It can be challenging to transfer and immobilize fruit flies. Using these techniques and tools, you can make routine fly transfer and immobilization easier. The materials needed to make these tools are inexpensive and easy to obtain.
Advisory and teaching or research lab can make them with ease. Before beginning this procedure, use an electric sodlering iron to make a hole in the centers of two sponge plugs that are larger than the internal diameter of the vials that will be used to transfer the flies. Next, use a sharp knife to cut one one-millimeter micropipette tip transversely in half and to cut 1.5 centimeters off the tip of a second one-millimeter micropipette tip.
Glue the pieces of the two pipette tips together with an all-purpose adhesive. Then insert the elongated micropipette tip into one sponge plug, and insert a funnel into the other sponge plug, and cap the pipette tips with individual 100-microliter microcentrifuge tubes. To prepare the microdissection needles, select insect pins that match the diameters of the lead refills of ergonomic mechanical pencils, and use a pair of pliers to cut off the one-eyed ends of the pins.
Then file the cuts flat, and replace the lead in the pencils with the modified pins. To prepare a Drosophila egg collection case, use a snap cutter to cut a soft-plastic 500-milliliter drink bottle approximately 2/3 down the bottle, and use tape to secure a strip of card paper around a 60-millimeter diameter apple juice plate. To transfer the adult flies between vials, carefully check the stem of the funnel in the funnel stopper and the tip of the pipette in the tip stopper.
Tap down the flies in the first vial, and replace the plug with a tip stopper. Plug the second vial with a funnel stopper, and place the first vial over the second vial such that the pipette-tip end of the tip stopper is inserted into the funnel opening of the funnel stopper. Knock the edge of the first vial to allow the flies to slip out of the pipette tip and through the stem of the funnel into the second vial.
If any old food in the first vial becomes less compact and drops when the vial is inverted, invert the second vial over the first vial, and allow the flies to crawl up into the second vial. When the desired number of flies has been transferred, separate the tip stopper from the funnel stopper, and replace the tip stopper with the original plug. Then replace the funnel stopper in the second vial with an appropriate plug.
For adult fruit fly immobilization by chilling, place a refreezable ice pack, that has been chilled at minus 80 degrees Celsius for at least 24 hours, at room temperature, and chill an empty vial in crushed ice. 20 minutes later, slightly moisten a piece of non-aseptic medical gauze, that is slightly smaller than the chilled ice pack, with running water. Place the gauze onto the surface of the ice pack so that the gauze clings closely to the surface of the pack.
Next use a piece of absorbent paper to wipe the water from the chilled, empty vial, and transfer the adult flies that need to be immobilized into the vial. When all of the flies have been transferred, cover the chilled vial, and knock the vial against the crushed ice two to three times to tap all of the flies to the bottom of the vial. The flies will immediately become immobilized.
Place the vial back on ice for one minute before pouring the flies onto the gauze covering the ice pack. Then use a paintbrush to spread out the overlapping flies. To examine the immobilized flies under a stereo microscope, remove the stage clips from the dissecting microscope, and cover the stage with a piece of plastic film.
Place the ice pack onto the stage, and turn on the cold light source of the microscope as available. Then focus the stereo microscope, and move the ice pack until the chilled flies can be seen clearly. After viewing the flies, transfer each fly, by the wing, into a new culture vial.
To microwave-sacrifice the adult flies for counting, sorting, or discarding, transfer the adult flies of interest into an empty vial, and cover the vial with a Petri dish. Next invert the vial inside a microwave, and heat the vial at standard temperature for 80 seconds. The dead flies will drop into the Petri dish.
Wearing protective work gloves, pour the flies onto a white, paper card, and transfer the card under a stereo microscope for examination and/or counting with the previously prepared microdissection needles. The flies can be discarded at the end of the examination. For bottle-shaped egg collection cage transfer, insert the modified egg collection cage bottle into the apple juice plate.
Use a strip of paraffin film to seal the joint around the two components. Next plug the cage with a funnel stopper, and invert the culture vial onto the stopper. Then add the flies to the cage, through the funnel stopper, before plugging the cage with the culture vial stopper.
To change the food, transfer the flies to an empty vial before replacing the apple juice plate with a new plate. Then transfer the flies from the vial back to the cage. At the end of the egg collection, transfer the flies into an empty vial before transferring the flies into several new culture vials.
Using this modified chill anesthetizing method, the flies are kept immobile on the ice pack, and the medical gauze that absorbs the condensing water keeps the flies dry and allows phenotyping or handling of the chilled flies with ease. At room temperature of 25 degrees Celsius, the temperature of the surface of a chilled, hard ice pack increases dramatically, from minus 19 degrees Celsius to minus two degrees Celsius, within 20 minutes, quickly reaching a plateau that is safe for both old and newly-hatched flies. Microwave dielectric heating is a faster, more convenient agent for killing adult flies compared to over-etherizing or deep freezing.
Similar to over-etherized flies, microwaved flies extend their wings at right angles from the body. Generally, fly carcasses killed by microwaving are significantly lighter in weight than those killed by ether or freezing, but the heat does not distort the body shape, and the carcasses do not become crisp or turgid. Be sure to squeeze as much water out of the moistened gauze as possible before pressing it onto the ice pack, and to make sure that the gauze clings closely to the ice pack.
It is also important to wipe the condensed water off the chilled, empty vial before you transfer flies into the vial.
