The frequency with which new subcultures need to be established depends on the health and fecundity of the genotype, the temperature at which it is raised, and the density of the cultures. Temperature has a large effect on the rate of Drosophila development. Generation time (from egg to adult) is approximately: 7 days at 29°C, 9 days at 25°C, 11 days at 22°C, 19 days at 18°C. For most purposes stocks are maintained by live culture, transferring adults to fresh medium every few generations. Stocks kept at room temperature should be transferred to fresh food every 20 to 30 days. Mites and mold are more likely to be a problem in older stocks, so it is good policy to set 30 days as an absolute upper limit for room temperature stocks. This period can be extended by keeping stocks at lower temperature. 18°C buys more time than 22°C, for example, but a significant number of genotypes fail to thrive at 18°C, and mold can be a serious problem. It is wise to keep a room temperature backup of stocks to be maintained at low temperature for the first two or three transfers in case the stocks do poorly. If the quality of your fly food is unreliable it is wise to have at least two cultures for each stock, staggered to assure the use of different batches of medium (at least until you find a new cook).
Cryopreservation of ovaries (see Ashburner, 1989) or embryos (Cole et al., 1993; Steponkus and Caldwell, 1993) are viable alternatives to continuous culture for some purposes. Genotypes that are unstable due to reversion, breakdown, or accumulation of modifiers, especially those with non-visible phenotypes that are time consuming to select, are good candidates for freezing. Also, if you are generating hundreds of stocks that will not be in use but must be kept for many years it might be cost-effective to maintain these as frozen stocks. For most routine stockkeeping purposes, however, live culture remains the preferred route.
Identify stocks with tags showing the complete genotype of the stock, sans shorthand. Writing the genotype on the vial or bottle at each transfer invites transcription errors and takes longer than moving a tag. Don't use a stock center stock number or other potentially cryptic symbol as the only identifier of a stock. Stocks are often kept for many years and what is obvious to you now may be meaningless even to you in a few years, and is easily misinterpreted by someone inheriting your stocks. Unless you are careful to maintain complete stock data elsewhere, record all relevant information on the tag.
2. Balanced stocks and balancers
In most cases, balanced lethal schemes work only if one of the lethal chromosomes is itself a balancer chromosome. Recombination between lethal (or sterile) mutations on different homologues can produce one homologue with both mutations and one wild-type homologue. The wild-type chromosome will rapidly predominate or become fixed in the stock. Balancers are structurally rearranged chromosomes that prevent recombination between homologues in females (meiotic recombination is absent in D. melanogaster males and in the tiny 4th chromosome in females). This is accomplished in part by reducing recombination directly and in part by preventing transmission of recombinant chromosomes. The most commonly used balancers carry overlapping sets of inversions and prevent recombination throughout most of the length of the chromosome. Some special purpose balancers work well only for specific regions of a chromosome. Suppression of recombination is less effective when balancers for two or more heterologues are present in a stock.
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.
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
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.
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.
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.
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.