Fig. 2: Incubation with 3H3 alters the biofilm architecture.

a S. Typhimurium biofilms were formed in the absence of mAb (untreated) or in the presence of 0.5 mg/ml 6A, 0.5 mg/ml 3H3, or anti-CsgA serum. After 72 h, biofilms were stained with Syto9 (green) and visualized using Leica TCS confocal microscopy at ×63. 3D surface plots were created in ImageJ and all Syto9 particles of the biofilms appear as green. Particles are colored black at the bottom of the z-plane and are increased in green intensity from 0 to 220 μm on the z-plane. b Particles above the mean biofilm mass of the untreated sample appear in white using the 3D surface plot application of ImageJ. c Number of particles above the mean biofilm mass (white particles) enumerated using ImageJ. d S. Typhimurium biofilms formed in the absence of mAb (untreated) or in the presence of 0.5 mg/ml 6A, 0.5 mg/ml 3H3, or anti-CsgA. After 72 h, biofilms were stained with Syto9 (green) and incubated with 10 μl Crimson FluoSpheres 1 μm red-fluorescent glyoxylate beads (red). Biofilms were imaged using confocal microscopy and biofilm projections of z-stacks were created using ImageJ (3D projections application). Scale bars represent 25 μm. Fluorescently labeled beads (red) were visualized by removing the Syto9 green channel. ImageJ was used to create 3D reconstructions of z-stacks. White arrows indicate location of beads in the untreated sample. e Number of red Crimson FluoSpheres within biofilms enumerated using ImageJ. Mean and SE were calculated from results of at least three independent experiments. *p < 0.05, **p < 0.01 as determined by Student’s t-test.