Theses and Dissertations

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Defense Date

2-14-2007

Document Type

Thesis - NSU Access Only

Degree Name

M.S. Marine Biology

Department

Oceanographic Center

First Advisor

Patricia Blackwelder

Second Advisor

Tomohiro Kawaguchi

Third Advisor

Joshua Loomis

Abstract

Microbial attachment to contact lenses is utilized in this study as a model to examine microbial/substrate interactions. These interactions commonly occur on a biofilm, containing microbes and exopolymeric secretions (EPS). EPS commonly contains sugars for attachment. Silica hydrogel contact lenses were studied with relation to microbial attachment. Scanning and transmission electron microscopy (SEM, TEM) and Fourier Transform Infared Spectrometry (FTIR) were utilized to examine this mechanism. A novel method was developed to prepare lenses and attached microbial communities for SEM imaging. A common marine fixative was utilized with extended fixation times which resulted in fixation osmolarities that preserved the delicate lens polymers as well as the attached microbial communities. In addition, a 24 hemisphere shaped well assaylike tray was developed that aided in lens shape retention during dehydration. SEM analysis of P. aeruginosa EPS yielded information on its 3-dimentional morphology on the attached lens. TEM analysis using gold nanoparticles confirmed that active sites on EPS secreted for attachment were comprised mainly of highly supported structures on the periphery of the EPS, with strut like supports crossing the surface. FTIR analysis of the EPS revealed chemical signatures related to the nature of its bonds as spectral peaks. When heated to 34 °C and then cooled to 20 °C the EPS did not return to its original chemistry. Compared to some polymers, which retain “memory” and return to original chemistry and shape, the bacteria EPS appeared to denature to the point that major components of its molecular structure went into solution. These results have possible implication to the in vivo behavior of bacteria. Assessment of several Acanthamoeba species attachment success found that cysts were in generally more successful in attachment compared to trophozoite stages. Results indicate that in all species and experimental conditions, cyst forms of Acanthamoeba sps. were more successful at attachment then the trophozoite forms. Furthermore, there was a suggested differential pattern in attachment if pathogen and non-pathogen species are compared. The results suggest that in pathogen forms (between 1/2 and 12 hours after inoculation) attachment was greatest at 8 hours compared with non-pathogen species, which had lower success attachment at 8 hr, and higher relative success attachment at ½ and 12 hours. It was also found that one species in which pathogeniety is not known (FL 32) was intermediate in attachment success. These findings warrant further study of pathogen Acanthamoeba sps. in terms of attachment success.

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