## 5 Discussion
It should be pointed out that the issue of a possible interference of various volatile compounds in a single experimental set-up, resulting in a potentially biased outcome, is often neglected in other in vitro tests. For example, in a test referred to as the aromatogram, not only various concentrations, but also various types of EOs are analyzed in a single Petri dish [38; 39]. Although the aromatogram is designed basically to assess the antimicrobial activity of liquid fractions of EOs, one should be aware that during incubation lasting for 24 h at 37 degC, also volatile fractions of EOs will be released and will interact with the microbial cells. Thus, the observed outcome of the aromatogram (expressed as the halo zone) would be an effect of a combined activity of liquid EO fractions and mixed-up plethora of volatile compounds. Therefore, a general principle with regard to standard Petri dish-based tests should be to apply a single type of EO, in a single concentration, per one Petri dish. This statement significantly supports the considerations concerning the advantage of AntiBioVol vs. Petri dish-based tests in the aforementioned context of space savings in the microbiological incubator. Figure 6: Cells of _P. aeruginosa_ attached to agar surface (magnification 5000x). Yellow arrows indicate pores within agar facilitating the adhesion process; red arrows indicate the cells’ adhesive structures anchored to the pores in the agar. SEM Zeiss Auriga 60. As already mentioned above, the variety of Petri dish-based assays on antimicrobial testing translates into inconsistent results reported in the literature. For example, [PERSON] et al. indicated that E-EO vapors are effective against _P. aeruginosa_[21], while [PERSON] et al. [22] reported that the volatile E-EO had no activity against this pathogen. The standard disc volatilization method, and the modification of this method, where one Petri dish was divided into four parts, were applied by the first and the second team, respectively. [PERSON] et al. [40] observed a reduction in the viability of _P. aeruginosa_ cells following exposure to TT-EO, while the study performed by [PERSON] et al. showed no effect of volatile TT-EO against _P. aeruginosa_[28]. Among other methods, both teams applied disc-diffusion methods. These opposite outcomes concerning EOs activity may not only be a result of variances in EOs' composition and various testing methods; they may be a consequence of differences in such seemingly trivial technical parameters as Petri dish diameter and the height of agar poured. These parameters correlate with the volume of air between the agar surface and the lid, translating into the concentration of volatilized compound inside the dish, and finally into the obtained size of the microbial growth inhibition zone. It should be noted that commercially available Petri dishes of the same diameter often vary in height (from 14 through 16 to 20 mm [41; 42; 43], most typically), and this difference has a substantial impact on the air volume within them and on the final concentration of the applied volatilized compound. To investigate the relationship between the above parameters and the obtained halo zone size, we have performed a disc volatilization method using Petri dishes of various diameters, agar volumes, and heights (Table 1) or of the same diameter but of various agar volumes (Table 2). As can be seen in Table 1, along with the decrease in air volume, the final outcome (halo zone) increased. It is worthwhile to note that the outcomes obtained for dishes of 9 vs. 15 cm were opposite (confirmation of antimicrobial activity vs. lack of antimicrobial activity, respectively). Moreover, a similar discrepancy of results was observed if Petri dishes of the same diameter but of various agar volumes were scrutinized (Table 2). Agar heights differed from each other by 2 mm only, which may easily be overlooked during the test performance.