Motivated by observations of cell orientation at biofilm–substrate interfaces and reports that cell orientation and adhesion play important roles in biofilm evolution and function, this thesis investigated the influence of surface chemistry on the orientation of Escherichia coli cells captured from flow onto surfaces that were cationic, hydrophobic, or anionic. We characterized the initial orientations of nonmotile cells captured from gentle shear relative to the surface and flow directions. The broad distribution of captured cell orientations observed on cationic surfaces suggests that rapid electrostatic capture of cells to oppositely charged surfaces preserve the instantaneous orientations of cells as they rotate in the near-surface shearing flow. By contrast, on hydrophobic and anionic surfaces, cells were oriented slightly more in the plane of the surface and in the flow direction compared with that on the cationic surface. This suggests slower development of adhesion at hydrophobic and anionic surfaces, allowing cells to tip toward the surface as they adhere. Once cells were captured, the flow was increased by 20-fold. Cells did not reorient substantially on the cationic surface, suggesting a strong cell–surface bonding. By contrast, on hydrophobic and anionic surfaces, increased shear forced cells to tip toward the surface and align in the flow direction, a process that was reversible upon reducing the shear. These findings suggest mechanisms by which surface chemistry may play a role in the evolving structure and function of microbial communities. The viability and growth of captured Escherichia coli cells on cationic and hydrophobic surfaces were tested. This thesis confirms cells can grow on both cationic and hydrophobic surfaces, especially for cationic surfaces which contrast to fact that cationic surfaces are often used as bactericide. When bacteria adhere to surfaces, the chemical and mechanical character of the cell-substrate interface guides cell function and the development of microcolonies and biofilms. Alternately on bactericidal surfaces, intimate contact is critical to biofilm prevention. The direct study of the buried cell-substrate interfaces at the heart of these behaviors is hindered by the small bacterial cell size and inaccessibility of the contact region. Here, this thesis presents a total internal reflectance fluorescence depletion approach to measure the size of the cell-substrate contact region and quantify the gap separation and curvature near the contact zone, providing an assessment of the shapes of the near-surface undersides of adhered bacterial cells. Resolution of the gap height is about 10%, down to a few nanometers at contact. Using 1 and 2 µm silica spheres as calibration standards we report that, for flagella-free Escherichia coli (E. coli ) adhering on a cationic poly-L-lysine layer, the cell-surface contact and apparent cell deformation vary with adsorbed cell configuration. Most cells adhere by their ends, achieving small contact areas of 0.15 µm2, corresponding to about 1-2% of the cell’s surface. The altered Gaussian curvatures of end-adhered cells suggest the flattening of the envelope within the small contact region. When cells adhere by their sides, the contact area is larger, in the range 0.3-1.1 µm2 and comprising up to ∼12% of the cell’s total surface. A region of sharper curvature, greater than that of the cells’ original spherocylindrical shape, borders the flat contact region in cases of side-on or end-on cell adhesion, suggesting envelope stress. From the measured curvatures, precise stress distributions over the cell surface could be calculated in future studies that incorporate knowledge of envelope moduli. Overall, the small contact areas of end-adhered cells may be a limiting factor for antimicrobial surfaces that kill on contact rather than releasing bactericide. Furthermore, this thesis studied the swimming patterns of bacteria in quiescent condition which is the pre-step to investigate bacterial swimming in flow near surfaces. Bacterial swimming in flow near surfaces is critical to the spread of infection and device colonization. Understanding how material properties affect flagella- and motility-dependent bacteria surface interactions is a first step in designing new medical devices that mitigate the risk of infection. In this part, the thesis reported statistics of motile swimmers and no-motor, nonmotile E. coli cells in the quiescent bulk solution. The run and tumble character of the motile swimmers was confirmed by particle tracking. The swimming velocities were consistent for multiple run phases of each cell, but the swimming speed itself was cell dependent, with some cells swimming faster than others, mostly in the range 3-6 µm/s. Molly Shave further reported the run and engagement motion pattern of motile swimmers in steady flow near the interfaces which driven by tumbling via flagellar unbundling.