Theses and Dissertations at Montana State University (MSU)
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Item Investigation of Staphylococcus epidermidis biofilm resistance to rifampin(Montana State University - Bozeman, College of Engineering, 2001) Zheng, ZhilanItem Nuclear magnetic resonance microscopy of Staphylococcus epidermidis biofilms(Montana State University - Bozeman, College of Engineering, 2004) Gjersing, Erica Lee; Chairperson, Graduate Committee: Philip S. Stewart; Joseph Seymour (co-chair)Nuclear Magnetic Resonance (NMR) microscopy is well-suited for investigating living systems since it is innocuous and non-invasive. In addition to imaging internal structures of systems, NMR microscopy techniques can be used to obtain information about transport phenomena, such as fluid velocities and diffusion. The goal of this research was to determine the applicability of NMR imaging techniques to studying transport properties in biofilms. Staphylococcus epidermidis biofilms were investigated because of their importance in medical implant infections. NMR experiments were used to image biofilm structure and the fluid flow patterns in one millimeter square glass capillaries. Results showing the heterogeneous structure of biofilms are congruent with confocal laser microscopy images. The advantage of using the NMR techniques to image biofilm structures is that there are no light or laser penetration barriers and the innermost regions of the structures can be easily revealed. In addition to imaging the biofilms, velocity distributions have been mapped for the one millimeter capillary system. Laminar flows in clean, square capillaries display axial velocities that are both uniform and symmetrical while non-axial components of velocity are not present. In contrast, a biofilm fouled capillary displays irregular flow patterns in the axial direction along with distinct non-axial secondary flow perturbations. These results demonstrate that biofilms impact bulk flow in ways that cannot be ignored when modeling their impact on transport in bioreactors and medial devices. This work establishes NMR microscopy as an important biofilm research tool which can spatially resolve structural characteristics and transport processes in biofilm fouled systems.Item Spatial and temporal patterns of antimicrobial action against Staphylococcus Epidermidis biofilms(Montana State University - Bozeman, College of Engineering, 2008) Davison, William Marshall; Chairperson, Graduate Committee: Philip S. Stewart; Joseph D. Seymour (co-chair)This study investigated the spatio-temporal patterns of antimicrobial action against Staphylococcus epidermidis planktonic and biofilm bacteria. Bacteria were stained with a fluorogenic esterase substrate, Calcein-AM, which allowed for the visualization of cells that possessed intact cell membranes. Four different antimicrobial agents were tested for their effect upon cell viability as associated with membrane integrity. The four biocides were Barquat®, glutaraldehyde, chlorine, and nisin. Planktonic bacteria were analyzed with flow cytometry, observing fluorescence loss during 1 h antimicrobial treatment. Treatment with Barquat resulted in initial fluorescence loss, which increased during the treatment period to levels which were present prior to the introduction of biocide, along with a decrease in cell density. Treatments with glutaraldehyde and chlorine resulted in increased average fluorescence intensity for the cell population, accompanied by decreased cell density for chlorine and increased cell density for glutaraldehyde. Nisin treatment resulted in a decrease in CAM fluorescence with an increase in cell density. Viable cell plate counts showed average log reductions in CFU/mL of 3.61, 3.83, 4.12, 4.26, and 4.67 for Barquat, glutaraldehyde, high and low concentrations of chlorine, and nisin treatments, respectively. There was no apparent correlation between plate counts and flow cytometry data. Biofilm bacteria were analyzed with time-lapse confocal scanning laser microscopy, observing fluorescence loss during biocide treatment. Biofilms treated with Barquat lost an average of 91.5% of their initial fluorescence, and clusters decreased in areal coverage by 9%. Fluorescence loss during Barquat treatment suggested the presence of a tolerant subpopulation of bacteria in the interior regions of the biofilm. Glutaraldehyde treatment reduced the average fluorescence by 16%, and cluster area did not change. There was CTC staining after glutaraldehyde treatment only. The high and low concentrations of chlorine treatment showed averages of 100% and 79% reductions in CAM staining, with liquefaction of biomass causing erosion events which reduced areal coverage by 90% and 43%, respectively. Nisin treatment reduced CAM staining by an average of 100%, while shrinking the cluster area by 8%. Corner biofilms showed qualitative differences during treatment than isolated clusters. Mathematically-predicted biocide diffusion times were much faster than experimentally observed fluorescence loss in biofilms.