Conical tubes containing 5 ml of 0.2% TTB (triclosan test broth; tryptic soy broth (Difco) supplemented with 2 g l31 triclosan) were inoculated with 3U106 cells of an overnight culture of A. xylosoxidans TR1, P. putida TriRY, or E. coli K12, and incubated with shaking at 225 rpm and 24³C. Periodically, tubes were removed and centrifuged at 6000 rpm for 5 min. Ethanol (3 ml) was added to the pellets ; the samples were vortexed for 30 s, and centrifuged for an additional 5 min