Confocal scanning microscopy.

Confocal laser scanning microscopy was performed to determine the presence of curli fimbriae in O111 cells grown in LBHS broth. A small aliquot of bacterial aggregates or of cells sampled from the bottom of the culture tubes in the case of nonaggregative strains was resuspended in sterile water, spotted onto microscope slides, and air dried. The slides were rinsed in double-distilled water (ddH₂O) and then incubated in BSA-TBS-Tween 20 solution to eliminate nonspecific binding of the antibody to glass. The bacterial cells were fluorescently stained with Hoechst 33342 nucleic acid stain (Life Technologies, Grand Island, NY) at 100 μg/ml for 40 min. The curli proteins were labeled by incubation in polyclonal anti-CsgA antibody diluted in BSA-TBS-Tween 20 solution followed with goat anti-rabbit secondary antibody labeled with Alexa Fluor 488 (Life Technologies). The cells were observed under a Leica SP5 scanning confocal microscope at 405-nm and 488-nm excitation wavelengths. The signals from the Hoechst stain and the anti-CsgA antibody were assigned the pseudocolors blue and green, respectively.