The Kirby-Bauer technique was used to determine the antibacterial activity of the extracts [11]. A total of 11 bacteria strains were used for this test. Mueller Hinton agar (pH 7.2 & 4 mm depth) plates were inoculated with test organisms (prepared in a sterile saline tube) by streaking the loop in a back-and-forth motion to ensure an even distribution of inoculum. MHA with 5% sheep blood was used for Streptococcus pyogenes. A circular 6 mm diameter well was punched aseptically with a sterile cork borer. Then, a volume of 100 μL methanol extracts of A. aspera, L. inermis and A. indica leaves (at concentrations of 100 mg/mL, 200 mg/mL, and 400 mg/mL) were dispensed into the wells. Similarly, 5% Di-methyl-sulfoxide (DMSO) was dispensed into the control well, and reference antibiotic discs were placed on the surface of the plate and incubated for 24 h at 37 °C. For Streptococcus pyogenes, a carbon dioxide incubator was used for incubation. Each experiment was done three times.

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extracts were determined using the p-iodonitrotetrazolium chloride (INT) colorimetric assay method [12]. The test was done according to the recommendations of the Clinical Laboratory Standard Institute [13]. Bacteria were sub-cultured on Mueller Hinton agar (pH 7.2) and incubated at 37 °C for 24 h. Bacterial colonies were inoculated into a sterile saline solution and used before 30 min. The bacterial suspension was evenly mixed and diluted to meet the turbidity of 0.5 McFarland standard (1 × 108 CFU/mL). Further dilution was performed to obtain the final concentration of inoculum (5 × 105 CFU/mL) in each well. A stock solution of plant extracts was prepared in DMSO. Serial dilutions of the working solution were prepared by diluting the extract solution in sterile Mueller Hinton Broth. The final concentration of DMSO in the solution was less than 2.8% in the solution. The test was performed in a sterile 96-well plate. Methanol extracts of A. aspera, L. inermis, and A. indica leaves were tested in triplicate in one plate for each bacterium. Mueller Hinton Broth (100 μL) was dispensed to all wells. A working solution of extracts (100 μL) and solvent controls (MH broth and 2.8% DMSO) were dispensed into their respective wells. Serial dilutions were performed from columns one to nine, and 50 μL of excess medium was discarded from column nine. The last column served as a blank containing only broth. Columns 10 and 11 served as negative controls, which only contained medium and bacterial suspension, and media DMSO and bacteria, respectively. 50 μL of test bacteria were added to each well except for the last row, which served as a blank. The concentration of plant extracts ranged from 0.78 mg/mL to 200 mg/mL. The plates were sealed and incubated for 24 h at 37 °C. After 24 h incubation, 40 μL (0.2 mg/mL) p-iodonitrotetrazolium chloride (INT) was added to all wells and incubated again at 37 °C for 30 min. The MIC of the samples was detected after 30 min of incubation. Viable bacteria reduced the yellow dye to pink. MIC was defined as the sample concentration that prevented the colour change of the medium and exhibited complete inhibition of microbial growth. The MBC was determined by adding 50 μL aliquots from the wells that did not show growth after incubation for the MIC test to 150 μL broth in the well plate, and incubated for 48 h at 37 °C. Then, MBC was observed as the lowest concentration of extracts which did not produce a colour change after the addition of INT as mentioned above.

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