Full Description

Currently-employed techniques for the detection of Cryptosporidium sp. oocysts in the environment are unable to distinguish C. parvum from other non-human-pathogenic Cryptosporidium species. In addition, they are not broadly suitable for routine determinations of oocyst viability. This study evaluated 18S ribosomal RNA (rRNA) Fluorescence-in-situ Hybridisation (FISH) probes for the simultaneous determination of C. parvum species and viability during routine analytical procedures. It also examined: the correlation between FISH and in-vitro excystation assays for determinations of viability during chlorine disinfection, as well as species specificity for C. parvum; the stability of the probe target rRNA during immunomagnetic separation (IMS) and flow cytometric isolation of oocysts from water concentrate detrital material, and upon laboratory storage in PBS; and, the effects of chlorine on immunofluorescence staining. Data indicated a good general correlation between excystation and FISH for determinations of oocyst viability over 15 days exposure to 0-15 ppm sodium hypochlorite. Probe rRNA target was stable through IMS and FACS purification of oocysts. Target half-life was estimated at 55 h after oocyst permeabilization in PBS at room temperature. The addition of RNAse significantly reduced, or eliminated probe binding. The effects of RNAse appeared to be significantly reduced by addition of RNAsein to bulk water concentrate samples. The utility of FISH for use in routine environmental analysis laboratory protocols is discussed.

Product Details

Published: 01/01/1998 ISBN(s): 0898679796 Number of Pages: 7File Size: 1 file , 49 KB