Results: Biofilms were cultured in static liquid and visualized with fluorescent cell membrane dyes and by
engineering cells to express green fluorescent protein (GFP). Analysis by confocal scanning laser microscopy
showed that H. volcanii cells formed microcolonies within 24 h, which developed into larger clusters by 48 h and
matured into flake-like towers often greater than 100 μm in height after 7 days. To visualize the extracellular matrix,
biofilms formed by GFP-expressing cells were stained with concanavalin A, DAPI, Congo red and thioflavin T. Stains
colocalized with larger cellular structures and indicated that the extracellular matrix may contain a combination of
polysaccharides, extracellular DNA and amyloid protein. Following a switch to biofilm growth conditions, a
sub-population of cells differentiated into chains of long rods sometimes exceeding 25 μm in length, compared to
their planktonic disk-shaped morphology. Time-lapse photography of static liquid biofilms also revealed wave-like
social motility. Finally, we quantified gene exchange between biofilm cells, and found that it was equivalent to the
mating frequency of a classic filter-based experimental method.