Millonig osmium tetroxide buffered solution

For SEM, S. aureus biofilm former B+ (1053) was grown on plastic cover slips (Falcon) for 48 h as described above. After biofilm cultivation slips were replaced, they were washed three times in PBS, fixed in 3% Millonig phosphate-buffered gluteraldehyde 3x for 10 min (Serva, Germany), postfixed in 2% Millonig osmium tetroxide buffered solution for 1 hour (Serva, Germany), and then washed 3x for 10 min in Millonig phosphate buffer. The samples were subsequently dehydrated in increasing concentrations of acetone (50, 70, 90, and 100%), every step for 20 min, and dried in hexamethyldisilazane (Sigma-Aldrich, Czech Republic) for 3 h in a hood at RT. Then, the samples were placed on carbon tabs attached to an aluminium holder and coated with platinum/palladium (Cressington sputter coater 208 HR, UK). The structure and interaction of biofilm cells were observed under a Hitachi SU 8010 scanning electron microscope (Hitachi High Technologies, Japan) at a magnification of 6000x (at 15 kV, wd 10.9 mm).

For SEM, dual‐species biofilms were grown on the plastic coverslips as described above. Samples were removed from the dishes at 24, 48 and 72 h, washed three times in PBS and fixed in 3% Millonig phosphate‐buffered glutaraldehyde three times for 10 min (Serva, Heidelberg, Germany) and postfixed in 2% Millonig osmium tetroxide‐buffered solution for 1 h (Serva, Germany). Samples were washed three times for 10 min in Millonig phosphate buffer. The samples were subsequently dehydrated in increasing concentrations of acetone (50, 70, 90 and 100%), every step for 20 min, and dried in hexamethyldisilazane for 3 h in a hood at room temperature (Sigma‐Aldrich, Praha, Czech Republic). Then, the samples were placed on the carbon tabs attached to the aluminium holder and coated with platinum/palladium (Cressington sputter coater 208 HR, UK). The structure and interaction of dual‐species biofilms formed by S. aureus and Gram‐negative bacteria were observed under a Hitachi SU 8010 scanning electron microscope (Hitachi High Technologies, Tokyo, Japan) at a magnification of 1500× (at 17 kV, SE+BSE detector, working distance wd 8.4 mm); 6000× (at 15 kV, wd 10.9 mm); 13 000× (at 17 kV, wd 8.4 mm); 30 000× (at 17 kV, wd 13.6).