2.4. Inoculum Preparation

All bacterial isolates were grown to the exponential phase in tryptic soy broth (TSB) (Difco Laboratories, Detroit, USA) at 37°C for 18 h. The bacterial growth was estimated as turbidity using spectrophotometer to measure the light absorption of the microbial mass as determined by the optical density readings at 620 nm (OD₆₂₀). Growth was checked every 30 minutes, and the exponential phase of bacterial growth was identified by the increased OD₆₂₀ reading. Then, the inoculum density of each bacterial suspension was adjusted to a final density equivalent to 0.5 McFarland Standard (1.5 × 10⁸ CFU/mL) in sterile saline (0.84% NaCl).

Antimicrobial Testing. The antimicrobial activity of miswak extracts was carried out using the agar diffusion and minimal inhibitory concentration (MIC) methods.

The antimicrobial testing was performed on Mueller Hinton agar plates (Difco Laboratories) using the agar diffusion method. Briefly, 100 µL of bacterial suspension was spread smoothly on the agar plates. The required numbers of wells, each 3 mm in diameter, were cut out of the agar using a sterile glass capillary ensuring proper distribution of holes in the periphery and one in the center for each agar plate. Then, wells were filled with 50 µL of sterile extract (aqueous or methanol) made from Salvadora persica stock solution (400, 200, 100, and 50 mg/mL). This was followed by 2 h preincubation at room temperature for proper diffusion of the plant extract into the media. Then, the plates were incubated at 37°C for 24 h [25]. The mean diameter of complete growth inhibition zone (in mm) was measured without the well's diameter and considered as the inhibition zone. Antibiotic discs (Difco Laboratories, Detroit, USA), 30 µg vancomycin for Gram-positive isolates and 10 µg tobramycin for Gram-negative strains, were used as positive controls, and water (for aqueous extract) or methanol (for methanol extract) was used as negative control. The test for each microorganism was repeated three times to ensure reproducibility. The average zones diameter values from three repeats were taken in determination of the final inhibition zones. This was done to ensure that all inhibition zones within each experiment were obtained under the same experimental conditions.

The MIC of the Salvadora persica extracts was determined using the standard microdilution method in 96 multi-well microtiter plates, as previously described [26], with slight modifications. Briefly, the dissolved extracts were first diluted to a concentration of 50 mg/mL, then 50 µL from each of the aqueous and methanol extracts was pipetted into the first well of each microtiter plate row, and 50 µL of TSB was distributed from the 1st to the 12th well of each row. Twofold serial dilution was achieved by transferring 50 µL of scalar dilution from the first to the subsequent wells of each row. The final concentration of the extracts adopted to evaluate antibacterial activity was included from 25 mg/mL to 0.003 mg/mL. Finally, 10 µL of each bacterial suspension was added to each well. Two row lines in each plate were used as controls: one row line with vancomycin as a positive control for Gram-positive isolates and another row line with tobramycin for Gram-negatives strains (in a serial dilution of 32–0.015 µg/mL). Plates were incubated at 37°C for 18–24 h. The lowest concentration at which no turbidity occurred was taken as the MIC value. Plates were analyzed individually to determine MIC and the average MIC values from three repeats were taken in determination of the final MIC values for each extract to ensure accuracy and reproducibility.