Quantitative microinjection of Candida albicans in zebrafish embryos for infection monitoring and survival analysis

This protocol describes a versatile in vivo zebrafish embryo infection model with Candida albicans that enables precise microinjection and monitoring of defined infection readouts to compare experimental conditions (e.g., injection site and incubation temperature). The core workflow consists of quantitative microinjection of DiI-labeled C. albicans into the hindbrain ventricle of 30-48 hpf embryos, incubation at 23 deg C, and daily survival scoring to evaluate condition-dependent mortality. We calibrate the inoculum by hemocytometer counts and by plating the same injection suspension to determine colony-forming units (CFU) per pulse, allowing CFU per embryo to be reported and experiments to be compared across laboratories. Survival is analyzed with Kaplan-Meier curves and, when groups are compared, Cox models. DiI fluorescence verifies successful delivery, excludes mis-injections, and permits semi-quantitative burden comparisons between injection sites and temperatures at 24-48 hpi; beyond [~]48 hpi, signal becomes unreliable due to dye dilution and redistribution. The protocol is designed to be reproducible without transgenic reporters or confocal microscopy and includes practical guidance on randomization, blinding, sample-size planning, and reporting.