M Beerens

62 CHAPTER 4 were captured by examiners who were calibrated before the start of the investigation and who were blinded with respect to the treatment group. One QLF device was used for all measurements. All analyses were performed by a single examiner (M.W.B.), who was trained and calibrated by an experienced QLF examiner (M.H.V.) at the start of the study on a data set of 500 QLF images. In cases where lesion visibility at the baseline was hampered as a result of swollen gingiva, the visible area of the lesion at the baseline was the part of the lesion analysed at subsequent timepoints using QLF. The QLF images obtained for each subject were analysed blind with regard to treatment group; the group allocations were added to the exported data file after completion of all analyses. Plaque processing Plaque was sampled from the buccal surface of the lower right first premolar at T0, T2, and T3. Plaque samples were labelled with consecutive numbers that were recorded on the subjects sheets. Plaque samples were spun down in a centrifuge (Eppendorf centrifuge 5415 D; Eppendorf, Hamburg, Germany) for 30 seconds at 16,200 g, then stored at 80° C after the addition of 1 ml of cysteine–peptone water (CPW) containing 10% glycerol (as transport medium) until further processing. Plaque samples were analysed blind with respect to subject number, visit, and group allocation. Microbiology data was coupled to Subject numbers, visit numbers, and group allocation after completion of all analyses. Plaque samples were sonicated (ultrasonic processor; Sonics vibra- cell, Newtown, CT, USA) for 2 min (amplitude 40, 1-s pulse duration) to disperse the cells. The samples were diluted in CPW and 50-ml aliquots were distributed on agar plates using a spiral-plater (EDDY JET; IUL Instruments, Barcelona, Spain). The samples were incubated anaerobically (in an atmosphere of 10% H 2 , 10% CO 2 , 80% N 2 ) at a temperature of 37° C for a period of 72 h on tryptic soy blood agar (at cell dilutions of 10 -3 , 10 -4 , and 10 -5 ), on brain–heart infusion (BHI) agar at a pH of 5.0 (at cell dilutions of 10 -3 , 10 -4 , and 10 -5 ), on trypticase yeast-extract cystine sucrose bacitracin agar (at cell dilutions of 10 -1 , 10 -2 , and 10 -3 ), and on Rogosa agar (at cell dilutions of 10 0 , 10 -1 , and 10 -2 ), to obtain the total numbers of colony-forming units (CFUs) per sample, and the proportions of aciduric bacteria, S. mutans , and Lactobacillus spp., respectively.