Graduation Year

2005

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Biology

Major Professor

Daniel V. Lim, Ph.D.

Keywords

Seawater, Enterococcus, Enrichment, Marine, Microorganisms

Abstract

Development of a rapid method for the detection of fecal enterococci and pathogenic microorganisms in beach water was attempted utilizing an evanescent wave fiber optic biosensor. Various assay formats including a sandwich immunoassay were tested in the development of a rapid assay. Fluorophore labeled antibodies were used for specific detection of bacteria captured or adsorbed directly to the surface of a polystyrene fiber optic waveguide. Binding of the fluorescent labeled antibody to its specific target or binding of a fluorescent labeled anti-IgG within 100-1000 nm of the waveguide surface caused excitation of the fluorescent conjugate resulting in a quantifiable signal. Enterococcus faecalis, Staphylococcus aureus, Escherichia coli O157:H7, and Vibrio cholerae were used as model organisms for biosensor detection in phosphate buffered saline and seawater. Seawater samples were selectively enriched for the presence of these model organisms, which were later dete

cted on the biosensor. The sensitivity and specificity of the biosensor was examined by testing various assay formats, sample preparations, and molecules for capture and detection. Finally, an enrichment protocol combined with filter concentration was utilized to enhance detection of low levels of enterococci. The fiber optic biosensor has the potential to be a sensitive and specific system for the detection of fecal enterococci. The lower limit of detection in seawater and phosphate buffered saline was 2.8 x 106 CFU/ml. As few as 6 CFU/100ml (0.06CFU/ml) could be detected in seawater following a 14-24 hour enrichment and concentration step. Vibrio alginolyticus was found to grow under the same enrichment conditions as the enterococci. V. alginolyticus crossreacted with the polyclonal anti-Strep group D antibody used in the immunoassay at high cell concentrations. Staphylococcus aureus was the only other organism which showed significant cross-reactivity with this antibody.

The biosensor was also able to detect other bacterial pathogens in PBS and seawater. The lower limit for detection of E. coli O157:H7 was 3.6 x 105 CFU/ml. The lower limit for detection of Vibrio cholerae O1 was 1.3 x 108 CFU/ml. The antibodies used in these assays were found to crossreact with other gram negative microorganisms. The biosensor was not able to detect Staphylococcus aureus.

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