Thesis (M.S.)--University of Michigan, 1972.

Thesis (M.S.)--University of Michigan, 1972.

Monitoring and controlling pH is of utmost importance in bioprocessing as it directly affects product yield and quality. Multiplexed experiments can be performed in nanobioreactors for optimisation of yield and cell heterogeneity in a relatively quick and inexpensive manner. In this thesis, a pH holographic sensor (holosensor) is miniaturised to 3.11 nL in volume and integrated into a PDMS-glass microfluidic chip for monitoring the growth of Lactobacillus casei Shirota. Although other established methods for monitoring cell cultures can be utilised, miniaturised holosensors enable real-time and non-consumptive monitoring of the bacterial cell culture growth medium. The 2-hydroxyethylmethacrylate (HEMA)-co-2-(trifluoromethyl) propenoic acid (TFMPA) holosensor was fabricated using an adapted technique from photolithography, coupled with the use of a polymerisation inhibitor to control the gel polymerisation with diameters not exceeding a standard deviation of 0.067. The hologram brightness was optimised to 1.05 ms integration time with 36X magnification using a low power (0.290 mW) 532 nm green continuous wave (CW) laser with a devised beam-offset technique. The holosensor was characterised with ionic strength balanced (9.50 mS/cm) McIIvaine pH buffers and a calibration curve plotted together with measured ionic strength, optical density at 600 nm (OD600) and pH. Correspondingly, RGB-xyY transformed values were plotted in the CIE 1931 chromaticity diagram. Later, a miniaturised 0.4φ HEMA-co-TFMPA holosensor and array was also demonstrated. Together with the 3.0φ holosensor, an accuracy parameter for the 0.4φ spot and array holosensors were calculated to be 99.08%, 99.38% and 97.77% respectively. Further work involved studying the issues associated with fabricating gels with unusually flat gel profiles. Other preliminary results suggested the alternative of utilising polymers as a holosensor substrate, together with a dye-free method for hologram fabrication, outlined the prospective possibility of a miniaturised holosensor integrated into a polymer microfluidic chip with the flexibility of hologram colour customisation for cell culture monitoring.

Orientadores: Francisco Carlos Groppo, Pedro Luiz Rosalen / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-06T14:44:39Z (GMT). No. of bitstreams: 1 Cogo_Karina_M.pdf: 1252832 bytes, checksum: c6cff0a881e182ed79ce20ff045dd117 (MD5) Previous issue date: 2006 / Resumo: Existem evidências de que o biofilme presente na região subgengival é o principal agente etiológico das doenças periodontais. O uso do cigarro tem sido associado com a progressão da doença periodontal bem como com a redução da resposta à terapia aplicada a essa doença. Alguns estudos têm indicado uma forte relação entre o hábito de fumar e o consumo de café. No entanto, os mecanismos pelos quais o cigarro e o consumo de café podem afetar o tecido periodontal ainda não foram totalmente esclarecidos. Dessa forma, o objetivo do presente estudo foi avaliar in vitro os efeitos da nicotina, cotinina e cafeína na viabilidade de algumas espécies bacterianas da microbiota subgengival. Biofilmes mono-espécie de Streptococcus gordonii, Porphyromonas gingivalis e Fusobacterium nucleatum e as combinações de biofilme com duas espécies, S. gordonii + F. nucleatum e F. nucleatum + P. gingivalis foram desenvolvidos em discos de hidroxiapatita banhados em saliva artificial. Um total de sete espécies foi avaliado como células planctônicas, incluindo as espécies acima mencionadas e o Streptococcus oralis, Streptococcus mitis, Propionibacterium acnes e Actinomyces naeslundii. As espécies bacterianas foram incubadas com ou sem nicotina, cotinina e cafeína nas concentrações que variaram de 0,37 a 400 µg/mL para células planctônicas e 400 µg/mL para biofilme. O crescimento das células planctônicas e do biofilme foi avaliado pelos testes de susceptibilidade e "time-kill", respectivamente. Os resultados do teste de susceptibilidade mostraram que a nicotina reduziu o crescimento da S. gordonii (400 µg/mL) e S. oralis (0,37-400 µg/mL); a cotinina estimulou o crescimento das espécies A. naeslundii (0,37 µg/mL) e F. nucleatum (0,37-400 µg/mL) e reduziu o crescimento da S. oralis (400 µg/mL); e a cafeína estimulou o crescimento da F. nucleatum (400 µg/mL). Nos' testes de "time-kill" foram observados um aumento do crescimento do biofilme mono-espécie de F. nucleatum e uma redução da viabilidade do biofilme mono-espécie de S. gordonii, após 24 horas e 48 horas de exposição à cotinina e à cafeína, respectivamente. Es~es resultados indicam que a nicotina, cotinina e cafeína podem afetar, embora em pequena extensão, o crescimento e a viabilidade das espécies bacterianas orais estudadas / Abstract: There are significant evidences that subgingival accumulation of bacterial biofilm is the etiologic agent in periodontal diseases. Cigarette smoking might result in progression of periodontitis and in impaired response to periodontal therapy. Some studiesindicated a strong relationship between cigarette smoking and coffee drinking. However, the mechanisms by which smoking and coffee consumption affect the periodontium are not clear. The purpose of this in-vitro study was to evaluate the effects of nicotine, cotinine, and caffeine on the viability of some bacterial species from the oral microbiota. Single-species biofilms of Streptococcus gordonii, Porphyromonas gingivalis, and Fusobacterium nucleatum and dual-species biofilms of S. gordonii + F. nucleatum and F. nucleatum + P. gingivalis were grown on hydroxyapatite discs. Seven species were studied as planktonic cells, including Streptococcus oralis, Streptococcus mitis, Propionibacterium acnes, Actinomyces naeslundii, and the species mentioned above. Bacteria were incubated in either O or 0.37 to 400 µg/mL of nicotine, cotinine or caffeine for planktonic cells and O or 400 µg/mL for biofilm. The viability of planktonic cells and biofilms was analyzed by susceptibility tests and time-kill assays, respectively. "Susceptibility Tese' showed that nicotine reduced the growth of S. gordonii (400 µg/mL) and S. oralis (0.37-400µg/mL); cotinine stimulated the growth of A. naeslundii (0.37 µg/mL) and F. nucleatum (0.37 -400 µg/mL) and reduced the growth of S. oralis (400 µg/mL), and caffeine stimulated the growth of F. nucleatum (400 µlg/mL). Results of "Killing Assays" showed an enhanced growth of F. nucleatum in single-species biofilm and a reduced viability of S. gordonii in single-species biofilm, 24 h and 48 h after exposure to cotinine and caffeine, respectively. These findings indicated that nicotine, cotinine and caffeine could slightly affect the growth and viability of some oral bacterial strains / Mestrado / Farmacologia, Anestesiologia e Terapeutica / Mestre em Odontologia

Orientador: Pedro Eduardo de Felicio / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-03T18:35:57Z (GMT). No. of bitstreams: 1 Gomes_TerezaCristinaSeger_M.pdf: 4002767 bytes, checksum: 54286ae4b65fbd30ba53abff3eca4b83 (MD5) Previous issue date: 2003 / Resumo: Porções de peso médio de 550 g de carne, em bifes de 10 mm de espessura, cortados a partir de 12 peças resfriadas de contrafilé, foram acondicionadas em bandejas de poliestireno expandido, envoltas em filme poliolefínico termoencolhível, 8 bandejas foram agrupadas formando um conjunto denominado masterpack.A modificação da atmosfera na embalagem masterpack foi obtida com injeção de 100% CO2 à razão de 0,8 L gás/kg de carne. O lote foi estocado à temperatura de 2±2 ºC. Foram comparados cinco períodos de estocagem em masterpack: 4, 10, 12, 17 e 19 dias e três tempos de espera para oxigenação da embalagem primária: 3, 24 e 48 horas a 4±2 °C, após a abertura e retirada do masterpack. Foi avaliada a cor regenerada durante a exposição à oxigenação, por análise instrumental e sensorial. Foram avaliadas a estabilidade microbiológica e físico-química, com medidas de pH, Eh e maciez. Verificou-se o efeito da atmosfera com 100% de CO2 sobre a flora aeróbia e as bactérias lácticas. Apesar do residual de oxigênio inicial obtido de 300 ppm, o ar ocluso nas embalagens primárias que migrou para o masterpack durante a estocagem, expondo o produto a uma atmosfera contendo entre 10000 e 34000 ppm de O2, desde o primeiro dia de estocagem, permitiu uma coloração inaceitável e, manchas de cor marrom, que limitaram a vida útil em 10 dias de estocagem em masterpack a 2±2 °C, e mais 24 horas de exposição aeróbia a 4±2°C / Abstract: Samples of steaks longissimus dorsi, approximately 10 mm thickness, were master-packaged in sets of 8 retail-ready polystyrene trays (550g each) under 4 liters volume of CO2. The modified atmosphere contained O2 at concentrations between 300 and 34000 ppm. The masterpackaged samples were stored at temperature of 2±2 °C for 4, 10, 12, 17 and 19 days, then subsequently displayed aerobically for 3,24 and 48 hours, at 4±2°C for reblooming. After each reblooming time the samples were evaluated in terms of color and microbial stability and pH, redox potential and tenderness were measured. The composite results clearly indicated color stability was progressively lost during both chilled storage and subsequent aerobic display, even though, the excellent oxygen residual obtained initially (300 ppm), the occluded air inside the trays allowed discoloration. Storage life was limited by the development of undesirable color which were independent of microbial presence due to microbial development was quite controlled by the CO2 including the lactic acid bacteria. But residual oxygen up to 34000 ppm during the storage life was critical to the success. The most attractive storage option was 10 days and additional 2 days aerobically exposed. / Mestrado / Mestre em Tecnologia de Alimentos

The adenylate energy charge values of twenty bacteria isolated from soil and cultured in a medium consisting of soil and distilled water were determined by the luciferin-luciferase bioluminescense method. The purpose of this study was to examine the growth and energy charge values of these organisms in soil extract medium, and to determine what effect the addition of glucose has on their energy charge values. Three of the organisms employed in this study showed energy charge values similar to those reported for bacteria grown in enriched media. The remainder of the isolates demonstrated low energy charge values, and scant growth in the soil medium.

The problem in this investigation is to determine the effects of various concentrations of sodium chloride upon the growth of three species of bacteria. An effort has been made to solve this problem, not only by a study of the relevant literature, but also by laboratory research consisting of cultivation and observation of the three organisms which were arbitrarily chosen for this study.

Ten bacteria capable of growth on the metal-cyano complex, tetracyanonickelate (II) {K2 [Ni(CN)J } (TCN), supplied as the sole nitrogen source, were isolated. Seven isolates were identified as pseudomonads while the remaining three were classified as Klebsiella species. In addition to TCN, all isolates were able to utilize KCN although it was significantly more toxic. The degradation of TCN was most complete when supplied at growth-limiting concentrations, did not occur when ammonia was present, and resulted in the formation of nickel cyanide [Ni(CN)2] as a degradation product.

Cette thèse porte sur l’étude de la variabilité de la croissance de bactéries en gouttes micro-fluidiques. Dans un premier temps, la puce micro-fluidique utilisée au cours de la thèse est présentée. Elle permet d’encapsuler des bactéries individuelles dans 1500 gouttes d’un nano litre, et de suivre leur croissance en parallèle grâce à la mesure de leur fluorescence par microscopie. La relation entre fluorescence mesurée et nombre de bactérie est discutée, et plusieurs questions techniques, comme la variabilité de taille des gouttes, l’hétérogénéité de fluorescence des bactéries, sont mesurées et leurs conséquences sur les mesures de croissance quantifiées. Dans un second temps, nous développons un modèle probabiliste qui permet, à partir de la variabilité des temps de divisions des bactéries, de prédire la variabilité de croissance entre les gouttes. Pour ce faire, nous adaptons le modèle classique de Bellman-Harris. La distribution aléatoire du nombre initial de bactérie par gouttes, ainsi que les temps de divisions différents des premières générations de bactéries sont ajoutées au modèle pour l’adapter à notre système expérimental. Les contributions de ces différentes sources de variabilité à la variabilité inter-gouttes de croissance des populations de bactéries sont quantifiées, et le modèle permet bien d’expliquer la variabilité de la croissance entre les gouttes. Dans un troisième temps, nous proposons un schéma d’inférence pour résoudre le problème inverse, qui est de retrouver, à partir des courbes de croissance, la variabilité des temps de division des bactéries individuelles. Le modèle précédent ne peut être utilisé à cause des sources externes de variabilité, nous proposons donc un schéma d’inférence basé sur le suivi dans le temps de chacune des trajectoires des gouttes. Grâce à des simulations reproduisant les conditions expérimentales, nous prouvons que l’inférence est possible. Elle ne peut être appliquée à nos expériences en raison de la précision insuffisante de notre mesure de fluorescence. Enfin, la même puce micro-fluidique est utilisée pour quantifier l’action d’antibiotiques sur des bactéries, notamment la réponse SOS qui est induite lorsque l’ADN de la bactérie est endommagé. La technologie d’encapsulation en goutte est utilisée pour mesurer l’hétérogénéité de réponse des bactéries et la relier à leur capacité à survivre au stress dû à l’antibiotique, et à reformer une colonie. / This thesis presents some results about the variability of the growth of bacteria in microfluidics droplets. In the first chapter, the microfluidic chip used throughout the PhD is presented. It allows to encapsulate bacteria in an array of 1.500 nano-liter sized droplets, and to follow their growth in each droplet in parallel through fluorescence microscopy. The link between the measured fluorescence and the number of bacteria in a droplet is discussed, and other technical questions are addressed, such as the variability in droplet size and the cell-to-cell fluorescence variability. Next, we develop a stochastic model to account for the observed variability of population size in the droplet during the exponential phase of growth. A well-known stochastic model, the Bellman-Harris model, is adapted to take into account the external sources of randomness due to our experimental system (initial distribution of bacteria per droplet, different division time of the first generations). They are taken into account, along with the effects of the cell-to-cell variability of division times in our model, which is successful to predict the variability observed in the microfluidics experiments. Then we tackle the inverse problem, which is to recover the cell-to-cell variability from the observation of the growth in droplets. We propose an inference scheme based on following each droplet in time. The deviation from pure exponential growth is linked back to the cell-to-cell variability, and this inference scheme is proven to be successful on simulations that mimic the experimental constrains. However, we cannot completely apply it to our experiments because of a lack of accuracy in our fluorescence measurements. Finally, we demonstrate how our chip can represent a gain of space and time to quantify the effect of antibiotics on a bacterial strain compared to classical susceptibility measurement methods. We also show how it can be used to study the variability of the SOS response of bacteria, which is a bacterial stress response induced when the DNA of the cell is damaged, and relate it to the ability to survive an antibiotic treatment.

An interaction of the commensalism type between P. vulgais and S. cerevisiae was discovered and verified. A continuous flow apparatus for investigation of this interaction was described. A Coulter Counter was used for detecting the number of the microorganisms grown in mixed cultures. Size distribution curves were prepared and used as a means for determining the Coulter Counter settings for counting each organism. Coulter Counter counts were compared with Petroff-Hausser counts; the results showed approximately 20 per cent discrepancy which is reasonable since the Petroff-Hausser method is quite crude. P. vulgaris in mixed culture and in the media of Table I was shown to be completely dependent upon S. cerevisiae. S. cerevisiae was found to be unaffected by P. vulgaris, thus, the definition of commensalism is fully satisfied. The search for the biochemical causing this interaction led to the trial of the following: 1 per cent yeast extract, 0.01 per cent DPN, 1 per cent Nicotinamide, and 0.01 per cent Niacin; all sterilized by filtration. The slug addition of these biochemicals to the culture vessels caused the P. vulgaris to be independent of the S. cerevisiae. As a result, Niacin or near biochemical relatives are believed to be the essential compounds causing the interaction between P. vulgaris and S. cerevisiae. The steady state population of the P. vulgaris was found to increase with Niacin concentration up to a certain limit above which further Niacin has no effect. The concentration of Niacin-like activity formed per yeast cell was also calculated; this concentration per yeast cell was found to vary with the flow rate. Concentration of the media and aeration were also found to have an effect on the steady state populations. Inoculating cultures of S. cerevisiae with a faster growing bacterium, E. coli, showed E. coli increasing in number until it reached a steady state while S. cerevisiae decreased in number to a null value. This result was in agreement with that predicted by published theories of contamination, Golle (1953). / Ph. D.