Wells were sealed with one drop of molten agar (0.8% agar) to prevent leakage of nanoparticles from the bottom of the wells.Using a micropipette, 100 ml (50 mg) of the sample of nanoparticles suspension was poured onto each of five wells on all plates. After overnight incubation at 35 C ± 2 C, the different levels of zone of inhibition were measured. Non-functionalized Ag3O4 nanoparticles and antibiotic tetracycline was used as control.