A sampling of bacteriophage, viruses that infect bacteria, was performed in the Mount Vernon, Iowa area. Sources included water and soil samples from the water treatment plant, Ink Pond, and Abbe Creek. The samples were screened for the presence of phage that infect Escherichia coli, Bacillus cereus, Pseudomonas fluorescens or Staphylococcus epidermidis. The techniques used were modified as better and more efficient methods were developed. After overnight culture, phage were isolated by forcing the sample through a .45um filter, and then plated via the double-layer agar method. A single plaque was then selected and grown to high titer with vigorous stirring to aerate the culture. The virus was isolated by centrifugation and extracted with phenol to yield the DNA, which was then precipitated with ethanol and analyzed with a spectrophotometer from 320 to 240nm to determine its purity. Restriction endonuclease digestion and agarose gel electrophoresis were performed to assess the size of the DNA. It was found that soil samples had to be treated with 1M NaCl and 0.01M EDTA in order to maximize phage release for isolation. Filtration, which allows phage to be collected from a water sample without growing it to a higher concentration, required a treatment of .5% polyethyleneimine (PEI) to be applied to the filter prior to filtration. Phage was successfully isolated that infected E. coli, B. cereus, and P. fluorescens, but none was found that infected S. epidermidis.
Jeff Ruttencutter, ’03 Marshalltown, IA
Major: Biochemistry and Molecular Biology
Sponsor: Jeffrey Cardon