Bottles or vials are tapped on the pounding pad to shake flies away from the plug, the plug is rapidly removed and the old culture inverted over a fresh bottle or vial. Flies are tapped into the new vessel, or some shaken back into the old one, as necessary, and the two are rapidly separated and replugged. Good tossing technique combined with plugs that are easily removed and replaced result in very few escapees. You will learn from experience which stocks require a medium or large inoculation of adults and which do better with only a few. 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.
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 o C buys more time than 22 o C, for example, but a significant number of genotypes fail to thrive at 18 o C, and mold can be a serious problem.