Raman analysis

On the 3rd day after inoculation, microbial suspension was discarded after the incubation period. Sterile saline was used to gently irrigate dentine disc for one minute to remove the loosely bound E. faecalis. Following this, dentine discs were placed into a new 50 ml test tube with saline for sonication at 37 kHz (5 min). This procedure effectively dislodged loosely bound E. faecalis and the biofilm. Twenty-four dentine discs were randomly assigned into 4 groups. Group I dentine discs (n = 6) were treated with saline and not treated with any intracanal medicament. Groups II, III and IV, (n = 6/group) were treated with 2% k21, calcium hydroxide and chlorhexidine respectively by immersing dentine discs into medicaments in a 24-well culture plate for 30 min. After treatment, dentine discs were rinsed gently with saline to remove the medicaments. Three random dentine discs from each group were placed in a new 24-well culture plate and fixed with 2.5% glutaraldehyde (2 ml) for 2 h at 4 °C. The specimens were then sent for Raman analysis. Remaining dentine discs from each group were then returned to separate 50 ml test tubes containing fresh TSB medium and placed inside anaerobic orbital shaker incubator for 4 more days to allow for redevelopment. TSB medium was changed every 3 days. On the 7th day, dentine discs from each group were washed gently for one minute with saline, placed into separate 50 ml test tube with saline and sonicated at 37 kHz for 5 min. Following this, dentine discs were placed inside 24-well plates, fixed with 2 ml of 2.5% glutaraldehyde for 2 h at 4 °C and sent for second Raman analysis.