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A Comparison of Three Assay Procedures for Determining Chlorine Inactivation of Waterborne Pathogenic Bacteria

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Abstract: One criterion on which chlorine treatment of water may be based is the concentration (C) in mg/l multiplied by the time (t) in min of exposure or Ct values. We compared different Ct values on waterborne pathogenic bacteria by cultural assay for viability and 2 assays that measured metabolic activity. Total counts were measured by 4'6-diamidino-2-phenylindole dihydrochloride (DAPI). Metabolic activity was based on the presence of esterase as detected by a modified fluorescein diacetate fluorophore designated V6 and respiration by the fluorophore 5-cyano-2,4-ditoyl tetrazolium chloride (CTC). V6 results were obtained using a solid phase cytometer and counts were validated with a microscope. CTC and DAPI results were obtained by applying a mathematical formula to determine microscopic counts of cells per ml. The bacteria included Escherichia coli 0157:H7, Legionella pneumophila and Helicobacter pylori. Each bacterium was added to 3 separate chlorine demand free water samples. Three different Ct values per sample were examined at 5oC and pH 7.0. Counts were obtained before and after chlorine treatment.

No significant differences were found between the DAPI controls and the corresponding chlorine treated samples. In the Chick-Watson theory of disinfection the rate of die-off of microorganisms is hypothesized to be proportional to the number of organisms remaining and a power function of the disinfectant concentration. This theory was shown to be reasonable for E. coli 0157:H7, L.
pneumophila and H. pylori where no plate growth or CTC counts were found after exposure to Ct

ranges of 7 to 95. V6 counts for these 3 treated bacteria were present but as Ct values increased,

counts were significantly reduced. After chlorine treatment each bacterium decreased by at least 2 to 3 log10 for all assays. V6 activity persisted when other assays were negative and would thus be

more apt to indicate any lingering activity within the cells. It may be a conservative indicator of

metabolic activity within bacteria.
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Citation:Mcdaniels, A. E., E. W. Rice, R. A. Tally, L. J. Wymer, and G. N. Stelma Jr. A Comparison of Three Assay Procedures for Determining Chlorine Inactivation of Waterborne Pathogenic Bacteria. Presented at 103rd General Meeting American Society for Microbiology, Washington, DC, May 18-22, 2003.
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Contact: Mary P. O'Bryant - (919)-541-4871 or obriant.mary@epa.gov
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Division: Microbiological & Chemical Exposure Assessment Division
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Branch: Microbial Exposure Research Branch
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Product Type: Abstrct/Oral
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Presented: 05/18/2003
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Related Entries:
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Bullet Item Exploratory Occurrence Study of Newly Emerging Pathogens in Potable Water
spacer Relationship Reason:   A Project of the Product
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Last Updated on Monday, October 22, 2007
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