In Vivo Analysis of Implant-Associated Bacterial Infections Using Zebrafish Embryos

Begin with a dish containing an agarose chamber molded with grooves.

Each groove holds anesthetized zebrafish embryos submerged in an embryo medium supplemented with an anesthetic agent.

Under a light microscope, insert a microinjection needle into the muscle tissue.

Inject a suspension containing S. aureus and polystyrene microspheres to model an implant-associated infection.

Transfer the injected embryos into individual wells of a multi-well plate filled with a fresh medium and incubate.

Microspheres offer a physical surface that supports bacterial colonization and promotes infection.

To assess implant-associated bacterial infection, transfer each embryo into a separate tube and wash it with a buffer. Add buffer and ceramic beads.

Homogenize to lyse the embryos and release internal contents, including bacteria.

Plate the diluted homogenates, then incubate to allow colony formation.

Embryos injected with the microsphere–bacterial suspension show higher colony counts than those injected with the bacterial suspension alone. This indicates that the microspheres increase infection susceptibility.

After waiting five minutes for the embryos to be anesthetized, align the embryos within the grooves