title: Purification of Enterocytozoon bieneusi spores from stool specimens by gradient and cell sorting techniques.
abstract: A three-step method for the purification of Enterocytozoon bieneusi spores from stool specimens was developed. The primary process of purification of the spores from bacterial contaminants involved Percoll gradient centrifugation followed by additional separation using cesium chloride density gradient centrifugation. The cesium chloride-isolated spores were further purified using a flow cytometer with cell sorting capabilities. Sorting was performed without the use of antibodies, fluorochromes, or dyes, leaving the sorted spores in their native state, which appears to be less destructive for spores. When quantified by flow cytometry using tubes with known numbers of highly fluorescent polystyrene beads, the sorted material showed a slight decrease in light scatter characteristics compared with the slightly larger Encephalitozoon species spores. Although the overall recovery of the E. bieneusi spores was low, calcofluor and Gram chromotrope staining, indirect immunofluorescence assay, and transmission electron microscopy revealed that the sorted material was highly purified and contained large numbers of E. bieneusi spores and relatively few bacteria and other debris. The sorted material appeared to be sufficiently pure and could be used for in vitro culture and for the development of a variety of diagnostic reagents as well as in studying the genome of E. bieneusi and host-parasite interactions.
authors.affiliation: Department of Physiology, Morehouse School of Medicine, Atlanta, GA 30341, USA. ZIK0@CDC.GOV
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Kucerova
authors.forename: Hercules
authors.initials: H
authors.lastname: Moura
authors.forename: Gordon J
authors.initials: GJ
authors.lastname: Leitch
authors.forename: Rama
authors.initials: R
authors.lastname: Sriram
authors.forename: Caryn
authors.initials: C
authors.lastname: Bern
authors.forename: Vivian
authors.initials: V
authors.lastname: Kawai
authors.forename: Daniel
authors.initials: D
authors.lastname: Vargas
authors.forename: Robert H
authors.initials: RH
authors.lastname: Gilman
authors.forename: Eduardo
authors.initials: E
authors.lastname: Ticona
authors.forename: Aldo
authors.initials: A
authors.lastname: Vivar
authors.forename: Govinda S
authors.initials: GS
authors.lastname: Visvesvara
mesh_terms: D002469:Cell Separation; D002499:Centrifugation, Density Gradient; D021862:Enterocytozoon / Q000302:isolation & purification*; D005243:Feces / Q000469:parasitology*; D005434:Flow Cytometry; D006801:Humans