Bacterial viability determination in a dentinal tubule infection model by confocal laser scanning microscopy

Dentinal tubule invasion protects bacteria from chemo-mechanical disinfection and frequently results in root canal treatment failures. Enterococcus faecalis is a primary causative agent, particularly in persistent, asymptomatic, and chronic apical periodontitis. In order to assess and compare the efficacies of endodontic antimicrobial agents and application strategies, we have developed a convenient and robust method to measure bacterial viability and assess distribution in an ex vivo tubule infection model. Following infection and antimicrobial treatment of prepared ex vivo roots, the tubule bacteria are exposed to nucleic acid-binding fluorescent stains (LIVE/DEAD BacLight stain), sectioned, and examined by confocal laser scanning microscopy. The proportion of red-fluorescing (dead) and green-fluorescing (live) bacteria is then visualized in situ and quantified with image analysis software.