Bacterial research has been flourishing ever since 17th century. Bacteria play an
important role in clinical microbiology, food microbiology and environmental microbiology. Therefore, the rising need to detect bacteria in clinical, food and environmental samples is emerging onto a craving research frenzy. There are some established methods to study bacteria, such as genetic techniques like PCR, culturing and RNA based analysis, but these require time, resources and labor. This thesis concentrates on highlighting the importance of MALDI-MS in rapid, direct, and simple detection of bacteria from food and clinical samples. The method is to enable detection of bacteria in real samples.
First attempts were made to detect bacteria from yogurt sample, because as already known commercial yogurt samples contain bacteria. And yogurts were used in this study to demonstrate the capability of MALDI-MS technique to detect the bacteria directly by culture-free methods. Since the bacterial signals were quenched because of the strong milk peaks, we had to employ nanoparticles and ionic solution for affinity capture of the bacteria for bacterial detection. The results showed that addition of ionic solution and Ag nanoparticles lead to enhanced bacterial detection. The deterioration of the yogurt bacteria on improper storage ( at room temperature) and after extended storage in refrigerator beyond expiry date was studied using MALDI-MS. The day by day deterioration in the microbiological quality of the yogurt was also detected. A special selective media for growth of the yogurt bacteria, DifcoTM Lactobacilli MRS Broth (Agar) was used to quantify the bacteria in various experiments. MALDI-MS results were useful in understanding the shelf life and quality control on extended storage.
The second part of this thesis concentrates on the use of MALDI-MS in clinical analysis. The major goal in this work was to use MALDI-MS to detect bacteria in mousy blood and urine. Phase I of this study involved detection of the bacteria by direct MALDI-MS in blood and urine samples spiked with bacteria. We call these experiments as in vitro experiments. Analysis using MALDI was a challenge since the blood peaks interfered with the bacterial peaks and subdued them. Ionic liquid is a pretty excellent extraction solvent when combined with SDME (Single-drop Microextraction). Just one drop of 1-butyl-3methylimidazolium hexafluorophosphate can extract bacteria from spiked samples. What¡¦s more, the LOD can be lower to ~105 cfu / mL using SDME with ionic liquid. It¡¦s definitely showing that 1-butyl-3methylimidazolium hexafluorophosphate, that is to say, the ionic liquid has good ability to trap pathogenic bacteria.
This bacterium is injected into mouse and the growth kinetics of bacteria in vivo was studied as a function of time. Samples taken at specific time points were simultaneously analyzed using standard plate count method as well as MALDI-MS. Analysis using MALDI was a challenge since the blood peaks interfered with the bacterial peaks and subdued them. Therefore, we employ the use of silver and zinc oxide nanoparticles and ionic liquid.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0901111-122509 |
| Date | 01 September 2011 |
| Creators | Lee, Chia-hsun |
| Contributors | Hay-yan Wang, Hui-fen Wu, Jing-yueh Jeng |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0901111-122509 |
| Rights | user_define, Copyright information available at source archive |
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