M Beerens

DGGE FOR CARIES RISK ASSESSMENT IN ORTHODONTICS 3 47 yeast-extract cysteine sucrose bacitracin (TYCSB) agar (at cell dilutions of 10 −1 , 10 −2 , and 10 −3 ), Rogosa agar (at cell dilutions of 10 0 , 10 −1 , and 10 −2 ) and Bismuth glucose glycine yeast (Biggy) agar (undiluted). BHI and TSB agar, both at pH 7, were used to obtain total numbers of colony-forming units (CFU’s) per sample; where TSB agar was used as control to verify that BHI at pH7 was an appropriate non-selective growth medium. BHI at pH 5 was used to determine aciduric flora, TYCSB agar was used to determine S. mutans , and Rogosa was used to determine Lactobacillus spp. Biggy agar (undiluted) was used to detect C. albicans . The relative proportions of aciduric flora, S. mutans , Lactobacillus spp. and C. albicans , with respect to total counts, were compared between the groups of subjects with WSL or without WSL to overcome differences in amount of plaque sampled in subjects. Statistical analysis Intra-class correlation coefficients (ICC) between visually determinedbands and each of the automated band detection settings were calculated to determine automated band detection settings for minimum profiling. Either, absolute or relative to the most intense band, with the highest level of agreement with visual band detection. The Mann Whitney U test was used to analyse the group medians for the band numbers in the DGGE pattern for visual band detection and the optimum automatic ‘absolute’ and ‘relative minimum profiling’ settings. To assess the similarities between whole profiles, gel images were subjected to bandmatching and a binary matrix of band classes was produced. The binary matrix was analysed through clustering using an unweighted pair group method with arithmetic averages (UPGMA) (Gafan et al. , 2005) based on the Pearson correlation. Microbiological data was also statistical analysed using the independent samples T-test with equal variances. RESULTS A total of 37 subjects were included in this study (mean age 15.4 years, SD 1.6 years). A total of 14 subjects were male and 23 subjects were female. WSL on the previously bracketed surfaces were clinically confirmed in 28 subjects through QLF and clinical examination. The remaining 9 subjects were confirmed to have no WSL developed on the previously bracketed surfaces.