M Beerens

DGGE FOR CARIES RISK ASSESSMENT IN ORTHODONTICS 3 43 Subjects without WSL had to have no WSL on former bracketed surfaces. Subjects with WSL had to have two or more buccal WSL on former bracketed surfaces, seen without prolonged air drying as a distinct visual change in enamel ICDAS score 2. Lesions were without localized enamel breakdown and without clinical visual signs of dentinal involvement. Collection of plaque samples Plaque samples were collected immediately prior to debonding. The subjects abstained from oral hygiene for at least 12 hours prior to plaque collection. Plaque samples were collected from the gingival margin with a plastic spatula by one stroke on the buccal surfaces of the first and/or second pre-molar in the third quadrant for microbiological assessment and the fourth quadrant for DGGE assessment. The plaque samples were collected aseptically in sterile Eppendorf tubes. The plaque samples were centrifuged for 1 minute in a centrifuge (Eppendorf 5414D, Germany) at 16,100 rpm and placed on ice. One millilitre of cysteine peptone water (CPW) containing 10% glycerol was added to the plaque samples for microbiological assessment. The plaque samples for DGGE were stored in a mixture of 125 µL of TE buffer and 125 µL of 0.5 M NaOH. All plaque samples were stored at -80 °C until further processing. DNA isolation and DNA extraction DNA was extracted from the collected plaque samples using the DNA isolation Qiagen DNeasy ® KIT (Qiagen, Hilden, Germany) (Muyzer and Smalla, 1998; Pham et al. , 2009). Dental plaque pellets were re-suspended in 1 ml of ATL buffer and transferred to sterile Beadbeater tubes containing 0.5 grams (± 0.01 grams) of 0.1 mm glass beads. The Beadbeater tubes were processed in a Mini-Beadbeater Fast-Prep machine ® (Qbiogene, Bio 101, Strasbourg, France) at a speed of 5.5 and immediately placed on ice to enhance microbial lysis of diverse Gram-positive microorganisms (de Boer et al. , 2010). A NanoDrop™ Spectrophotometer (Thermo Fisher Scientific, Waltham, MA USA) was used to detect if any contamination with e.g. proteins had occurred. For DNA the absorption is measured at 260 nm and for protein at 280 nm. The ratio of extinction is a measure of purity. For pure DNA this is ~1.80. For the samples amedian was found of 1.9 (range 1.6 - 2.2) for the ratio 260nm/280nm.