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Counting Cryptosporidium, An Analysis of the Utility of Various Cytometric Techniques

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Abstract:To develop, evaluate and implement methods to detect C. parvum oocysts in water, samples must be seeded with known concentrations of oocysts. Methods for counting oocysts are inaccurate and highly variable. To address this, several cytometric methods were tested: flow cytometry, solid phase cytometry, electric resistance particle characterization, hemacytometry, chamber slides, and epifluorecent well slide. These methods were compared on the basis of accuracy, variability, and practicality. One hundred oocysts were enumerated and sorted using the flow cytometer and directly deposited onto a 13 mm, 0.8 micron porosity membrane. For all other techniques, the oocyst suspension was counted and a volume calculated to contain 100 oocysts was delivered by pipette to this type of membrane. The membranes were stained with fluorescent conjugated antibody, and oocysts counted by epifluorescent microscopy. Other parameters influencing the selection of cytometric methods were recorded. Flow and solid phase laser cytometric methods were the most accurate; flow cytometry gave the most consistent results. Practical concerns may influence the selection of a method other than flow or solid phase cytometry. Electric resistance cytometry overestimates the concentration of oocysts due to its non-specific nature; this method also requires a large suspension volume. Hemacytometry overestimates the concentration of oocysts, but is useful for highly concentrated material. The epifluorescence well slide method is the most costly and most time consuming method based on microscopy. This method consistently underestimates the concentration of oocysts, has high variability, and is the least accurate of all methods. Where this method has been used for enumeration of material to test detection methods, the reported recovery overestimates the actual recovery potential, due to the underestimation of spike oocysts concentrations inherent with this method.
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Citation:Lindquist, H. A., J. W. Bennett, K. Broomall, G. Glover, and F. W. Schaefer III. Counting Cryptosporidium, An Analysis of the Utility of Various Cytometric Techniques. Presented at Annual Meeting of American Society of Parasitologists, Monterey, CA, July 5-10, 1999.
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Contact: Mary P. O'Bryant - (919)-541-4871 or obriant.mary@epa.gov
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Division: Microbiological & Chemical Exposure Assessment Division
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Branch: Biohazard Assessment Research Branch
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Product Type: Abstrct/Oral
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Presented: 07/06/1999
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Related Entries:
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Bullet Item Detecting Ccl-Related, Emerging and Regulated Waterborne Human Protozoa for Exposure Assessment
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