Scanning electron microscopy (SEM) was used to monitor the topographical features of the biofilm. In preparation for SEM, the dentine blocks with biofilm were rinsed in 4% formaldehyde followed by 1% PBS; they were then placed in 1% osmium tetroxide solution for 60 min. The blocks were washed in distilled water and dehydrated in a series of ethanol solutions at increasing concentrations (70% for 10 min, 95% for 10 min, and 100% for 20 min). The blocks were dried in a desiccator and sputter-coated with gold. The surface topographies of biofilms were studied under SEM (Hitachi S-4800 FEG Scanning Electron Microscope, Hitachi Ltd., Tokyo, Japan) at 12 kV in high-vacuum mode . Two dentine blocks per group were used in this qualitative experiment.