Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 137-152).

No description available.

Quality Control (QC) reference materials are widely used in microbiology to demonstrate the efficacy of testing methods and culture media. The current method for preparation of QC materials is by serial dilution of a microbial broth culture to obtain a suspension that contains an estimated number of colony forming units (cfu). Commercial reference material products are available with dried microbial cells, however, the numbers of cells are variable between batches as the production processes are reliant on cell suspensions of estimated cell number. This study developed a method to produce precise microbial reference materials with a accurate number of viable cells. Flow cytometry was used to count and dispense precise numbers of cells into a single droplet of fluid. The droplets were then mixed with a lyoprotectant solution and subjected to freeze-drying. The resultant freeze-dried pellets showed consistent average cfu counts between 28-33 cfu with a standard deviation < 3 cfu. The freeze-drying methodology and developed conditions of cell growth enabled > 90% of the cells to survive freeze-drying and remain viable for one year at a storage temperature below -18??C. The methodology for the production of freeze-dried pellet was applied to a range of genera including, different E. coli strains, Gram positive bacteria such as Listeria and Staphylococcus, the yeast Candida albicans and a spore-producing Bacillus cereus. The precision of cell numbers was comparable between different microbial genera and strains and a consistent standard deviation below 3 cfu was achieved. The same freeze-dried pellet method was used for the different micro-organisms, except for changes to preparation of cell suspensions. Different methods of broth culture were developed to ensure freeze-dried cell survival. A measurement of method reproducibility was obtained when 99 batches of pellets were produced, and within batch and between batch variation was determined.

Quality Control (QC) reference materials are widely used in microbiology to demonstrate the efficacy of testing methods and culture media. The current method for preparation of QC materials is by serial dilution of a microbial broth culture to obtain a suspension that contains an estimated number of colony forming units (cfu). Commercial reference material products are available with dried microbial cells, however, the numbers of cells are variable between batches as the production processes are reliant on cell suspensions of estimated cell number. This study developed a method to produce precise microbial reference materials with a accurate number of viable cells. Flow cytometry was used to count and dispense precise numbers of cells into a single droplet of fluid. The droplets were then mixed with a lyoprotectant solution and subjected to freeze-drying. The resultant freeze-dried pellets showed consistent average cfu counts between 28-33 cfu with a standard deviation < 3 cfu. The freeze-drying methodology and developed conditions of cell growth enabled > 90% of the cells to survive freeze-drying and remain viable for one year at a storage temperature below -18??C. The methodology for the production of freeze-dried pellet was applied to a range of genera including, different E. coli strains, Gram positive bacteria such as Listeria and Staphylococcus, the yeast Candida albicans and a spore-producing Bacillus cereus. The precision of cell numbers was comparable between different microbial genera and strains and a consistent standard deviation below 3 cfu was achieved. The same freeze-dried pellet method was used for the different micro-organisms, except for changes to preparation of cell suspensions. Different methods of broth culture were developed to ensure freeze-dried cell survival. A measurement of method reproducibility was obtained when 99 batches of pellets were produced, and within batch and between batch variation was determined.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [92]-97).

Thesis (Ph.D.) - University of St Andrews, February 2008.

The effects of altered membrane fatty acid composition on the toxic interactions of heavy metals with Saccharomyces cerevisiae were examined. Saccharomyces cerevisiae was enriched with the polyunsaturated fatty acids (PUFAs) linoleate (18:2) and linolenate (18:3) by growth in 18:2- or 18:3-supplemented medium. Incorporation of the exogenous PUF As resulted in them comprising greater than 65% and 40% of the total fatty acids in whole-cell and plasma membrane lipids, and nuclear membrane lipids, respectively. Incorporation of the exogenous PUF As had no discernible adverse effects on cell division. However, inhibition of cell division in the presence of Cd(N03)2 was accentuated by growth in the presence of the di-unsaturated fatty acid linoleate. Furthermore, susceptibility to both Cd2+ - and Cu2+ -induced plasma membrane permeabilisation and whole cell toxicity was markedly accentuated in PUF A-enriched cells, and increased with the degree of fatty acid unsaturation. The increased sensitivity ofPUFA-enriched cells to membrane permeabilisation and whole-cell toxicity was correlated with increased levels of lipid peroxidation in these cells. Cu2+ - and Cd2+_ induced lipid peroxidation was rapid and associated with a decline in plasma membrane lipid order, detected by fluorescence depolarization measurements. Levels of the lipid peroxidation products thiobarbituric acid-reactive substances (TBARS) and conjugated dienes were markedly higher in PUF A-enriched cells, compared with unsupplemented cells, following exposure to cadmium or copper. Thus, lipid peroxidation was demonstrated as a major means of heavy metal toxicity in a microorganism for the first time. In addition, the effects ofPUFA-enrichment on the interactions of heavy metals with cellular nucleic acids were examined. Exposure ofPUFA-enriched cells to the redox-active metals chromium and copper resulted in the uncoupling of DNA synthesis from cell division, leading to sequential S phases. For example, DNA levels of up to 8C were evident in 18:3-enriched cells after only 4.5 h exposure to 100 JJ.M Cu(N03h. Using flow cytometry, the heterogeneity in susceptibility to copper toxicity of exponential phase S. cerevisiae was also examined. Susceptibility towards copper toxicity was demonstrated to be cell cycle stage-dependent, whereby G2/M phase cells were found to be the most susceptible towards copper toxicity. Staining with the oxidantsensitive probe 2',7' -dichlorodihydrofluorescein diacetate (H2DCFDA) revealed that the greater copper sensitivity of G2/M phase cells correlated with elevated endogenous levels of reactive oxygen species in these cells.

Monitoring phytoplankton species composition and their abundance are routine tasks in marine ecological research and environmental monitoring. As phytoplankton populations are highly heterogeneous in terms of size, morphology, and most significantly, their abundance can change drastically in a very short time, it is extremely difficult to quantify and monitor them and there are demands on the instrumentation. Conventional optical microscopy and flow cytometry are the main tools to enumerate and identify phytoplankton, but they have a compromise between spatial information and acquisition speed. While imaging flow cytometry has the potential to integrate the benefit of high spatial resolution from optical microscopy and the advantage of high throughput from flow cytometry, two intrinsic blur sources, motion blur and out-of-focus blur, prevent imaging flow cytometers from obtaining high spatial resolution images with high throughput. To address these limitations, in this work, a novel light sheet based fluorescence imaging flow cytometer has been proposed, constructed, and tested for phytoplankton analysis. Both 2D and 3D imaging mode of the light sheet based fluorescence imaging flow cytometer have been investigated. In the 2D imaging mode, the instrument can screen untreated costal water samples at a volumetric throughput up to 1 ml/min. The instrument demonstrated shows a high immunity to motion blur, and all-in-focus fluorescence images are captured with a lateral resolution of 0.75 ± 0.06 µm for a wide size range ~ 1 µm to ~ 200 µm that includes pico-, nano-and microphytoplankton. This is made possible by suppressing the out-of-focus blur using thin light sheet illumination and image deconvolution, and by precluding the motion blur with a unique flow configuration. With these abilities, the instrument demonstrated has high potential as a practical field instrument for monitoring phytoplankton. In the 3D imaging mode, the instrument can scan a large number of phytoplankton cells in a short time with spatial resolution as achieved by light sheet microscopy. The lateral resolution is 0.81 ± 0.07 µm, and axial resolution in terms of FWHM of the axial scattering PSF is 1.42 ± 0.15 µm. The volumetric throughput of the instrument is 0.5 µl/min. This is benefitted from the improvement that 3D images can be acquired without the need of sample immobilization, in contrast to existing 3D imaging approaches, such as confocal fluorescence microscopy. Preliminary results from untreated coastal water samples and cultured samples show promising potentials of the instrument for phytoplankton monitoring and scientific research.

M.Tech.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy