Typescript (photocopy). / Digitized by Kansas Correctional Industries

Advanced laboratory techniques for Microbial and Molecular Biology at the graduate level are presented in this thesis. The procedures for the laboratory experiments are set forth in detail. This laboratory is conducted as two parts, each by a different professor. Part 1 covers the experiments conducted by Dr G.A.O. Donovan. These experiments include an introduction, staining procedures, biochemical reactions, mutagenesis experiment, essays,. preparation and analysis of plasmid DNA and various other topics. Part 2 covers the experiments conducted by Dr. Daniel Kunz and includes various topics like media preparation, phenotyping strains, conjugative transfer of plasmids, SDS-PAGE, induction and measurement of enzyme and transposon mutagenesis

Call number: LD2668 .T4 1985 L43 / Master of Science

No description available.

The infrared spectra of 14 bacteria were recorded by Fourier Transform Infrared (FTIR) spectroscopy. The effects of changes in growth conditions, such as temperature, growth medium, and incubation time, sampling conditions on the reproducibility of the spectra were investigated. The spectra of bacteria suspended in saline solution, in D$ sb2$O-saline and on plate films were obtained and stored in separate spectral libraries. The application of library search routines for differentiation and identification of the 14 bacteria was investigated. The spectral regions used for the library search include 1750-1560 cm$ sp{-1}$ and 1480-960 cm$ sp{-1}$, which contain bands mainly associated with proteins (secondary structure), lipids, and polysaccharides. An index produced by the library search, which indicates how much an "unknown spectrum" matches a library spectrum, is suggested as a criterion for the differentiation and identification of bacteria. A correct identification of five unknown samples shows the feasibility of using spectral library searching routines for identification of bacteria down to the species level by FTIR spectroscopy. This technique is rapid, easier, and more efficient than conventional microbiological and biochemical methods. Quantitative analysis of a mixture of Escherichia coli and Staphylococcus aureus by the partial-lest-square (PLS) technique was also evaluated. The analytical error was about 10%.

[Truncated abstract] The applications of biosensors are rapidly expanding with the increased emphasis placed on the use of technology in the evaluation of food safety and also in military use. The United States food industry carried out 144.3 million microbiological tests in 1999 (Alocilja and Radke, 2003). These numbers are expected to rise with the recently implemented regulatory measures for food safety in the United States. In fact, similar trends in food safety are occurring on a global scale. Furthermore, with the recognition and establishment of Microbial Forensics as a new field of forensics, the interest in biosensor development for the detection of microbes will thrive. Moreover, the recent spate of biocrimes, notably the anthrax scares, has called for newer and improved techniques for the sensitive, rapid and reproducible detection of microbes. Biosensors have the capability to fill this role as an efficient device for microbial detection. There is a wide range of biosensors available for different purposes. In addition, their versatility allows for their overlap in many fields. The quartz crystal microbalance (QCM) is a biosensor that is cost-efficient, sensitive, field-deployable with the ability to perform automated, real-time assays within minutes. The QCM is a mass sensitive device that works on the principle where a change in mass deposited on the crystal is inversely proportional to the change in the resonant frequency of the crystal. Therefore, frequency decreases with increasing mass deposited. The QCM has been used in several studies as a biosensor for the detection of a number of viral and bacterial species. ... High antibody incubation concentration required a shorter antibody incubation duration. Conversely, low antibody incubation concentration required a longer antibody incubation duration. Furthermore, regardless of antibody incubation concentration, a distinct pattern in the rate of antibody binding with time was observed. One hour antigen incubation at ambient room temperature (22.5oC) was sufficient for the efficient binding of the antigens to the immobilized antibody layer. Extension of antigen binding time to 15 hours produced inconsequential differences in readings. The binding efficiency of the quartz crystals after a storage period of 2 to 4 weeks at ambient room temperature (22.5oC) fared better than the crystals that were refrigerated at 4oC. Results showed that 0.2M glycine hydrochloride is a poor reagent for the removal of the antigen layer on the quartz crystals for repeated assay use. The 16-mercaptohexadecanoic acid (MHDA) layer and adsorbed proteins on the quartz crystals can be removed by a mixture of sulphuric acid and hydrogen peroxide, known as a piranha process. This allows the crystals to be repeatedly recoated and reused. Overall, this research provides new insights into the preparation process of the quartz crystals for the specific detection of E. coli O157:H7. Conclusive results have been obtained for several tested parameters and suggestions have been raised for further studies in the optimization of the QCM for the E. coli O157:H7 detection process. With improved knowledge and recognition in the capability of the QCM as a biosensor, the QCM may soon be used in conjunction with conventional techniques for the rapid detection of E. coli O157:H7.

"June, 2008". / Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Dept. of Chemistry & Biomolecular Sciences, 2009. / Bibliography: p. 227-259. / Literature review -- Materials and methods -- Sampling sites and sampling material -- Enrichment cultures and molecular analyses -- Optical and binding characterisation of the QDs -- Applications of the QDs -- Concluding remarks. / White island, the most active volcano in New Zealand, is a poorly studied environment that represents an ideal site for the investigation of acidophilic thermophiles. The microorganisms present on here are continually exposed to extreme environmental conditions as they are surrounded by steamy sulphurous fumaroles and acidic streams. The sediment temperature ranges from 38°C to 104°C whilst maintaining pH values below 3. A survey of the volcanic hydrothermal system of White Island was undertaken in order to gain insights onto the microbial diversity using culture-dependant techniques and molecular and phylogenetic analyses. A novel liquid medium based on "soil-extract" was designed which supported growth of bacterial and archaeal mixed cultures. Molecular analyses revealed that the dominant culturable bacterial species belong to the Bacteroidetes, Firmicutes and α-Proteobacteria groups. Several previously uncultured archaeal species were also present in the mixed cultures. The knowledge gained from these studies was intended to help in the development of a novel microbial detection technique suitable for community analysis. -- Conventional molecular techniques used to study microbial biodiversity in environmental samples are both time-consuming and expensive. A novel bead-based assay employing Quantum dots (QDs) was considered to have many advantages over standard molecular techniques. These include high detection speeds, sensitivity, specificity, flexibility and the capability for multiplexed analysis. QDs are inorganic semiconductor nanoparticles made up of crystals about the size of proteins. It has been claimed that the physical and chemical properties of the QDs have significant advantages compared to organic dyes, including brighter fluorescence and resistance to photo-bleaching. Their optical properties facilitate the simultaneous imaging of multiple colours due to their flexible excitation and narrow band emission. Functionalised QDs are able to bind to different biological targets such as DNA, allowing high-throughput analysis for rapid detection and quantification of genes and cells. -- The optical and physical characteristics of the QDs as well their interaction with biomolecules are shown to be suitable for the development of a novel bead-based technique able to target the key microbial species and identify them by flow cytometric measurements (FCM). The broad absorption and narrow emission spectra of the QDs, as well as their fluorescence intensity and specify to target biomolecules, was compared to other organic fluorophores. The potential advantages and limitations of QDs as a fluorophores for biological applications are discussed. -- The data acquired during this study provides a broad overview of the microbial diversity and ecology of the volcanically-active hydrothermal systems of White Island and constitutes the baseline for the development of a novel bead-based technique based on QDs. / Mode of access: World Wide Web. / xvii, 259 p. ill. (some col.)