Computer-Assisted Laser Scanning and Video Microscopy for Analysis of Cryptosporidium parvum Oocysts in Soil, Sediment, and Feces

Author:

Anguish L J,Ghiorse W C

Abstract

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(sup2) oocysts(middot)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 sections 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 (A. T. Campbell, L. J. Robertson, and H. V. Smith, Appl. Environ. Microbiol. 58:3488-3493, 1992) was modified by adding an immunostaining step after application of the fluorogenic dyes propidium iodide and 4(prm1),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.

Publisher

American Society for Microbiology

Subject

Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology

Reference50 articles.

1. American Society for Testing and Materials. 1992. Proposed test method for Giardia cysts and Cryptosporidium oocysts in low-turbidity water by a fluorescent antibody procedure. Proposal P229 p. 925-935. In 1992 annual book of ASTM standards section 11. American Society for Testing and Materials Philadelphia Pa.

2. Anguish L. J. M. J. Walker D. D. Bowman W. C. Ghiorse and M. B. Jenkins. Dye permeability and determination of survival kinetics of Cryptosporidium parvum oocysts. Submitted for publication.

3. Distribution and activity of microorganisms in subsurface sediments of a pristine study site in Oklahoma;Beloin R. M.;Microb. Ecol.,1988

4. Fully automatic determination of soil bacterium numbers, cell volumes, and frequencies of dividing cells by confocal laser scanning microscopy and image analysis;Bloem J.;Appl. Environ. Microbiol.,1995

5. .Brush C. (Cornell University). Personal communication.

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3