## 5 Discussion
In conclusion, such technicalities as dish height (or dish diameter) contributing to the final parameter (halo zone) are frequently not reported in the methods sections of manuscripts [44; 45; 46], and we believe it may be one of the reasons for the observed result discrepancies. The above-presented consideration explicitly indicates the need to standardize the test, which the AntiBioVol setting offers. The differences in the diameter to height ratio (which serves to calculate air volume) in the wells of the 24-well plate used in the AntiBioVol test are too faint to have a substantial impact on the outcome, and hence at least one important factor of variability is eliminated. Moreover, we have explicitly indicated the volume of agar needed to be poured to the well (2.5 mL, please refer to Section 2.2.1 of Materials and Methods). Although the AntiBioVol test provides many advantages as compared to standard Petri dish tests, we are aware of certain limitations of our methodology. First of all, AntiBioVol is not designed to analyze anaerobic microorganisms, which are a frequent etiological factor of oral, bone, and chronic wound infections [47]. An adaptation of the AntiBioVol methodology for the analysis of anaerobic biofilm would require an implementation of expensive anaerobic chambers and nucleic acid sequencing methods, significantly increasing the time and cost of the procedure. Secondly, the biofilms cultured in laboratory conditions, as described in Section 2.2.2 of Materials and Methods, do not fully resemble the ones infecting a patient's body in terms of cell number, matrix composition, and dimensional structures [48]. Therefore, one should be careful with translating the results obtained by means of AntiBioVol into clinical conclusions. Thirdly, the basic AntiBioVol setting relies on a TTC-based assay whose sensitivity differs depending on the specific microbial strain applied (and its ability to reduce tetrazolium salts). However, as we have shown in Figure S3, although the TTC test is able to detect from 10 to up 10\({}^{10}\) microbial cells, the performance of additional tests to evaluate a strain's ability to metabolize TTC may be required, especially for strains able to switch their metabolism from aerobic to anaerobic. Another limitation of our study, strictly related to the previous one, is the fact that microorganisms form various types of biofilm (as regards cell number and yield of the exopolymericmatrix) in various types of culturing media [49]. This fact makes us lean toward a conclusion that a more labor-consuming version of AntiBioVol (in which quantitative culturing (QC) is performed, please refer to Figure 3C) would be of higher usability than a quick, TTC-based version of AntiBioVol (please refer to Figure 3A,B). In such case, a plausible solution would be to use the TTC-based version of AntiBioVol for the rapid screening of a high number of strains and, subsequently, the QC-based version of AntiBioVol for further analyses of selected strains. Thus, the AntiBioVol methodology and study is of preliminary character and should be investigated further. Despite the above-mentioned limitations, the AntiBioVol test set provides a powerful tool for a consisted, rapid analysis of already used or newly designed volatile antimicrobials of known or suspected anti-biofilm activity. Bearing in mind the fact that biofilm often exists in body sites hardly reached by liquid or stable medicinal products, the application of EOs and the data provided by AntiBioVol on their efficacy may be of importance for not only basic but also clinical studies on the eradication of biofilm formed in the pathogenesis of infections.