Isolation and Cultivation of Bacteria from Zebrafish Embryos to Assess Infection Load

Take a zebrafish embryo infected with Mycobacterium abscessus, a pathogenic bacterium that targets the central nervous system and forms abscesses containing bacteria and host immune cells.

Incubate the embryo on ice to immobilize it, then expose it to an overdose of anesthetic to stop cardiac and neural activity, resulting in death.

Wash the embryo to remove residual anesthetic.

Add a detergent solution to disrupt cellular membranes and lyse the embryo.

Shear the lysate through a needle to homogenize the tissue and release the bacteria.

Centrifuge, discard the supernatant, and resuspend the pellet in a surfactant solution to prevent bacterial clumping.

Serially dilute the bacterial suspension and spread it onto agar plates.

The medium is supplemented with antibiotics to inhibit the growth of other microbes while the Mycobacterium cells engineered for antibiotic resistance proliferate.

Incubate to promote colony formation, enabling quantification of the embryo's bacterial load.

To enumerate colony-forming units or CFUs at the desired time point, collect five embryos per infection condition and transfer each embryo into a 1.5 milliliter microcentrifuge tube. Cryoanesthetize the embryos by inc