*2.5. Antimicrobial*

All extracted samples were screened for their antimicrobial properties using agar disc diffusion assay. Bacteria grown at 35 ◦C for 24 h and yeasts at 25 ◦C for 48 h prior to the day of the experiment. A volume of 100 μL suspension of culture medium (10<sup>5</sup> cell per mL) adjusted to the appropriate density of 0.5 McFarland standard using a cuvette spectrophotometer at absorbance reading of 540 nm was inoculated on a solid media plate where standard plate count agar (Oxoid, London, UK) was used for bacteria and Sabouraud dextrose agar (Oxoid, London, UK) for yeasts. Dried sample extracts were dissolved in ethanol and 2 mg loaded onto sterile 6 mm paper discs under the fume hood and allowed to dry out before placing on the inoculated plates. Standard antibacterial chloramphenicol (Oxoid, London, UK) at 30 μg/disc and antifungal fluconazole (Sigma-Aldrich, St. Louis, MO, USA) and amphotericin B (Sigma-Aldrich, St. Louis, MO, USA) at 20 μg/disc were included as positive controls. Ethanol and water used to dissolve the standard antimicrobial drugs and assayed as negative controls. Incubation was done at 35 ◦C for 24 h for bacteria and at 25 ◦C for 48 h for yeasts. The experiments were done in duplicate. Zones of inhibition were measured using a digital caliper and expressed in millimeters. Criteria for antimicrobial strength were divided into three ranges according to Ahmad et al. [32]: weak activity (inhibition zone <10 mm), moderate activity (inhibition zone 10 to 15 mm) and strong activity (inhibition zone >15 mm).

#### *2.6. Yeast Cell Staining and Fluorescence Microscopy*

Yeast cells, *S. cerevisiae*, were grown at 25 ◦C for 24 h in tryptone soya yeas<sup>t</sup> extract broth (Oxoid, London, UK). Yeast cells suspension was adjusted to the appropriate density of 0.5 McFarland standard using a cuvette spectrophotometer at absorbance reading of 540 nm. The adjusted suspension divided into control and treatment groups. The treatment group was centrifuged at 2500 rpm for 3 min, supernatant was removed, 4% (*v*/*v*) lemon myrtle hexane extract (dissolved in sterile water containing 0.4% tween-80) was added and allowed to stand for 30 and 60 min. The control group treated in the same manner except 0.1 M phosphate buffer added instead of lemon myrtle extract. Suspensions of treatment and control groups centrifuged at 2500 rpm for 3 min and washed with 0.1 M phosphate buffer. Cells fixed and stained according to Shimada et al. [33] with few modifications. Cells fixed for 30 min by the addition of 4% paraformaldehyde (4 mL), centrifuged (2500 rpm, 3 min) and washed twice with 0.1 M phosphate buffer. Cell suspension was mixed with 1:1 *v*/*v* of 50 ng/mL DAPI (4,6-diamidino-2-phenylindole) (Thermo-Fisher Scientific, Waltham, MA, USA) and 8 μL of the mixture was added on a glass microscope slide and covered with a coverslip. Cell fluorescence images observed using a Leica DM6000B microscope with Leica Microsystem LAS AF6000 software (Leica, Hamburg, Germany) at 100× objective using a Leica DFC420C digital camera (Leica, Hamburg, Germany).

#### *2.7. Total Phenolic Content*

Total phenolic content (TPC) of ethanol, hexane, methanol and water extracted herb samples were spectrophotometrically analyzed according to Folin–Ciocalteu colorimetric technique [34]. Samples were diluted (10–1000 times) with Mili-Q water and 25 μL from each dilution added to the 96-well polystyrene plate (Sarstedt, Nümbrecht, Germany). Several concentrations of gallic acid (3,4,5-Trihydroxybenzoic acid ≥98%, Sigma-Aldrich, St. Louis, MO, USA), 0–100 mg/L, were prepared to construct the standard calibration curve and 25 μL of gallic acid was loaded into the plate. All wells were loaded with 125 μL of Folin-Ciocalteu's phenol reagen<sup>t</sup> (Sigma-Aldrich, St. Louis, MO, USA), followed by the addition of 125 μL Sodium carbonate (Chem-Supply, Gillman, Australia). The 96-well plate placed in a microplate-reader (Tecan, Grödig, Austria) and shaken for 15 s and the absorbance reading measured at 750 nm after 15 min of incubation in the dark. Calculated results expressed as milligram of gallic acid equivalents per gram of sample dry weight (GAE/g DW).

#### *2.8. DPPH Radical Scavenging Activity*

The radical scavenging activity of the ethanol, hexane, methanol and water extracted herb samples were evaluated using DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay according to Nirmal and Panichayupakaranant [32] with slight modifications. On the day of the experiment, DPPH (Sigma-Aldrich, St. Louis, MO, USA) concentration of 0.15 mM was prepared in 95% ethanol and mixed with different sample concentrations at 1:1 ratio (*v*/*v*) in a total volume of 3 mL. All reagents brought to room temperature before mixing. The mixing was carried out in the dark at room temperature for 30 min and the absorbance measured at 517 nm using a cuvette spectrophotometer (Thermo-Fisher Scientific, Waltham, MA, USA). Sample blank was prepared in the same manner except ethanol was used instead of DPPH solution, while control included DPPH solution and ethanol but without the addition of any sample. The percentage inhibition capacity of scavenging property for the DPPH radicals was calculated using the following formula:

$$\% \text{ inhibition} = (1 - \text{(sample absorber/control absorbance))} \times 100. \tag{1}$$
