## 2 Materials and Methods
### In Vitro Antibacterial Activities Assay  
#### 2.3.1 Disc Diffusion Assay  
One hundred mg of the dried plant extracts were dissolved in 10% dimethylsulfoxide (DMSO) (Sigma, Hamburg, Germany) to a final concentration of 100 mg/mL and sterilized by filtration through 0.22 \(\upmu\)m cellulose membrane filter (Indiamart, Vadodara, India). The modified disc diffusion assay was performed using 6 mm filter paper disc (Oxoid, UK) that was impregnated with 50 \(\upmu\)L plant extract (10 mg/disc) at a concentration of 200 mg/mL and kept at room temperature for one hour [13]. The bacterial broth culture was adjusted to (0.5) McFarland turbidity, and then streaked evenly onto Muller Hinton agar. The plant extract discs and antibiotic discs were applied on the surface of Mueller Hinton Agar MHA (Oxoid, UK) plates and incubated under aerobic conditions at 37 \({}^{\circ}\)C for 24 h. The tests were carried out in triplicate. Zone of inhibition \(\geq\)11 mm indicates susceptibility of the tested bacteria and consider as high antibacterial activity [22]. The inhibition of bacterial growth was evaluated by measuring the diameter (mm) of the clear zone around each disc [23]. Vancomycin (10 \(\upmu\)g/disc) (Oxoid, UK) and gentamycin (10 \(\upmu\)g/disc) (Oxoid, UK) were used as a positive control, while DMSO was used as a negative control [13]. .3.2 Minimum Inhibitory Concentration Assay (MIC) and Minimum Bactericidal Concentration Assay (MBC)  
The plants that showed antibacterial activity by disc diffusion assay were further tested by microdilution assay. The initial plant extracts concentration (100 mg/mL) was diluted by two-fold dilution manner in MHB in 96 well plates (50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, and 0.19 mg/mL). One hundred \(\upmu\)L bacterial inoculum size of 1 \(\times\) 10\({}^{6}\) CFU/mL was added to each well except the negative control, so the final inoculum size become \(5\times 10^{5}\) CFU/mL. The positive control was vancomycin for Gram positive bacteria and gentamycin for Gram negative bacteria. Wells containing the bacterial inoculum were used as positive control and wells containing plant extract as a negative control. Each plant extract was run in triplicate. The MIC considered as the lowest concentration of plant extract dilution that inhibited any visible growth of the tested bacteria. Bacterial growth in each sample was confirmed by adding 40 \(\upmu\)L indicator solution (0.2 mg/mL) p-iodontrotetrazolium chloride (INT) (ZellBio, Lonsee, Deutschland, Germany) to every well and incubated at 37 \({}^{\circ}\)C for 30 min. The microbial activity was indicated by the formation of a pink-red color while inhibition of the growth was observed by the continuation of purple color, and the color changes were assessed visually. The lowest concentration that prevented the purple color change was considered as the MIC [19; 24]. The MBC was determined by adding 50 \(\upmu\)L aliquots of the extract preparations that did not show any growth after incubation during MIC assays, to 150 \(\upmu\)L broth in the 96-well plate (Sigma, Hamburg, Germany). These preparations were incubated at 37 \({}^{\circ}\)C for 48 h. The MBC was regarded as the lowest concentration of extract, which did not produce a color change after addition of INT as mentioned above. Bacterial growth in each sample was confirmed by adding 40 \(\upmu\)L indicator solution (0.2 mg/mL) p-iodontrotetrazolium chloride (INT)