Biofilm Staining and Confocal Imaging

Pre-formed 24-h biofilm formations, generated as described above, were incubated for 45 min at 37°C in a solution of 25 μg/ml CAAF 488 (Concanavalin A conjugated to Alexa Fluor 488, CAAF; Molecular Probes, Inc. PO Box 22010, Eugene, United States) and 10 μg/mL FUN 1 (Molecular Probes) (Martinez and Casadevall, 2005 (link)) generated by combining 4 μl of FUN 1 (10 mM) and 15 μl of CAAF 488 (5 mg/ml) with 3 ml of sterile PBS (Martinez and Casadevall, 2005 (link); Tavares et al., 2019 (link)). Subsequently, the coverslips were washed with distilled water taken from the wells and inverted over 4 μl of Fluoromount-G (Sigma-Aldrich, United States) that was previously deposited on microscope slides for observation under a confocal microscope (LSM 510 META, Zeiss).