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EPIFLUORESCENCE MICROSCOPY AND SOLID PHASE CYTOMETRY AS CONFIRMATORY METHODS FOR THE ENUMERATION OF PROTOZOA BY FLOW CYTOMETRY
Citation:
Bennett, J W. AND H.D A. Lindquist. EPIFLUORESCENCE MICROSCOPY AND SOLID PHASE CYTOMETRY AS CONFIRMATORY METHODS FOR THE ENUMERATION OF PROTOZOA BY FLOW CYTOMETRY. Presented at Annual Meeting of American Society of Parasitologists, Monterey, CA, July 5-10, 1999.
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 detection of infective protozoan parasites contained in large volume environmental samples represents a unique challenge in environmental parasitology. Compounding this problem is the fact that infective stages of many protozoan parasites do not readily replicate in media or cell cultures without intensive effort. Since the infective dose for many protozoan parasites may be 1-10 protozoan cells there is a need for the development of methods to detect low numbers of protozoan infective stage organisms. Flow cytometry is an important tool for detection method development, because it provides the most accurate method of enumeration at lower concentrations of parasites. One humdred C. parvum oocysts can be enumerated and sorted with less than a 10% deviation between samples. However, a confirmation step must be done in order to be certain that the number of parasites sorted agrees with the number of parasites counted via the flow cytometer. In this study, several cytometric methods were used to enumerate C. parvum oocysts and giardia lamblia cysts. First, parasites were sorted onto a polycarbonate membrane based on their forward and side scatter properties using a flow cytometer. the sorted protozoa were then labeled with fluorescent antibodies. Solid phase cytometry and direct microscopic observation were then used as methods for confirming the counts obtained via flow cytometry. In theory, the use of solid phase cytometry provides an automated means to increase the accuracy of flow cytometry by eliminating both the possibility of over-counting as well as the likelihood of overlooking a field on the membrane. The results indicated, however, that there is no benefit to using solid phase cytometry when standard epifluorescence is available.