Development of bacterial oxidative stress assays : towards using fluorescence methods
- WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
- Amanda Marie Schoonover (Creator)
- Institution
- Western Carolina University (WCU )
- Web Site: http://library.wcu.edu/
- Advisor
- Lori Seischab
Abstract: Several classes of bactericidal antibiotics increase oxidative stress in bacteria by
upregulating the production of reactive oxygen species. Reactive oxygen species are also
produced by host immune cells as protection against infectious bacteria. Superoxide
dismutase (SOD) is a defensive enzyme that protects bacteria from the damage caused by
reactive oxygen species. It is hypothesized that the inhibition of SOD would increase
bacterial cell death caused by oxidative stress. The goal of this study was to establish a
fluorescence-based assay for the determination of the viability of Escherichia coli that
have been treated with antibiotics and SOD inhibitors. The fluorescence-based assay was
compared to traditional methods of assessing bacterial viability including spread plating
and measuring growth by optical density. The concentration of E. coli cells and the type
of 96-well plate used in the fluorescence microplate reader affected the ability of the
fluorescence assay to accurately assess the bacterial viability. Quercetin, a Cu,Zn-SOD
inhibitor, interfered with measurements due to fluorescence it emits when bound to
targets in the cell. Diethyldithio carbamate (DDC), also a Cu,Zn-SOD inhibitor, caused a
decrease of bacterial viability at high concentrations of inhibitor (100 µM and 1000 µM
DDC) which was noted when using both fluorescence measurement methods and
traditional optical density growth measurements. The effects of ampicillin, kanamycin
and norfloxacin, representatives of three classes of bactericidal antibiotics, on cell
viability were measured using fluorescence methods and spread plate methods. The
affects of the antibiotics on the bacterial cells could not be accurately measured because
using the fluorescence methods. E. coli incubated with DDC and antibiotic showed little
to no difference from cells grown with DDC alone or antibiotic alone.
Development of bacterial oxidative stress assays : towards using fluorescence methods
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Created on 7/1/2009
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Additional Information
- Publication
- Thesis
- Language: English
- Date: 2009
- Keywords
- fluorescence, oxidative stress, Superoxide dismutase
- Subjects
- Antibiotics -- Analysis
- Quercetin
- Fluorimetry