Computer-assisted laser scanning and video microscopy for analysis of Cryptosporidium parvum oocysts in soil, sediment, and feces
- Cornell Univ., Ithaca, NY (United States)
A computer-assisted laser scanning microscope equipped for confocal laser scanning and color video microscopy was used to examine Cryptosporidium parvum oocysts in two agricultural soils, a barnyard sediment, and calf fecal samples. An agar smear technique was developed for enumerating oocysts in soil and barnyard sediment samples. Enhanced counting efficiency and sensitivity (detection limit, 5.2 x 10{sup 2} oocysts {center_dot} g [dry weight]{sup -1}) were achieved by using a semiautomatic counting procedure and confocal laser scanning microscopy to enumerate immunostained oocysts and fragments of oocysts in the barnyard sediment. An agarose-acridine orange mounting procedure was developed for high-resolution confocal optical sectioning of oocysts in soil. Stereo images of serial optical section revealed the three-dimensional spatial relationships between immunostained oocysts and the acridine orange-stained soil matrix material. In these hydrated, pyrophosphate-dispersed soil preparations, oocysts were not found to be attached to soil particles. A fluorogenic dye permeability assay for oocyst viability was modified by adding an immunostaining step after application of the fluorogenic dyes propidium iodide and 4{prime},6-diamidino-2-phenylindole. Comparison of conventional color epifluorescence and differential interference contrast images on one video monitor with comparable black-and-white laser-scanned confocal images on a second monitor allowed for efficient location and interpretation of fluorescently stained oocysts in the soil matrix. This multi-imaging procedure facilitated the interpretation of the viability assay results by overcoming the uncertainties caused by matrix interference and background fluorescence. 48 refs., 4 figs., 1 tab.
- DOE Contract Number:
- FG05-90ER75580
- OSTI ID:
- 530659
- Journal Information:
- Applied and Environmental Microbiology, Journal Name: Applied and Environmental Microbiology Journal Issue: 2 Vol. 63; ISSN AEMIDF; ISSN 0099-2240
- Country of Publication:
- United States
- Language:
- English
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