Thesis
Novel applications of advanced optical microscopy for microbiology
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2020
- Thesis identifier
- T15692
- Person Identifier (Local)
- 201686042
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- The study of bacteria often requires visualisation by optical microscopy, but the use of advanced optical microscopy methods is uncommon by many microbiologists. Therefore, there remains many areas of microbiology which require exploration using newly developed techniques. This thesis describes the application of advanced optical microscopy methods to three distinct microbiological questions centred on bacterial gliding motility, the spatial organisation of biofilms, and the growth of bacteria in a mimetic three-dimensional (3D) culture environment. The gliding motility of Myxococcus xanthus has been described as a lateral process, and it was unclear if single bacteria were capable of moving in three dimensions. This was due to three-dimensional imaging of bacteria often being unachievable by optical methods due to the height of a bacterial cell being on the order as the axial resolution of the conventional optical microscope. To overcome this a novel variant of the livecell label-free technique, interference reflection microscopy (IRM), was developed. This method relies on the interference of multiple wavelengths of incident and reflected light and results in a series of intensity maxima and minima which encode 3D information. A specimen of known geometry was used to characterise this method before application to gliding M. xanthus cells. Multi-wavelength confocal IRM revealed that M. xanthus exhibited aperiodic oscillations during gliding, which challenged the theory that gliding motility was a lateral phenomenon. By use of deleterious mutants, it was deduced that the oscillatory behaviours were not linked to the main driving force of gliding, proton motive force. A hypothesis was proposed which suggested that these behaviours were caused by recoil and force transmission along the cell body following firing of the Type IV pili. Bacterial biofilms have been studied by conventional microscopy methods for over 50 years; however due to a technology gap, the structure of large microbial aggregates remained unclear. The development of the Mesolens, an optical system which uniquely allows simultaneous imaging of individual bacteria over a 36 mm2 field of view, enabled the study of mature Escherichia coli macro-colony biofilm architecture like never before. The Mesolens enabled the discovery of intra-colony channels on the order of 10 µm in diameter that are integral to E. coli macro-colony biofilms and form as an emergent property of biofilm growth. These channels have a characteristic structure and reform after total mechanical disaggregation of the colony, facilitate transport of particles, and play a role in the acquisition of and distribution of nutrients through the biofilm. Furthermore, intra-colony channels potentially offer a previously unobserved route for the delivery of dispersal agents or antimicrobial drugs to biofilms, which would ultimately lower their impact on public health and industry. The practice of bacterial culture has remained unchanged for over a century. Therefore, almost all observations of bacterial behaviour have been made using synthetic laboratory condition which are not representative of the natural environment. To address this, a mimetic 3D transparent soil culture medium was fabricated and designed specifically for bacterial culture. This novel culture medium was optimised for two wide-ranging genera of soil bacteria, Streptomyces coelicolor and Bacillus subtilis. Following careful design of the transparent soil platform, each strain was imaged using the Mesolens to provide a better understanding of how they colonised their natural habitat. Each species was found to colonise the surface of soil independently of their growth behaviours on traditional two-dimensional culture methods. Moreover, the viability of bacteria grown in transparent soil was found to be uncompromised. Therefore, transparent soil stands as a readily tailored platform for bacterial culture and is compatible with any optical microscope to study bacterial behaviours in a mimetic soil environment.
- Advisor / supervisor
- Hoskisson, Paul A.
- McConnell, Gail
- Resource Type
- Note
- This thesis was previously held under moratorium from 30th October 2020 until 30th October 2022
- DOI
- Date Created
- 2020
- Former identifier
- 9912922288502996
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PDF of thesis T15692 | 2021-07-02 | 公开 | 下载 |