Serial 10-fold dilutions of homogenized biofilm samples in 1% PBS were collected from dentine blocks and then plated in duplicate with a spiral plater (Autoplate 4000; Spiral Biotech Inc., Norwood, MA, USA) onto horse blood agar (Defib Horse Blood; Hemostat Laboratories, Dixon, CA, USA). The plates were incubated aerobically for 72 h to assess the bacterial colony-forming unit (CFU) per mL. Biofilms on six dentine blocks per group were studied.