Study of bacterial growth and adhesion in environments subjected to flow or surface vibration

Abstract

This thesis presents investigations into the adhesion of E.coli cells and the growth of such cells into colonies on substrates that are subject to external mechanical perturbations. The first perturbation we introduce is in the form of an applied flow through a microfluidic channel that contains adhered E.coli cells. We show that when the types of appendages present at the cell membrane vary, the flow rate at which the cells become dislodged changes, indicating a change in the strength of the adhesion of the cells to the substrate. This allows for a maximum drag force that cells can withstand before detaching for each E.coli strain to be quantified. We also investigate how the elasticity of the substrate of the microfluidic device plays a role in the adhesion of the cells to the surface. This is achieved by altering the elastomer ratio of the device material, PDMS, and comparing maximum drag forces of the cells for different ratios. This allows for insight into how the stiffness of a surface affects the adhesion of cells, and we ultimately show that a more deformable substrate favours prolonged attachment of cells. The dependence on the cell's orientation with respect to the direction of flow is investigated and linked to the likelihood that a cell will remain attached when exposed to an increasing applied flow rate. Using an agar microfluidic device we further investigate, the role cell appendages play in surface adhesion when bacteria are subjected to flow. We next describe the design of an experiment in which an agar plate is periodically deformed, at frequencies in the range 5-50 Hz, by coupling it to a loudspeaker cone. Colonies grown on the deformed plates were found to exhibit two main effects that differ from control colonies grown on static plates. These effects were: an increase in colony perimeter roughness and an increase in the final diameter of the colony after a fixed period of growth. It was shown that these effects increased with increasing driving frequency. In addition to the acceleration which accompanies the vibration, when the boundary of the plate is fixed there is an induced elastic strain field that varies across the plate. Work was carried out to quantify the effect of the strain and acceleration on colony perimeter roughness and final diameter and attempt to relate each one separately to the observed effects. It was determined that the presence of the strain field was related to the increase in diameter whilst the large increase in acceleration resulted in rougher colonies forming. We hypothesise that both of these effects contribute to a modification of the forces on the bacteria that allow cells at the perimeter of the colony to switch their relative position and orientation which then leads to an increase in colony perimeter roughness and diameter. When colonies formed from two bacterial strains (fluorescent/non-fluorescent) it was observed that the peak sector size remains the same but the distribution of sectors angular size broadens, so that there is an increase in finding larger sectors. Finally, this thesis closes by detailing the development of an in-situ microscope to directly observe bacterial colonies on a deformed agar plate. The microscope was used to quantify the velocity at which the colony perimeter advances and the width of the growing layer. This work was expanded to measure these parameters on agar plates with different elastic properties. It was shown that as the agar becomes stiffer, the expansion velocity of the growing front decreases and with this the total roughness of the colonies also decreases. This was explained as the increasing stiffness of the agar results in a stronger polymer network being formed that offers greater resistance to the applied deformation, reducing the strain field, which results in a decrease in the radial expansion velocity. We also measured the height profiled of colonies grown on static and deformed plates. The observed behaviour is found to be consistent with a computational model in which decreasing the friction coefficient between the cell and the substrate leads to an increase in the colony diameter.