PhD Thesis Presentation: Quantifying Bacterial Motion in Twitching Colonies and the Effect of the Agar-Glass Interface on Bacterial Twitching Motility

PhD Candidate

Erin Shelton

Abstract

We have used bright-field optical microscopy, together with a custom-built, temperature- and humidity-controlled environmental chamber, to study the growth f colonies of Pseudomonas aeruginosa PA01 due to twitching motility driven by type IV pili. The advancing front of the colonies consisted of finger-like protrusions (fingers) containing many bacterial cells, with the cells within the expanding colony moving within a lattice-like pattern. We studied the expansion of twtiching colonies at the interface between agar and glass for a range of agar concentrations \(\mathrm { 1.0 \% \; w/v < CC < 1.9 \% \; w/v,}\) and we interpreted the microscopy results by characterizing the adhesion and local agar concentration at the interface using micropipette deflection and Attenuated Total Relectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy. To analyze the collective morphology and dynamics of the fingers, we used a combination of custom particle image velocimetry and Fourier analysis techniques. For agar concentrations below \(\mathrm { CC\;^<_- \; 1.5 \% \; w/v,}\) the average finger width and the density of the lattice region increased with increasing \(\mathrm{CC,}\) whereas the average edge speed remained constant. We observed a transition at \(\mathrm {CC \sim 1.6\% \; w/v}\) in which the average edge speed dropped significantly while the average finger width remained constant. For \(\mathrm {CC > 1.7\% \; w/v,}\) all measured quantities remained constant and the colonies were visually indistinguishable. We attribute this transition to a corresponding increase in teh agar-glass adhesion that occured because of an enhanced local concentration of agarose helices at the agar-glass interface at large agar concentrations.

During the outward expansion of fingers along the interface, cells can vertically displace the agar to form multilayered regions. We observed a transition from monolayer to stable multilayer coverage within fingers at  \(\mathrm {CC = 1.5\% \; w/v.}\)  We studied this transition by characterizing multilayer formation and dissolution, and transient and stable multilayer regions with fingers. We observed that a minimum finger width was required for multilayer stability, and we described the dependence of multilayer on finger width using a simple nucleation model. 

Examination Committee 

• Dr. Hermann Eberl, Chair
• Dr. John Dutcher, Advisor
• Dr. Robert Wickham, Advisory Committee
• Dr. Leonid Brown, Graduate Faculty
• Dr. Benjamin Hatton, External Examiner (University of Toronto)