Calcofluor white m2r cw

At each sampling time, a small square of the bottom of each well of 24-well microtiter plates was cut off and the biofilms were inspected by an Olympus BX51 epi-fluorescence microscope (Olympus, Tokyo, Japan) with different stains: calcofluor white M2R (CW) (Invitrogen/ Molecular Probes, Leiden, the Netherlands), FUN-1 (In-vitrogen/Molecular Probes), and FDA and acridine orange (AO) (Sigma-Aldrich). Washed biofilms were stained with 15 μl of 25 μM of FUN-1 at 30°C for 30 min and 10 μl of 25 μM CW at room temperature for 15 min, in the dark. Other fluorochrome combinations were tested, namely FDA with CW, and AO with CW. In this case, washed biofilms were stained with 15 μl of FDA (0.05 mg l -1 ) plus 10 μl of 25 μM CW; or 15 μl of AO (0.04 mg l -1 ) plus 10 μl of 25 μM CW, at room temperature for 15 min, in the dark. After staining biofilm samples were observed under an epifluorescence micro-scope using UV light equipped with 10 × /0.30 and 40 × / 0.75 objective lenses. The optical filter combinations used for FUN-1, FDA and AO were a 470-490 nm excitation filter, a LP516 nm emission filter and a 500 nm barrier filter and for CW were a 365-370 nm excitation filter, a LP421 nm emission filter and a 400 nm barrier filter. The biofilm images were acquired with a microscope camera (Olympus) using the CellB software.

At each sampling time, a coupon of polystyrene (1 cm 2 ) that had been inserted in a 24-well microtitre plate was inspected by an Olympus BX51 epifluorescence microscope (Olympus, Tokyo, Japan) with different stains: calcofluor white M2R (CW) (Invitrogen/ Molecular Probes, Leiden, the Netherlands) to stain the fungi and 4,6-diamino-2-phenylindole (DAPI) (Sigma-Aldrich) to stain the bacteria. Washed fungal biofilms were stained with 10 ll of 25 lM CW at room temperature for 15 min, in the dark. Washed bacterial biofilms were stained with 10 ll of 100 lg ml À1 DAPI at room temperature for 15 min, in the dark. Washed inter-kingdom biofilms were stained with 5 ll of 25 lM CW and 5 ll of 100 lg ml À1 DAPI at room temperature for 15 min in the dark. After staining, biofilm samples were observed under epifluorescence microscopy. The optical filter combination used for CW and DAPI were a 365-370 nm excitation filter, a LP421 nm emission filter and a 400 nm barrier filter. Biofilms images were acquired with a microscope camera (Olympus DP71) using the cellSens software (Olympus Corporation).