Three-dimensional imaging of bacterial microcolonies

Previous research into microbial colonies and biofilms shows a significant gap in our current understanding of how bacterial structures develop. Despite the huge body of research undertaken into the formation, genetic makeup, composition, and optimal growth conditions of colonies, no study has been successful in identifying all individual bacteria in a colony in three-dimensions as a function of time. This lack of bacterial cell lineage in such a simple class of organisms is conspicuous in the light of what is known about other organisms, such as Caenorhabditis elegans [1]. In this thesis I show that using laser scanning confocal microscopy in conjunction with developments in sample preparation and post acquisition image analysis, it is possible to fully reconstruct all individual bacteria within an Escherichia coli (E. coli ) microcolony grown in viscoelastic media. Additionally, I show that by further pushing the resolution of confocal microscopes, commercial systems are capable of extracting three-dimensional information on protein structures inside bacteria at early stages of growth. This thesis is in three parts. The first part shows that by pushing the resolution of a commercial laser scanning confocal microscope system it is possible to achieve single cell resolution of a bacterial colony growing in three dimensions in a viscoelastic medium (agarose) from a seed bacterium. The growth of individual bacteria is examined as the concentration of agarose in the media is altered. Results show there is a nonlinear dependence between the rate of growth of a bacterium and the concentration of the agarose in the media with a peak in growth rate at 3% (weight) concentrations of agarose in M9 media. The second part of this work presents a study of how an initially two-dimensional colony growing between a glass slide and agarose gel suddenly invades the third spatial dimension by buckling. The results show that the cells within the centre of the colony flex and buckle, due to confinement by their neighbours, creating additional layers. Indeed, flexing is not limited to the buckling event but occurs throughout the early growth cycle of a colony. The final part of this thesis shows that by further pushing the resolution of confocal microscopes, commercial systems are capable of extracting three-dimensional information about the temporal evolution of the spatial distribution of the FtsZ septation ring within the cell. As the bacterial colony grows from a seed bacterium to a microcolony, the error in placing the division accurately at the cell centre is seen to increase as the number of bacteria within the colony increases and spatial confinement occurs