Capstone Title

Quantitative Microbial Assessment in Marine Organisms: Applications and Methodologies

Defense Date

12-2013

Document Type

Capstone

Degree Name

M.S. Marine Biology

First Advisor

Jose V. Lopez

Second Advisor

Patricia Blackwelder

Abstract

Microbes dominate the world oceans, making up 70-75% of the total biomass and contributing 98% to Earth’s primary productivity. They are ubiquitous and play an essential role in the food chain, as well as being responsible for various human illnesses and biological processes. Integration of both molecular and microbial assessment methodologies are examined. This study compares quantitative real-time PCR, culture-dependent methods, flow cytometry (FCM), and Fluorescent In Situ Hybridization methods for quantifying bacteria by reviewing the application of these methods to microbial ecology. qPCR methods are relatively straightforward, specific, and have a greater sensitivity than other methods. However, SYBR Green I qPCR can result in primer dimer bindings and Taqman qPCR is expensive and primer/probe design complicated. Culture-based methods assess viable cells; however individual bacterial species cannot be easily grown on media and less than 1% of bacteria can be cultivated. FCM can reveal structural and morphological cellular details, as well as provide a rapid way to count cells; however solid phase cytometry can be inaccurate due to background and autofluorescence. Enumeration of microbes with FISH may not be very accurate due to interferences by background and autofluorescence, as well as photobleaching. Although there are advantages and limitations to each individual method, when combined valuable information can be obtained and predictions made about a microbe’s structure and function. Efforts to quantify microbial populations and determine relative distribution are imperative to better understand how microbes contribute to specific niches within the ecosystem.

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