M Beerens

46 CHAPTER 3 FIGURE 1 . The DGGE banding patterns and the dendrogram grouping after clustering using UPGMA. Automated band detection settings used were: minimum profiling of 4% absolute to the most intense band, 0% grey zone, minimum area between 0.1% and 0.5% and shoulder sensitivity 0. The lines above the gel indicate where the bands are detected by automated band detection. These lines clearly show the mismatch with visual detection. Microbiological data The plaque samples were subjected to blind analysis with respect to group allocation (WSL or No WSL). Subject group allocation was combined with the microbiology data after the completion of all analyses. The plaque samples were sonicated (ultrasonic processor; Sonics Vibra-Cell, Newtown, CT, USA) for 2 minutes (amplitude 40, 1-s pulse duration) to disperse the cells optimally. The samples were diluted in cysteine peptone water (CPW) and 50-μl aliquots were dispensed in duplo onto agar plates using a spiral plater (EDDY JET; IUL Instruments, Barcelona, Spain). The samples were incubated anaerobically (10% H 2 , 10% CO 2 and 80% N 2 ) at a temperature of 37 °C for 72 h on tryptic soy blood (TSB) agar (at cell dilutions of 10 −3 , 10 −4 , and 10 −5 ), brain–heart infusion (BHI) agar (at pH 7 and pH 5, at cell dilutions of 10 −3 , 10 −4 , and 10 −5 ), trypticase