Date of Award


Degree Type


Degree Name

Master of Science


Integrated Environmental Sciences

First Advisor

David W. Buckalew. Ph.D.

Second Advisor

Mrs. Thelma Dalmas

Third Advisor

Mrs. Frances Zaun


This study compares two different analytical methods for the determination of coliform bacteria within natural waters. The two methods are: 1) membrane Filtration, and 2) multiple tube fermentation. Water samples from ten different locations within the Appomattox River watershed were collected by technicians from the Piedmont Soil and Water Conservation District and the Piedmont Region TMDL Initiative in Farmville, Virginia.

The Membrane filtration (MF) method utilizes m-FC broth to isolate and differentiate total fecal coliform bacteria (E.coli, Klebsiella, Pseudomonas) in natural water samples. A 1% dilution of each water sample is filtered through a 0.45 micron (um) Millipore filter and placed in petri plates containing m-FC broth. All petri plates are incubated for 24 hours +/- 2 hours at 45° C +/- 0.5° C.

Isolation and enumeration of total fecal coliform bacteria using MF method depends upon lactose fermentation and the production of dark blue colonies. The number of colony forming-units {CFU's) times 100 (the dilution factor) determines the number of total fecal coliform bacteria in al 00 ml water sample.

The Multiple Tube Fermentation (MTF) process uses IDEXX Colilert Defined Substrate Technology (DST). Three series of dilution 100%, 10%, 1% (five tubes each) are produced. All fifteen tubes are incubated with the DST at 35 ° C +/- 0.5 ° C for 24 hours +/- 2 hours. The results are obtained from the Most Probable Number (MPN) table in the Standard Methods for the Examination of Water and Wastewater, 19th ed.

Enumeration of total coliform bacteria and E. coli using the MTF technique depends upon the fermentation products of DST metabolism. Observation of a yellow coloration equal or greater than a stock control determines the presence of total coliform bacteria in water sample. Observation of fluorescence equal or greater than a stock control confirms the presence of E. coli.



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