Bacteria grown in bacteria-hydrogel composite materials have distinct clustering and growth behaviors compared to bacteria in biofilms grown without hydrogel confinement. Bacteria-hydrogel composites have applications ranging from studies of living self-healing materials to in vitro infection models. Here we create a living material containing bacteria and alginate hydrogels for studying the colloidal microstructures of bacteria that emerge in response to the mechanics of their microenvironment. We use quantitative image analysis techniques frequently used in colloidal science to measure the size and morphology of bacterial clusters grown in alginate hydrogels with moduli ranging across five orders of magnitude (0.01 – 100 kPa). Bacteria have viable growth within alginate hydrogels after 24 hours across the full range of moduli studied. Bacterial aggregates that form after 24 hours of growth are evenly distributed throughout each hydrogel. Bacterial growth in hydrogels stiffer than bacterial biofilm matrix materials results in the formation of dense aggregates with oblate, spherical morphologies containing > 25 cells per aggregate. The cellular density of bacteria within aggregates formed in the hydrogels is higher than that of bacterial biofilms. As hydrogel stiffness increases, aggregates become more spherical and cellular density increases, while the mean number of cells per aggregate and Euclidean diameter decrease. We discuss the significance of these results for the development of living materials capable of responding to external environment cues as well as the development of high-throughput in vitro models of bacterial infection.
