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Journal of Clinical Microbiology, July 2004, p. 3256-3261, Vol. 42, No. 7
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.7.3256-3261.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Purification of Enterocytozoon bieneusi Spores from Stool Specimens by Gradient and Cell Sorting Techniques

Zuzana Kucerova,^1,2,3* Hercules Moura,² Gordon J. Leitch,¹ Rama Sriram,² Caryn Bern,² Vivian Kawai,⁴ Daniel Vargas,⁴ Robert H. Gilman,⁵ Eduardo Ticona,⁶ Aldo Vivar,⁷ and Govinda S. Visvesvara²

Department of Physiology, Morehouse School of Medicine,¹ Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention,² Atlanta Research and Education Foundation, Atlanta, Georgia,³ Asociacion Benefica PRISMA,⁴ Hospital Dos de Mayo,⁶ Hospital Arzobispo Loayza, Lima, Peru,⁷ Bloomberg School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland⁵

Received 12 December 2003/ Returned for modification 27 January 2004/ Accepted 27 February 2004

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.

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* Corresponding author. Mailing address: Division of Parasitic Diseases, Mail Stop F-36, Centers for Disease Control and Prevention, Atlanta, GA 30341. Phone: (770) 488-7119. Fax: (770) 488-4253. E-mail: ZIK0{at}CDC.GOV.

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Journal of Clinical Microbiology, July 2004, p. 3256-3261, Vol. 42, No. 7
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.7.3256-3261.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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