Culture contaminants
Drosophila is relatively pestilence-free, but mites, fungi and bacteria can be problems in laboratory cultures. It is good practice to clean your bench top and fly pushing equipment regularly. This is particularly important if a problem is evident. Clean the bench top and all equipment that comes into contact with potentially contaminated stocks with 10% bleach, 70% ethanol or soap and water after use. Sharing pounding pads, CO2 pads, fly pushers and sorting plates can aid the spread of contaminants. If sharing is unavoidable, the need for cleanliness should be understood by all and enforced.
1. Mites
"I have mites" is not an admission you want to have to make to your fly colleagues (but you must make it, if true). The most dangerous species are egg predators, but even those that simply feed on the medium can out-compete weak genotypes and compromise experimental observations. Frequent stock transfer, tight plugs, and zero mite-tolerance by all the fly workers in a building are the best defenses. In general, cultures grown at 24-25°C should never be kept for more than 30 days. If mites are known to be a problem in your lab or building, cultures should be checked and discarded after 18 to 20 days. Lining stock trays with benzyl benzoate-treated cheese cloth (soak cloth in 10% benzyl benzoate in 95% ethanol, air dry; replace the cloths every 6 months) may help prevent infestation. Some kinds of plastic are dissolved by benzyl benzoate, so test first if you use plastic vials or trays (the cloth will fuse with the plastic within 24 hours) and protect paper items such as stock tags from direct contact with the cloths.
To prevent the importation of mites from outside sources all stocks new to your lab should be quarantined for at least two generations. Never open a foreign bottle or vial at your fly bench (or your neighbor's) without first inspecting the culture for mites. Using a microscope, examine the surface of the medium and the walls of the container, especially around pupae or pupal cases. If no mites are evident, replace foam or paper stoppers with tight cotton plugs and isolate cultures in a quarantine tray. As an added precaution, cultures can be wrapped in the mite cloths described above. Keep the original bottle or vial for about 20 days, even though you have established fresh cultures, rechecking for mites every 5 to 10 days. We check the new cultures too, just to be safe, but we have never found mites in a subculture when the parental vial was mite free.
Any culture found to contain mites should be frozen or autoclaved immediately if it can be replaced from a mite-free source. If replacement is not possible, use one of the methods described in Ashburner (1989) to disinfect the culture, such as daily transfer of adults for about a week, using only the final transfer to establish a new and mite-free, it is hoped, culture. Keep infected cultures wrapped in mite cloths until they have been mite free for three generations.
2. Fungi and Bacteria
If mold is a problem in isolated cultures it can usually be eliminated by daily transfer of adults for 7-10 days. Visually inspect cultures from the later transfers for hyphae (look around the pupal cases) and use one that appears to be free of fungal growth for further subculture. In extreme cases this process may need to be repeated for an additional generation. If fungal contamination is a wide spread problem be sure that fungal inhibitor (p-hydroxy-benzoic acid methyl ester) is being added to the medium after it is cooked (boiling destroys the inhibitor), add a small amount of live baker's yeast to every culture (the yeast tends to inhibit the growth of unwanted fungi) and check for sources of infection in the lab, such as incubator fan housings. Clean any contaminated or suspicious areas with disinfectant.
A variety of bacterial contaminants can occur in fly cultures. The most common problems are caused by mucus producing bacteria. Although not directly toxic, larvae, and to some extent adults, become trapped in the heavy layer of mucus that coats the surface of the food. Large numbers of larvae overcome the effects of the bacteria in a healthy stock, but weak stocks or pair matings can be seriously compromised. A widespread bacterial problem may indicate that the pH of your medium is too high; try lowering the pH to about 5. Individual stocks can be treated with antibiotics for one generation. A quick approach that often works: add 100 µl of penicillin-streptomycin solution (10,000 u/ml and 10,000 µg/ml, respectively) to the surface of the medium in a vial and allow it be absorbed. Add a small amount of yeast and transfer flies to the treated medium. Discard adults before progeny eclose; subculture progeny on untreated medium. Other antibiotics may be tried if the contaminant proves to be resistant to penicillin and streptomycin.
