Date of Award

2003

Degree Name

Biological Sciences

College

College of Science

Type of Degree

M.S.

Document Type

Thesis

First Advisor

Charles C. Somerville

Second Advisor

Dan K. Evans

Third Advisor

Frank L. Binder

Abstract

Molecular investigations of microbial community structure and dynamics involve costly and time-consuming methods. This approach is limiting when rapid assessment and detection of microbial organisms are needed. In aquatic environments, especially freshwater environments which may be used as a water source, rapid detection of pathogenic microbes is essential. Likewise, monitoring for the presence or absence of various functional genes can be used to indicate the type of microbial community present in the environment of interest. Therefore, the aim of this study was to develop and optimize methods necessary for the rapid assessment of freshwater microbial community structure and dynamics. Primers for several genes of interest (i.e. small subunit (SSU) rRNAs, nifH, rbcL, stx I & II, and primers targeting Cryptosporidium parvum, Giardia lamblia, Entamoeba histolytica, and Listeria monocytogenes) were either constructed de novo or synthesized from previously published sequences. These primers were used to establish optimal PCR amplification parameters and to create probes from type-strain cultures representing the twelve main divisions of bacteria (Actionmycetes, Aquificales, Low GC Gram positives, Cytophaga/ Flavobacteria/ Bacteroides [CFB], Cyanobacteria, Deinococcus, Green Non-Sulfur, Green Sulfur, Planctomyces, Proteobacteria [α, β, γ, and δ], Spirocheta, and Thermotogales), Archaea (kingdoms Crenarchaeota and Euryarchaeota), and Eukarya (Brown Algae, Green Algae, Red Algae, Diatoms, Dinoflagellates, Cryptomonas, Euglenozoa). Water samples from Mill Creek, in southern Wayne County (a fresh water environment) were collected seasonally. DNA extraction by sonication was optimized and used to collect total DNA from the water samples. Environmental DNA was labeled with 35S using the random prime label method for hybridization to target genes immobilized on nylon membranes. This method is called Reverse Sample Gene Probing (RSGP). PCR amplification of specific gene targets was significantly improved for some template DNAs by including either a touchdown PCR method, titrating the Mg 2+ concentration and/or diluting the DNA. However, some templates could not be amplified and were, therefore, eliminated from the study. Hybridization by RSGP was attempted twice, once using lower stringency conditions, and once using higher stringency conditions. In both cases the optimum conditions were not obtained and thus, it was concluded that the optimum parameters must lie somewhere between the parameters attempted. With further optimization and development, the application of RSGP can provide a rapid and inexpensive alternative to current methods used in microbial ecology studies of aquatic environments.

Subject(s)

Microbial ecology.

Aquatic ecology.

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