Bdellovibrio bacteriovorus is a famously fast, flagellate predatory bacterium, preying upon Gram-negative bacteria in liquids; how it interacts with prey on surfaces such as in medical biofilms is unknown. Here we report that Bdellovibrio bacteria "scout" for prey bacteria on solid surfaces, using slow gliding motility that is present in flagellum-negative and pilus-negative strains.Surface-associated, Gram-negative bacterial biofilms are an increasing health problem along with resistance to commonly prescribed antibiotics (7). The small, naturally predatory, soil deltaproteobacterium Bdellovibrio bacteriovorus preys upon and kills a wide range of such pathogens in liquids and on biofilms (1). Bdellovibrio bacteria are famously fast and flagellate in liquids, swimming at high speeds (60 to 160 m/s) by rotating a single polar flagellum, using chemotaxis to locate regions rich in prey bacteria (5,9,20). We showed previously that although flagellum-based swimming allows encounters with prey-rich regions, it is nonessential for prey entry once prey are encountered and cannot account for surface interactions with prey in biofilms where liquid is scarce (9). Prey bacteria are invaded in a process that involves type IV pili; Bdellovibrio bacteria pass through the outer membrane, which is then resealed (2, 10). They develop internally in the periplasm of the prey, sequentially degrading prey macromolecules, killing prey in a few minutes and digesting their contents over 2 to 4 h (16, 17). Escape of progeny Bdellovibrio cells from exhausted prey has been presumed to involve both lytic enzymes and flagellar rotation (13).To study interactions between Bdellovibrio bacteriovorus HD100 and prey on surfaces, we applied 10-l samples of predatory Bdellovibrio bacteriovorus HD100 and mutant strains (⌬pilA, ⌬fliC3, and mreB monomeric teal fluorescent protein [mTFP] tagged) mixed with Escherichia coli prey bacteria on solid 1% agarose pad surfaces in 2 mM CaCl 2 -25 mM HEPES (pH 7.6) buffer, and microscopic images were acquired over several hours at room temperature every 150 s as described previously (3). The fliC3 mutant was constructed as described elsewhere (9), and the mreB mTFP fusions were constructed as described elsewhere (4). Silent, in-frame deletions of the pilA and Bd0416 genes were constructed by a modification of the method of Steyert and Pineiro (19).The microscopic images were then encoded into time-lapse movies at 7 frames per second (fps). Speeds of moving Bdellovibrio bacteria were measured by determining the path of specific cells using the montage function of the SimplePCI imaging software program (Compix Inc. Imaging Systems), and then the measuring tool and time signatures were used to acquire speeds. Standard deviations of speeds are shown in Table 1, and the Student t test was used to determine significance of differences using the Microsoft Excel software program.When Bdellovibrio progeny lysed their digested prey and emerged to other immobilized prey cells on the agarose pad, they were unable to swim to...