Evaluation of an alternative IMS dissociation procedure for use with Method 1622: detection of Cryptosporidium in water

Authors

Michael W. Ware, U.S. EPAFollow
Larry Wymer, U.S. EPA
Alan Lindquist, U.S. EPA
Frank W. Schaefer III, U.S. EPA

Date of this Version

2003

Citation

Published in Journal of Microbiological Methods 55 (2003) 575– 583. DOI:10.1016/j.mimet.2003.06.001

Abstract

U.S.EPA Methods 1622 and 1623 are used to detect and quantify Cryptosporidium oocysts in water. The protocol consists of filtration, immunomagnetic separation (IMS), staining with a fluorescent antibody, and microscopic analysis. Microscopic analysis includes detection by fluorescent antibody and confirmation by the demonstration of 1–4 sporozoites or nuclei after staining with 4V,6-diamidino-2-phenyl indole dihydrochloride (DAPI). The purpose of this study was to evaluate a new IMS dissociation, a 10-min incubation at 80 °C. Heat dissociation improved the average oocyst recovery from 41% to 71% in seeded reagent water, and from 10% to 51% in seeded river samples. The average DAPI confirmation rate improved from 49% to 93% in reagent water, and from 48% to 73% in river samples. This modification improved both oocyst recovery and confirmation.