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Item Single cell encapsulation, detection, and sorting of Pseudomonas syringae using drop-based microfluidics(Montana State University - Bozeman, College of Engineering, 2023) Lindsay, Travis Carson; Chairperson, Graduate Committee: Abigail Richards; Connie Chang (co-chair)Bacteria can survive antibiotic or bactericidal treatment through genetic mutations. Even within bacterial populations that are fully susceptible to treatment, a small proportion of cells can have enhanced survival capacity in a phenomenon called persistence. Traditional microbiology methods can fail to identify or isolate these persister cells present within the population. A novel method for high-throughput single cell analyses of microbial populations is that of drop-based microfluidics, in which individual cells can be isolated within picoliter-sized drops. In this work, fluorescent detection and dielectrophoresis-based sorting of drops was developed for isolating Pseudomonas syringae persister cells following antimicrobial treatment. We demonstrate: (1) the dielectrophoresis-based sorting of dye-filled 25 micron drops based upon two colors, (2) differences between laser-induced fluorescent detection of dyes compared to single bacterial cells, (3) single-cell isolation of P. syringae into 25 micron droplets with ~10% of droplets containing singlecells, and (4) the treatment, staining, and fluorescent characterization of P. syringae at 0.5x, 5x, and 50x the minimum inhibitory concentration of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), an antibiotic which resulted in 6.2%, 10.2%, and 88.6% cell death of the population, respectively. These results provide the groundwork for studying antibiotic-treated P. syringae and the isolation of surviving cells that will lend insight into the molecular basis of persistence for preventing recurrent infections and decreasing the likelihood of antibiotic resistance.Item Ultra high-throughput fluorescence detection for single cell applications in drop microfluidics(Montana State University - Bozeman, College of Engineering, 2016) Schaefer, Robert Willman; Chairperson, Graduate Committee: Connie ChangConventional methods in microbiology can be limited by assay execution and analysis times, phenotypic dominance within bulk communities, reagent volumes, and single-use supply costs. These limitations can be overcome using drop-based microfluidics. In this discipline, pico-liter sized, water-in-oil emulsions serve as independent 'test tubes,' allowing for the compartmentalization of community constituents and interrogation at the single cell level. Furthermore, two-phase, continuous flow microfluidic devices enable drop populations to be manipulated and analyzed at kilohertz rates according to experimental needs. In this research, a fluorescence-based method for drop analysis and sorting was developed and applied, in conjunction with other microfluidic techniques, to perform assays in microbiology. The applications explored include cell dormancy within P. aeruginosa subpopulations, microalgae lipid accumulation for the production of biofuels, optimization of microbially-induced calcite precipitation (MICP), and human norovirus infectivity. Results from each application include: 1. The hibernation promoting factor (Hpf) was found to play a key role in the maintenance of P. aeruginosa viability during planktonic starvation. 2. Progress was made on a Nile Red based, ultra high-throughput, single cell algal lipid detection platform. 3. MICP was demonstrated at the single cell level. 4. A drop based human norovirus infection platform was attempted using human B cells as the viral host.Item A high-throughput, multiplexed microfluidic method utilizing an optically barcoded drop library(Montana State University - Bozeman, College of Engineering, 2016) Zath, Geoffrey Kane; Chairperson, Graduate Committee: Connie ChangThe power of drop-based microfluidics promises reduced biological assaying times and greater sample throughput; however, current drop-based microfluidic methods focus on single-input single-output techniques to provide these benefits. In order to achieve truly high-throughput analysis of biological assays, a multiple-input approach must be taken. This thesis is focused on developing and validating a drop-based microfluidic method that is capable of encapsulating, in parallel, 96 assay samples in drops and optically tracking them in a barcoded drop library. The advantage of the method presented here is its ability to be integrated with current biological assays performed on a 384-well plate. The first step was to fabricate a three-dimensional microfluidic device capable of accepting 96 sample inputs. Second, formation of drops within the device was characterized by creating a state diagram using Capillary and Weber numbers of the two phase flow. Finally, the use of fluorescent microbeads was investigated for the purpose of optically barcoding drops. A barcoding scheme was developed to allow for fluorescent and spatial labeling of 96 wells of a 384-well plate. The three-dimensional microfluidic device was successfully used to encapsulate 50 microns diameter drops from 24 wells barcoded with fluorescent microbeads at a drop formation rate of 3 kHz per well. Fluorescent detection of the barcoded drop mixture was performed at a rate of 200 Hz and density-based clustering algorithm DBSCAN was used to identify barcoded drop clusters from the fluorescent signal data. Validation of this method was achieved by adding known concentrations of fluorescent blue microbeads to barcoded wells and detecting for their presence in barcoded drop clusters. The barcoding method can be expanded to fully incorporate the 96 inputs of the microfluidic device by adding a spatial barcoding component to each quadrant of 24 optically barcoded wells. The results presented here show the microfluidic platform has the potential to be a useful tool in biological assays involved with tracking a large number of samples in a well plate format.