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A CRITICAL EVALUATION OF A FLOW CYTOMETER USED FOR DETECTING ENTEROCOCCUS FAECIUM AND ENTEROCOCCUS FAECALIS IN RECREATIONAL WATERS
Citation:
STANG, D., K. P. BRENNER, AND M. R. RODGERS. A CRITICAL EVALUATION OF A FLOW CYTOMETER USED FOR DETECTING ENTEROCOCCUS FAECIUM AND ENTEROCOCCUS FAECALIS IN RECREATIONAL WATERS. Presented at 2005 American Society of Microbiology General Meeting, Atlanta, GA, June 05 - 09, 2005.
Impact/Purpose:
Develop a rapid (same day), sensitive and accurate method for detecting and measuring fecal contamination of recreational water.
Description:
The current U. S. Environmental Protection Agency-approved method for Enterococci (Method 1600) in recreational water is a membrane filter (MF) method that takes 24 hours to obtain results. If the recreational water is not in compliance with the standard, the risk of exposure to enteric pathogens will occur before the water is identified as hazardous. Because flow cytometry combined with specific fluorescent antibodies has the potential to be used as a rapid detection method for microorganisms, this technology was evaluated as a rapid, same-day method to detect Enterococci in bathing beach waters. A comparison of the viable MF counts with flow cytometry counts of pure Enterococci cultures in phosphate buffer and sterile-filtered recreational water showed fairly good agreement between the two methods. However, when the flow cytometer was used with spiked and unspiked natural water samples, the counts were several orders of magnitude higher than the MF counts with no correlation to Enterococcus spike concentrations, and the unspiked sample controls frequently had higher counts than the samples spiked with Enterococci. Particles within the spiked water samples were probably counted as target cells by the flow cytometer because of autofluorescence or non-specific adsorption of antibody, and carryover to subsequent samples occurred often. Carryover can cause acceptable water samples to exceed the recreational water Enterococci limits and result in unnecessary beach closings. In order to eliminate this problem, several steps were added to the manufacturer's method to clean the system between samples. This significantly decreased the number of samples that could be processed in one day. For these reasons, this technology may not be suitable for Enterococci detection in recreational waters.