Detection of Cryptosporidium oocysts in water: techniques for generating precise recovery data

When determining the recovery efficiency of a procedure for the detection o f Cryptosporidium or the removal efficiency of a treatment process, it is n ecessary to accurately enumerate a 'seed dose'. Conventional techniques for this are highly variable and consequently, can result in misleading data. In this study, a flow cytometric method was del eloped for the production o f suspensions of Cryptosporidium oocysts in which the number of organisms c ould be precisely determined. A Becton Dickinson FACScalibur flow cytometer was employed to produce oocyst suspensions containing 100 oocysts. Analysi s of these suspensions resulted in a mean dose of 99.5 oocysts (s.w. = 1.1, %cv = 1.1). These results indicate that the use of such suspensions to see d test systems generates far more accurate data than is presently possible using conventional techniques. In addition, the use of immunomagnetic separ ation (IMS) for the isolation of oocysts from three different water matrice s, after seeding with oocysts counted using flow cytometry, was investigate d. The recovery efficiency of the IMS procedure was found to be high, with the percentage recovery of oocysts ranging from 82.3 to 86.3%, and the use of precise numbers of oocysts allowed accurate recovery efficiency data to be generated. A laser scanning instrument (ChemScan RDI) was employed for t he rapid detection and enumeration of oocysts after capture using membrane filtration. This technique Mras found to be faster and easier to perform th an conventional epifluorescence microscopy. These findings demonstrate that the ChemScan RDI system may be used as alternative procedure for the routi ne examination of IMS supernatant fluids for the presence of Cryptosporidiu m.