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Method prepared by Grace Panganiban: gepangan@facstaff.wisc.edu


Panganiban lab Drosophila husbandry guidelines

General practices
The life cycle of the fly is 10 days at 25oC and 21 days at 18oC.
Fly stocks should be turned over every 14-17 days if they're being reared at room temperature, and every 30 days if they're being reared at 18oC.

When brooding the lab stock collection, wipe the bench and pounding pad with 70% ethanol before and after you turn over the flies. To avoid cross contamination between stocks, please do not set the rayon/cotton balls down on the bench while you're pounding the vials. If you drop the rayon/cotton on the floor, throw it away and get a clean piece. If there is visible debris in the trays, please wipe them out with 70% ethanol.

Females for crosses must be virgins. Females remain virgins for ~6 hrs at 25oC, and for ~18 hrs at 18oC. Virgins can be most efficiently collected by use of a twice a day procedure in which vials/bottles are cleared in the morning, placed at at 25oC for 6 hrs, the females (who should all be virgins) are collected and the vials/bottles are placed at 18oC overnight, and females/virgins are collected again the following morning. If you have missed a collection, or you need only a few virgins for a particular cross, virgins can be identified by the presence of a dark spot in their abdomens.

When setting up crosses, use ~5 females, and 2-5 males/vial or 30 females and 10-20 males/bottle. To maximize the number of progeny, crosses should be brooded every 2-4 days, and a few yeast granules should be sprinkled into each new vial/bottle.

Mites
The single biggest threat to Drosophila stocks is a mite infestation. While some mites eat only the flies' food, others eat embryos and pupae, and can completely wipe out a fly lab. Mites are very small, and therefore difficult to detect. The adults are smaller than Drosophila embryos, and can most easily be seen (under the dissecting microscope) walking on empty Drosophila pupal cases inside old vials. Mite embryos are even tinier, and often are found in pearly strands of 10-20 eggs on the Drosophila pupal cases.

Mites love the detritus that accumulates in fly trays and incubators, so keeping things clean is essential. Since mites and their eggs can be killed by ethanol, wiping down your dissecting scope, bench, CO2 pad, and brushes with 70% ethanol before or after you handle your flies is always a good idea. The mite life cycle is slightly longer than that of the fly, so if flies are turned over as soon as they eclose, mites will be unable to overcome a stock. The best insurance against mites is: a) to quarantine incoming stocks; and b) to turn over fly stocks regularly.

If you find mites in a stock, you should immediately discard the vial somewhere outside of the lab, wipe down everything with which the vial has come in contact with 70% ethanol, and carefully check all the stocks housed near it for mites. If you need to rescue a mite infested stock, you can select 2-3 females and 1-2 males from it, making sure their bodies aren't carrying any mites or mite eggs, and place them in a clean vial in quarantine.

Quarantine
To guard against mites, flies coming into the lab from other labs or stock centers should be quarantined for 6 weeks in an office or room where no other flies are being maintained. Quarantine procedure:
a) write the date on the original vial (and keep it till the 6 weeks are up);
b) turn the flies over every 14-17 days;
c) periodically inspect the pupal cases in the original vial under a dissecting microscope, looking for adult mites and/or mite eggs;
d) wipe down the microscope and the bench around it with 70% ethanol;
e) at the end of 6 weeks, carefully inspect the original vial and some of the more recent ones for adult mites and/or mite eggs; if none are seen, the stock can be brought into the lab.
f) if mites are detected, you can rescue the stock as described above, but will need to keep the flies in quarantine for 6 weeks following the attempted rescue, and to check them carefully at the end of the six weeks.

If you are anxious to work with a stock that is in quarantine, you can set up your crosses in the quarantine room. If at the end of 6 wks, the original stock vial is mite free, you can move your entire experiment into the lab/incubators.