Calcofluor White staining of the biofilm matrix

Calcofluor White staining (Calcofluor White M2R and Evans Blue 0.5 g/L, Sigma-Aldrich) was used to stain the biofilm matrix. Biofilms were grown and processed as described in section 2 and plasma-treated for 0, 3, 15, and 30 min. Then, 15 μL of a 1:1 mixture of Calcofluor White and 10% w/v potassium hydroxide were added to each coupon which was further incubated for 1 min in the dark. Stained biofilms were visualized with a Leica epifluorescence microscope using a 355/433 nm excitation/emission filter. Images were acquired with the Leica Application Suite (2.5.0 R1) with a 400X magnification and image analyses were performed with the ImageJ free software [44].