Microinjection of Bacteria into the Caudal Vein of Zebrafish Embryos

Take early-stage zebrafish embryos suspended in fish water and remove the chorion, the protective membrane surrounding the embryos.

Transfer the embryos to a V-shaped positioning chamber filled with fish water containing an anesthetic agent to immobilize them for microinjection.

Position the embryos within the chamber.

Next, dilute a homogeneous suspension of fluorescently labeled pathogenic bacteria in a buffer containing detergents to minimize bacterial clumping.

Add a tracer dye to facilitate monitoring of the injection.

Load the suspension into a microinjection needle, mount the needle onto a micromanipulator, and break the tip to create a small opening.

Reposition the embryo with the ventral side facing the needle and bring the needle tip near the urogenital opening.

Gently insert the tip into the caudal vein region and inject the bacterial suspension.

Transfer the embryos to a multi-well plate containing fish water for subsequent host-bacterial interaction studies.

To carry out microinjection of M. abscessus at about 24 hours post fertilization or hpf, use tweezers to dechorionate embryos in a 100 millimeter Petri dish and keep them at 28.5 degrees Celsius.

Transfer 30-48 hpf embryos to a V-shaped positioning chamber filled with 25 milliliters of fish water containing 270 milligrams per liter of tricaine. With a clipped micro loader tip, lay the embryos properly in the channels for easier micro manipulation.

Use PBST with 0.05% Tween 80 to dilute the microbacterial inoculum to the desired CFU to be delivered, and include 10% phenol red to check proper injection. With a microloader tip, load a microinjection needle with 5 to 10 microliters of the bacterial inoculum.

Connect the microinjection needle into the holder of a micro manipulator connected to the injector, and use fine tweezers to break off the top of the needle to obtain an opening diameter of 5 to 10 micrometers.

Calibrate the injection volume by adjusting the microinjection pressure and time. Then, to obtain the required injection volume, measure the diameter of a droplet expelled into the yolk of one embryo.

To microinject into the caudal vein, position the embryo with the ventral side facing the needle and place the needle tip close to the urogenital opening and gently push the tip of the needle into the embryo until it just pierces the caudal vein region.

Then deliver the desired volume of bacterial suspension, usually 1 to 3 nanoliters containing around 100 CFUs per nanoliter. Incubate and monitor the embryos according to the text protocol.