CHAPTER III

MATERIALS AND METHOD

3.1 Method

3.1.1 Plant

1kg of ginger (Zingiber officinale) rhizomes were bought from Janiuay

Local Market in Janiuay, Iloilo last December 23, 2017 at exactly 6:30 in the
morning. The specimen was kept inside a screen bag.

3.1.2 Plant preparation for extraction

On the same day, at the School’s Laboratory, the fresh ginger rhizomes were
washed with tap water and distilled water. Firstly, the rhizomes were peeled and then
were cut into smaller pieces using chopping boards and knives.

(a) (b) (c)

Figure 1 (a) washing of rhizomes (b) peeling of rhizomes (c) cutting of rhizomes
3.1.3 Plant extraction
500 ml of 90% ethanol and 250g of ginger rhizomes were put inside a
blender and were mixed. The mixed solution were then transferred inside a
sterilized glass jar. After that, the mixtures were shaken for 1 hour in every 3
hours for 24 hours. The shaking started at exactly 7:30 in the evening.

(b)

(a)

Figure 2 (a) blending of plant specimen (b) transferring to container

3.1.4 Preparation of Test organisms

The pure cultures of S.aurues, and E. coli were obtained from West
Visayas State University, Lapaz, Iloilo City. After such, a 13g/L concentration
of Nutrient Broth was made and 5 mL of which was dispensed into the two
pre-labeled sterile test tubes. The bacterial and fungal inocula of pathogenic
microorganisms (E. coli, and S. aureus, ) were prepared by having a loopful
of bacteria from the agar plate diluted in the designated test tube containing the
nutrient broth. The mixture was swirled around to mix thoroughly. The test
tubes were covered with a cotton plug and then with Parafilm. These were then
put inside a test tube rack and incubated at 37°C overnight. The bacterial and
fungal inocula were standardized with McFarland’s Standard Table swabbing
them on the agar plates.

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) (b)

Figure 3 (a) preparation of agar (b) incubating test organism for 24 hours

3.1.5 Preparation of Inoculant

The bacterial plates had been incubated for 18-24 hours at 37°C. Then the
ZOI was measured and recorded in millimeter using a ruler. It is done with
triplicates.

3.2 Production of discs (discs of the extracts)

By using whatman filter paper No. 1, Discs of 5 mm in diameter were
produced by using of a paper puncher. After that, the prepared disks were put in a
petri dish. Then, the discs were subjected to autoclave in order to sterilize the
disks (adjusting the conditions of autoclave to be 1210C for 15 mins ) and left to
become cold. Later on, the discs were allowed to suck up the extract filtrate and
maintained for later assay. The produced discs (each one) have the ability to
absorb 0.01ml.

3.3 Antimicrobial susceptibility test

The antibiotics susceptibility procedure for bacterial spices has been done
through using a method that depends on the ability of disc to permit the
penetration of antibiotics through the medium which is also called Kirby-Bauer
method 15. The overall steps of the procedure should be produced through
entirely sterilized status. Plates of Mueller Hinton agar have been inoculated;
each alone, by the test bacterial isolates -107 CFU (compared with Mcfarland

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turbidity standard), then the bacterial suspension was uniformly distributed
overall the area of the plate. After a while, sterilized discs (measuring five
milliliters in diameter) were put under sterilized conditions in various extracts
(for about 1 min), then fixed on Mueller Hinton plates (petridishes) inoculated
previously by suspension of the bacteria. After this step, all plates had been put
aside (at 250C for about 15 mins). After that, all cultured plates were placed in
the incubator at 36°C for 18-20 hrs; the area of inhibition has been examined and
calculated in millimeters (mm). The organisms under the experiment were
evaluated, as well, for their susceptibility toward organisms were reactivated by
culturing in sterile nutrient broth for 16 hrs at 37°C. After incubation and
turbidity comparison with Mcfarland turbidity standard, sterile cotton swabs were
used to transfer the bacterial cultures aseptically and swabbed over Mueller
Hinton agar petridishes. A sterilized forceps was used to fix the antibiotic disc
aseptically over the cultured petridishes. Then, the petridishes were placed in the
incubator at 37°C for 20-24 hours and subsequently all diameters of inhibition
zones could be determined.

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