Elimination of AH133 BLS is due to the bactericidal effect of PAO1. PAO1/p7605 (red) gradually eliminates previously-formed AH133 (green) BLS. ASM+ was inoculated with AH133 and the cultures were grown for 8 h to allow for the partial development of AH133 BLS. (A) One culture was continued without addition of PAO1 for a total of 64 h. (B) The other culture was inoculated with PAO1/p7605 (starting density similar to that used to initiate the AH133 culture). Incubation was continued for an additional 56 h and cultures were analyzed at 8- to 16-h intervals by CLSM (exact time points indicated above and below micrographs). (C) PAO1 is bactericidal to AH133. Two sets of wells containing ASM+ were inoculated with AH133 and incubated for 8 h. PAO1/pMP7605 was added to one set of wells and incubation of both sets was continued for 56 h. At the specified time points, the gelatinous mass was obtained and the CFU/ml of each species was determined using selective media (Methods). White bars: AH133 CFU/ml in single culture; green bars, CFU/ml of AH133 in the co-culture; red bars, CFU/ml of PAO1/pMP7605 from the co-culture. Values represent the means of at least three independent experiments ± SEM.