A Horizontal Sequential Cutting Method to Estimate the Effectiveness of Dentin Disinfection by Using Confocal Laser Scanning Microscopy

Introduction: This study aimed to develop a technique to create sequential slices, allowing the fluorescent visualization of bacterial viability in all parts of an infected dentin. Methods: Cylindrical dentin blocks were prepared from freshly extracted human teeth with a single-rooted canal. Each block was immersed in 5% sodium hypochlorite (NaOCl) and 17% EDTA for 5 minutes before being infected with Enterococcus faecalis. The bacteria were allowed to develop inside dentin specimens for 28 days under anaerobic conditions. The specimens were exposed in 2% NaOCl for either 2 minutes or 20 minutes at 20 degrees C, 37 degrees C, and 45 degrees C, respectively. After staining with calcein AM (Thermo Fisher Scientific, Waltham, MA) and propidium iodide, the samples were cryoembedded, mounted on an adhesive film, and sectioned at a thickness of 10 mu m along the running of the dentinal tubules. Stacks of fluorescent images were collected in the z dimension using confocal laser scanning microscopy, and the maximum affected distance from a root canal was measured from the 3-dimensional reconstructed image. The reliability of this technique was verified by comparison with a dye bleaching test. Results: Horizontal sequential sections preserving 3-dimensional bacterial distribution and their viabilities could be made without decalcification. The treatment time contributed to the penetration of NaOCl into dentinal tubules, whereas temperature did not significantly affect the penetration. The judgment by confocal laser scanning microscopic analysis was consistent with that of a dye bleaching test. Conclusions: The horizontal sectioning method has the advantage of creating sequential sections, allowing information to be imaged at every portion.