Live Imaging of Host-Pathogen Interactions in Zebrafish Embryos Infected with Mycobacterium abscessus

Begin with anesthetized, transparent, transgenic zebrafish embryos expressing fluorescently labeled macrophages.

The embryos are pre-injected into the tail muscle with fluorescently labeled Mycobacterium abscessus rough strain to induce a localized infection.

The absence of surface glycopeptidolipids in these bacteria promotes their aggregation.

Immobilize the embryos in low-melting-point agarose and orient them to expose the infection site.

Once the agarose solidifies, add fish water containing an anesthetic agent to maintain anesthesia and prevent dehydration.

Monitor infection in real time via fluorescence microscopy.

Following infection, endogenous macrophages migrate toward the bacteria and phagocytose individual cells.

However, some bacteria form cords—elongated extracellular aggregates that replicate, spread, and resist phagocytosis, causing host cell death and immune infiltration.

Despite this, immune cells fail to eliminate the cords, allowing bacteria and dead host cells to accumulate and form an abscess.

This reveals cording as a key M. abscessus immune evasion strategy.

For live imaging of M. abscessus infection, mount tricaine anesthetized embryos in 1% low-melting-point agarose in a 35-millimeter glass-bottom Petri dish for an inverted microscope or a single cavity depression slide for upright confocal microscopy. Orient the embryo to the desired position and cover the solidified agarose with fish water containing tricaine. Finally, use a fluorescence microscope with a 10X objective or a fluorescence confocal microscope with 40X or 63X objectives for sequential fluorescence acquisition and transmission imaging of the embryo.