## 5 Discussion  
The aim of this work was to develop a reliable and easy-to-perform test for the evaluation of antibiofilm activity of volatile fractions of antimicrobial compounds. We have chosen EOs as an example because, due to their plant-derived origin resulting in a variability of microbiological outcomes, EOs present a higher challenge than compounds obtained in a strictly defined process of chemical synthesis [20]. Another reason behind choosing EOs were promising reports on their antimicrobial and antibiofilm activity [9; 27]. To stay in line with methodological requirements, we have also scrutinized ethanol fumes of known antimicrobial activity (usability control). Moreover, to allow other research teams to replicate our experiment, we have confirmed the presence of antimicrobial substances in the analyzed EOs and presented the results in Figure S1 and Table S1. While the methodology for liquid EOs' antimicrobial activity is practically based on the protocol for antibiotic-susceptibility testing provided by EUCAST [15], the assessment of EOs' volatile fraction activity against settled forms of microbial aggregates is performed by means of diverse approaches, which are fraught with certain disadvantages [28; 29; 30; 31]. Therefore, a methodology is needed allowing the performance of a high number of technical repeats within a single screening test, providing results in the form of a parametric ratio of microbial cells' eradication (and not in the form of the size of the halo zone, as is the case for the inverted Petri dish method, see Section 2.2.4.1 of Materials and Methods). Such methodology should provide standardized test conditions and the ability to analyze the impact of volatile compounds against (among others) microbial biofilm, which is basically missing in the standard test methods. The prerequisite condition for AntiBioVol performance was to obtain a robust biofilm in in vitro conditions. As can be seen in Figure 2, the Gram-positive bacteria, Gram-negative bacteria, and the fungus formed multicellular biofilms on the agar surface. A confirmation of this fact allowed us to perform the analyses presented in Figures 3 and 4. It should be emphasized that methods simpler than Scanning Electron Microscopy can also be used to visualize biofilm presence, including dyeing with tetrazolium salts or quantitative culturing. These methods are sufficient for the above purpose, as we have indicated in our previous research [31; 32; 33]. Although we have performed an analysis of EOs' activity specifically against biofilms, the AntiBioVol methodology (thanks to using agar discs) also enables testing such surface-attached cellular aggregates as microbial lawns or even single agar colonies, unlike standard Petri dish-based methods, where only planktonic cells seeded on agar are used. The results presented in Figure 3 indicate that the application of AntiBioVol translates into low standard errors of mean (expressed as box whiskers in Figure 3) in outcomes of technical repeats. The number of _S. aureus_ biofilm-forming cells of the growth control differed maximally by 27.6% \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Plate Diameter (cm)** & 9.0 & 9.0 & 9.0 \\ \hline
**Plate Height (cm)** & 1.4 & 1.4 & 1.4 \\ \hline
**Agar Volume (mL)** & 30 & 20 & 10 \\ \hline
**Agar Height (cm)** & 0.7 & 0.5 & 0.3 \\ \hline
**Air Volume (cm3)** & 45 & 57 & 70 \\ \hline
**Inhibition Zone (cm)** & **2.7** & **0.5** & **0.0** \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Dependence between agar height in the Petri dish and the size of _S. aureus_ growth inhibition zone after exposure to T-EO. Inverted Petri dish method (also known as disc volatilization method). between six biofilm samples used to produce the average value presented in Figure 3A. The difference in outcomes measured for four out of these six samples did not exceed 8% (please refer to the corresponding raw data of the experiment in Tables S3 and S4).