In conducting the study, the following steps will be followed for the procedures and

ethical considerations by the researchers to get necessary data in order to determine the

antibacterial property of star anise fruit.

First, the researchers will ask permission to conduct the study. The researchers will

present a formal letter to the Senior High School Program Head to allow them to conduct the

study in the laboratories of San Pedro College.

Second, the researchers will ask the panelists to review some ethical considerations.

The researchers will write a letter to the panelists, allowing us to check the ethical

considerations of the study.

Third, the researchers will ask for permission to use the San Pedro College laboratory –

where the researchers will perform the whole antibacterial experiment. The researchers will

make a letter to the laboratory head to allow, help and request laboratory equipment and

chemicals needed in the conduct of the experiment.

After asking permission and gaining approval to use one of the laboratories of San

Pedro College, collection of samples will take place. Star anise fruits will be bought at local

public markets within Davao City. The water samples will be collected in Bankerohan River.

Utilizing a sterilized plastic bottle (APHA, 2005), the water samples would be taken at a site

near the household, but not too near the bank, as stated in the Standard Methods for the

Examination of Water and Wastewater (APHA, 2005). The sterilized bottle will be immersed

at approximately 30 cm below surface as stated in the Microbiology Practical Guide (2010).

After which, an initial testing for the pH level of the water will be done using a litmus paper on

the site. Accordingly, the pH level of the water sample will also be tested in the laboratory for

accuracy.
Procedure for Extraction of Star Anise

The procedure for extraction of star anise fruits will be adapted from the methods of Alo,

Anyim, Igwe, Elom & Uchenna (2012), Wong, Lee & Nurdiyana (2014), Marwaha, Khan &

Abhyankar (2015) and Tian & Li (2015), following their specific measurements but with some

modifications. The star anise fruit samples will be washed with tap water and rinsed with distilled

water. After which, the star anise will be dried at fruits at 60 °C for 48 hours using an electric air

blow drying oven.

Subsequently, 50 grams of star anise fruits will be weighed using analytical balance.

After which, the whole star anise fruits will be divided into separate carpels and will be

pulverized to a fine powder using a blender. The ethanol extract will be obtained by weighing 20

grams of the powdered star anise fruits and dissolve in 100 ml of the 60% ethanol for 24hrs with

occasional shaking (Aly, Bassem, Shaheen & Hathout, 2014). After the extraction time, the

extracts will then be filtered using Whatman No. 1 filter paper and air dried at room temperature.

The ethanolic extract will be stored in an amber bottle and kept in a refrigerator at 4°C.

Procedure for Preparation of the Water Samples

After testing for the pH level of the water samples, a three-fold serial dilution of the water

samples will take place. 3 test tubes will be filled with 9ml of distilled water. 1ml of the water

sample will be taken to be serially diluted in 9ml distilled water. Polluted water samples will be

serially diluted from 10-1 to 10-3 with the help of a sterile pipette. 10-3 dilution will be taken. 1ml of

the dilution will be taken using a separate sterile pipette to transfer to an empty petri dish. Two

replicate plates will be prepared for the dilution used (APHA, 2015). After which, 10 ml of potato

dextrose agar (PDA) will be poured unto the petri dish containing the dilution. PDA will be mixed

with the test portions in the petri dish by rotating and tilting the dish and let the medium solidify

on a level surface. After the medium solidifies, the plates will be inverted, and will be placed in
an incubator. The plates will be incubated for 24 hours at 37 °C to encourage growth of bacteria

and 22 °C for 72 hrs. enumerate the bacteria (Bartram, Balance & World Health

Organization, 1996). After incubation, the number of bacterial colonies will be counted. This will

be recorded as the initial bacterial count before applying the star anise fruit ethanolic extract.

Procedure for Application of Star Anise Fruit Extract

Preparation of the disc will be done. Each disc will be about 10mm in diameter and will

be cut from Whatman No. 1 filter paper. The discs will be put into a petri dish and then undergo

autoclaving for 160 (degree) C for 2 hrs. for sterilization. The discs will then be soaked in the

star anise fruit ethanolic extract for 24 hours. The amount of ethanolic extract contained in the

disc will also be measured. Using sterilized forceps, each disc will be recovered from the extract

and held for a few seconds for some of the ethanol to evaporate (Alo, Anyim, Igwe, Elom &

Uchenna (2012), Wong, Lee & Nurdiyana, 2014) before applying aseptically unto the agar

surface in a plate. The plates will be left to stand for 1 hr. at room temperature to allow proper

diffusion of the extract to occur. All the plates will then be incubated at 37 °C for 24 hours and

will be observed for zones of inhibitions.

A zone of clearance will signify inhibition. Thus, diameter of such zones will be

measured in millimeters (mm) using a digital caliper. At the end of the incubation period,

antibacterial property of star anise will be evaluated by measuring the zone of inhibition against

the test. Moreover, the number of colonies will also be determined to compare if there will be

changes from the initial count of bacteria before applying the extract.