Drosophila Intestinal colonization assay

Triplicates of 20 female Oregon-R flies, 5–7-days-old, are fed for 2 days on an ‘infection mix’ added on an autoclaved cotton ball (Genesee Scientific Cat.#51–101) placed at the bottom of a fly vial (Genesee Scientific Cat.#32–120). Each ‘infection mix’ contained 50 μl of bacterial culture in LB medium at OD600nm=2, added in 5ml of 4% sucrose. The flies are then incubated at 25oC. Infected flies are transferred into 50-ml Falcon tubes perforated eight times along their length with a heated 0.9-mm needle to enable aeration, while the cup was perforated four times with a heated 1.2-mm needle and covered with a 2.3-cm whatman disc (Fisher #09-820AA). Sucrose solution (4%, 0.2 ml) with or without drugs are added onto the whatman disc and covered with parafilm to provide food to the flies while reducing contamination of the food by the flies. New fly tubes were prepared daily and flies were incubated at 25oC. To assess bacterial load inside the flies, live flies were briefly immersed in 95% EtOH, dried on a sterile napkin, ground with a 1.5-ml tube pestle and plated onto LB agar. Only live flies were used because dead flies desiccate within hours and bacterial load is gradually eliminated.