## 4 Discussion
There is inconsistency of data regarding the recommended concentration of TB [15]. There were reported concerns about the possible occurrence of tooth staining and discoloration after PS application, particularly with MB. However, these concerns would be limited with appropriate PS concentrations and pre-irradiation time [13]. In prior studies, the concentrations of TB were between 15 \(\upmu\)g/mL and 15 mg/mL [15]. There is controversy about the pre-irradiation time in the literature [15]. In fact, the pre-radiation time is a key element of PDT, in which the PS takes its time penetrating the dental tubules, diffusing inside the bacteria, exerting its antibacterial effect, and consequently resulting in a deeper antimicrobial effect after irradiation [13]. In our study, the increase of pre-irradiation time did not lead to greater reduction of bacterial load. Appropriate selection of laser wavelength is a crucial factor in PDT technique. The selected wavelength should match the highly absorbed wavelength by the selected PS to produce the photochemical cascade leading to cell death [32]. The wavelength of maximum absorption is 630 nm for TB [13]. In literature, the output power of lasers used for PDT with wavelengths between 600 and 805 nm ranged from 40 mW to 5 W and the irradiation time ranged from 0.5 to 10 min [1]. [PERSON] et al. found that the use of optical fiber did not play a crucial role in eliminating bacteria in root canals [33]. Some authors believe that utilization of an optical fiber may improve the antimicrobial efficacy of PDT through the uniform distribution of light along the root canals [15]. Concerns were reported regarding this issue because of the possible extrusion of microbial pathogens. Therefore, it is recommended not to apply the fiber to the full length of the root canal [13]. There are several techniques for the evaluation of bacteria in root canal systems [4]. BTA is an easy and sensitive protocol for estimating the bacterial count. The main advantage of this assay is that BTA does not require sample manipulation, thereby overcoming some limitations related to biofilm-counting methods [20]. This study showed a significant reduction of _E. faecalis_ with the chemo-mechanical procedures alone (with 5% NaOCl irrigation) when compared to the negative control group or PDT groups with 0.9% NaCl irrigation (groups B and C). Irrigation is a complementary procedure of instrumentation in endodontic treatment. It facilitates and contributes in the elimination of pulp tissue, the smear layer, and the remaining biofilm [34]. The most commonly used and studied irrigants are NaOCl and chlorhexiidine (CHX) [35]. NaOCl is characterized by having antibacterial property and tissue dissolving ability [36]. The commonly used concentrations of NaOCl are from 0.5% to 6% [35]. The duration of action ranges in the literature between 15 s and 15 min [32]. Its action and toxicity may be directly proportional to its concentration [37]. One of the main disadvantages of NaOCl is its tissue toxicity, and another is its adverse effect on periradicular tissues when extruded from the apex [37]. CHX has a bactericidal effect and substantivity property that may contribute to prolonging the duration of the antibacterial effect [36]. CHX has been proposed as an alternative of NaOCl for open apex cases and patients with NaOCl allergy [37]. The major disadvantage of CHX is its limited ability to dissolve organic tissues [34]. There is a diversity of the outcomes of in-vitro and ex-vivo studies that compare between the antibacterial effects of NaOCl and CHX [10; 28; 37]. An in-vitro study compared the antibacterial effects of different irrigants against _E. faecalis_ and _Candida albicans_ and demonstrated that 2% CHX had a greater antibacterial effect than 2.5% NaOCl [37]. Other study compared different disinfection methods including PDT, 2.5% NaOCl, and 2% CHX against different microbial pathogens. The most satisfactory results were observed with 2.5% NaOCl, and followed by 2% CHX [28].