<p>During bio-technical processing it is important to monitorbiological parameters such as cell growth, viability andproduct formation. Many of the analyses traditionally used areslow to perform and provide only average data for thepopulation. Flow cytometry is a laser-based technique, whichmeasures physical properties of a cell in a flowing stream, ata rate of several thousand cells per second. It offers theprospect of an at-line, multi-parameter analysis of individualmicroorganisms in a population.</p><p>In this project several methods for at-line measurements ofbioprocesses were developed such as protocol's for measuringcell concentration, viability and product formation. Theprimary focus was on prokaryotic organisms (<i>E. coli</i>) but eukaryotic organisms (<i>P. pastoris</i>) were included.</p><p>The possibility to use volumetric cell counting to measurecell concentration (cell number) was evaluated. It was shownthat the method was applicable for high cell density processesof both<i>E. coli</i>and<i>P. pastoris</i>.</p><p>The combination of Bis- (1,3-dibutylbarbituric acid)trimethine oxonol (depolarised membranes) and propidium iodide(loss of membrane integrity) as fluorescent markers was usefulto measure viability at-line of cells in high cell densityprocesses. The protocol was shown to be reproducible for<i>E. coli</i>and<i>P. pastoris</i>.</p><p>The viability staining was used to study the kinetics ofweak organic acids (food preservatives). The protocol provideddata about cell functions such as membrane depolarisation andloss of membrane integrity caused by introducing weak organicacids to shake flask cultures of<i>E. coli.</i></p><p>Labeling inclusion bodies with fluorescent antibodiesprovided a method, which could specifically monitor theincreased accumulation of recombinant promegapoetin proteinwith process time. This technique was further developed forintracellular staining by application of a permeabilising stepbefore labeling with antibodies. Staining of inclusion bodiesdirectly inside permeabilised cells gave information about thedistribution of protein expression in the cell population.</p><p>In conclusion, flow cytometry provides an at-line, singlecell technique for measurement of several biological parametersin bioprocesses.</p><p><b>Key words</b>: flow cytometry, Partec PAS, propidium iodide(PI), bis- (1,3-dibutylbarbituric acid) trimethine oxonol(BOX), Alexa fluor 488, bioprocess,<i>E. coli</i>,<i>P. pastoris</i>, inclusion body, food preservatives,viability, membrane potential</p>

During bio-technical processing it is important to monitorbiological parameters such as cell growth, viability andproduct formation. Many of the analyses traditionally used areslow to perform and provide only average data for thepopulation. Flow cytometry is a laser-based technique, whichmeasures physical properties of a cell in a flowing stream, ata rate of several thousand cells per second. It offers theprospect of an at-line, multi-parameter analysis of individualmicroorganisms in a population. In this project several methods for at-line measurements ofbioprocesses were developed such as protocol's for measuringcell concentration, viability and product formation. Theprimary focus was on prokaryotic organisms (E. coli) but eukaryotic organisms (P. pastoris) were included. The possibility to use volumetric cell counting to measurecell concentration (cell number) was evaluated. It was shownthat the method was applicable for high cell density processesof bothE. coliandP. pastoris. The combination of Bis- (1,3-dibutylbarbituric acid)trimethine oxonol (depolarised membranes) and propidium iodide(loss of membrane integrity) as fluorescent markers was usefulto measure viability at-line of cells in high cell densityprocesses. The protocol was shown to be reproducible forE. coliandP. pastoris. The viability staining was used to study the kinetics ofweak organic acids (food preservatives). The protocol provideddata about cell functions such as membrane depolarisation andloss of membrane integrity caused by introducing weak organicacids to shake flask cultures ofE. coli. Labeling inclusion bodies with fluorescent antibodiesprovided a method, which could specifically monitor theincreased accumulation of recombinant promegapoetin proteinwith process time. This technique was further developed forintracellular staining by application of a permeabilising stepbefore labeling with antibodies. Staining of inclusion bodiesdirectly inside permeabilised cells gave information about thedistribution of protein expression in the cell population. In conclusion, flow cytometry provides an at-line, singlecell technique for measurement of several biological parametersin bioprocesses. Key words: flow cytometry, Partec PAS, propidium iodide(PI), bis- (1,3-dibutylbarbituric acid) trimethine oxonol(BOX), Alexa fluor 488, bioprocess,E. coli,P. pastoris, inclusion body, food preservatives,viability, membrane potential

Recent studies suggest that lymphocyte apoptosis serves to regulate pulmonary inflammation. Equine COPD, an allergic disease of the lower airway, is likely due to dysregulation of the pulmonary immune response. In this study, the hypothesis tested was COPD affected horses would have less apoptotic airway lymphocytes than control horses during clinical disease. To achieve this, 3 methods of measuring apoptosis, Vindelov's propidium iodide with Triton-X (PI/Triton-X), 7-aminoactinomycin D (7-AAD), and Annexin V with propidium iodide (Annexin/PI) were evaluated in equine airway lymphocytes. A significant linear relationship was found for equine bronchoalveolar lavage (BAL) lymphocytes stained with 7-AAD and Annexin/PI . No relationship was identified with cells stained with PI/Triton-X and Annexin/PI, and 7-AAD and PI/Triton-X indicating that methods which preserve cell membrane characteristics are more comparable when measuring BAL lymphocytes apoptosis in a heterogeneous population of cells. Additionally, all stains appear to perform the same in COPD and normal horses in remission and disease. Comparison of predominately BAL lymphocyte apoptosis using the above methods were performed at baseline, after natural challenge, and after dexamethasone administration in nine horses, five of which were affected with COPD. No differences in bronchoalveolar lavage lymphocyte apoptosis between COPD and control horses were detected either before or after dexamethasone administration, although numerical trends in COPD horses identified less apoptosis after natural challenge indicating that defective apoptosis may play a role in equine COPD pathogenesis. Dexamethasone administration was associated with trends of improvement in the pulmonary gas exchange and increased apoptosis toward baseline in the COPD horses. / Master of Science

This research focused on the cytotoxic properties of RRR-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) in transformed and cancerous cell lines. We used RAW264.7 macrophage and prostate cancer (LNCaP) cell lines in this study. TPGS caused cell death and decreased cell viability in a dose and time dependent manner. Cell death was evaluated fluorimetrically by employing the nucleic acid-binding fluorophore; propidium iodide. A colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate cell viability. Cell death can occur through necrosis or apoptosis. Our results suggested that TPGS triggered apoptotic cell death. Induction of apoptosis, as measured by caspase 3 enzymatic activity, was dependent upon the TPGS dose and incubation time. Caspase 8 was activated before caspase 9, suggesting the importance of the death receptor pathway in apoptosis. Our results indicated that TPGS cytotoxicity could also be due to one of its products of hydrolysis, alpha-tocopheryl succinate.

The antimicrobial properties of dextrose encapsulated gold nanoparticles (dGNPs) with average diameters of 25 nm, 60 nm, and 120 nm (± 5 nm) synthesized by green chemistry principles were investigated against both Gram-negative and Gram-positive bacteria. Studies were performed involving the effect of the dGNPs on the growth, morphology and the ultrastructural properties of bacteria. dGNPs were found to have significant dose dependent antibacterial activity which was directly proportional to their size and also their concentration. The microbial assays revealed the dGNPs to be bacteriostatic as well as bactericidal. The dGNPs exhibited their bactericidal action through the disruption of the bacterial cell membrane causing leakage of cytoplasmic content. The overall outcomes of this study suggest that dGNPs hold promise as a potent antimicrobial agent against a wide range of disease causing bacteria and can control and prevent possible infections or diseases.

Humans can be exposed to waterborne bacterial pathogens and numerous outbreaks have been reported involving these microorganisms around the world. Many different enteric pathogens can be found in source waters used for drinking water. Assessing these pathogens and their possible threat to public health has always been important. Waterborne pathogens can be difficult to detect, and despite a large variety of recognized microbial detection techniques, the cause of many outbreaks has not been unidentified. Effective and rapid pathogen detection techniques are required to achieve reliable data for microbial source water quality, outbreak investigations, and for drinking water treatment efficacy monitoring. Bacteria have long been detected using classical culture-based methods, with the rationale that living cells are able to grow/replicate. However, many pathogenic bacteria in source waters may turn into viable but not culturable (VBNC) cells and are thus undetectable by growth-based methodologies. Alternatively, PCR-based techniques have been developed to detect both non-culturable and culturable bacteria. Yet with these techniques, post-death DNA persistency can inaccurately overestimate the number of viable cells. This problem may be circumvented by an alteration to the PCR procedure that is reported to be able to block PCR amplification of DNA that originates from dead cells. This alteration involves a chemical pre-treatment step prior to PCR using a photoreactive intercalating dye, propidium monoazide (PMA). In this research, a successful modification was made to the PMA-PCR method that can result in substantial suppression of the PCR signal from dead cells, and provide results that can more accurately measure bacterial pathogen viability. PMA-PCR was applied to high concentrations (1 × 107 cells mL-1) of heat-killed cells of Salmonella enterica and Campylobacter jejuni. Using PMA-PCR in combination with primers that amplified a relatively short fragment of the S. enterica invA gene (119 bp), only a 3-log reduction of the dead cell PCR signal was obtained. Similarly, for C. jejuni using PCR primers that amplified a relatively short fragment of DNA (174 bp of cpn60 gene), only a 1-log reduction of the PCR signal was observed for dead cells. Therefore, PMA treatment followed by PCR amplification of short DNA fragments resulted in incomplete signal inhibition of heat killed Salmonella and Campylobacter. To further investigate how PCR conditions can affect the ability of PMA to inhibit PCR amplification, primers were then used that could amplify a larger fragment of DNA. PCR amplification of a longer DNA fragment (1614 bp of invA gene for S. enterica and 1512 bp of cpn60 gene for C. jejuni) strongly suppressed the signal (7 log reduction) for both heat-killed Salmonella and Campylobacter. For UV-treated S. enterica and C. jejuni, short amplicon PMA-PCR showed no or very low PCR signal reduction, in part due to intact membranes directly after UV irradiation. Long amplicon qPCR, however, resulted in dead cell signal removal and PMA pretreatment had no effect on PCR signal suppression. This study used quantitative PCR and the PMA-PCR viability assays to evaluate the levels and occurrences of four groups of pathogenic bacteria in surface water samples from two locations on the Grand River, Ontario, Canada, to demonstrate the reliability of the PMA-PCR technique for the enumeration of viable cells. The bacterial groups investigated included S. enterica, thermophilic Campylobacter, Escherichia coli O157:H7, and Arcobacter butzleri. Small numbers of dead cells (not more than 0.5 log 100 mL-1) were present, detected as the difference between PMA-PCR and PCR without PMA treatment. In this particular river, pathogen enumeration by PCR was only slightly influenced by false positive signal detection due to the presence of dead cells or extracellular DNA and reliable bacterial pathogen detection could be attained by PCR without PMA pretreatment. Viable A. butzleri were detected at elevated concentrations (up to 4.8 log cells per 100 mL) in the Grand River. Arcobacter has not been previously studied in the Grand River and this is one of the few studies that have quantitatively assessed Arcobacter in the environment. This suggests that additional research is required on the pathogenicity of this organism and its occurrence in water. In the next stage of this research, both the improved viability assay (long amplicon PMA-PCR) and conventional quantitative PCR were applied to investigate the survival trends of selected enteric bacterial pathogens including Yersinia enterocolitica, S. enterica, C. jejuni, and A. butzleri. The target bacteria were inoculated into sterile or non-sterile river water to study the impact of background microbiota on cell survival. These experiments were perfomed at 3 different temperatures (5, 15, and 25°C) and at high/low dissolved oxygen (DO) concentrations (for C. jejuni, and A. butzleri only) to evaluate the effect of these potential environmental stresses on bacterial survival trends. The results indicated that the autochthonous microbiota in river water had a significant effect on the bacterial die-off. Although lower temperatures enhanced bacterial survival in non-sterile river water, it was found that PCR may overestimate the effect of temperature on survival and that the PCR viability assays (PMA-PCR) could more accurately measure the impact of temperature. The survival of viable C. jejuni was adversely affected by high DO levels only at a low temperature (5°C) and this effect was observed only when the PMA-PCR viability assay was applied. A. butzleri survival was not affected by water DO levels. This research provides an improved understanding of viable/active enteric waterborne bacteria and their survival in the aquatic microcosms as well as reliable data to better elucidate the effect of environmental factors on the occurrence of pathogenic bacteria. It can also offer valuable information for microbial risk assessments used by regulators and decision makers.

This diploma thesis deals with the possibilities of stress resistance analysis of the Cupriavidus necator H16 and PHB-4 bacterial cells by spectroscopic methods and by testing the suitability of acridine orange as a viable dye. Based on research in literature, suitable analytical methods have been proposed, namely flow cytometer and fluorescence microscope. The first part of the experimental work was focused on the fluorescence microscope, which confirmed the basic character of acridine orange. Three stress factors, 50% and 70% ethanol, and acidic pH (pH = 1) were selected for viability monitoring. The bacteria fluoresced with green color after exposure to ethanol and red spots were found next to the cells, indicating their loss of integrity. In an acidic environment, the bacteria fluoresced red because of a partial DNA breakdown. The results were verified by the combination of propidium iodide with SYTO9 and the acridine orange suitability proved to be useful in this method. Image records were processed using image analysis. In the second part, acridine orange was used to monitor fluorescence using a flow cytometer. The result of the measurement was fluorescence expressed as histograms for individual channels, where fluorescence was characterized by median and mean intensity. By comparing the methods used, the acridine orange appears to be a more suitable fluorescent dye for the microscope than for a flow cytometer in which it was more difficult to obtain cell viability information. In the last part of the experimental work interesting photophysical properties of acridine orange were investigated.

The ability to deliver sequences of DNA and other molecular loads across the membrane of a cell and into its nucleus is an area of interest in the medical community. One of its many applications is that of gene therapy. In contrast to other forms of treatment, gene therapy seeks to treat diseases at the cellular level. The success of these treatments depends on the technologies for cell transfection that are available. Physical methods are sometimes able to overcome poor efficiencies of chemical methods and the safety concerns of viral methods, but are usually impractical due to the limited number of cells that are able to be transfected at a time, isolation, and immobilization of the cells. Nanoinjection is capable of using millions of small lances in an array to inject hundreds of thousands of cells simultaneously with relatively high efficiencies and viabilities. The solid nature of the lances also allows them to be smaller than their hollow-needle counterparts, which results in higher cell viability. Propidium Iodide (PI), a dye whose fluorescence increases greatly when bound to nucleic acids, was used as an injection molecule for testing the efficacy of the nanoinjection process on HeLa 229 cancer cells in a portion of the experiments, with a GFP plasmid of DNA being used in the rest. After injection, flow cytometry was used to detect the concentration of PI or the expression of the GFP in the injected cells. Since PI cannot normally penetrate the membrane of living cells, those found with high concentrations of PI were either successfully injected or dead, which can be determined by the flow cytometry. Investigation of the parameters that affect the efficiency of the nanoinjection process will help improve it for further research. Some of these parameters that were investigated include the force of injection, the material used for the lances (silicon versus carbon nanotubes), and the injection speed of the lance arrays. An injection device capable of small changes in deflection was designed to ensure accurate increments in force for testing, as well as a pulsed current control injection system. Results for injections of varying forces indicate a slow rise in PI uptake from 0 to 1.8 Newtons where it reaches a maximum uptake of 4.11 when normalized to the PI uptake of the positive controls. The PI uptake then remains relatively level as the force continues to increase, averaging an uptake of approximately 3.1. The slow rise is likely due to more of the cells being punctured as the force increases until most have been punctured and the PI uptake levels off. The viability of the injected cells was close to that of the controls with no clear trend. A comparison of lance arrays made from silicon and carbon nanotubes using DNA as the molecular load shows little difference between materials. Different injection speeds tested show that only 1-5% of the cells in the injection process are lost for speeds in the range of 0.08-0.16 mm/sec, whereas 49-69% of the cells are lost using speeds between 0.6-3 mm/sec.

Delivering foreign molecules into living cells is a broad and ongoing area of research. Gene therapy, or delivering nucleic acids into cells via non-viral or viral pathways, is an especially promising area for pharmaceutics. All gene therapy methods have their respective advantages and disadvantages, including limited delivery efficiency and low viability. Nanoinjection, or delivering molecules into cells using a solid lance, has proven to be highly efficient while maintaining high viability levels. In this thesis, an array of solid silicon lances was tested by nanoinjecting tens of thousands of HeLa cancer cells simultaneously. Several molecule types were injected in different tests to understand cell uptake efficiency and cell viability. Voltage was used to determine the impact of an electric field on molecule delivery. Propidium iodide, a dye that fluoresces when bound to nucleic acids and does not fluoresce when unbound, was delivered into cells using the lance array. Results show that the lance array delivers propidium iodide into up to 78% of a nanoinjected HeLa cell culture, while maintaining 78%-91% viability. Using similar protocol as in propidium iodide experiments, plasmid DNA containing the code for a fluorescent protein was nanoinjected into HeLa cells, resulting in an average expression rate of up to 0.21%. Since gene expression only occurs in cells which have integrated DNA into the genome in the nucleus, a different DNA detection method was developed to determine total DNA count in cells following nanoinjection. DNA strands tagged with a radioactive isotope were nanoinjected into HeLa cells. Liquid scintillation was employed to quantify and discriminate between DNA delivered to cells and DNA that remained in solution around cells following nanoinjection. The largest average amount of DNA delivered to cells was 20.0 x 10^3 DNA molecules per cell. Further development of the radioactive nanoinjection process is needed to more fully understand the parameters that affect DNA delivery efficiency. In all experiments with propidium iodide and DNA molecules, low accumulation voltage, coupled with a short pulsed release voltage, resulted in the greatest molecule delivery efficiencies when compared to tests without voltage or with a constant voltage only. Lastly, an automated nanoinjection system was developed to eliminate variability in user applied nanoinjection force. The automated system was found to reduce variability in average propidium iodide uptake values by 56%. In conclusion, experimental testing of the multi-cell nanoinjection process has shown promising molecule delivery results into human cells, suggesting that further optimization of the process would have positive implications in the field of academic and clinical gene therapy.

Genomic analysis provides a substantial amount of information on evolutionary history, novel genes, transcriptomic expression and regulation in response to environmental stimuli, how efficiently organisms utilize their genome, and directional genome evolution. Genome size analysis serves as the first step in the sequencing process, because sequencing and annotation costs are directly correlated with genome size. Invertebrates represent the vast majority of faunal diversity on the planet, and, to a greater extent, the marine environment, although they are vastly understudied when compared to vertebrate genomes. Flow cytometry is a widely used, reliable, and accurate means of estimating genome sizes and has yielded valid measurements in this comparatively broad taxonomic study. This methodology quantifies genome sizes by measuring the fluorescent re-emission from nuclei that have been saturated with DNA- intercalating dyes, such as propidium iodide. Genome sizes of 19 species across five phyla were estimated by comparison with the known genome size of chicken (Gallus domesticus). Several estimates reported here are the first for their species or class. In addition to estimating new marine invertebrate genome sizes, analyses of some common preservation methods of tissue viability for flow cytometric estimations were performed. Generally, in comparison to RNAlater or ethanol, DMSO-based storage buffer was most successful at preserving nuclear membrane integrity, a requirement for flow cytometric genome size estimations. Recommendations of cost-effective species eligible for current next-generation sequencing technology (<3.5 Gb) are given for invertebrate genomicists seeking potential novel species to sequence.