Masters Degrees (Microbiology)

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Browsing Masters Degrees (Microbiology) by browse.metadata.advisor "Bessarabov, D. G."

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    Characterization of the early stages in biofilm development
    (Stellenbosch : Stellenbosch University, 2004-04) Marais, Susara; Wolfaardt, Gideon M.; Bessarabov, D. G.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Complex biofilm communities have extensively been studied in the past. Less work has been done on the early stages of biofilm formation. This study aimed to assess initial colonization patterns of biofilms on different surfaces and under different environmental conditions with application of novel methods describing biofilm surface profiles. Biofilms were cultivated on glass, polyvinylchloride (PVC) and polished stainless steel. Results from microscopy, followed by mathematical analysis and contact angle measurements proved that glass was the most appropriate substrate for this study. More extensive extracellular polymeric substances (EPS) production and apparently less cell attachment were observed on PVC and polished stainless steel surfaces. Two different series of experiments were conducted where biofilms were cultivated on the glass. Biofilm morphology was analysed under various conditions of temperature and nutrient concentration. Different temperature conditions were 8°C, 22°C and 37°C and different nutrient concentrations were 0.1%, 1% and 10% Tryptic Soy Broth (TSB). After obtaining samples after 1, 2, 3 and 4 days respectively, the biofilm surfaces were visualised using atomic force microscopy (AFM) and epifluorescence microscopy. Less cell attachment was displayed at lower temperatures and nutrient limitations. The roughness profile of the early stages of biofilm development was explored by the novel application of various existing statistical methods. Benoit software was applied for the statistical analysis of various data sets obtained from AFM imaging, using power spectrum, variogram and wavelet methods to determine the Hurst exponent. The variogram method proved to be the most suitable to describe biofilm surface profiles with consistent values of ± 0.9, indicating that biofilm growth behaviour will continue in a similar pattern. Fractal dimension values of images obtained from epifluorescence microscopy was determined by the box dimension method. The values described the self-affine patterns displayed by biofilms. Using the results of these investigations, a series of models concerning the initial stages of biofilm formation was compared to describe the development of colony patterns. This study showed that the AFM and epifluorescence microscopy can be used as analytical tools for raw data assembly. It also demonstrated a novel application of existing statistical methods in order to describe the early stages of biofilm formation. Using this approach it was shown that the early stages of biofilm formation display certain colony patterns that can be described and predicted. Such information may be used in efforts to control biofilm formation.
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    Microbial response to oxidising biocides
    (Stellenbosch : Stellenbosch University, 2003-03) Jackson, Vanessa A. (Vanessa Angela); Wolfaardt, Gideon M.; Bessarabov, D. G.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Biofouling of water systems is a problem extensively experienced in industry. Although this subject is the focus of many studies, the ability of microorganisms to survive exposure to biocides is still poorly understood. This study aimed to assess the biocidal effect of ozone on planktonic cells and biofilm communities, to evaluate different ozone generation techniques, and to follow population shifts within the biofilm community. Specific objectives included determining the effect of different ozone concentrations, the effect of different exposure times, and an assessment of microbial responses after exposure to sub-lethal ozone concentrations. Typically, 300 ml of an ovemight bacterial culture was exposed to ozone that was generated by anodic oxidation (0.3% wt or 18- 20% wt, respectively) or silent electric discharge (3.5% wt 03). The ozone was purged into the culture for 5-, 7-, 10- and 15 min., respectively. Enumeration of cells following ~10 min. exposure to 18-20% wt ozone showed a significant reduction in viable cell numbers. In contrast, when exposed to the two lower 03 concentrations, there was little change in the viable cell numbers even after prolonged exposure (30- and 60 min.). To evaluate biofilms, ozone was bubbled into the irrigation that was pumped through replicate flow cell channels. Response to ozone exposure was evaluated after staining the biofilms with the Baclight Viability probe, observation with fluorescence microscopy, and image analysis. The higher ozone concentration (18-20% wt 03) more effectively disrupted the biofilm structure of denser biofilms than the lower concentration, especially after 90 min. exposure. When compared to the controls, the 90 min. exposure resulted in a notable reduction in viable cells from 69% to 38% and a corresponding increase in nonviable cells from 29% to 62%. The lower concentration ozone (3.5% wt 03) was effective against the less dense, thinner biofilms evaluated, but not effective against the thicker biofilm. An analysis of the differences between continuous culture biofilms and batch culture biofilms showed that the biofilms in the batch system were less rigid. To evaluate microbial response to biocides, techniques such as Biolog whole-community metabolic profiles and terminal restriction fragment length polymorphisms (T-RFLP) were used. Biolog analysis of planktonic cells revealed changes following exposure to sub-lethal biocide concentrations, however carbon utilisation profiles resembled that of the controls after 24-48 hours. For biofilm communities, no carbon utilization differences could be detected under these conditions. There was, however differences in T-RFLP patterns between treated and untreated biofilm communities.