Mature colony biofilms were stained by transferring a
staining solution in Hv-starve medium directly on top of
the colony biofilm and incubating at room temperature
for 60 min. Cross sections of stained colony biofilms
(e.g., Figure 1B) were cryo-processed by rapid freezing
on dry ice and cross-sectioned using a Leica CM1850
cryostat microtome as described previously [23]. Then,
the 5-μm transects were placed onto microscope slides
and visualized immediately.