The bacterium Pseudomonas aeruginosa is ubiquitous in the environment and is also an opportunistic human pathogen. A hallmark characteristic of P. aeruginosa is its ability to readily develop attached-growth biofilms on many surfaces under various growth conditions. During colonization of a surface, initial biofilm development for P. aeruginosa is aided by surface motility mediated by both type IV pili (TFP) and flagella. Understanding the environmental cues that influence motility could provide insight into biofilm formation. This thesis examines how cations act as environmental signals to affect P. aeruginosa bacterial TFP motility in a concentration dependent manner.  Low levels of calcium, magnesium, iron, potassium, or sodium stimulated an increase in P. aeruginosa TFP surface motility at the single cell level. More specifically, there was an increase in the TFP motility mode called crawling, mean square displacement, and mean velocity. This TFP-specific behavior is further exaggerated in the ΔfliM mutant, as this flagellar mutant is strictly TFP-dependent. Calcium starvation was found to affect the resultant biofilm phenotype as homogeneous flat biofilms formed under calcium starvation as compared to the structured architecture of biofilms that formed under ample calcium conditions. The influence of these cations upon TFP motility at the community level as determined using standard twitch and chemotactic twitch assays was unclear, suggesting that the effect of cations on TFP motility are complicated and dependent upon multiple factors.  The affect of calcium starvation on TFP was further characterized since calcium has direct links to virulence and TFP. Several types of experimental approaches were utilized in an effort to pinpoint a specific gene and/or regulatory mechanism that would link calcium response with TFP action. There is a clear population effect under calcium-starved conditions as less TFP-dependent motility is observed at high cell density. Swarming motility, which is a flagellar motility influenced by TFP, is reduced under calcium-starved conditions. This calcium effect was determined not to be related to acyl homoserine lactone quorum sensing, rhamnolipid production, or the TFP retraction gene pilU.  In separate experiments, methods were developed to allow chemical analysis of P. aeruginosa biofilms. These methods allowed for a combination of confocal Raman Microspectroscopy (CRM) and matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) to analyze biofilms. A method was developed to enable visualization of individual twitching cells with confocal fluorescence microscopy using a plate complex that has been used to observe TFP-dependent social motility patterns of Myxococcus xanthus. This new approach enables researchers to better characterize twitching and its role in biofilm formation.