Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

The overall goal of this procedure is to micro inject zebrafish embryos with fluorescent bacteria for live imaging of the interaction with host immune cells. This is accomplished by first preparing and loading the injection needle with bacterial suspensions. Next, the embryos are staged and aligned on an injection plate.

Then the bacterial inoculum is injected using a route to achieve either a local or a systemic bacterial infection. Finally, the embryos are mounted and the embryos are imaged. Ultimately, results can be obtained that show intracellular localization of bacterial pathogens inside fluorescent phagocytes of the transparent zebrafish embryonic host through fluorescence and confocal microscopy.

This method can help answer key questions in the immunology field because the zebrafish embryo model is very powerful for life imaging of host pathogen interactions, Generally, individuals new to this method will struggle because it requires a lot of practice To reproducibly micro inject bacteria into the blood circulation of zebrafish embryos for systemic infection or into specific compartments to achieve local infections. After pulling glass micro capillary needles and beveling the tips to a 45 degree angle, stage the zebra fish embryos at 28 hours post fertilization by checking for consistent blood circulation. The start of pigmentation in the eye, a straight tail and the heart being positioned just ventrally to the eye.

Once the embryos are anesthetized, use a micro loader tip to load the needle with a previously prepared bacterial inoculum. Mount the loaded needle onto a micro manipulator connected to a stand and position it under a stereo microscope. Set the injection time to 0.2 seconds and the compensation pressure to 15 Hector Pascals.

Adjust the injection pressure between 700 and 900 Hector Pascals to obtain the correct injection volume for the needle used. Set the micro manipulator with the loaded needle into the correct position prior to injecting. Place the anesthetized embryos on a flat 1%agarose injecting plate and remove any excess egg water.

Next, use a hair loop tool to line up the embryos for each injection. Move the plate by hand during injections to orient the embryos with their tails pointing towards the needle tip. Place the needle tip directly above the coddle vein close to the urogenital opening.

Pierce the periderm with the needle tip and inject the desired dose of fluorescently labeled bacteria. We use approximately 250 colony forming units or CFUs of salmonella typhimurium and approximately 120 CFUs of mycobacterium marum. The injected bacterial suspension will follow the blood flow through the coddle vein towards the heart monitor if the injection was performed correctly by checking for an expanding volume of the vascular system directly after the pulse.

Frequently check that the injection volume remains the same during the experiment. To provide a control for the consistency of the injections throughout the experiment, inject a drop of bacteria directly onto a drop of sterile PBS on bacterial growth medium. After approximately every 30th embryo injection, plate out this drop and count the bacterial colonies after incubation.

To determine the CFU in the injection volume, use a fluorescent stereo microscope to observe individual fluorescent styria cells circulating in the bloodstream directly after injection and discard embryos that are not properly injected. Fluorescent aggregates of M marum bacteria should be visible by two days post infection and grow larger over time. To inject into the duct of cuvier, line up anesthetize two to three days Post fertilization embryos.

As for coddle vein injection at a 45 degree angle from the dorsal side of the embryo, insert the needle into the starting point of the duct of cuvier. Just dorsal to the location where the duct starts broadening over the yolk sac for hindbrain ventricle injecting position. Anesthetize 32 hours post fertilization embryos with their dorsal side towards the needle tip.

Insert the needle into the hindbrain ventricle from an anterior position without touching the neuro helium to inject into tail muscles. Position anesthetized one to two days post fertilization embryos with their tail pointing toward the needle tip and with the needle at an approximately 65 degree angle, inject into the muscle above the urogenital opening for otic vesicle injecting. Orient anesthetize two to three days post fertilization embryos with the tails pointing to towards the needle.

Inject at a 65 degree angle and at low pressure to inject into the node cord using one to two days. Post fertilization embryos with the tail pointing away from the needle. Insert the needle through the tail muscle tissue into the cord.

For a yolk injection of the 16 to 1000 cell stage embryos pierce the needle through the corion into the center of the yolk to image the infection. Anesthetize the infected embryos in a 1%augurous layered Petri dish covered with egg water containing trica. Using a hair loop tool aligned the embryos in the correct position for imaging under a fluorescent stereo microscope.

Individual fluorescent s tym cells can be observed circulating in the bloodstream directly after injection. If a different position than the lateral view is required. Mount the embryos in 1.5%methyl cellulose and use a hair loop tool to manipulate the embryo into the required position to image the infection using an inverted confocal microscope.

Place a drop of low melting point aeros on a glass bottom dish. Place the anesthetized embryo into the aero drop with limited amount of egg water and use a hair loop tool to manipulate the embryo into position. Let the aero solidify and submerge the aero drop in egg water containing trica.

The sample is now ready for confocal imaging injection of salmonella typhimurium or mycobacterium marum bacteria into the blood island of embryos at one day. Post fertilization results in the rapid phagocytosis by macrophages dissemination of the relatively large and brightly fluorescent DS.Red labeled styrian bacteria can be imaged directly with stereo fluorescence and confocal imaging at two hours. Post-infection shows that many bacteria are phagocytose by fluorescent macrophages.

An injection dose of 250 CFU of wild type styria will induce a strong pro-inflammatory response and is lethal within a day. In contrast, the intravenous injection of erum leads to a persistent infection where infected macrophages form tight aggregates that are considered as the initial stages of granulomas, which are the hallmark of tuberculosis. Confocal imaging of such a granuloma like aggregate in the transgenic EG one EGFP line at five days post-infection shows the intracellular growth of m cherry labeled M num bacteria.

Inside the green fluorescent macrophages, bacteria can be injected into the hind brainin ventricle, which is a compartment devoid of macrophages at 32 hours post fertilization injection of 20 to 100 M cherry labeled M marum bacteria into this compartment leads to the rapid infiltration by macrophages that phagocytose the bacteria as shown here. By using the transgenic M-P-X-E-G-F-P injection of approximately 20 CFU of Styria into the otic vesicle leads to the attraction of neutrophils at three hours post infection. While this response is not observed in PBS control injections, the notochord, which appears to be resistant to infiltration by leukocytes, is a permissive compartment for the growth of erum mutants that are strongly attenuated when injected in other tissues.

Following yoke injection of a dose of 2240 CFU erum bacteria spread over several days into the embryonic tissues and form granuloma like aggregates. Similar to those observed with the conventional intravenous injection method. While attempting this procedure, it's important to remember that proper staging of the zebrafish embryos is critical because the embryonic immune system becomes increasingly competent during development.

Following this procedure, other methods like transcriptome profiling and immunohistochemistry can be performed in order to answer additional questions like how the embryonic innate immune system responds to pathogenic infections.