Small Molecule Screening and Toxicity Testing in Early-stage Zebrafish Larvae

To begin, gather early-stage zebrafish embryos at three days post fertilization in a Petri dish. With a 1000 microliter micro pipette set to a 100 microliter volume, transfer one healthy-looking hatched embryo into each well of a 96 well plate. Now, pipette the necessary amount of the test chemical into each well of a 12 well plate.

Then, adjust the total volume to 2.4 milliliters with E3 medium. Prepare the vehicle control according to the solvent used to dissolve the stock test chemical. With a 200 microliter pipette, remove any existing E3 solution from each well of the 96 well plate.

Use an eight channel micro pipette to replace it with 100 microliters of the prepared test medium for each concentration. Ensure that a total of 24 larvae is tested per concentration. After 24 hours, use a stereo light microscope to perform morphological scoring.

Score the morphology using a binary method, where absent indicates normal morphology and present indicates a morphological abnormality. When scoring is complete, remove 50 microliters of the medium from each well. Replace it with 50 microliters of freshly-prepared test medium.

Remove and record any dead larvae, then incubate the larvae at 28.5 degrees Celsius with a 14 hour light and 10 hour dark cycle. Zebrafish at five days post-fertilization exhibited diverse phenotypes after two days of drug exposure. Unaffected zebra fish were observed as normal, while mild pericardial edema was evident in some fish.

Severe pericardial edema, accompanied by yolk hemorrhage and yolk extension was noted in others. Delayed development with reduced pigmentation, swim bladder, and total length was also observed. Additional phenotypes included zebrafish with swollen yolk and kinked caudal fin, absent caudal fin, and curved notochord.

Severe cases exhibited truncation and death with necrosis.