2.3. Determination of Minimum Inhibitory and Bactericidal Concentrations (MIC, MBC) of Each Compound against Planktonic Bacteria

The MIC of each compound (i.e., CAR, THY, EUG, and BAC) against the planktonic cells of each Staphylococcus species was determined using the broth microdilution method, as previously described [46]. Briefly, on the day of application, ten different concentrations for each compound were prepared by appropriately diluting its stock solution (i.e., 10% and 1%, for terpenoids and BAC, respectively) in fresh TSB. For terpenoids, the tested concentrations ranged from 19.5 to 10,000 ppm (two-fold dilutions), while for BAC those ranged from 1 to 10 ppm. Subsequently, 180 μL of each dilution were transferred to a well (in duplicate) of a sterile flat-bottomed 96-well polystyrene (PS) microtiter plate (transparent, hydrophobic, Ref 655101; Greiner bio-one GmbH, Frickenhausen, Germany) and 20 μL of a 10-fold dilution of the appropriate bacterial suspension (A_{600 nm} = 0.1) in quarter-strength Ringer’s solution were then added, so as to have an initial bacterial concentration in each well of ca. 10⁵ CFU/mL. Wells without bacteria and wells without any added compound served as negative and positive growth controls (for bacterial growth), respectively. The plates were sealed with parafilm and statically incubated at 37 °C for 24 h. The growth in each well was finally turbidimetrically assessed by naked eye observation and confirmed by measuring absorbances at 620 nm using a computer-controlled microplate reader (Halo Led 96; Dynamica Scientific Ltd., Livingston, UK). The MIC value was considered as the lowest concentration of each compound that totally inhibited the visible bacterial growth. To calculate MBCs, from all the wells showing no visible growth, 10 μL were aspirated and spotted on TSA and the number of colonies was counted following incubation at 37 °C for 48 h. MBC for each compound was defined as its lowest concentration, reducing the initial inoculum by at least three logs (i.e., no appearance of colonies).