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Assessment of the effects of superoxide-generating agents on the growth and viability of Escherichia coli using traditional microbiological methods and fluorescence methods

WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
Jennifer Lynn Patterson (Creator)
Institution
Western Carolina University (WCU )
Web Site: http://www.wcu.edu/404.asp
Advisor
Lori Seischab

Abstract: The effects of superoxide-generating agents on the growth and viability of Escherichia coli was investigated using two different strains, a lab strain (ER2566) and a clinical isolate (ATCC 4157). Endogenous superoxide was generated using the redoxcycling agent paraquat, while exogenous superoxide was generated using the xanthine/xanthine oxidase (X/XO) enzyme system. Using optical density measurements to monitor culture growth, the bacteriostatic effect of paraquat was tested in three different growth media: Luria-Bertani broth, double strength Luria-Bertani broth, and nutrient broth. For both strains, paraquat toxicity was greatest in nutrient broth, with toxicity in each medium dependent upon the time of paraquat addition following inoculation. Protection against paraquat toxicity by salts and yeast extract was suggested by the differences observed between growth rates of ER2566 treated cultures in each medium. Addition of the copper/zinc superoxide dismutase inhibitor diethyldithiocarbamate decreased paraquat toxicity, consistent with its role in induction of the superoxide response regulon (soxRS). Based on colony-forming unit (CFU) counts, the toxicity of X/XO-generated superoxide on ER2566 was found to be altered by pH, with cell viability lower at a pH of 6.5 than at 7.5. Using CFU counts obtained with the spread-plate method and total cell counts obtained with a hemacytometer, the relationship between optical density and cell number was found to be different between cultures of the two strains at both log and stationary phases. Reliable cell counts were necessary to avoid the inner filter effect otherwise encountered during optimization of the BacLight Bacterial Viability Kit for ATCC 4157. Assessment of cell viability following X/XO treatment with the BacLight kit indicated this method was more sensitive than the traditional spread plate method for determining cell viability.

Additional Information

Publication
Thesis
Language: English
Date: 2010
Subjects
Escherichia coli -- Growth
Superoxide dismutase