P. aeruginosa is commonly associated with infections in individuals with compromised host-defenses. Critical to persistence within the hostile host environment is the ability to form a biofilm, consisting of a microbial community enmeshed in extracellular polymeric substances. Much of our understanding regarding biofilm has been acquired via confocal microscopic monitoring of flow cell cultures. However, the relevance of these in vitro studies to the complex host environment remains uncertain due to the lack of a vertebral animal model amenable to such detailed visual analyses. This thesis describes the development of a larval zebrafish hindbrain ventricle infection model for the analysis of biofilm formation in vivo. Infection with wild-type P. aeruginosa PAO1 was compared with that of a psl mutant (Δpsl promoter), incapable of producing the critical biofilm exopolysaccharide Psl. Δpsl PAO1 was found to be less fatal, to produce fewer and smaller microcolonies, and to persist poorly throughout infection in comparison to the wild type. The unprecedented microcolony quantification achieved herein is dependent on the optical transparency of the zebrafish, thus demonstrating the power of the zebrafish as a tool for in vivo microcolony analyses.