Quantifying Bacterial Infection in Zebrafish

Zebrafish larvae at 96 hpf were immersed in E3 media containing ~1 × 10^{9 (link)} CFU/mL of BA1250, BA1250ΔecpA, or BA1250ΔecpRABCDE that were transformed with pGEN-mCherry. At 24, 48, and 72 hpi, larvae were carefully washed with PBS to remove any external bacteria, and then immobilized in 3% methylcellulose placed on 3% agar within a Petri dish. Animals were visualized using a fluorescent Olympus SZX10 microscope equipped with an Olympus DP72 camera. Fluorescent signals from captured images were quantified using Fiji/ImageJ 2 2.3.0/1.53f^{87 (link)}. The corrected total cell fluorescence (CTCF) was calculated using the formula: CFCT = integrated density − (selected area × mean fluorescence of background readings)^{88 (link)}. For each image, three selected and background areas were used to normalize against autofluorescence.