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Fluorescent labelling methods for detecting microorganisms in water have limited sensitivity partly due to the natural autofluorescence from environmental particles. The aim of this study was to examine the autofluorescence of water samples to determine the optimal excitation source and fluorescent labels for minimising background autofluorescence and therefore enhancing sensitive detection of Cryptosporidium oocysts. Particles concentrated from water were examined using fluorimetry at a wide range of excitation wavelengths to determine their autofluorescent properties. Two major peaks were identified emitting at 390 to 510 nm and at 640 to 700 nm. Flow cytometry was used to define the optical properties of oocysts immunofluorescently labelled with a range of fluorochromes. Concentrated water samples were analysed using flow cytometry and the number of particles with fluorescence and light scatter properties similar to the fluorescently labelled oocysts recorded. Fluorescein isothiocyanate exited at 488 nm was the most suitable label for oocysts in untreated water with less than 70 particles having optical properties similar to labelled oocysts, detected in 10 litre concentrates. The fluorochromes CY3, phycoerythrin (PE), and tetramethylrhodamine B thioisocyanate (TRITC) excited at 542 nm were the most suitable labels for oocysts in drinking water with less than 40 particles having optical properties similar to labelled oocysts, detected in 100 litre concentrates.

Fluorescence-activated cell sorting was used to isolate five spores of the soil amoeba Dictyostelium discoideum that carried new glycosylation mutations, which were produced by restriction enzyme-mediated integration (REMI)-induced gene disruption and which occurred at frequencies of around 10(-5). These mutations were identified by the loss of an O-glycosylation epitope found on surface proteins of wild type D. discoideum spores that is recognised by the monoclonal antibody MUD62. A secondary antibody conjugated to the fluorochrome fluorescein isothiocyanate identified MUD62 bound to spores. Spores lacking this epitope did not fluoresce, allowing this population to be separated. Samples were found to contain around 0.1% of viable spores that were wild type but lacked the MUD62 epitope at the time of sorting. To remove these spores from the unlabelled population, samples were labelled with monoclonal antibody MUD50, which recognises surface proteins on immature spores and proteins exposed from an inner coat layer. Double labelling with MUD50 and MUD62 reduced the unlabelled viable population to less than 0.002%, allowing the glycosylation-defective spores to be isolated. This is the first use of a selective approach to isolate nonmorphological REMI-induced mutants in D. discoideum. This study also characterises the surface properties of spore types found in mature fruiting bodies of D. discoideum.