1. Preparing Tools and Accessories
<ol>
	<li>Tip/funnel stoppers
	<ol>
		<li>Obtain two sponge plugs (the diameter of the plugs must be slightly larger than the internal diameter of the vials used to transfer flies). Make a hole in the centers of the sponge plugs with a heated electric soldering iron.</li>
		<li>Obtain two 1 mL pipette tips, cut one in half transversely with a sharp knife, and discard the pointed end. Then, cut 1.5 cm of the pointed end off the second pipette tip. Glue the remains of t...

<ol>
	<li>Jennings, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+&#8211;+a+versatile+model+in+biology+&#38;+medicine.">Drosophila &#8211; a versatile model in biology &#38; medicine.</a> Materials Today. 14 (5), 190-195 (2011).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology I: yeast, Drosophila and C. elegans. An Introduction to Drosophila melanogaster. , (2018).</li><li>Ashburner, M., Roote, J., Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+Culture+of+Drosophila.">Laboratory Culture of Drosophila.</a> Drosophila Protocols. , (2000).</li><li>Greenspan, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fly+pushing:+The+theory+and+practice+of+Drosophila+genetics.">Fly pushing: The theory and practice of Drosophila genetics.</a> Cold Spring Harbor Laboratory Press. , (2004).</li><li>Ashburner, M., Thompson, J., Ashburner, M., Wright, T. R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+laboratory+culture+of+Drosophila.">The laboratory culture of Drosophila.</a> The genetics and biology of Drosophila. 2a, 1-109 (1978).</li><li>Ratterman, D. M., O&#39;Donnell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eliminating+ether+by+using+ice+for+Drosophila+labs.">Eliminating ether by using ice for Drosophila labs.</a> Tested Studies For Laboratory Teaching. , 259-265 (2003).</li><li>. Culturing techniques for Drosophila Available from: <a target="_blank" href="https://www.ptbeach.com/cms/lib/NJ01000839/Centricity/Domain/113/ap%20biology%20Labs/Culturing%20techniques%20for%20Drosophila.pdf">https://www.ptbeach.com/cms/lib/NJ01000839/Centricity/Domain/113/ap%20biology%20Labs/Culturing%20techniques%20for%20Drosophila.pdf</a> (2019)</li><li>Markow, T. A., O&#39;Grady, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: A Guide to Species Identification and Use. , (2006).</li><li>Artiss, T., Hughes, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+the+Headaches+Out+of+Anesthetizing+Drosophila:+A+Cheap+&#38;+Easy+Method+of+Constructing+Carbon+Dioxide+Staging.">Taking the Headaches Out of Anesthetizing Drosophila: A Cheap &#38; Easy Method of Constructing Carbon Dioxide Staging.</a> The American Biology Teacher. 69 (8), e77-e80 (2007).</li><li>Qu, W. -. H., Zhu, T. -. B., Yang, D. -. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Modified+Cooling+Method+and+its+Application+in+Drosophila+Experiments.">A Modified Cooling Method and its Application in Drosophila Experiments.</a> Journal Of Biological Education. 49 (3), 302-308 (2015).</li><li>. Egg-laying cages for drosophila Available from: <a target="_blank" href="https://www.kisker-biotech.com/frontoffice/product?produitId=0H-19-17">https://www.kisker-biotech.com/frontoffice/product?produitId=0H-19-17</a> (2018)</li><li>Roberts, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: a practical approach. , (1998).</li><li>Tang, M., Peng, Q. -. F., Yang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+devices+for+Drosophila+experiments+(in+Chinese).">Two devices for Drosophila experiments (in Chinese).</a> Bulletin of Biology. 45 (11), 49-50 (2010).</li><li>Zhou, T. -. y., Gan, J., Yang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+sponge+plug+and+sponge+plug+based+fly+transferring+device+for+Drosophila+experiments+(in+Chinese).">Preparation of sponge plug and sponge plug based fly transferring device for Drosophila experiments (in Chinese).</a> Bulletin of Biology. 46 (6), 49-50 (2011).</li><li>Yang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genetics laboratory investigation. , (2016).</li><li>Yang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carnivory+in+the+larvae+of+Drosophila+melanogaster+and+other+Drosophila+species.">Carnivory in the larvae of Drosophila melanogaster and other Drosophila species.</a> Scientific Reports. 8, (2018).</li><li>Stocker, H., Gallant, P., Dahmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+Started:+An+Overview+on+Raising+and+Handling+Drosophila.">Getting Started: An Overview on Raising and Handling Drosophila.</a> Drosophila: Methods and Protocols. , (2008).</li></ol>

Some homemade tools for handling basic activities involved in Drosophila rearing and experimentation are described in this paper. These tools are simple but rather effective. Virtually, any lab can make these tools with ease, and a research or a teaching laboratory does not need to find a ready-made alternative that is perhaps not available locally.
Fly transferring is the most common practice and a difficult task in Drosophila experiments. Unfortunately, until now, there hav...

The fruit fly, Drosophila melanogaster, is a model organism widely used in biological research and biology education to study a wide range of topics1,2. The basic problems of handling Drosophila are the transfer of adults from vial to vial and immobilization of the flies so they are easier to handle, as all adults (except for some mutants3,4) can fly.
Conventionally, a researcher transfers flies from one vial to another by holding two vials mouth-to-mouth, tapping the flies down or allowing flies to fly up into another vial, then separating and replugging both vials4. Obviously, this requires that the opening of two vials with the same diameter, and it is hard to control the quantity of flies transferred. Meanwhile, this requires quick hands to get the job done, and escaping stray flies can result in problems for the laboratory or classroom. Adding extra virgin flies or male flies to an already prepared cross is another routine task in Drosophila experiments. Conventionally, flies must be immobilized in the cross vial before the addition of extra flies.
Adult Drosophila are routinely anaesthetized by ether, CO2, or chilling5. Compared to ether and CO2 exposure, chilling is the most cost-efficient agent for immobilizing adult Drosophila and the least harmful to both the flies and researchers (especially young students)6,7. However, water that condenses continuously on the cold surface or chamber wets the flies. It is difficult to determine the phenotypes of wet flies, and they can easily become damaged during manipulation8,9. This has kept the chilling method from becoming more widely accepted.
Tools for fly transferal and a method for fly cooling have been previously described10. Herein, a modified chilling anesthesia technique is reported that is safe, reliable, and feasible for Drosophila experiments. Also described in this paper are 1) methods for killing adults for counting, sorting, or discarding, 2) labor-saving devices and protocols for cleaning glass culture vials, and 3) a simple cage for collecting eggs. The easily designed and cost-effective tools described here can be used to address the difficult issues of fly handling, and these methods have been tested and are proven to be robust, reliable, and easy-to-handle for experienced and novice researchers.

<table>
	<tbody>
		<tr>
			<td>a pair of pliers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>cordless drill driver</td>
			<td></td>
			<td></td>
			<td>max speed: 500 rpm</td>
		</tr>
		<tr>
			<td>electric soldering iron</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>file</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>funnel</td>
			<td></td>
			<td></td>
			<td>diameter of disk&#60;60mm</td>
		</tr>
		<tr>
			<td>ice box</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>insect pins</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>infrared thermometer</td>
			<td></td>
			<td></td>
			<td>HCIYET HT-830</td>
		</tr>
		<tr>
			<td>long cuff rubber gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mechanical pencils</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>medical gauze</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>microcentrifuge tube</td>
			<td></td>
			<td></td>
			<td>100 ul</td>
		</tr>
		<tr>
			<td>microwave oven</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>peri dish</td>
			<td></td>
			<td></td>
			<td>internal diameter 60 mm</td>
		</tr>
		<tr>
			<td>pipette tips</td>
			<td></td>
			<td></td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>plastic film</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>plastic peri dish</td>
			<td></td>
			<td></td>
			<td>&#934;36 mm used to cover the empty vial</td>
		</tr>
		<tr>
			<td>point tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>protective work gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>re-freezable hard icepacks</td>
			<td></td>
			<td></td>
			<td>26.5&#215;14.5&#215;2.5 cm or larger</td>
		</tr>
		<tr>
			<td>rubber air blower</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>snap cutter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>soft drink bottle</td>
			<td></td>
			<td></td>
			<td>500 ml, internal diameter c.a. 65 mm</td>
		</tr>
		<tr>
			<td>sponge stopper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>stainless steel sponges</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tube brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>vial</td>
			<td></td>
			<td></td>
			<td>&#934;34 mm &#215; 90 mm</td>
		</tr>
	</tbody>
</table>

simple homemade tools to handle fruit flies&#8212;drosophila melanogaster

The fruit fly, Drosophila melanogaster, is widely used both in biological research and biology education. Handling adult flies is common but difficult in practice, as adult flies fly. Demonstrated here is how to make some simple and cost-effective tools to address difficult issues in the handling of Drosophila. Holes in foam stoppers are made and pipette tips or funnels are inserted into the holes. Flies then move only in one direction into the pipette tip/funnel assemblage, allowing efficient control of the transfer of adult Drosophila into or out of a vial. Existing protocols have been modified for cool-anesthetizing flies by chilling in crushed ice and transferring them onto a cold, hard icepack surface. The icepack is covered with a piece of medical gauze that keeps immobilized flies from the condensed water when examined under a stereomicroscope. The flies are finally euthanized for counting and sorting or discarded by microwaving. A bottle-shaped cage has also been developed for collecting eggs, as well as a labor-saving device and accompanying protocol for cleaning glass culture vials.

The T- and F-stoppers were developed as a set of simple tools that can be adapted and used in any fly transferring activities. Transferring flies from an old culture into several fresh cultures involves removing the plugs of the fresh vials, replacing them with F-stoppers, then tapping down the flies in the old vial, quickly removing its plug, and replacing it with a T-stopper. If the old food is compact, then it is important to flip the old vial and insert the tip of T-stopper into the o...

Watch this Scientific Journal Video about Simple Homemade Tools to Handle Fruit FliesÃ¢â‚¬â€Drosophila melanogaster at JoVE.com

Simple Homemade Tools to Handle Fruit Flies&#8212;Drosophila melanogaster

Described here is the use of several homemade tools to transfer, chill, and kill adult Drosophila, as well as to clean glass culture vials and collect eggs. These tools are easy to make and are rather efficient in handling Drosophila.

The fruit fly, Drosophila melanogaster, is widely used both in biological research and biology education. Handling adult flies is common but difficult in ...

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.

Research

JoVE Journal

Biology

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Isolation and Biophysical Study of Fruit Cuticles

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and ...

Fruit Volatile Analysis Using an Electronic Nose

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit ...

Measurement of Lifespan in Drosophila melanogaster

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Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...