Extended Data Fig. 4: Microscopy images of chitin particles from growing cultures.

Samples were incubated with two fluorescent dyes: FITC-WGA, here shown in the green channel and FM 4-64, a cell membrane dye, shown in the red channel, and fixed with glutaraldehyde. Imaging of the samples was done in custom-built chambers (see microscopy in Methods) under a confocal microscope with 40x magnification. Sample representative images were chosen but microscopy sessions were performed to image 10-50 particles per sample, which all showed similar colonization trends. a) Sterile chitin flake incubated with both dyes. FITC-WGA specifically binds to chitin particles, though some FM 4-64 can also be seen in the background. b) Chitin particles were isolated from an exponentially increasing chitin culture at \(O{D}_{600}^{plank}=0.04\) and resuspended in fresh media. Red (membrane dyed) cells can be seen to bind to chitin and form microcolonies. c) A sample including both planktonic cells and chitin particles from an exponentially increasing chitin culture at \(O{D}_{600}^{plank}=0.4\). Consistent with bulk measurements, more planktonic cells are observed than surface-associated cells. d) From the same growing culture as in Panel c, chitin particles were isolated and resuspended in fresh media to remove planktonic cells. In both c) and d), the surface of particles is not saturated with cells even at this moderately high OD. This suggests that cell detachment from particles is not a result of a ‘space limitation’ on the particle surface.