Assessment of antimicrobial efficacy via CLSM and SEM.

To assess the antimicrobial efficacy, two well-established quantitative and qualitative methods were used: confocal laser scanning microscopy (CLSM) and SEM, respectively. For CLSM analysis, 4 dentin slices from each group were stained using fluorescent LIVE/DEAD BacLight Bacterial viability Kit L-7012 (Molecular Probes, Eugene, OR, USA) composed of SYTO 9 and propidium iodide (PI) dyes. Two random areas in each dentin slice were analyzed using a mosaic technique with 3D reconstruction, wherein 9 subareas (300 × 300 μm) were merged, totaling 18 areas per sample (Fig. 2B). The areas were selected starting from the root canal space toward the cementum side for imaging on CLSM using a 40 × lens (Leica SP2 CL5Mt, Leica Microsystems Inc., Heidelberg, Germany). The sequence of segments through the depth of tissue (Z-stacks) was collected by using optimal step size settings (0.35 μm); the images were composed of 512 × 512 pixels and were evaluated and quantified using a dedicated software (Imaris 7.2 software, Bitplane Inc., St. Paul, MN, USA). The excitation emission maxima for the dyes were approximately 480/500 nm for SYTO 9 and 490/635 nm for PI, respectively. The percentages of live/dead bacteria were compared to establish statistical significance in differences of dead bacterial cells between treatment groups using a mixed-model ANOVA, with a fixed effect for group and a random effect for sample, to account for measurements at multiple areas on each specimen.³¹ All tests were performed with an alpha level of 5%.

For SEM analysis, the dentin slices were carefully removed from the wells of 24-well culture plates using sterile forceps and gently washed with PBS to remove non-adherent bacteria. Next, they were fixed in 2.5% glutaraldehyde for 24 h and dehydrated in increasing concentrations of alcohol solutions. To visualize bacterial presence/absence, the dentin slices were split in the middle and mounted face-up on Al stubs, sputter coated with Au-Pd prior to imaging.^31,32