albicans strains and a S. aureus strain using AFM. Figure 1 Schematic illustration of the principle of atomic force microscopy and definition of different hyphal regions. (A) Schematic presentation of AFM set-up. A sample with attached C. albicans cells is positioned

by a xyz piezo scanner, while a bacterium attached to a tipless AFM cantilever is brought into contact with the hyphal surface. The deflection of the cantilever upon retract is a measure NVP-LDE225 of the adhesion forces between a bacterium and the hyphal surface and is detected by an optical laser. The laser beam is focused on the very end of the cantilever and reflected onto a position sensitive detector from which the adhesion forces can be calculated, provided the mechanical properties of the cantilever are known. (B) Schematic indication of the different hyphal regions defined for bacterial-hyphal adhesion force measurements. Methods Strains, growth conditions and harvesting C. albicans SC5314 (a commonly used, wild type reference strain), C. albicans MB1 (a biofilm-associated, clinical isolate [27]) and bacterial strain S. aureus MLN0128 cell line NCTC8325-4 (wild type) were used. To generate green fluorescent protein (GFP)-expressing S. aureus NCTC8325-4, pMV158GFP [28] was introduced into competent bacterial cells by electroporation [29]. Selection of subsequent transformants was performed on tryptone soya broth with 1.5% bactoagar (TSB, Oxoid,

Basingstoke, UK) plates containing 10 μg/mL tetracycline. S. aureus NCTC8325-4 click here that received pMV158GFP (S. aureus NCTC8325-4GFP) showed constitutive GFP expression that could be visualized using fluorescence microscopy. Strains were grown on TSB agar plates, supplemented with tetracycline when appropriate. Single colonies were inoculated in 5 mL TSB containing 10 μg/mL tetracycline for bacterial pre-cultures or 5 mL yeast nitrogen

base acids (YNB; Difco, Sparks, USA) pH 7, containing 0.5% D-glucose for C. albicans pre-cultures. S. aureus was routinely grown at 37°C while C. albicans was grown at 30°C to prevent hyphal formation for 24 h with rotation (150 rpm) and used to inoculate a main culture (1:50 dilution of pre-culture). Main bacterial cultures were grown for an additional 18 h under the same conditions. C. albicans hyphae were induced by growing a culture (1:50 dilution) for 4 h with rotation (150 rpm) at 37°C in 12 wells tissue culture polystyrene plates (Costar, Corning Inc., NY, USA). Hyphal formation was obtained at 90-95% efficiency under these conditions, as confirmed by phase contrast microscopy. Main cultures were harvested by centrifugation for 5 min at 6,250 x g and 14,800 x g for S. aureus and C. albicans, respectively, followed by two washes with phosphate buffered saline (PBS: 10 mM potassium phosphate, 0.15 M sodium chloride, pH 7) and resuspended in PBS. Adhesion of staphylococci to hyphae and yeast using fluorescence microscopy Adhesion of S. aureus NCTC8325-4GFP to C.