Date of Award

Spring 2014

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Ocean & Earth Sciences

Program/Concentration

Oceanography

Committee Director

Alexander B. Bochdansky

Committee Member

Fred C. Dobbs

Committee Member

John R. McConaugha

Abstract

Sucrose gradient centrifugation and Field-flow fractionation (FFF) are two different particle separation methods that overcome the problems of similar size microorganisms clumping together during standard filtration methods. FFF separates particles based on size and density via the parabolic velocity profile of laminar flow in a ribbon-like channel. The sucrose method separates particles via centrifugation in a density gradient. Both techniques worked well in separating eukaryotic from prokaryotic microbes, with the preferred method depending on the type and relative abundance of organisms to be separated. Minicells were separated from mother cells in transformed Escherichia coli cultures, heterotrophic flagellates (e.g., Diplonema papillatum) from their bacterial prey in cultures, and various eukaryotes from prokaryotes in natural seawater communities. These separated clean fractions were further analyzed for their biochemical composition (using radiolabeling and subsequent biochemical fractionation) revealing significant differences in the bulk biochemical makeup between prokaryotic and eukaryotic microbes. Even within domains, the sucrose gradient method was able to separate prokaryotic and eukaryotic microbes by size. Variables that influence and improve these separation techniques were explored, including the effect of aldehyde fixation.

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DOI

10.25777/y8jj-7c04

ORCID

0000-0002-5270-1437

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