Development of fluorescently labelled Cryptosporidium oocyst surrogates to test the efficacy of sand filtration processes

Abstract

The objectives this thesis were to: (i) develop and produce suitable labelled surrogates for Cryptosporidium oocysts for sand filter performance studies; (ii) evaluate the surface interaction effects of labelling on surrogate behaviour; and (iii) compare the relative performance of the developed surrogates with respect to sand media interactions, by simulating some aspects of conventional sand filtration. Novel oocyst surrogates were developed by using fluorescence resonance energy transfer (FRET) labelled Cryptosporidium parvum oocysts and Saccharomyces cerevisiae (yeast) cells. FRET-labelling overcame conventional flow cytometric analysis problems associated with autofluorescent particles that interfere with the assay of target cells in environmental water samples. FRET-labelled oocysts were easily detected and quantified (even at 10 oocysts per 10-L) amongst a range of environmental autofluorescent particles by conventional analysis-only flow cytometry. The surface properties of FRET-labelled surrogates were compared against unlabelled oocysts by using hydrophobic and electrostatic interaction assays. Overall, hydrophilic and electrostatic surface properties dominated the behaviour of oocysts and FRET-labelled surrogates, and FRET-labelling appeared to have no significant effect on the surface properties of the oocysts (compared to unlabelled oocysts of about the same age). Age of oocysts, however, did appear to change these physical interaction effects. The removal of oocysts and novel surrogates were studied using a bench-scale sand filtration system with modified sand media. FRET-labelled oocysts (of the same aged as unlabelled oocysts) consistently showed similar removal behaviour, measured by comparing log removal, and FRET-labelled oocysts showed similar detachment behaviour compared to unlabelled oocysts in chemically coated sand media. In conclusion, (heat-inactivated) FRET-labelled oocysts appear to be a suitable surrogate to demonstrate the removal of Cryptosporidium oocysts by sand filtration.