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 in a single orientation for their injection. Set the microinjector to the appropriate injection settings, and insert the needle into the muscle tissue of the first embryo at a 45 to 60-degree angle under a stereo microscope.

Gently move the needle back and forth to adjust the position within the tissue as necessary, and use the microinjector foot pedal to inject the loaded suspension. When injecting a bacteria microsphere suspension into the tissue of zebrafish embryos, the needle should be inserted gently but with a steady hand. After a successful insertion, a space should be created for the materials before injection.

And maintain the embryos in E3 medium without tricaine in individual wells of 48-well plates with daily medium changes. To monitor the infection progression by colony-forming unit quantification, randomly transfer five to six viable infected embryos into individual two-milliliter microtubes shortly after injection, and gently wash the embryos with sterile PBS. After discarding the washes, add 100 microliters of sterile PBS to each tube, followed by two to three sterile two-millimeter-diameter zirconia beads.

Then crush the embryos in a homogenizer at 3,500 rpm for 30 seconds, and culture the homogenate as demonstrated.