Fig 6. Biofilms and EPS production by the A. brasilense Sp7, 2116, and 2117 strains.

(A) CR staining assay. Cells were grown on agar-solidified CR medium inoculated with 10 μL of 1.0 OD₆₀₀ nm of each culture strains for 72 h at 30 ºC. For EPS quantification, the strains were cultured as described in Material and Methods and incubated at 30°C for 5 days. CR binding was expressed as mg CR/OD₆₀₀ nm. (B) For biofilm production cells were grown statically for 5 days at 30 ºC on NFb* with 27mM malate as a C source + 10mM D-alanine. Biofilm formation was visualized by Crystal violet staining as well quantified and normalized per OD₆₀₀ nm of growth. All data are the average from three independent experiments performed in duplicate. Error bars indicate standard errors of the means as compared with values observed for WT. For all data, P was < 0.001 as assessed by Student´s t-test. (C) Calcofluor white colorant M2R (CWC) staining. Cells were grown on agar-solidified NFb* supplemented with CWC and inoculated with 10 μL of 1.0 OD₆₀₀ nm of each culture strains for five days at 30 ºC. The cell fluorescence was observed under was observed with a Nikon Eclipse Ti-E C2+. Bars represent 10 μm. The white arrows indicate EPS.