Quantitation of fluorescence intensity in S. epidermidis-injected embryos using the COPAS system. Panel A: Bright field /fluorescence overlay images of mCherry-labelled S. epidermidis. Wild type zebrafish embryos injected with 100 CFU of S. epidermidis O-47 into the yolk at 2 HPF were imaged at 5 time points from 1 to 5 DPI, scale bar is 250 µm. Panel B: CFU counts of S. epidermidis-infected embryos. Groups of 10 embryos were homogenized and plated directly after injection until 5 DPI. Panel C: The graphs represent the average fluorescence intensity from the entire group of non-injected and S. epidermidis-injected embryos, from 2 DPI until 5 DPI. An increase in fluorescence intensity is visible during this infection period. (Error bars = SEM). Different letters indicate statistical significant differences (P<0.001). Panels D and E: Correlation between CFU counts and fluorescence intensity of embryos infected with mCherry-labelled (D) and GFP-labelled (E) bacteria. Pools of 10 infected embryos between 2 and 5 DPI were homogenized and plated. The average fluorescence intensity is plotted against the CFU count.

Invasion of S. epidermidis into the zebrafish embryo body. Confocal z-stacks are shown as transmission/fluorescence overlay (a &c) and fluorescence images (b &d). Panel 1: at 3 DPI mCherry labelled S. epidermidis is observed inside the body (1a &1b, scale bar: 50 µm), and intracellular in the hematopoietic region (1c &1d, scale bar: 10 µm). Panel 2: at 4 DPI bacteria are found inside the vasculature (2a &2b, scale bar: 50 µm), including the intersegmental vessels (2d &2d, scale bar: 10 µm). Panel 3: at 5 DPI bacteria are still persisting in the vasculature (3a &3b, scale bar: 25 µm) and in the intersegmental vessels (3c &3d, scale bar: 10 µm). Panel 4: bacteria being taken up by mpeg1:KAEDE positive cells and extracellular in the hematopoietic region at 3 DPI (scale bar: 10 µm).