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.

FIG. 1.

Staining of E. bieneusi spores during various stages of the spore purification procedure. Stool preparation: calcofluor (a), Gram chromotrope (b), and IIF (c). Cesium chloride gradient centrifugation: calcofluor (d), Gram chromotrope (e), and IIF (f). Sorted material: calcofluor (g), Gram chromotrope (h), and IIF (i).

FIG. 2.

FSC-versus-SSC and FL1 (515 to 545 nm)-versus-FL31 (633 to 677 nm [FL3 in laser 1]) fluorescence dot plots on unsorted material from cesium chloride density gradients performed on samples from two patients (patient A [A and B, respectively]; and patient B [C and D, respectively]). (E and F) FSC-versus-SSC and F2-versus-F31 dot plots, respectively, on sorted material from patient B. Boxed areas region 1 (R1) and region 2 (R2) enclose concentrations of E. bieneusi spores. Of the sorted events, 98% were contained in R1 of the FSC-versus-SSC dot plot (E) and 99% were contained in R2 of the FL1-versus-FL31 dot plot (Fig. 2F).

FIG. 3.

TEM of a stool preparation from an AIDS patient with confirmed E. bieneusi infection (A) (bar = 500 nm) and from sorted material from the same patient (B) (bar = 200 nm).