| Summary: | 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. === Graduation date: 2002
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