## 2 Materials and Methods
### Analytical Procedures
#### 2.3.3 Adherent Biofilm Inhibition Formation Assays  
Pathogenic biofilm inhibition formation activity expressed as Minimum Biofilm Inhibition Concentration (MBIC) of EOs was assessed using a microtiter plate-based crystal violet staining assay [4; 11; 23]. Specifically, 200 \(\mu\)L of 10\({}^{7}\) CFU/mL bacterial suspensions in 14 Ringer's solution (VWR International S.A.S, Darmstadt, Germany) were transferred into a well of a 96-well polystyrene flat-bottomed microtiter plate and placed at 30 \({}^{\circ}\)C for 2 h under static conditions, allowing bacteria to adhere to the well surface. After the incubation, the suspensions were carefully removed, and the wells were washed once with 14 Ringer's solution (VWR International S.A.S) to remove any remaining planktonic bacteria. The adherent biofilm was treated with EOs, as previously described by [PERSON] et al. [4], for 48 h. Two hundred \(\mu\)L of Brain Heart Infusion broth (LABM) supplemented with EOs or their main components or WSTRTc extracts at concentrations tested for MIC and MBC determination (Section 2.3.2), followed by incubation at 30 \({}^{\circ}\)C for 48 h. After 48 h of incubation, the wells were washed once with 14 Ringer's solution (VWR International S.A.S) to remove all planktonic bacteria, and the growth medium was renewed with fresh medium without the addition of EOs or main compounds or WSTRTc extracts. After medium renewal, biofilms were incubated again for 48 h in order to reach their maximum formation [24; 25; 26; 27].  
Afterward, the supernatant was carefully removed, and the wells were washed once with 14 Ringer's solution (VWR International S.A.S). The biofilms were then fixed with methanol (Chem-Lab NV, Zedelgem, Belgium), stained with 1% \(w/v\) Crystal Violet (Sigma-Aldrich) for 30 min, washed once with distilled water to remove any excess Crystal Violet (Sigma-Aldrich) and quantified by eluting Crystal Violet with an ethanol-acetone mix (80/20 \(v/v\)). The optical absorbance of the eluted dye was determined at 620 nm using a microplate reader (Tecan, Sunrise, Program XFLUOR4, Mannendorf, Switzerland). Wells containing only Brain Heart Infusion broth (LABM) and wells containing Brain Heart Infusion broth (LABM) and pathogens with no treatment served as negative and positive controls, respectively.  
MBEC is defined as the lowest concentration of an antimicrobial substance that eradicates 99.9% of biofilm-embedded bacteria. The Minimum Biofilm Excitation Concentration (MBEC) was determined as described by [PERSON] et al. [28] with some modifications. MBEC determinations were performed on biofilms grown in triplicate for 2 h under static conditions and rinsed once with 14 Ringer's solution to remove planktonic cells keeping the biofilm on the bottom of the wells intact. The growth media was replaced with 200 \(\mu\)L of Brain Heart Infusion broth containing the EOs or the main compounds, or the WSTRTc extracts (antimicrobials), at various concentrations. After 48 h of incubation, the wells were washed once with 14 Ringer's solution to remove all planktonic cells and the growth medium was renewed with fresh medium without the addition of the antimicrobials. Following medium renewal, biofilms were incubated for another 48 h to allow maximum formation. Then, the biofilms were rinsed four times with 200 \(\upmu\)L Brain Heart Infusion broth to minimize residual antimicrobial agents. Biofilms were then removed from each well by scraping with sterile transfer pipettes (Thermo Fisher Scientific, Waltham, MA, USA). The collected biofilm fragments, after scraping, were placed in 3 mL of Brain Heart Infusion broth for subculturing. The subcultures were then incubated at 37 \({}^{\circ}\)C for 21 days with aliquots plated onto Brain Heart Agar every 48 h for colony enumeration. The MBEC was recorded as the lowest concentration, and at least two of the three replicates were culture-negative after subculture incubation. The resulting data were noncontinuous, ordinal in nature, and characterized by variable intervals between concentrations [28].