2.5. Minimum Inhibitory Concentration (MIC) Test

This test was performed to determine the lowest concentration of antibacterial agents at which about 90% of the bacterial growth is inhibited. The MIC was estimated for synthesized nanoparticles against the four tested bacterial strains E. coli, S. aureus, K. pneumoniae, and P. aeruginosa. A bacterial suspension of 300 μL at a concentration of 0.5 McFarland (1.5 × 10⁸ CFU/mL) was seeded into each tube of 9 mL nutrient broth individually, following the addition of 1 mL of different concentrations of synthesized nanoparticles at 2, 0.2, 0.02, 0.002, 0.0002 mg/mL and DMSO, which was used as the negative control, then incubated at 37 °C overnight. The values for the MIC have been recorded by comparing tube turbidity using the naked eye with that of a 0.5 McFarland standard medium. The concentration in the transparent tube, which indicated the growth prevention by antibacterial agents, is considered as the MIC values [17].