Multiple assessment methodologies in determining the antibiofilm actions of sodium hypochlorite mixed with clodronate or etidronate in endodontic irrigation

Abstract

This study aimed to use multiple methodologies, including a novel usage of scanning electron microscopy (SEM), to evaluate the antimicrobial actions of sodium hypochlorite (NaOCl) admixed with clodronate or etidronate in root canal irrigation. The study also examined the usefulness of colony counting as a biofilm assessment methodology. Seven day Enterococcus faecalis biofilms were grown on hydroxyapatite discs. The discs were disinfected with 0.26 M clodronate-5% NaOCl, 0.26 M etidronate-5% NaOCl, 5% NaOCl, or treated with phosphate buffered saline (PBS). Assessments were performed using colony counting, SEM and the XTT reduction assay. The XTT assessment used the same groups but with 2.5% NaOCl. For colony counting, bacteria were removed from the discs by vortex mixing, followed by plating. The discs were subsequently fixed for SEM imagining and evaluators scored the SEM micrographs for remaining bacteria. Antibiofilm actions were assessed with the Kruskal-Wallis and Dunn's multiple comparison tests. SEM micrographs and the XTT assay revealed no differences between the NaOCl controls and the clodronate or etidronate mixtures with NaOCl (P > 0.05). It was concluded that the chelator mixtures with NaOCl had antibiofilm actions comparable to NaOCl. Furthermore, vortex mixing incompletely removed biofilm from HA discs in the PBS controls and hence colony counting using E. faecalis biofilms on hydroxyapatite discs could not be used for intergroup comparisons involving PBS. Additionally, colony counting could not be used for comparisons between the NaOCl treatment groups because the removal of bacteria from the substrate by vortex mixing was affected by the irrigant type.

Keywords: Biofilm assessment; Clodronate; Continuous chelation; Endodontic irrigation; Etidronate; Sodium hypochlorite.