Fig. 4

Development on nutrient agar and in suspension. A On agar. Wild-type and gsk3⁻ cells were cultured in darkness with live E. coli on nutrient agar until, about 24 h after clearing bacteria, fruiting bodies had fully formed. Plates were photographed at low magnification (left, Bar: 1 mm). The agar surfaces were then wetted with 0.001% Calcofluor and photographed under phase contrast (centre) and epifluorescence (right). Bar: 100 µm, inset bar: 10 µm. B In suspension. Wild-type, gsk3⁻ and gsk3⁻/GSK3 cells were incubated in suspension with autoclaved K. aerogenes for 120 h, with bacteria being cleared at 50–60 h. Cells were then stained with Calcofluor and photographed under phase contrast and UV illumination. Bar: 100 µm. C Quantitation. From the experiment shown in B, cells were sampled at the indicated time points and stained with Calcofluor. The numbers of fluorescent cysts and unstained amoebas were counted under UV and phase contrast illumination, respectively, and the percentage of cysts over total cells was calculated. Means and SD of 3 independent experiments, each counting 300–500 cells/sample, are presented