One-hundred and forty-four single-rooted human teeth with no cavities and resorption were selected for this study. The teeth were soaked in 5.25% NaOCl for 30 minutes. Calcified root canals with more than one canal were excluded. Access cavity was prepared and working length was determined. Root canals were instrumented to the apical size #35 using Mtwo rotary files (VDW, Munich, Germany) according to the manufacturer’s instructions. All canals were irrigated after using each file by 2 mL of 2.5% NaOCl. To prevent bacterial leakage, apical foramina were sealed with wax. Nail varnish was applied on the surface of roots in two layers. Three mL of 17% EDTA (Meta Biomed Co. Ltd., Mandaluyong, Korea) was used for one minute to remove the smear layer. Then, the canals were irrigated with saline and dried. Finally, 108 teeth (for test groups and positive control) were rinsed with 5 mL of 2% CHX (Consepsis®, Ultradent, South Jordan, UT, USA) for 10 minutes. Thirty-six remaining samples that were not irrigated with CHX were used as negative controls.
The teeth were randomly divided into the following groups:
The teeth were randomly divided into the following groups: