## 4 Discussion
The observed differences were in the scope of a single dilution of the octenidine concentration (i.e., 7.812 vs. 15.625 mg/L). The applied microdilution method (using the 96-well plate model) is derived directly from the binding European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology on the evaluation of the activity of antibiotics. The EUCAST annotations concerning this particular methodology indicate that MBC (and MIC) values differing by a single dilution are not conclusive because of the specific accuracy of the micro-dilution method [21]. Therefore, it may be stated that within this particular experimental setting (96-well plate, contact timeof 24 h), the results obtained for the two formulations of antiseptics were comparable. Such an outcome may be explained by the composition of the OCT, in which star arise essential oil and peppermint essential oil are present. These products of plant origin also possess antibacterial/ antifungal activity and their mechanism of action relies, similarly to octenidine, on compromising microbial cell walls/membranes, leading to their disfunction [22]. It may be hypothesized that these essential oils increased the overall antimicrobial activity of the OCT to a level similar to the one displayed by the OL comparator. Next, the scrutinized strains were tested regarding their ability to form biofilms on HA (the main mineral of bones, including the jaws) and on a soft agar surface. This approach was dictated by the seemingly obvious statement that pathogenic biofilms in vivo do not grow on flat, smooth surfaces (such as the surface of a well on a 96-well plate) and that the functionality of biofilms grown in laboratory conditions resembling the surfaces on which these biofilms develop in reality may be more similar to the functionalities of clinical biofilms. As indicated, all strains formed strong biofilm structures on both types of surfaces (Figure 2). In our earlier research [23], we showed that the type of fluid in which biofilm is cultured is also of high importance regarding the tolerance of a biofilm toward antiseptics. Therefore, we decided to apply the artificial saliva (AS) instead of a standard microbiological medium. Nevertheless, contrary to our earlier study, in this case, there were no differences observed in the reduction in biofilm regarding the type of surface used for its growth (HA or agar disks) (Figures 3-6). This may be related to the fact that the highest possible concentration (65 mg/L), at least in this particular experimental setting, was applied. It was at least two times higher than the MBEC concentration recorded for the 96-well plate model (Table 1). It can thus be assumed that such a relative abundance of octenidine during the 20 min of contact time was sufficient to penetrate through the biofilm and damage this structure to the extent which made it impossible to observe any differences in the biofilm's tolerance to the antimicrobial between the two different surfaces used. Nevertheless, the application of this specific contact time was crucial for the assessment of antimicrobial activity as it reflected the factual time of the full dissolution of the lozenge and, at the same time, the complete release of octenidine from the carrier. These obtained results are to a major extent comparable with earlier results of ours as well as to the data presented by other researchers concerning octenidine activity. We have already shown, by means of a Biofilm-Oriented Antiseptic Test, that at least 15 min is needed for the working solution of octenidine to eradicate the _P. aeruginosa_ biofilm; a significantly shorter time is needed to achieve the same result against _S. aureus_ biofilm [24]. A similar conclusion with regard to the _P. aeruginosa_ biofilm was provided by [PERSON] et al. [25]. The highest tolerance of _C. albicans_ toward octenidine (among other tested microbial biofilms) was in turn indicated by [PERSON] et al. [26].