Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 127-134).

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

No description available.

An all-electrical system is developed to actuate and detect single biological cells in a microfluidic channel for diagnostic applications. Interdigitated electrodes fabricated on the channel floor transfer a high frequency signal for capacitance detection and a low frequency signal for dielectrophoretic actuation. In the fluid-filled channel, a pressure-driven flow propels single biological cells, which induce time-dependent capacitance signatures as they pass over the electrodes. With a sub-attofarad (~0.15 aF RMS, 53 Hz bandwidth) capacitance resolution, this system detects biological cells (e.g., 1 yeast cell ~ 50 aF) and their deflections (1 micrometer ~ 5 aF) from exerted dielectrophoretic forces (> 5 pN). Electrical detection of cell actuation by strong DEP forces provides an avenue for both inducing and monitoring the deformation of viscoelastic cells. A strong and repulsive dielectrophoretic force can be used to press a biological cell into a channel wall. When this occurs, the mechanical properties of the cell can be investigated by capacitively monitoring the cell-to-wall interaction. The nature of the resulting interaction is shown to depend on the mechanical properties of the cell (surface morphology and viscoelastic properties). Various mammalian cell types such as Chinese Hamster Ovary (CHO) cells, mouse fibroblasts, human blood cells, human breast cells and their tumorogenic phenotypes are investigated using this system. Between these populations, the effective Young's modulus varies widely from 20 Pa (neutrophils) to 1-2 GPa (polystyrene microspheres). The viability and phenotype of a biological cell are known to reflect its mechanical and electrical properties. Consequently, this work investigates whether dielectrophoretically induced cell deformations are correlated with corresponding variations in capacitance, which could be used for discriminating cell phenotypes in the future.

An all-electrical system is developed to actuate and detect single biological cells in a microfluidic channel for diagnostic applications. Interdigitated electrodes fabricated on the channel floor transfer a high frequency signal for capacitance detection and a low frequency signal for dielectrophoretic actuation. In the fluid-filled channel, a pressure-driven flow propels single biological cells, which induce time-dependent capacitance signatures as they pass over the electrodes. With a sub-attofarad (~0.15 aF RMS, 53 Hz bandwidth) capacitance resolution, this system detects biological cells (e.g., 1 yeast cell ~ 50 aF) and their deflections (1 micrometer ~ 5 aF) from exerted dielectrophoretic forces (> 5 pN). Electrical detection of cell actuation by strong DEP forces provides an avenue for both inducing and monitoring the deformation of viscoelastic cells. A strong and repulsive dielectrophoretic force can be used to press a biological cell into a channel wall. When this occurs, the mechanical properties of the cell can be investigated by capacitively monitoring the cell-to-wall interaction. The nature of the resulting interaction is shown to depend on the mechanical properties of the cell (surface morphology and viscoelastic properties). Various mammalian cell types such as Chinese Hamster Ovary (CHO) cells, mouse fibroblasts, human blood cells, human breast cells and their tumorogenic phenotypes are investigated using this system. Between these populations, the effective Young's modulus varies widely from 20 Pa (neutrophils) to 1-2 GPa (polystyrene microspheres). The viability and phenotype of a biological cell are known to reflect its mechanical and electrical properties. Consequently, this work investigates whether dielectrophoretically induced cell deformations are correlated with corresponding variations in capacitance, which could be used for discriminating cell phenotypes in the future.

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.