## 2 Materials and Methods
\begin{table}
\begin{tabular}{l l l} \hline \hline
**Control Group** & 9 teeth & No treatment \\ \hline
**Group A** & 8 teeth & Chemo-mechanical debridement (irrigation with 5\% NaOCl) \\ \hline \multirow{2}{*}{**Group B**} & \multirow{2}{*}{8 teeth} & Mechanical instrumentation (irrigation with 0.9\% NaCl) \\  & & + 15 \(\upmu\)g/mL of TB with T1 = 120 s + Diode laser 635 nm with \\  & & T2 = 150 s \\ \hline \multirow{2}{*}{**Group C**} & \multirow{2}{*}{8 teeth} & Mechanical instrumentation (irrigation with 0.9\% NaCl) \\  & & + 15 \(\upmu\)g/mL of TB with T1 = 30 s + Diode laser 635 nm with \\  & & T2 = 30 s \\ \hline \multirow{2}{*}{**Group D**} & \multirow{2}{*}{8 teeth} & Chemo-mechanical debridement (irrigation with 5\% \\  & & NaOCl) + 15 \(\upmu\)g/mL of TB with T1 = 120 s + Diode laser 635 nm with T2 = 150 s \\ \hline \multirow{2}{*}{**Group E**} & \multirow{2}{*}{9 teeth} & Chemo-mechanical debridement (irrigation with 5\% \\  & & NaOCl) + 15 \(\upmu\)g/mL of TB with T1 = 30 s + Diode laser 635 nm with T2 = 30 s \\ \hline \end{tabular} NaOCl: sodium hypochlorite; NaCl: sodium chloride; TB: Toluidine Blue; T1: pre-irradiation time; T2: irradiation time of diode laser 635 nm.  
\end{table} 
Table 1: Description of the study groups. The dental roots of PDT groups (groups B, C, D, and E) were filled with about 15 \(\mu\)L of a 0.05 mM solution of TB (Figure 1). The TB was applied by a Hawe irrigation probe 30 gauge (Kerr). The TB solution was agitated by a K-file #15. Before laser application, the TB solution was left in situ for different pre-irradiation times (T1), and precisely, T1 was 30 s for groups C and E and T1 was 120 s for groups B and D.  
After that, a diode laser with wavelength of 635 nm (LAMBDA SpA, Brendola (Vi), Italy) was applied to all the PDT groups (group B, C, D, and E) with the following parameters: Power of 100 mW in continuous mode (CW) (Figure 2). An optical fiber (diameter of 200 \(\mu\)m) was used to deliver the laser beam. A rubber stopper was used and positioned on the laser fiber at the designed working length (1 mm from the apex). Two irradiation times (T2) were carried out, and more precisely, T2 was 30 s for groups C and E and T2 was 150 s for groups B and D. During the irradiation, a helical movement from the apical to the cervical portion of each canal was performed. To quantitatively evaluate the biofilm on the inner walls of the root canals, BioTimer Assay (BTA) was employed. BTA measures bacterial metabolism and employs Phen  
Figure 1: (**a**) Mechanical preparation with a file F2 of the ProTaper® system (\(\O\) = 0.25 mm); (**b**) irrigation of a root canal with 5% of sodium hypochlorite (NaOCI); (**c**) application of 15 \(\mu\)g/mL of Toluidine Blue (TB). Figure 2: (**a**) The diode laser with a wavelength of 635 nm (LAMBDA SpA, Brendola (Vi), Italy); (**b**) irradiation of a root canal. reagent, whose color shifts red-to-yellow due to bacterial metabolic acidification [17; 18; 19; 20]. The time (in hours) required for the reagent shift was inversely related to the _E. faecalis_ planktonic concentration through a correlation line whose equation and linear correlation coefficient were y = -0.4767x + 10.022 and \(\mathbf{r}^{2}\) 0.9977, where \"y\" corresponds to log\({}_{10}\) (CFUs/mL) and \"x\" corresponds to the hours required for color switch [6]. To evaluate the _E. faecalis_ populations colonizing the inner walls of the roots, infected teeth were placed in 1.5 mL Eppendorf tubes containing 1 mL of BT-PR reagent and incubated at 37 \({}^{\circ}\)C. The time for red-to-yellow color switch was recorded and used to evaluate the number of _E. faecalis_ through the correlation line. To normalize the CFU values obtained with the different teeth, the data were correlated to the areas of the root canals. The area of each root canal was calculated by assuming that the instrumented root canal was a truncated cone, with a surface equal to the canal master.