The overall goal of this procedure is to demonstrate techniques for infecting zebrafish larvae using the fungal pathogen candida albicans. First, a fluorescent fungal culture is prepared and fish embryos are coated. Next, the fish are lined up on an aros injection dish.
Candida albicans is micro injected through the ear of each fish into the hind brainin ventricle fisher, then embedded in low melt aeros and visualized by epi fluorescence microscopy. Ultimately, this method can be used to non-invasively image, the host pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish. The main advantage of this technique over existing methods like coddle vein injection is that you create a localized infection that results in lethal disseminated disease.
Further, this technique allows for efficient injection of the inherently large C bucan cells into small zebrafish larva. Begin by streaking a candida albican stock on a yeast pone. Dextrose agar plate incubate the plate overnight at 37 degrees Celsius to allow colonies to grow.
Once the colonies are visible, use a sterile wooden dowel to pick an isolated colony and dissolve the yeast in five milliliters of fresh yeast Extract Pepto dextrose broth in 16 by 150 millimeter culture tubes. Place the culture on a tissue culture roller drum, equipped with a test tube wheel and incubators overnight at 37 degrees Celsius. The next day, transfer one milliliter of the culture to a 1.5 milliliter eend off tube and centrifuge it at 14, 000 times, G for one minute to pellet the cells then discard the supinate and vortex the pellet to loosen it.
Resuspend the cells in one milliliter of PBS vortex and centrifuge them again. Then discard the supinate and repeat this wash. Step three times to remove any residual culture medium.
Once the cells have been washed, count them in a hemo cytometer at a one to 100 dilution in PBS. For a 12 hour culture, you should expect three to four times 10 to the eight cells per milliliter. Reese's bend the yeast at a final concentration of 10 to the seven cells per milliliter.
Use a sieve to collect zebra fish embryos from a spawning tank on the day before the injection and store them in an egg water solution containing 60 milligrams per milliliter of instant ocean salts in sterile deionized water. Then place the embryos at 33 degrees SIUs for 24 hours with a 12 hour light dark cycle. This permits development of the embryo to the prim 25 larval stage, the optimal stage for infection the day of the infection.
Transfer the embryos with a transfer pipette in 60 milliliters of egg water to an extra deep Petri dish. Then user dissecting microscope and dumont tweezers to dec coate the embryos. This can be achieved by gently pulling the Corian apart like a bag of potato chips until the fish pops out.
Once all the embryos have been dec coated. So while the Petri dish to move the fish towards the center, transfer the fish to the lid of the dish, remove the medium and replace with fresh egg water. Add fish to fresh egg water.
Prepare a 200 microgram per milliliter solution of trica methane sulfonate to anesthetize the fish using a transfer pipette. Collect 20 to 50 embryos and replace them in the anesthetic solution for one to two minutes or until they stop moving. In the meantime, turn on the injection unit, making sure the pressure switches on pulse, and the pulse duration is set to nine with three PSI for the back pressure unit.
Gradually open the valve on the nitrogen tank until the injection pressure reaches 30 PSI. Next load pooled micro pipette with five microliters of vortex candida albican solution and place it in the micro pipette holder. Place an extra deep Petri dish containing water on the dissecting microscope stage and adjust the micropipets position until it is in view and its tip is only partially submerged in the water.
Then using tweezers, clip the needle of the pooled pipette about three millimeters from the tip. If the needle has been successfully clipped, pressing the injection unit's foot switch should allow the liquid to be dispensed. Using a graduated slide, measure the volume dispensed, then adjust the pressure so that the volume of liquid dispensed is 0.21 millimeters in diameter or just under the size of a zebra fish embryo's pupil.
Once the microscope is set up and the fish are anesthetized, swell the dish to draw the fish to the center and take them up into a transfer pipette. Carefully tap the side of the pipette so that the fish settle at the tip. Then using as little volume as possible, gently deposit the fish onto a larval aose injection dish prewarm to 28 degrees Celsius.
Using a smooth glass rod, carefully line the fish up. Then with a paper towel, remove as much liquid as possible from the dish. Place the aros dish with fish under the microscope until both the needle and first fish are in clear view.
Ensure that each fish is still alive by observing the presence of a visible heartbeat. If it is dead, discard it and move on to the next one. Move the glass needle towards the fish zooming in.
As you do so, then carefully insert the needle into the fish's ear circular structure with two ear stones located just behind the eye. Once the needle is in position, inject the yeast using the foot switch. If the needle is correctly inserted, the fish is hind brainin ventricle will sw slightly after the injection, retract the needle and repeat the procedure with the next fish.
The whole process should take no longer than 15 or 20 minutes, or the fish will dry out and die after this time. The yeast will also settle into the bottom of the micropipet and lead to uneven infection, doses and or clogging of the pooled micro pipette. Once all the fish have been injected, use egg water to wash them off the aros and into a fresh Petri dish containing 60 milliliters of egg water.
Then place the dish at 28 degrees Celsius. Close the nitrogen valve, set the injection switch to continuous to relieve pressure in the tank line. Then when the pressure falls to zero, set the switch back to pulse and turn off the injection unit.
Begin by anesthetizing the infected fish in trica methane sulfate as described earlier. Once the fish are anesthetized, use a pipette to transfer one fish at a time into a Petri dish containing 0.4%low melt agar. Then using a transfer pipette and carrying over as little agros as possible.
Transfer each fish from the Petri dish into one well of a glass bottom imaging dish at approximately 0.2 milliliters and 0.4%Low melt arose supplemented with 200 micrograms per milliliter of anesthetic on top of the fish so that the inner circles are filled and a thin layer of aros spreads across the bottom of the well. The fish can now be imaged for up to 24 hours using an inverted microscope with a laser scanning confocal system. Five hours after infecting zebra fish with red fluorescent candida albicans.
The yeast is visible in the fish's hind brain in FL I one EGFP fish, such as these, the vascular endothelium and macrophage like cells throughout the fish's body are fluorescent. They're shown here in green, five hours after infection. The yeast can be seen within macrophage like cells by 24 hours.
The infection has disseminated along the dorsal tail tissue where the yeast can again be detected within FTIC cells. After watching this video, you should have a good understanding of how to micro inject candida albicans into the hindbrain ventricle of Prim 25 stage zebrafish, including one preparing the fungal culture, two anesthetizing zebrafish larvae. Three micro injecting into the hindbrain ventricle.
And four, preparing infected larvae for confocal imaging.
