Theses and Dissertations at Montana State University (MSU)
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Item Bacterial cultivation in microscale drops and capsules to resolve single-cell growth physiology(Montana State University - Bozeman, College of Engineering, 2023) Pratt, Shawna Leigh; Chairperson, Graduate Committee: Ross Carlson; This is a manuscript style paper that includes co-authored chapters.Single-cell heterogeneity contributes to the complex population dynamics of infectious microbial communities. Improving our understanding of single-cell physiology and heterogeneity may aid in mitigating microbial infections; however, assaying large populations of single cells can be challenging. Despite recent developments in single-cell assaying, tracking the physiology of large numbers of individual cells and their lineages over time is difficult to achieve using current technologies. Here, I apply drop-based microfluidics to develop microscale tools for improving high-throughput single-cell microbial growth assays. Drop-based microfluidics is a technology that generates and manipulates microscale drops. In this work, I create water-in-oil drops and hydrogel-shelled microcapsules using drop-based microfluidics to study the growth of P. aeruginosa bacteria, a key pathogen implicated in chronic lung infections and wounds. The growth of single bacterial cells inside drop microcompartments is observed via time-lapse confocal microscopy. Bacteria were cultured in water-in-oil drops and prepared for long-term storage in a novel microfluidic device environment, which we call a DropSOAC (Drop Stabilization on a Chip) chamber. The DropSOAC method prevents drop destabilization by saturating microfluidic devices with equilibrated water and oil, maintaining phase equilibrium in the drop emulsion. Using DropSOAC, the single-cell growth of starved P. aeruginosa wildtype and hibernation promotion factor mutants were characterized, revealing significant growth heterogeneity in the mutant strain. Finally, we present a method for generating hydrogel-shelled microcapsules that enables the culturing of single cells in microscale environments where nutrients and waste can diffuse in and out of the microculture environment. A 3-D microfluidic device and capsule generation protocol are designed, resulting in an optimized approach for capsule production using phase-separating polymer systems and rapid hydrogel crosslinking. The growth of hundreds of individual P. aeruginosa cells is observed over time with the hydrogel- shelled microcapsules. Due to the permeability of the microcapsules, antibiotics can be introduced at various times during growth to investigate single and biofilm P. aeruginosa physiology. Overall, this work introduces novel approaches for high-throughput, single-cell microbial growth characterization that enables a deeper understanding of the role of heterogeneity in bacterial populations.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 Microgels for single-cell culturing of neurons and chondrocytes(Montana State University - Bozeman, College of Engineering, 2023) Fredrikson, Jacob Preston; Chairperson, Graduate Committee: Abigail Richards; This is a manuscript style paper that includes co-authored chapters.Tissue engineering is a multidisciplinary field that combines engineering and life sciences to restore, improve, or generate biological substitutes to replace damaged tissues or organs. This is often performed using hydrogels that serve as scaffolds for the growth and maintenance of target tissues. Hydrogels, crosslinked polymer networks composed primarily of water, are excellent tissue mimics with highly tunable mechanical and biochemical properties. Hydrogels can be fabricated at the microscale, termed microgels, using drop-based microfluidics, which enables the precise control of cell density within the microgels down to a single cell. Encapsulating cells in microgels allows for the manipulation of microgels after production for single cell analyses. In this dissertation, human articular cartilage (HAC) cells and neurons are cultured within and upon microgel particles that serve as microscale tissue models for the study of chondrocyte matrix production and Herpes Simplex Virus type -1 (HSV-1) infection studies. HAC is the load-bearing tissue that lines the interfaces of joints and is responsible for shock and wear resistance. Chondrocytes, the cells in HAC, are responsible for producing and maintaining HAC. The chondrocyte pericellular matrix (PCM) regulates the metabolism and mechanical strain of the cells, which is critical to cellular function and cartilage homeostasis. However, the PCM is challenging to produce in vitro. The first half of this work applies microgels for PCM formation in chondrocytes. Immunofluorescence and high-performance liquid chromatography-mass spectrometry data demonstrate that chondrocytes grown in alginate microgels form a collagen VI-rich PCM, significantly altering the cells' metabolic response to dynamic compression. Atomic force microscopy data demonstrates that when chondrocytes are grown in alginate microgels for ten days, the elastic modulus of the PCM increases an order of magnitude. HSV-1 is a human pathogen that invades the peripheral nervous system. Understanding the complexities of HSV-1 infection at the single-cell level could lead to better therapeutics and reduced disease outcomes. Drop-based microfluidics (DBM) has recently been adapted for studying single-cell viral infection but has not been applied to neurons and HSV-1. The second half of this work develops a method for growing individual neurons in microgels. These microgel-embedded neurons are isolated, encapsulated with precise inoculating doses of HSV-1 using DBM, and the kinetics of viral gene expression are tracked in individual neurons using a fluorescent-recombinant HSV-1 virus. The data demonstrate that microgels provide a solid scaffold for neuronal development that supports single-cell productive HSV-1 infection within droplets.Item Development of drop-based microfluidic methods for high-throughput biological assays(Montana State University - Bozeman, College of Engineering, 2021) Zath, Geoffrey Kane; Chairperson, Graduate Committee: Connie Chang; This is a manuscript style paper that includes co-authored chapters.Drop-based microfluidics allows single-cell biological assays to be performed by encapsulating samples in picoliter scale drops. Adapting biological assays to drop-based microfluidics requires novel approaches to meet the method requirements of each assay. For example, microtiter plates are a common tool for storing many unique samples in some assays. An equivalent strategy for drops involves labeling samples with a barcode prior to drop encapsulation and storing the barcoded drops in a single mixture, thereby creating a drop library. Other assay adaptions, such as drop-based reverse transcription quantitative polymerase chain reaction (RT-qPCR) require that drops be stabilized during the high temperatures used for thermal cycling. Drop-based RT-qPCR is useful for studying single-cell dynamics in drops, such as influenza A virus (IAV) infection. Conventional methods for measuring IAV output from individual cells are labor intensive and low-throughput. Thus, there is a need to adapt RT-qPCR to drop-based microfluidics for the purpose of high-throughput single cell analysis of infected cells. The research presented here focuses on the characterization of the Pressure Cooker Chip (PCC) to rapidly encapsulate drop libraries and the development of a drop-based RT-qPCR method to measure IAV output from infected cells. The PCC was used to make drop libraries by rapidly generating drops of up to 96 different conditions in parallel by interfacing individual drop makers with a standard microtiter well plate. The drop library was optically barcoded using a two-color combination of fluorescent microbeads or quantum dots with 24 or 192 unique combinations, respectively. To adapt RT-qPCR in drops, known PCR additives were systematically tested to optimize drop stability and limit dye diffusion during thermocycling. A novel qPCR data analysis method was developed to convert drop fluorescence data collected at a single thermocycle to an initial RNA template concentration. Together, the additive screening and novel qPCR data analsyis method enabled the use of drop-based RT-qPCR to quantify the highly heterogeneous IAV burst size from single cells in thousands of drops. Our method is the first to measure single cell IAV burst size using a high-throughput, drop-based RT-qPCR assay.Item Microbially induced calcium carbonate precipitation: meso-scale optimization and micro-scale characterization(Montana State University - Bozeman, College of Engineering, 2020) Zambare, Neerja Milind; Chairperson, Graduate Committee: Robin Gerlach and Ellen G. Lauchnor (co-chair); Ellen Lauchnor and Robin Gerlach were co-authors of the article, 'Controlling the distribution of microbially precipitated calcium carbonate in radial flow environments' in the journal 'Environmental science and technology' which is contained within this dissertation.; Robin Gerlach and Ellen Lauchnor were co-authors of the article, 'Spatio-temporal dynamics of strontium partitioning with microbially induced calcium carbonate precipitation in porous media flow cells' submitted to the journal 'Environmental science & technology' which is contained within this dissertation.; Robin Gerlach and Ellen Lauchnor were co-authors of the article, 'Co-precipitation of strontium and barium' submitted to the journal 'Environmental science & technology' which is contained within this dissertation.; Nada Naser, Robin Gerlach and Connie Chang were co-authors of the article, 'Visualizing microbially induced mineral precipitation from single cells using drop-based microfluidics' submitted to the journal 'Nature methods' which is contained within this dissertation.Microorganisms have the potential to impact processes on a scale orders of magnitude larger than their size. For example, microbe-mineral interactions at the micro-scale can drive macro-scale processes such as rock formation and weathering. Many bioremediation technologies derive inspiration from microbial mineralization processes. Microbially induced calcium carbonate precipitation (MICP) can produce calcium carbonate (CaCO 3) precipitates which can be utilized as a biological cement to strengthen porous media by reducing fluid permeability in subsurface fractures or as an immobilization matrix to remove metal contaminants dissolved in groundwater. To make MICP a feasible and successful bioremediation technology in the world outside the lab, it is necessary to bridge the gap between the meso-scale research studies and macro-scale applications. This thesis focuses on such meso-scale studies but also contributes to bridging the gap in the other direction, i.e., meso-scale to micro-scale to gain a fundamental understanding of the cellular level processes behind MICP. The research presented here investigates two applications of MICP with a focus on controlling precipitate distribution and process efficiency in target environments. Subsurface precipitate distribution and metal partitioning during MICP were studied in novel reactive transport systems that mimic application-environment conditions. A radial flow reactor was used to study the spatial distribution of precipitates in conditions similar to subsurface injection well environments. The distribution and degree of metal partitioning during MICP was investigated in batch reactors and porous media flow cells to study kinetics and reactive transport effects on kinetics. In the radial flow environment, more precipitates formed away from the center injection zone. Results showed that longer reactant residence times and an equimolar ratio of calcium to urea were able to maximize precipitation efficiency. Metal partitioning could be maximized at low reactant flow rates and low metal concentrations. The novel flow cell set up used revealed a spatial decoupling between ureolysis and precipitation. A micro-scale investigation of the fundamental MICP process itself is presented wherein microbe-mineral interactions are observed at the cell level. A semi-correlative approach to investigating individual precipitates in microdroplets is presented, using a multitude of microscopy and microanalysis techniques. The presented studies capture MICP across a range of scales.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.