8 g/L Congo red (Prolabo, Leuven, Belgium) and without or with 5% sucrose (Merck, Darmstadt, Germany). Colony morphology and color were evaluated after incubation at 37°C for 24 h. Colonies with a dry crystalline (rough) morphology were considered deviant and slime producing positive [16], smooth round colonies were classified as low-slime producers. Detection of biofilm biomass with crystal violet staining The polystyrene crystal violet adherence assay was carried out as described previously [41], with some modifications. Briefly, overnight cultures in Trypticase Soy Broth (TSB) without dextrose (Becton find more Dickinson, Le pont de Claix, France) were diluted until 108 CFU/mL in TSB containing

0%, 0.1%, 0.25% and 0.5% glucose. Individual wells of polystyrene, flat-bottomed 96-well plates (Greiner Bio-One, Frickenhausen, Germany) were filled with 100-μL aliquots of the cultures.

As a negative control, uninoculated medium was used. S. aureus ATCC 25923 and one clinical S. aureus isolate www.selleckchem.com/products/prt062607-p505-15-hcl.html from our collection, known to form fully established biofilms (A 590 values within the highest range and stable) as observed during a pilot experiment, were added to each plate as reference standard [17] and positive control, respectively. After 4 h of adhesion at 37°C on a rocking platform at 25 oscillations min-1, the medium containing non-adhered cells, was replaced by 100 μL fresh broth and the plates were further incubated for 24 h. Next, the wells were washed three times with 200 μL 0.9% NaCl. Biofilms were fixed at 60°C during 1 h. Subsequently, 100 μl crystal violet solution (0.3% wt/vol) was added to all wells. After 15 min, the Methane monooxygenase excess crystal violet was rinsed off by placing the plates under Torin 1 nmr running tap water. Finally, after drying the plates, bound crystal violet was released by adding 100 μl 70% (vol/vol) ethanol with 10% isopropyl alcohol (vol/vol). Absorbance was measured spectrophotometrically at 590 nm (A 590) and was proportional to biofilm biomass. All assays

were performed in triplicate, and repeated on three occasions. The intra- and interday coefficients of variation for the assay were 14% and 23%, respectively. To obtain a threshold A 590 value for which strong biofilm formation commences, the A 590 values of all strains at the different glucose concentrations were sorted in ascending order and divided into quartiles. The distribution of A 590 values in the lower three quartiles was similar at glucose concentrations of 0%, 0.1% and 0.25% and therefore used to determine the cut-off value (two standard deviations above the mean A 590 value). The threshold A 590 value was 0.374. Bacteria with A 590 values above this value were considered strong biofilm formers. Determination of the agr type The agr types were determined by a real-time multiplex PCR assay, as described previously [42]. Statistical analysis SPSS version 15.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analyses.