Distribution of Fusobacterium nucleatum,
Porphyromonas gingivalis, and biofilm within the gingival
tissue. (A) Tissue sections were stained with hematoxylin
and eosin (H&E). Three areas (a, b, c) were examined under high
magnification (×1,000) with differential interference contrast
microscopy. Arrows indicate the potential directions of infection
spread. PE, pocket epithelium. (B, C) Tissue sections were
in situ hybridized with F. nucleatum– and
P. gingivalis–specific probes,
respectively. Arrows, bacterial signals; insets, areas with biofilm-like
structure are magnified. (D) Tissue sections were stained
with 1% alcian blue for acid mucopolysaccharide and counterstained with
nuclear fast red. Arrows, biofilm-like structures; arrowheads, mast
cells; asterisk, area with intact connective tissue fibers.
(E) Correlation plots between bacterial signals and
biofilm formation (r and P values by Spearman’s rank correlation test).
ROI, region of interest. See Appendix Figure 3 for DIC images taken by confocal
microscopy.