Ccd colour view 3 camera

Cells were fixed with ice-cold ethanol, washed with Aq_dest, and incubated with 40 mM Alizarin red solution (Sigma Aldrich, pH 4.1) for 20 min at RT. After thorough washing, the air-dried specimens were evaluated with a SZH10 microscope (Olympus, Münster, Germany) equipped with a CCD Colour view III camera and the resulting images were taken and analyzed using the CellSens software version 1.5 (both Olympus, Münster, Germany). For quantification, Alizarin red was extracted with 10% acetic acid for 30 min at RT. The cell layer was scraped, and the solution was covered with mineral oil (Sigma Aldrich) and incubated for 10 min at 85 °C. The supernatant was transferred into microplates, the absorbance was read at 420 nm in triplicates, and data were collected and analyzed using the Magellan v6.2 software (Tecan, Crailsheim, Austria).

For CFU assays, cells were seeded in limiting dilution, i.e., 2 cells/cm², incubated for 14 days, fixed with 4% formaldehyde, and stained with 0.1% Azur II (Sigma Aldrich, Taufkirchen, Germany) dissolved in Aq_dest for 20 min at room temperature (RT), air dried, and photographed with a SZH10 microscope (Olympus, Münster, Germany) equipped with a CCD Colour view III camera (Olympus, Münster, Germany). The resulting images were taken using the Cell Olympus cell Sens software version 1.5 (Olympus, Münster, Germany). Images were analyzed using the ImageJ plugin “analyze particles” following threshold adjustment. Stained colonies of ≥50 cell were scored as colony forming (CFU-F) and counted. CFU-F efficiency was calculated as follows: