Graduate Thesis Or Dissertation
 

High resolution digital imaging of bacterial cells

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/vd66w3296

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  • The most abundant clone found in ribosomal RNA clone libraries obtained from the world's oceans belongs to the SAR11 phylogenetic group of environmental marine bacteria. Imaging and counting SAR11 bacterial cells in situ has been an important research objective for the past decade. This objective has been especially challenging due to the extremely small size, and hypothetically, the low abundance of ribosomes contained by the cells. To facilitate the imaging of small dim oligotrophic bacterial cells, digital imaging technology featuring very small pixel size, high quantum yield scientific grade CCD chips was integrated with the use of multiple oligonucleotide probes on cells mounted on a non-fluorescing solid substrate. Research into the composition of bacterioplankton populations in natural marine systems follows a two-fold path. Increasing the culturability of microbes found in the natural environment is one research path. Identifying and enumerating the relative fractions of microorganisms in situ by culture-independent methods is another. The accumulation and systematic comparison of ribosomal RNA clones from the marine environment has resulted in a philosophical shift in marine microbiology away from dependence upon cultured strains and toward investigations of in situ molecular signals. The design and use of oligonucleotide DNA probes targeting rRNA targets has matured along with the growth in size and complexity of the public sequence databases. Hybridizing a fluorescently labeled oligonucleotide probe to an rRNA target inside an intact cell provides both phylogenetic and morphological information (a technique called Fluorescence in situ Hybridization (FISH)). To facilitate the imaging of small, dim oligotrophic bacterial cells, digital imaging technology featuring very small pixel size, high quantum yield, scientific grade CCD chips is integrated with the use of multiple oligonucleotide probes on cells mounted on a non-fluorescing solid substrate. This research develops the protocols necessary to acquire and analyze digital images of marine bacterial cells. Experiments were conducted with Bermuda Atlantic Time Series (BATS) environmental samples obtained during cruise BV21 (1998) and B138 (2000). The behavior of the SAR11⁴*Cy3 probe set when hybridized to bacterial cells from these samples was investigated to determine the optimal hybridization reaction conditions. The challenges of bacterial cell counting after cell transfer from PCTE membrane to treated microslides were addressed. Experiments with aged Oregon Coast seawater were performed to investigate the protocol used to transfer cells from membrane to microslides, and examined the distribution of cells and the statistics of counting cells using traditional epifluorescence microscopy and image analysis techniques.
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