Detection of Cryptosporidium oocysts and Giardia cysts in water samples with a Becton Dickinson FACSort flow cytometer

Current detection techniques for Cryptosporidium oocysts and Giardia cysts in water samples combine filtration of large volumes of water, concentration by centrifugation and flotation and immunofluorescense microscopy. The techniques are extremely labour-intensive and inefficient. The various steps in the sample processing procedures cause large losses of (oo)cysts resulting in an average overall recovery of about 3% in surface water samples. Microscopic interpretation of concentrates is time consuming and difficult due to the presence of debris. We used fluorescence activated cell-sorting with a flow cytometer prior to fluorescence microscopy in order to improve our standard method. We therefore incorporated the FACSort flow cytometer prior to epifluorescence microscopy in our current detection method to purify Cryptosporidium and Giardia (oo)cysts in concentrated river water, sewage water and secondary effluent samples. The (oo)cysts were stained with FITC-conjugated monoclonal antibodies and sorted by the FACSort on the basis of fluorescence and forward scatter characteristics on a 13 mm polycarbonate membrane filter. 28 environmental samples (including 10 seeded samples) were examined with both our standard method and the method with the FACSort flow cytometer. The FACSort flow cytometer proved to be user-friendly and easy to operate and it improved the performance of the standard method. With FACSort 96% of the samples were positive for Giardia, the geometric mean was 8.6 cysts per liter. The standard method detected Giardia cysts in 86% of the samples with a geometric meam of 4.1 cysts per liter. In 22/25 samples that were examined with both methods FACSort detected 14 (range 1.2-84) times as many cysts per liter as the standard method. Differences between FACSort and standard are significant (P