Confocal laser scanning microscopy (CLSM) (Fluoview FV 1000, Olympus, Tokyo, Japan) was employed to assess the viability of the biofilms. The bacteria on the dentine surfaces were labeled in situ using two fluorescent probes: propodium iodide and SYTO-9 dye (LIVE/DEAD BacLight Bacterial viability kit, Molecular Probes, Eugene, OR, USA). Five images from the middle layer of each biofilm were obtained using CLSM and were analyzed using Image J (Version 1.51a, National Institutes of Health, Bethesda, MD, USA). The red-to-green ratio was calculated to denote the ratio of dead-to-live bacteria, respectively . Two dentine blocks per group were used in this qualitative experiment.