碩士 / 國立成功大學 / 化學工程學系碩博士班 / 101 / This study presents the effects of alternating frequency and duration of electric field on the fluorescence labeling of Chlorella vulgaris by two lipid probes: Nile red and Lipid TOX® Green. The application of electroporation on releasing cellular contents is also investigated as well as the feasibility of this technique in quantifying cell compositions.
Conventional gravimetric methods for quantifying lipid amounts inside microalgae are complicated and time consuming. Fluorescence detection of cellular lipids has been established decades ago; however, it is limited by the inconsistences of fluorescence intensity due to variations in cell membrane compositions. Therefore, this study aims to use electroporation to facilitate the transport of fluorogenic dyes into cells for consistent labeling outcomes. It is found that 10KHz, 2000V/cm was optimal for enhancing the fluorescence labeling of Chlorella vulgaris and optimal durations in electric field were 90 sec for Nile red and 20sec for lipid TOX® Green. To avoid photo-bleaching, we applied flow-through electroporation and discovered that the decay of cell fluorescence was contributed by not only photo-bleaching but also the release of cell contents. When electric field strength was higher than 500V/cm at 10KHz, cell contents can be effectively released and high cell density can provide stable releasing efficiency. Because alternating current electric field can result in dielectrophoretic flow, this study also optimized the cell fixation method to prevent cell movement during observations. Different dispersants were used to disperse cellulose for well surface coating. The results indicate that the mixture of alcohol and cellulose had better abilities for cell fixation.
In additional to the electroporation of Chlorella vulgaris cells in high electric field strength, this study also investigates the effects of low electric field strength on the cell culture. We found that Chlorella vulgaris have larger amounts of pigment and chlorophyll contents inside electric field. The amount of cellular lipid depended on the starting cell density. When starting O.D.(at 682nm) value was 0.1, Chlorella vulgaris accumulated larger amount of lipid in the electric field; however, when starting OD value was 0.02, they had less cellular lipid amount than those cultured without external electric field.
| Identifer | oai:union.ndltd.org:TW/101NCKU5063154 |
| Date | January 2013 |
| Creators | Yi-HsiangHsu, 徐溢祥 |
| Contributors | Hsiang-Yu Wang, 王翔郁 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 92 |
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