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ENHANCED DAPI STAINING FOR CRYPTOSPORIDIUM IN WATER SAMPLES
Citation:
Ware, M W., F W. Schaefer III, AND H.D A. Lindquist. ENHANCED DAPI STAINING FOR CRYPTOSPORIDIUM IN WATER SAMPLES. Presented at American Society for Parasitologists Annual Meeting, Albuquerque, NM, June 29-July 3, 2001.
Impact/Purpose:
1) Refine new, practical methods for the detection of CCL-related and emerging waterborne human protozoa.
2) Perform field tests of devices or methods that have been developed under this task.
3) Evaluate these methods or devices in a variety of water matrices and parasite concentrations.
This work in this task supports CCL2 and 3 and is expected to be completed by 9/07.
Description:
The U.S. Environmental Protection Agency's Method 1623 is used to detect and quantify the presence of {ital Cryptosporidium} spp. oocysts in water. The protocol consists of concentrating a sample, staining this concentrate with a fluorescent antibody, and examining the sample microscopically. Since algae can be misinterpreted as positives using this procedure, confirmation may be done by either differential interference contrast microscopy, or by observation of the staining pattern when stained with 4,6-diamidino 2-phenyl-indole dihydrochloride (DAPI). Our research has shown that differential interference contrast is only able to confirm about 2% of oocysts with sporozoites in a fresh preparation. The rate of confirmation is improved to around 30% with DAPI staining when following the Method 1623 protocol. By a slight modification of the DAPI protocol, the confirmation rate improves to nearly 100%. A number of formats for optimizing slide preparation have been tested. Confirmation by both fluorescent antibody and DAPI staining is also possible using flow cytometry.