4.7. Broth Microdilution Susceptibility Testing against Natural Products

The broth microdilution (BMD) susceptibility test according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) international guidelines was performed. Muller Hilton broth (Oxoid, Basingstoke, Hampshire, UK) was used to test the antimicrobial activity of GR-OLI and O. vulgare EO against the Salmonella spp. strains. The BMD test was performed on a 96-well plate by adding 100 μL of a cell suspension equal to 5 × 10⁵ CFU/mL to a final volume of 200 μL. Scalar dilutions, between 16% v/v (equal to 40 μL of EOs content/ mL) and 0.125% v/v (equal to 0.3 μL of EOs content/mL) of GR-OLI and between 4% (40 μL /mL) and 0.03% (0.3 μL /mL) of O. vulgare EO, were tested. A concentration surfactant (Tween 80, Sigma Aldrich, Saint Louis, MO, USA) corresponding to that contained in the GR-OLI was tested together with O. vulgare EO to facilitate its solubilization in the hydrophilic medium. Plates were incubated overnight at 37 °C. After this period, MIC values were determined by spectrophotometric reading at 450 nm (EL808, Biotek, Winooski, VT, USA). To evaluate the MBC, 5 μL of the content of each well were seeded on standard medium agar plates, which were incubated for 24 h at 37 °C. Surfactants were tested separately. The MIC is defined as the lowest concentration that completely inhibits the growth of a given organism compared with the growth in the substance-free control; whereas the MBC is defined as the lowest concentration determining the death of 99.9% or more of the initial inoculum. Each test was performed in triplicate, and in each experiment suitable positive controls and blank were added. Surfactants were tested separately.