## 5 Discussion
One of the reasons behind these low deviations in outcomes is the fact that the AntiBioVol test is performed not in Petri dishes (as in standard methods) but in 24-well plates. This simple change allows performing 24 assays and obtaining 24 outcomes using only one test plate, while a similar assessment performed by means of the inverted Petri dish method requires 24 vessels. The high number of easy-to-achieve technical repeats gained owing to the AntiBioVol test correlates positively with the quality of data obtained and processed during post-laboratory statistical calculations. This is of paramount importance with regard to biofilm studies, where high deviations in outcomes is a common phenomenon hindering the obtaining of statistically significant results [34]. The above-mentioned reduction of experimental setting size observed for AntiBioVol vs. various Petri dish-based methods translates also into significant space savings in the microbiological incubator [Figure 5] and into materials' cost savings. One of the key challenges of in vitro biofilm testing is its low reproducibility of results due to aggressive pipette-based washing. It frequently causes a random de-attachment of large amounts of biofilm [35]. Therefore, in our test, we put special stress on the careful performance of the procedures concerning agar discs rinsing, washing, and transferring (please refer to Materials and Method Sections 2.2.2 and 2.2.4). Moreover, we have deliberately chosen agar discs in the AntiBioVol test as a surface for biofilm growth. While a majority of other in vitro biofilm-oriented tests apply various types of polished polystyrene/polpropylene inserts or plugs for this purpose [36], we hypothesized that a porous agar surface used in the AntiBioVol test may be more appropriate for enabling the cells' adhesion. The correlation between higher biofilm adherence and surface porosity has already been demonstrated by other research teams [37]. Indeed, as shown in Figure 6, the microorganisms used agar pores to anchor to the surface. Figure 5: Comparison of place and resource consumption for the conventional Petri dish method of EOs testing (**A**) and the AntiBioVol (**B**) test. Another issue related to the testing of volatile compounds activity against microorganisms is the possibility of obtaining biased results due to the natural tendency of fumes to spread in the air and to get across from one experimental setting to another. To prevent the passing of volatile EOs from the plates to the microbiological incubator, we have wrapped the two AntiBioVol plates up tightly (using an adhesive tape) (A and C) (Figure S2, II). After 24 h of AntiBioVol test incubation, no EO scent was organoleptically detected in the incubator. The aforementioned wrapping resulted in another beneficial effect, i.e., the relevant wells of plate A and C were placed directly one over another (Figure 1, Part III). We hypothesized that such a setup would prevent EO volatile fractions placed in a specific well from reaching another well and from cross-reacting with other biofilm-covered agar discs. To test this hypothesis, we have introduced three different concentrations (25%, 50%, and 100%) of TT-EO to a single AntiBioVol setting and checked whether the obtained results of biofilm reduction differed from the results obtained in settings where each of the aforementioned TT-EO concentrations was tested in separate plates (for TT-EO concentration distribution, please refer to Figure S4). Moreover, we have confirmed that EO solvent does not affect the microorganisms' viability [Figure S5]. As shown in Figure 4, the differences in these two set-ups with regard to the level of biofilm reduction were statistically insignificant (K-W test, \(p>0.05\)). Therefore, it can be concluded that AntiBioVol allows testing various concentrations of volatile compounds within a single experimental setting.