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 Investigation of a control strategy for manipulation and prevention of Pseudomonas aeruginosa PAO1 biofilms in metalworking fluids(Montana State University - Bozeman, College of Engineering, 2018) Ozcan, Safiye Selen; Chairperson, Graduate Committee: Christine Foreman; Markus Dieser, Albert E. Parker, Narayanaganesh Balasubramanian and Christine M. Foreman were co-authors of the article, 'Quorum sensing inhibition as a promissing method to control biofilm growth in metalworking fluids' submitted to the journal 'Environmental science & technology' which is contained within this thesis.Microbial contamination in metalworking fluid (MWF) circulation systems is a serious problem. Particularly water based MWFs promote microbial colonization despite the use of biocides. Inhibiting the quorum sensing mechanism (i.e. cell-cell communication) in bacteria is a promising approach to control and prevent biofilm formation. The objective of this study was (i) to determine the microbial community in MWFs from operational machining shops, (ii) to investigate the effect of well-known quorum sensing inhibitors on controlling biofilm formation, and (iii) to implement experimental data from selected enzymes to a computer simulation biofilm accumulation model (BAM). Planktonic and biofilm samples from two local machining shops in Bozeman, MT, were collected to determine the extent of microbial colonization. In both operations, microbial communities were dominated by Pseudomonadales (60.2-99.7%). Rapid recolonization was observed even after dumping spent MWFs and cleaning. Considering the dominance of Pseudomonadales in MWFs, the model organism Pseudomonas aeruginosa PAO1 was selected for testing the effects of quorum sensing inhibitor compounds on biofilm formation. From a variety of enzymes, natural, and chemical compounds screened for quorum sensing inhibition, Patulin (40microns) and Furanone C-30 (75microns), were found to be effective in reducing biofilm formation in MWFs when applied as single compound amendments and in combination with the polysaccharide degrading enzyme alpha-amylase from Bacillus amyloliquefaciens. Particularly Furanone C-30, as a single amendment and in combination with alpha-amylase decreased biofilm formation by 76% and 82% after 48 hours. Putatively identified homoserine lactones in MWFs treated with Furanone C-30 provided evidence for quorum sensing inhibition on biofilm formation. BAM was employed to study the effect of alpha-amylase (3 Units mL -1) on P. aeruginosa PAO1 biofilms in batch reactors for 24 and 48 hours. In the absence of alpha-amylase, biofilm thickness was predicted to be 23.11 and 31.37 microns, while its presence reduced thickness to 10.47 and 13.07 microns after 24 and 48 hours, respectively. The results presented herein highlight the potential effectiveness of quorum sensing inhibition as a strategy to reduce biofilms in MWFs.Item Evaluation of a computer model for biocide action against biofilms(Montana State University - Bozeman, College of Engineering, 1996) Sanderson, Sara ShermanItem Diffusion coefficients for Pseudomonas aeruginosa and Klebsiella pneumoniae(Montana State University - Bozeman, College of Engineering, 1990) Kim, Yeong-ChulItem Action of monochloramine on biofilms of Pseudomonas aeruginosa and Klebsiella pneumoniae(Montana State University - Bozeman, College of Engineering, 1994) Srinivasan, RohiniItem Transport of substrate and biomass in a packed bed bioreactor(Montana State University - Bozeman, College of Engineering, 1992) Lundman, Ross WadeItem Adhesion of Pseudomonas aeruginosa to surface modified and unmodified polystyrene with and without a conditioning layer(Montana State University - Bozeman, College of Engineering, 1999) Thompson, Jenny KathleenItem Growth kinetics of Pseudomonas aeruginosa(Montana State University - Bozeman, College of Engineering, 2001) Chen, Suet NeeItem Effects of ultrasonic exposure on growth and attachment rates of a capsule-forming bacterium(Montana State University - Bozeman, College of Engineering, 1991) Campbell, Teresa AnnItem Kinetics of biofilm detachment(Montana State University - Bozeman, College of Engineering, 1992) Peyton, Brent Michael