The detection of Cryptosporidium oocysts in water presently relies on the concentration of particulate matter from large volumes of water prior to staining with fluorescently labelled monoclonal antibodies. Until recently, detection and identification of fluorescently labelled oocysts required examination of the sample using epifluorescence microscopy. The tedious and labour intensive nature of this detection method,. in particular the amount of fluorescent microscopy required, limited the monitoring work which could be performed. The development of flow cytometric assisted detection methods has alleviated some of these problems and enabled the routine monitoring of water for the presence of Cryptosporidium oocysts (Vesey et al., 1994A). However, a major limitation of all these methodologies is the lack of oocyst viability measurements. Methods to determine viability such as animal infectivity and excystation, are impractical because of the low number of oocysts normally present in water samples and the tedious nature of such tests.
The presence of dead Cryptosporidium oocysts in drinking water is of little significance to public health, however if oocysts are viable the risk to public health is enormous. Moreover, of the seven species of Cryptosporidium; C. parvum, C. muris, C. meleagridis, C. serpentis, C. nasorum, C. wrairi and C. baileyi only one, C. parvum is infectious to humans (Rose et al., 1988), and yet all the currently available monoclonal antibodies are not species specific. There is therefore, an urgent requirement to develop an effective method for determining the viability of C. parvum oocysts in water.
Fluorescence in situ hybridisation (FISH) is a relatively new method by which microorganisms can be specifically labelled. The technique is reliant upon the identification of a specific sequence of nucleic acid within the target organism. Probes targeting a specific nucleic acid sequence are then synthesised and labelled with a fluorochrome. The cell is then permeabilised and the complementary sequence allowed to hybridise with the target sequence resulting in specific labelling of the target cell.
The use of ribosomal RNA (rRNA) targeted oligonucleotide probes with FISH and flow cytometry has been reported by Amann et al. (1990B). Molecules of rRNA are ideal targets for fluorescently labelled nucleic probes for several reasons: (1) high sensitivity can be achieved since the target molecules are present in very high numbers; (2) a denaturation step is not required during the procedure as the target region is single stranded; and (3) rRNA has a short half life and will only be present in a high copy number in viable cells.