Differential Dynamic Microscopy Studies of Bacterial Motility

Abstract

This thesis investigates the use of Differential Dynamic Microscopy (DDM) to measure bacterial motility, aiming to understand its advantages and limitations and to investigate its applications in microbial identification and in studying bacterial responses to environmental factors. The specific objectives are: (1) to optimize DDM for accurate and high-throughput measurement of bacterial motility; (2) to assess the ability of DDM to distinguish between different bacterial strains and species, including non-motile ones, based on their motility characteristics; (3) to investigate the effects of hypergravity on bacterial motility; and (4) to examine how antibiotics influence bacterial motility over time. The research is presented in several stages that correspond to these objectives. First, the inherent biological variability between samples prepared from the same strain is investigated. It is found that variability can develop during the growth process, requiring careful normalisation before comparing samples batch to batch. Furthermore, it looks into the effects of time and concentration. The study found that oxygen depletion reduces motility in sealed samples. Bacteria fresh from the flask maintain their motility for approximately 2 hours, which is the upper limit for each batch. Higher bacterial concentrations result in reduced swimming speed, diffusivity, and variability due to increased cell-cell interactions. Second, DDM is used to study the differences in motility between different strains of E. coli (BAA-2523, 43888, and DH52), and different bacterial species, including motile species (Pseudomonas aeruginosa; Enterobacter cloacae; Bacillus cereus) and a non-motile species (Staphylococcus epidermidis). DDM was able to distinguish between different strains and species and revealed variations in their motility patterns. Third, DDM was used to understand the effect of hypergravity on Enterobacter cloacae motility. It was found that exposure to 3Xg hypergravity lead to a suppression of swimming speed, but that further increases in hypergravity had little further effect. Fourth, the impact of antibiotics on bacterial motility was studied. The addition of sub-lethal concentrations of tetracycline caused a systematic reduction in motility over time, without destroying the bacteria. This detailed examination validates DDM's efficacy as a valuable tool in microbiological research and shows that it could be used more broadly in clinical diagnostics and the study of bacterial responses to environmental stresses.