After 4 h of hyphal formation, wells were washed once with PBS. Bacteria were added to a final optical density measured PS-341 mouse at 600 nm (OD600) of 0.1 in PBS. After 3.5 h of co-incubation with staphylococci at 37°C under static conditions,
wells were gently washed two times with PBS and C. albicans hyphae were counter-stained with Calcofluor White (35 μg/mL, 15 min at room temperature), known to bind to chitin-rich areas of the fungal cell wall. Note that PBS was used in order to avoid the influence of growth, while co-incubation was done at 37°C in order to mimic the human body temperature. Afterwards, images were taken at five randomly chosen locations in the wells using a 40x water immersion objective using filter sets for GFP and UV. All
experiments were performed in triplicate with separately grown cultures. Staphylococcal adhesion forces along hyphae using atomic force microscopy Adhesion forces between S. aureus NCTC8325-4GFP and hyphae were measured at room temperature in PBS using an optical lever microscope (Nanoscope IV, Digital Instruments, Woodbury, NY, USA) as described before [26]. Briefly, C. albicans was immobilized on glass slides (Menzel, GmbH, Germany), coated with positively charged poly-L-lysine. A fungal suspension was deposited onto the coated glass and left to settle at room temperature for 20 min. Non-adhering cells were removed by rinsing with demineralized water and the slide was kept hydrated prior to AFM analysis in phosphate buffer. To create a bacterial probe, S. aureus was immobilized Dibutyryl-cAMP order onto poly-L-lysine treated tipless “V”-shaped cantilevers (DNP-0, Bacterial neuraminidase Veeco Instruments Inc., Woodbury, NY, USA). Bacterial selleck products probes were freshly prepared for each experiment. AFM experiments were performed at room temperature due to the limitations of the equipment.
This is unlikely to have an effect on the outcome of physico-chemical measurements such as of adhesion forces, as here the absolute temperature scale, that is in Kelvin units, is relevant. On a Kelvin scale the change from 37°C to 22°C is very small, decreasing only from 293 Kelvin to 273 Kelvin. For each bacterial probe, force curves were measured after different bond-maturation times up to 60 s on the same, randomly chosen spot on a hyphal or yeast cell with a z-scan rate of less than 1 Hz. To ensure that no bacteria detached from the cantilever during the experiment, control force-distance curves were made with 0 s contact time after each set of measurements. Whenever the “0 s contact time” forces measured deviated more than 0.5 nN from the initial measurement, a bacterial probe was considered damaged and replaced. For each combination of a bacterial strain and fungal–coated glass surface, five different probes were employed on average and the number of bacterial probes used depended on the outcome of the control measurements.