Alternatively, clean cultures can be established from embryos dechorionated with 5.25% sodium hypochlorite (liquid household bleach, full strength). A convenient method is to transfer eggs to a bleach soaked wedge of filter paper, wick away bleach after chorions have dissolved (3-5 minutes), wash eggs several times with water and transfer to a fresh piece of filter paper (small enough to sit on the surface of the medium in a vial) moistened with water. Place the filter paper with eggs into a fresh vial of food and place a larger strip of filter paper along the wall of the vial. Wet this strip of paper to maintain high humidity in the vial until the eggs hatch.
Experimental populations
1. Matings
While mass transfer of adults works well for most stockkeeping purposes, it often results in overcrowded cultures. Overcrowding can effect the outcome of crosses and experimental procedures. Development time is slowed, different genotypes may be disproportionately affected by competition for food and pupation sites, and many pupae and adults will drown in the soup of larvae and liquified medium. The best yield of healthy adults is obtained from cultures established with an optimum number of animals. Expect 50-100 adults from a vigorous 8 dram vial culture, 300-600 from a comparable half-pint bottle culture. For most genotypes the optimum number of females will range from 1 to 3 per vial and 5 to 20 per bottle. Set up a few test bottles or vials to determine this number empirically for the genotypes involved; control the age of the food, the age of the females, and the number of days the females are left in the vials. One or two males are usually sufficient to rapidly inseminate several females, but some genotypes will require an equal or excess number of males to insure rapid mating. If necessary, the effects of overcrowding can be reduced mid-culture by adding baker's yeast and a tissue (such as a Kimwipe®) to provide additional nutrition and pupation sites, respectively, for the excess larvae. Extremely crowded cultures are best dealt with by distributing larvae (scoop them out with a spatula) to several fresh bottles or vials.
If you cannot distinguish parent from progeny by phenotype, parents should be discarded before the progeny begin to hatch. Experimental crosses maintained at 25°C should be discarded after 18 days to prevent recovery of second generation progeny. An effective schedule is to establish crosses on day 0 (start on a Friday if you want to begin virgin collection on a Monday), discard adults and add yeast and papers (optional) on day 7, collect virgins or score progeny on days 10 through 18.
Some mutant phenotypes are affected by temperature or genetic background. Before setting up a large scale effort such as a screen, make a test cross of the relevant genotypes under the conditions to be used and confirm that all phenotypes are scorable. It is also prudent to assure the absence of 'background' lethals in a stock to be used for mutagenesis by isogenizing a chromosome for use in a screen. To isogenize a ri e chromosome, for example, cross to an appropriate balancer stock, recover 10-20 progeny heterozygous for ri e and the balancer, backcross them individually to the balancer strain, cross sibling ri e/balancer progeny, and then recover ri e homozygotes from one of the lines and establish a stock. Only lines carrying lethal-free chromosomes will produce homozygotes among the progeny of the sib matings.
2. Virgins
Most experimental schemes require virgin females. D. melanogaster adults do not mate for about 10 hours after eclosion, allowing virgins to be collected within 8-12 hours after the culture has been cleared of adults. The timing of this virgin window appears to be genotype dependent. Collect females within 8 hours to be safe, or determine empirically for a given strain how long you can wait and still recover virgins. Ashburner (1989) describes a variety of environmental and genetic tricks to facilitate the collection of large numbers of virgin females.
For many mating schemes virginity is desirable for efficiency's sake, but not essential because the progeny of non-virgin females can be distinguished phenotypically from the progeny of interest. If your scheme requires virginity (e.g., male fertility testing, or the progeny of non-virgin females are indistinguishable from those of the intended mating), hold females for 3-4 days and check for larvae in the holding vial before using the females in matings. Don't overcrowd females in holding vials - 50 or so in an 8 dram vial, fewer if you aren't sure of their virginity (you'll have to discard all of the females in the holding vial if any have mated). The peak of female fertility is genotype-dependent, but on average females are best used between 4 and 10 days old.
