## 4 Discussion
The review showed that activation methods of the irrigants could provide significantly higher microbiological flora removal. However, activation methods, time of the application and volume of irrigating solutions should be standardized to find out the optimal irrigating protocols [37]. In this study some penetration of the sealer marked with fluorescein in control group (group 1) into dentinal tubules was observed. In this group the irrigation was carried out with sodium hypochlorite only. It is likely that there was some dye leakage of the fluorescein from the sealer, or the probability that the lubricant containing 17% EDTA used during the preparation may have resulted in partial removal of the smear layer. Passiveultrasonic activation could have influenced the initial and incomplete elimination of the smear layer. [PERSON] investigated ultrasonics to remove a smear layer and confirmed its usefulness in the process [38]. During the thermoplastic vertical gutta-percha compaction, some pressure is applied to the materials against the canal walls. It could have also influenced the mechanical pressing of the sealer into partly opened dental tubules after the irrigating process. It is important to point out that these explanations require long term research using additional and different methodologies to verify the results presented in this study. In the case of a porphyrin marked sealer, the penetration into the dental tubules in this group was undoubtedly limited by the smear layer in group 1. It is probably due to the different physio-chemical characteristics of a sealer marked with two different substances. Fluorescein is soluble in basic solutions, where it forms highly fluorescent salt. Thus, it could partially migrate from the sealer into dental tubules even in the samples of group 1. The authors supposed that the applied fluorescein dye may migrate and leak from the sealer, as it is soluble in water (in basic solutions). As mentioned by [PERSON] et al., rhodamine B is also highly soluble in water, making it susceptible to leakage to the hydrophilic environment, even disregarding its pH [16]. Thus, the use of two types of dye (hydrophilic and hydrophobic) was necessary to verify if the choice of a dye affects the results of this study. The applied porphyrin (TPP), unlike fluorescein and rhodamine B, is hydrophobic, which significantly limits its leakage to the surrounding aqueous medium, presumably making the presented results more reliable. Thus, using hydrophobic porphyrin as a fluorescent tracker seems to be more appropriate in this type of study, but it still seems to remain a weakness of the method. After filling, the roots were exposed to a humid environment and partial diffusion of fluorescein with the water vapor (the environment after NaOCl is alkaline) into the tubules may have occurred. The use of porphyrin appears to be more beneficial due to its higher sensitivity in comparison to the fluorescein (there is a smaller standard deviation, there is more emphasis on the differences in results between the groups and the statistical significance). The initial doubt concerned the question of whether the porphyrin would leach from the sealer into the alcoholic environment in the tubules of the samples from group 3, but the results showed that the penetration of the stained sealer was greater in an aqueous environment (sodium hypochlorite group). Also, the hydrophilic fluorescein did not leak into the aqueous environment as much as anticipated in the sodium hypochlorite group. The initial assumption that each dye could leak into its favorable medium was rejected. This might be correlated with the type of the sealer. In this paper, the well-documented, chemically stable and hydrophobic AH Plus epoxy sealer was used. [PERSON] et al. used three different sealers, and AH Plus yielded the least leakage of rhodamine B into the PBS solution in comparison to the two bioceramic sealers (BioRoot RCS and Total Fill BC) [16].