The effect of substratum topography on bacterial surface colonization was studied using a chemically homogeneous silicon coupon. "Grooves" 10 µm deep and 10, 20, 30, and 40 µm wide were etched on the coupon perpendicular to the direction of flow. Flow (Re = 5.5) of a bacterial suspension (10(8) cells/ml) was directed through a parallel plate flow chamber inverted on a confocal microscope. Images were collected in real time to obtain rate and endpoint colonization data for each of three strains of bacteria: Pseudomonas aeruginosa and motile and nonmotile Pseudomonas fluorescens. A higher velocity experiment (Re = 16.6) and an abiotic control using hydrophilic, negatively charged microspheres were also performed. Using a colloidal deposition expression, the initial rates of attachment were compared. P. aeruginosa attached at a higher rate than P. fluorescens mot+ which attached at a higher rate than P. fluorescens mot-. For all bacteria the rate was independent of groove size and was greatest on the downstream edges of the grooves. Only the motile organisms were found in the bottoms of the grooves. A higher fluid velocity resulted in an increase in the initial rate of attachment. In contrast, there was no adhesion of the beads. Attachment of the bacteria appears to be predominated by transport from the bulk phase to the substratum. Copyright 1998 Academic Press.