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- Return plate to incubator and leave to “rest” for 90 min.
- Pour off E3 and refill with fresh E3 (see Note 18).
- Transfer embryos into a 96-well plate (each embryo to a sepa-
rate well) topping up well to about 3/4 full with E3 (for sur-
vival and microscopy experiments). - In order to verify the inoculum dose, spot 10 μL of injected
samples of 1 mL of PBS (see Subheading 3.5, steps 6 and 8 )
into a BHI agar plate (in triplicate). - Observe infected zebrafish embryos at intervals for the end-
point of diminished blood circulation, visually identifiable bac-
terial lesions and collect for either determination of in vivo
bacterial numbers or fluorescence microscopy.
Fig. 2 Microscopy using zebrafish. (a). Microscopic image of zebrafish embryo at 30 hpf. The site and direction
of bacterial injection into the circulation is indicated by the red arrow. Scale bar indicates 500 μm. (b). In vivo
image of the yolk circulation valley of 32 hpf larvae, 2 h after injection with a mixture consisting of 750 CFU of
CFP-labeled (Cyan Fluorescent Protein) and 750 CFU of YFP-lablled (Yellow Fluorescent Protein) S. aureus. Left
panel shows DIC, middle panel—fluorescence, and right panel—merge image. Two differently labeled bacte-
rial strains were equally distributed within phagocytes at early stages of infection. Scale bar indicates 10 μm.
(c). Examples of In vivo images of terminally infected wild-type larvae (24–44 hpi) after injection with a mixture
consisting of 750 CFU of CFP-labeled and 750 CFU of YFP-labeled S. aureus. At later stages of infection, each
abscess-like structure seen was formed almost exclusively by bacteria with a single fluorescent label (CFP-
labeled bacteria for the left panel, and YFP-labeled bacteria for the right panel). In the middle panel, lesions are
formed by two bacterial strains, but they are spatially separated. Scale bar indicates 100 μm
Tomasz K. Prajsnar et al.