Bacillus subtilis

The metal precursors such as nickel nitrate hexahydrate [Ni(NO₃)₂.6H₂O; (99.99%)], sodium molybdate dihydrate [Na₂MoO₄.2H₂O; (99.5%)], sodium hydroxide [NaOH; (97%)], and Urea [CO(NH₂)₂; (99.5%)] were purchased from sigma Aldrich. All the chemicals were of analytical grade and used without further purification. Deionized water was used as a solvent throughout the synthesis of the material. Acridine orange (AO), 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), Hydrogen peroxide (H₂O₂), 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA), 2,3,5-triphenyl tetrazolium chloride (TTC), Propidium Iodide (PI), 4',6-diamidino-2-phenylindole (DAPI), potassium persulfate, trichloroacetic acid and other reagents and chemicals were purchased from HiMedia Laboratories Pvt. Ltd., (Mumbai, India). The pathogenic bacterial strains of Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli were obtained from the Microbial Type Culture Collection (MTCC), Chandigarh, India.

The disc diffusion method [19 (link)] was used to determine the antibacterial properties of RSALE and R-AuNPs. For antibacterial assay analysis, pure cultures of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), and Streptococcus pyogenes (ATCC 19615) were obtained from American Type Culture Collection. However, Bacillus subtilis (MTCC 8114) and Proteus vulgaris (MTCC 1771) were obtained from the Microbial Type Culture Collection and Gene Bank (MTCC), housed at the Institute of Microbial Technology (IMTECH), Chandigarh, India. The suspensions of the strains tested were standardized to 0.5 McFarland. The suspension was obtained from overnight trypticase soy broth (TSB) cultures and placed on the surface of Mueller–Hinton (M.H.) agar for the study, as mentioned earlier. During the experiment, 50 µL of various concentrations of R-AuNPs (10, 20, 40, 60 and 80 mg/mL) and various concentrations of crude RSALE (10, 25, 50, 75, and 100 mg/mL), negative control (PBS) and positive control amoxicillin (25 mg/mL) were added to the wells of MH agar plates. The experiments were conducted in triplicate, and the agar plates were incubated overnight at 37 °C. Following that, the diameter of the inhibitory zone was determined. The experiment was conducted in triplicates in the same experimental conditions.