*2.4. Mono- and Dual-Species Biofilm Formation*

For biofilm formation, the method described by Peeters et al. [33] was used. Briefly, polystyrene microtiter plates were inoculated with 200 μL of 24-h-old bacterial culture containing cell count of approximately 10<sup>8</sup> CFU/mL. Following 4 h of cell adhesion at the corresponding temperatures, the supernatant was removed, and the plates were rinsed with physiological saline. Subsequently, 200 μL of fresh medium containing the EO or its component to be examined was added at MIC/2 concentration to avoid total growth inhibition. Plates were further incubated for 24 h to allow a biofilm to form. Positive controls contained the inoculated growth medium but without any EOs or components, and negative controls contained EOs or their components in growth medium. Experiments were repeated at least twice, and six parallel measurements were conducted each time.

The inhibition of biofilm formation was detected by the crystal violet staining method. Briefly, after 24 h of treatment, the supernatant was removed, and the wells were rinsed with physiological saline. For fixation of the biofilms, methanol was added, and the supernatant was removed again. Then, 0.1% crystal violet (CV) solution was added to each well and, 20 min later, the excess dye was removed by washing the plates under running tap water. The bound CV was released by adding 200 μL of 33% acetic acid followed by an incubation for 10 min at room temperature. The absorbance was measured at 590 nm (SPECTROstar Nano Spectrophotometer).

For polymicrobial biofilms, except for those produced from the culture containing *L. monocytogenes* and *E. coli*, 24-h-old liquid monocultures containing approximately 10<sup>5</sup> CFU/mL were mixed at a 1:1 ratio in TSB broth. In contrast, the inoculum size for *L. monocytogenes* and *E. coli* was 10<sup>3</sup> CFU/mL because the cell count of the dual culture reached more than 1010 CFU/mL after 24 h when a larger inoculum was used. For dual-species biofilms, the EO concentrations were chosen according to the MIC values of the individual strains. For investigation of the possible interactions, for example, synergism or antagonism between the species, concentrations corresponding to half and double the MIC level were examined. When the MIC values were equal for the two species, the levels corresponding to one-quarter of MIC, half of MIC, MIC, and double MIC were used.
