University. Each petri dish has 20 mL of agar. By using a cotton swab, dip it into the bacterial inoculum and swab it in solidified Nutrient Agar and Eosine Methylene Blue Agar plate evenly. On a single petri dish, three filter disks are to be placed with equal zoning. Then, place in an incubator for 24 and 48 hours. To gather the data, Vernier caliper is to be used in measuring the zone of inhibition.

Data Gathering Techniques All petri dishes will be placed in an incubator for 24 and 48 hours. A ruler in millimeters is to be used in measuring the diameter of the zone of inhibition to the edge of the growth. The researchers will record all the results of all they observe in the experiment.

Statistical Tools For the presentation of the analysis and the interpretation of the data collected, the researchers will use One-Way Analysis of Variance (ANOVA) and Fisher’s Least Significance Difference to see up to what extent is the significant difference of the results.