(ii) Immunofluorescence (IF) microscopy.

The remaining half of the biofilm suspension was centrifuged to pellet cells, and the pellets were washed with distilled water before smears were prepared on slides for immunofluorescent staining as described previously (33). Slides were fixed with ethanol and blocked with 5% normal goat serum (NGS) (Vector Laboratories, Burlingame, CA, USA)–PBS prior to incubation with the rabbit anti-curlin diluted 1:10 in PBS at room temperature for 1 h. Subsequently, slides were washed with PBS at room temperature and incubated with secondary antibodies diluted in PBS at room temperature for 1 h. Alexa Fluor 594 (red)-labeled goat anti-rabbit IgG [H+L; F(ab′)₂ fragment] (Invitrogen) targeting the anti-curlin primary antibody and the fluorescein isothiocyanate (FITC; green)-labeled goat anti-O157 or anti-E. coli (KPL, Gaithersburg, MD, USA) antibodies that label O157 or E. coli, respectively, comprised the secondary antibodies. Following washes performed with PBS and distilled water, the slides were air-dried and added to coverslips with Prolong Gold antifade reagent (Invitrogen). All slides were analyzed using a Nikon Eclipse E800 fluorescence microscope (Nikon Instruments Inc., Elgin, IL, USA) equipped with fluorescence illumination and digital imaging.