(A) Experimental overview. Droplets containing Y. pseudotuberculosis were grown for 7 hr at 26°C prior to incubation with BMDMs for 4 hr at 37°C, CO2. The fixed microcolonies were visualized using brightfield and fluorescence microscopy and subjected to image analysis (Materials and methods). (B) Resting BMDMs (left) and LPS/IFNγ-primed BMDMs (right) were fixed, probed with α-iNOS and visualized by fluorescence microscopy. (C). C57BL/6 mice were inoculated intravenously (i.v.) with 103 WT gfp+ Phmp-mcherry bacteria, and spleens were harvested 3 days post-inoculation (PI) for fluorescence microscopy. A representative microcolony from tissue is shown. (D, E). Representative WT (D) and Δhmp (E) microcolonies incubated with LPS/IFNγ-primed BMDMs. (F). Microcolony area in presence of BMDMs, incubated as noted. (G) Periphery verses PLI ratios for microcolonies incubated with BMDMs treated as noted. Black and gray circles: 100 individual microcolonies from each condition. Line indicates median. (H). Flow cytometry of dispersed microcolonies after 4 hr of incubation with BMDMs. Dispersed bacteria were gated for GFP+ and mCherry fluorescence was determined (Materials and methods). Statistics: Mann-Whitney test, ****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05, ns: not significant.