Phenotype Variability Determination using Airy Disc Analysis
Penwill, Lynsey Ann
Date: 24 December 2013
University of Exeter
PhD in Biological Sciences
Rapid phenotype identification and screening is a relatively unexplored field compared with genotype screening probably owing to a lack of appropriate technology. The Lensless microscope has a large field of view and allows the capture of the diffraction pattern from a large number of cells simultaneously, its potential to screen ...
Rapid phenotype identification and screening is a relatively unexplored field compared with genotype screening probably owing to a lack of appropriate technology. The Lensless microscope has a large field of view and allows the capture of the diffraction pattern from a large number of cells simultaneously, its potential to screen growth phenotypes will be evaluated in this thesis. A simple algorithm has been developed to measure intensity changes in the Airy Disc First Fringe (ADFF) from which length and width dimensions can be derived from scattering objects with an accuracy of 5%, except for those lengths below 6 microns which have diffraction-limited measurements. A low refractive index growth medium was developed to allow growth phenotypes under normal and silver-stressed conditions to be measured for the three model organisms, S. pombe, E. coli and S. aureus. Phenotype classification parameters were derived from the growth curve of these organisms from which a total of 18 growth phenotypes were identified. All three cell populations exhibit survival phenotypes for both transitions from planktonic to surface growth, typically 98%, and from natural to stressed growth conditions at sub-lethal concentrations of silver. In S. pombe growth phenotypes of interest involve the movement into a possible G0 growth phase of the cell cycle on exposure to silver and a skewed ratio of monopolar to bipolar growth rate increase not previously observed. S. aureus growth under silver stress displayed asymmetric growth of the colonies under silver stress. Analysis of the lag period parameter in the normal growth population of S. aureus identified 4% of the population which have the characteristics of a known growth phenotype, Small Colony Variants. The lag period parameter also identified two cell populations of E. coli under normal conditions, with 20% of the colonies demonstrating a significantly shorter lag period length than the remaining 80%. More importantly, a high sub-lethal dose of silver ions induces two growth phenotypes in E. coli, called here ‘sub-bug’ with parameters indicating an increased resistance to the silver stress growing slowly and a second sub-population with similar enhanced silver resistance that grew rapidly, a ‘super-bug’, which has a shorter lag period, a faster growth rate and reaches a much larger colony size. Genomic analysis demonstrated that these two growth types were genetically identical and are therefore a silver resistant growth phenotype.
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