## 2 Materials and Methods
### Additional Control Experiments  
This part of the manuscript is deliberately presented at the end of this section because the techniques presented below were not part of the standard AntiBioVol test, and they were performed to provide additional data confirming the correctness of the AntiBioVol setting. .3.1 Assessment of Antimicrobial Activity of Liquid and Volatile EO Fractions Using Standard Methods  
To compare AntiBioVol with the methods previously developed for the assessment of antimicrobial activity of liquid EO fractions, we used the disc diffusion method [24], while to assess the activity of volatile fractions, we applied the inverted Petri plate method (also referred to as the Disc Volatilization Method [25]). This part of the experiment was performed as a proof of concept for _S. aureus_ biofilm vs. T-EO, because the activity of this oil was the highest against this particular pathogen, and it could be hypothesized that positive outcomes (with regard to the halo zone) might also be obtained. In the case of the disc diffusion method, microbial suspension at 0.5 MF density was cultured on a Muller-Hinton (BioCorp, Warsaw, Poland) agar plate (diameter of 9 cm, height of 1.4 cm). Next, standard paper discs (6 mm in diameter, thickness of 0.5 mm) saturated with EOs were placed on the agar plates (one disc per plate). Then, the plates were incubated for 24 h at 37 \({}^{\circ}\)C. After incubation, microbial growth inhibition zones were measured (in mm) using a ruler (Leniar, Krakow, Poland). In the inverted Petri plate method, paper discs saturated with EOs were placed on the inside of Petri-dish lids (one disc per plate). Next, the lids were placed on the base of the dish containing microbial-seeded agar. Subsequently, the whole dishes (lids and agar-containing bases) were incubated in such a manner that the lids were lying on the incubator shelf. Such a setting prevented EO-saturated discs and EO droplets from falling down on the microbial lawn formed on the agar. Inhibition of growth (if occurred) was visible as zones of reduced microbial growth and was measured using a ruler. Moreover, in order to evaluate the correlation between the parameters relating to Petri dish sizes and the antimicrobial activity of the EOs applied, this version of the experiment was performed on Petri dishes 15, 9, and 6 cm in diameter; plate height was equal to 2.5 cm (for plates 15 cm in diameter) and 1.4 cm (for plates 6 and 9 cm in diameter); poured agar height was 1.5, 0.5, and 0.3 cm resulting in agar volume equal to 100, 20, and 10 mL, respectively. When the test involved dishes 9 cm in diameter specifically, the range of agar volume was 30, 20, and 10 mm. 3.2 Scanning Electron Microscopy Analysis of the Strains' Biofilm-Forming Ability in the Applied In Vitro Setting  
The aim of this procedure was to confirm the microorganisms' ability to form biofilm on the agar surface. Fresh, 24-h liquid cultures of _S. aureus_, _P. aeruginosa_, and _C. albicans_ strains were diluted to 10\({}^{5}\) CFU/mL. Next, 2 mL of such a suspension was introduced to the wells of a 24-well plate containing agar discs, which was prepared as described in Section 2.2.1 of Materials and Methods of this manuscript. The plates with the discs and microbial suspensions were incubated for 24 h at 37 \({}^{\circ}\)C. Subsequently, the discs were carefully taken out and rinsed twice with 0.9% saline to remove non-adhered microorganisms. Next, the agar plugs were fixed by immersion in 2% glutamate (ChemPur, Piekary, Poland) for 4 h at 4 \({}^{\circ}\)C. After incubation, the samples were rinsed three times (for 2 min) with distilled water to remove the fixative. Subsequently, 10 min dehydration for each of the following ethanol concentrations (10%, 25%, 50%, 70%, 80%, 90%) was performed; the last dehydration lasted for 15 min and was performed in 100% EtOH.