A fluorescence in situ hybridization (FISH) technique has been developed for the fluorescent labelling of Cryptosporidium parvum oocysts in water samples. The FISH technique employs a fluorescently labelled oligonucleotide probe (Cry1 probe) targeting a specific sequence in the 18S ribosomal RNA (rRNA) of C. parvum. Hybridization with the Cry1 probe resulted in fluorescence of sporozoites within oocysts that were capable of excystation, while oocysts that were dead prior to fixation did not fluoresce. Correlation of the FISH method with viability as measured by in vitro excystation was statistically highly significant, with a calculated correlation coefficient of 0.998. Examination of sequence data for Cryptosporidium spp. other than C. parvum suggests that the Cry1 probe is C. parvum-specific. In addition, 19 isolates of C. parvum were tested, and all fluoresced after hybridization with the Cry1 probe. Conversely, isolates of C. baileyi and C. muris were tested and found not to fluoresce after hybridization with the Cry1 probe. The fluorescence of FISH-stained oocysts was not bright enough to enable detection of oocysts in environmental water concentrates containing autofluorescent algae and mineral particles. However, in combination with immunofluorescence staining, FISH enabled species-specific detection and viability determination of C. parvum oocysts in water samples.

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The polymerase chain reaction (PCR) has been applied to the identification of Escherichia coli and coliforms after overnight growth using two sets of primers described previously. The primer set for E. coli, which was derived from the uid A gene, correctly identified all E. coli strains tested. The sequence was also identified in five non-E. coli coliforms. The coliform primer set correctly identified approximately 70% of the coliforms tested. We conclude that PCR can be used for the rapid identification of E. coli using the primers described here but that further work is required accurately to define a new primer set for the coliform group.

The Colilert presence/absence test has been compared with membrane filtration using membrane lauryl sulphate broth for the detection of coliforms and Escherichia coli in a number of different types of water. Colilert appears to give results which are essentially the same as those obtained by membrane filtration whilst taking considerably less time to perform. The data generated in this study are in agreement with a large number of studies published in the United States and suggest that a large comparative study with drinking water samples is warranted.

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