Chapter 3

Methodology

3.1 Research Design

The researchers used an experimental experimental research for this study. An in-

vitro experimental study of fungicidal activity using phytochemical analysis of N. tabacum

leaves crude extracts against Candida auris. Positive and negative controls were used to

monitor the antimicrobial activities of the extracts in all the analysis.

3.2 Research Locale

This study was conducted at Science Laboratory of Tanza National

Comprehensive High School for the extraction and preparation of materials and

Department of Science and Technoogy for conducting the Kirby-Bauer test (disk diffusion

susceptibility test).

3.3 Collection and Processing of Materials

Fresh aerial leaves of N. tabacum L. was gathered from Pangasinan. The collected

tobacco leaves were washed three times under running tap water and rinsed twice with

sterile distilled water to remove the contaminants and other extraneous matter. The

cleaned plant materials will cut into small pieces and air dried under shade for one week
at room temperature. Completely dried leaves were weighed and ground finely using

mortar and pestle.

The Candida auris will be collected and cultured from the University of Santo

Thomas at España Blvd., Sampaloc, Manila.

3.4 Extraction of Chemical Compounds in N. tabacum

The powderized N. tabacum (g) will be extracted using pure Ethanol with the

measure of ml for the concentration of it. Researchers will use Manual Vacuum Pump. 10

ml of solvent were added to the powderized N. tabacum in each set up until the extracted

one is clear. The residue was extracted with same amount of solvent twice more. Then,

the extracts were combined to be dried and dissolved in 100 mL of 95% ethanol for further

fungicide activity.

3.5 Anti-Fungal Analysis of Extracts

The fungicidal activity of tobacco extract will determined by the inhibitory effect of

the Candida auris to the N. tabacum L. extract. The inhibition percentage is going to be

calculated with following formula: % Inhibition = (Colony diameter of control − Colony

diameter of sample) × 100/Colony diameter of control, in which Colony diameter (mm) =

Measured value − 6.0.

3.6 Anti-Fungal Determination Testing

 Disc diffusion is the method proposed for anti-fungal activity. A stock solution of

extract is prepared by dissolving 0.1 g of extract with 100 mL of their respective solvents

(distilled water and absolute ethanol) to produce a final concentration of 100 mg/mL. The

stock solution is going to be diluted to concentrations of 2.5, 5, 10, 20, 50, and 100 mg/mL

of extract. 20 μL of each dilution is impregnated into sterile, blank discs 6 mm in diameter.

Distilled water and ethanol-loaded discs were used as negative controls for aqueous and

ethanol extracts, respectively. All discs are fully dried before the application on bacterial

lawn. Antibacterial activity will be evaluated by measuring the diameter of the inhibition

zone (IZ) around the discs. Antibacterial activity is express as the mean zone of inhibition

diameters (mm) produced by the tobacco extract.