Congo red fluorescence (CRF) was used as it has been
proposed as the most sensitive and reliable method for
amyloid detection when staining with CR [60] and has
been applied to biofilm compositional studies [72]. CRF
was sharply defined and granular in appearance and was
colocalized with the large biofilm cellular clusters and
towers shown in Figure 2F,G,H. Overlays where single
GFP-producing cells are visible and images of CRF at
600× magnification (Additional file 1: Figure S3) indicated
that CR did not stain individual cells. CR-stained
biofilm aggregates were also orange-red under transmitted
light and were visible macroscopically (Figure 2I–L).
Further, green fluorescence signal was detected within
ThT-stained mature biofilms formed by a non-GFP strain
with blue excitation (Figure 2M; no signal detected in control
without ThT staining).