TY - JOUR
T1 - Purification of Enterocytozoon bieneusi spores from stool specimens by gradient and cell sorting techniques
AU - Kucerova, Zuzana
AU - Moura, Hercules
AU - Leitch, Gordon J.
AU - Sriram, Rama
AU - Bern, Caryn
AU - Kawai, Vivian
AU - Vargas, Daniel
AU - Gilman, Robert H.
AU - Ticona, Eduardo
AU - Vivar, Aldo
AU - Visvesvara, Govinda S.
PY - 2004/7
Y1 - 2004/7
N2 - 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.
AB - 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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U2 - 10.1128/JCM.42.7.3256-3261.2004
DO - 10.1128/JCM.42.7.3256-3261.2004
M3 - Article
C2 - 15243090
AN - SCOPUS:3142660272
SN - 0095-1137
VL - 42
SP - 3256
EP - 3261
JO - Journal of clinical microbiology
JF - Journal of clinical microbiology
IS - 7
ER -