AWWA WQTC55029

Enterococci detection is one of the two approved procedures by the US Environmental Protection Agency (EPA) used for the assessment of the microbiological quality of recreational waters. The action levels established by the EPA for enterococci are 35 per 100 ml in marine recreational waters and 33 per 100 ml in fresh water. Turn-around time of the method is over 24 hrs., and thus there is a clear need to reduce that time to allow a faster and reliable assessment of the safety/quality of the waters. The most abundant and prevalent species among the fecal enterococci (>90%) are E. faecalis and E. faecium. The objective of this research is to devise an efficient bacterial capture/concentration system in conjunction with a rapid detection method that will make possible the assessment of very low levels of fecal enterococci within a working day. In the first phase of the project described in this paper, the objectives were twofold: the procurement and/or production of antibodies able to capture efficiently the above mentioned microorganisms; and, the evaluation of various tagging alternatives (nucleic acid based generic dyes, specific antibody fluorochrome-conjugates, etc) for the detection of enterococci with the RBD2000, a specialized flow cytometer designed for bacterial detection. This initial part of the project was done in clean samples spiked with various bacterial loads. The next phase of the project tested the reagents produced and the methods developed in real samples, and the final goal was the full development and validation of a flow cytometry based method that will enable the detection and enumeration of low levels of fecal enterococci in recreational waters in less than 8 hr. The methods discussed in this paper include: bacterial cultures; immunization; antibody coated magnetic beads; immunomagnetic capture-separation; tagging and detection using Syto 62; tagging and detection using DRAQ5; and, tagging and detection using Cy5-antibody conjugates. Includes figures.

Product Details

Edition:

Vol. - No.

Published:

01/01/2001

Number of Pages:

7

File Size:

1 file , 260 KB