Anesthetization of flies is required when sorting out by sex or by genotype, and also when preparing tissue homogenate. Common methods are ether and carbon dioxide, but cooling can also be used in some cases.

Although the carbon dioxide method requires setting up of some devices (see Roberts and Standen, 1998), it has a number of advantages over the ether (ethyl ether or triethylamine) method; it is much less likely to kill or sterilize Drosophila. Additionally it is odorless and neither flammable nor reactive. With carbon dioxide, use of the diffusion pad enables application of continuous anesthetization. Flies can be kept unconscious for up to 20 minutes under the carbon dioxide air and still recover quickly once the carbon dioxide is removed, although too strong or too long exposure to carbon dioxide can kill flies. On the other hand, the effects of ether will wear off in less than 10 minutes. Thus, carbon dioxide is a preferred choice.

While they are anesthetized, flies can be placed on a glass plate or carbon dioxide plate to be examined under the microscope. The flies are moved around using a small paintbrush on the plate, and care must be taken not to damage the wings and so forth.

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