2.4. Antibacterial Activity

The antibacterial properties of the samples were evaluated by the agar well diffusion method by slight modification of the process reported in [37]. The tested bacteria (S. aureus CCM 4223 and E. coli CCM 3988) were obtained from the Czech collection of microorganisms (CCM, Brno, Czech republic). The procedure was as follows:

Bacteria were cultured overnight, aerobically at 37 °C in LB medium (Sigma-Aldrich, Saint-Louis, MO, USA) with agitation. After this, bacteria were mix with 50% glycerol (Mikrochem, Pezinok, Slovakia) and frozen glycerol stock cultures were maintained at −20 °C. Before the experimental use, cultures were transferred to LB medium and incubated for 24 h and used as the source of inoculum for each experiment.

Plate count agar (HIMEDIA, Mumbai, India) medium was cooled to 42 °C after autoclaving, inoculated overnight with liquid bacterial culture to a cell density of 5 × 10⁵ colony-forming units cfu/mL.

20 mL of this inoculated agar was pipetted into a 90 mm diameter Petri dish.

Once the agar was solidified, five mm diameter wells were punched in the agar and filled with 50 µL of samples prepared in the form of suspensions (either directly the nanosuspensions prepared by green synthesis or 50 µL of the suspensions prepared by dispersing 20 mg of LEV-Ag-MS samples in 1 mL of distilled water). Gentamicin sulfate (Biosera, Nuaille, France) with the concentration 30 mM was used as a positive control.

The plates were incubated for 24 h at 37 °C.

Afterwards, the plates were photographed and the inhibition zones were measured by the ImageJ 1.53e software (U. S. National Institutes of Health, Bethesda, MD, USA). The values used for the calculation are mean values calculated from 3 replicate tests.

The antibacterial activity was calculated by applying the formula reported in [37]:

where RIZD is the relative inhibition zone diameter (%) and IZD is the inhibition zone diameter (mm). As a negative control, the inhibition zones of distilled water equal to 0 were taken. The inhibition zone diameter (IZD) was obtained by measuring the diameter of transparent zone and subtracting the size of the wells (5 mm).