Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.798900
Title: Bacterial confinement in micro fluidic micro-environments
Author: Taylor, Daniel
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2019
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Abstract:
Microfluidic droplet systems have shown great promise in the study of biological systems. In this thesis I explore the application of a microfluidic droplet system to the study of small bacterial populations and their growth response to antibiotics. This thesis comprises of three primary results sections. The first section details the development and fabrication of a custom microfluidic system. This microfluidic system is designed to study the growth dynamics of small Escherichia coli (E. coli ) bacterial communities. The second section presents an image processing and analysis workflow that is designed to be used in conjunction with the microfluidic device to extract quantitative growth data of confined bacterial communities with single cell precision. The third section details a study that uses the microfluidic system to measure the growth dynamics of E. coli communities encapsulated within microfluidic droplets whilst uninhibited and under the effect of the antibiotic streptomycin. In the first section, I present the development, design and fabrication of a two-part microfluidic device that is later used to study the growth dynamics of E. coli bacteria. The first part of the microfluidic device consists of droplet generating microfluidic geometry that is designed to encapsulate small communities of E. coli bacteria (typically 1-5 cells in size) within microfluidic droplets around 30μm in diameter. The second part of the microfluidic device is composed of a micro fluidic reservoir that is designed to store microfluidic droplets. By imaging the droplets in the storage reservoir with a combination of brightfield and fluorescence microscopy, the growth dynamics of the encapsulated bacterial communities can be described. In the second section of the thesis, I present the development and operating principles of my image processing and analysis workflow. This workflow automatically extracts quantitative bacterial growth data from a gridded array of brightfield and fluorescence microscopy images taken across the microfluidic device during experimentation. The analysis algorithm is capable of processing upwards of 200 fields of view across 80 time frames. This makes it possible to detect, track and measure the size of upwards of 1000 encapsulated bacterial communities with single cell precision. Droplet boundaries are detected using brightfield microscopy images and each droplet is tracked from one time step to the next using a modified particle tracking algorithm. Bacterial community sizes are measured by counting individual bacteria in thresholded fluorescence microscopy images. In the final section of this thesis, a study of E. coli bacteria was conducted using the microfluidic device. Small communities of bacteria, typically 1-5 cells in size, were confined in microfluidic droplets and observed for 8 hours. The bacteria were grown in the absence of antibiotic, as well as in the presence of the antibiotic streptomycin at various concentrations. A number of metrics of the growth dynamics are extracted from the study and compared with traditional bulk growth techniques. It is shown that in the absence of streptomycin, the final droplet population size distributions feature extended tails due to large, fast growing bacterial populations. The tails of these distributions are reduced in length in the presence of streptomycin at a concentration below the Minimum Inhibitory Concentration (MIC). A positive correlation is found between initial and final bacterial population size, however this correlation is weak, indicating a highly stochastic growth process. It is also shown that at moderate streptomycin concentrations both around and above the MIC, some bacterial populations are inhibited in a non-lytic manner, whilst other populations underwent lysis. It is also shown that at moderate antibiotic concentrations around the MIC value, there is a bias against bacterial lysis in droplets with large populations. Finally, at low antibiotic concentrations below the MIC, it is shown that some bacterial populations are inhibited but not lysed, whereas some populations are not inhibited. The microfluidic device and image analysis workflow described in this thesis is a novel experimental system with widespread applicability. The system is able to partition an aqueous solution into a large number of small, compartmentalised liquid volumes and analyse these volumes using both brightfield and fluorescence time-lapse microscopy. This could be used to study the population dynamics of a variety of bacterial strains, and could also be used to explore more general questions relating to stochastic cell biology where encapsulation is experimentally beneficial.
Supervisor: Titmuss, Simon ; Allen, Rosalind Sponsor: Engineering and Physical Sciences Research Council (EPSRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.798900  DOI: Not available
Keywords: bacteria ; microfluidics ; antibiotics ; fluorescence microscopy ; microscopy ; image processing ; E. coli ; population dynamics ; statistical analysis ; minimum inhibitory concentration ; single cell variability ; cell heterogeneity
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