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

      Schets FM; Medema GJ; Boschman GD; LWL; Becton Dickinson Europe; Aalst; Belgie (Rijksinstituut voor Volksgezondheid en Milieu RIVM, 1995-03-31)
      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<0.05). FACSort detected Cryptosporidium in 74% of the samples, while the standard method detected oocysts in 64% of the samples. Geometric means were 0.95 and 0.38 oocysts per liter respectively. FACSort found 17 (range 1.1-91) times as many oocysts per liter in 15/27 samples that were examined with both methods ; differences are, however, not significant. FACSort sorted (oo)cysts onto membrane filters and microscopic preparations obtained were very clean and easy to interpret, thus giving more reliable counts. When sorting was performed onto 13 mm diameter membrane filters microscopy time could be reduced with approx. 70%. The overall recovery of both methods is relatively low. FACSort recovered 5.9% of the Cryptosporidium oocysts and 17.1% of the Giardia cysts and the standard method recovered 2.9% of the oocysts and 15.6% of the cysts. The low overall recovery is not due to the performance of the FACSort flow cytometer, for this instrument recovered 95% of oocysts present in the suspension. But FACSort as well as the standard method use the same sample processing procedure which includes various steps that cause large losses of (oo)cysts. Recommendations are made to improve the discrimination of (oo)cysts from the debris with other monoclonal antibodies or fluorochromes, in order to overcome the need for microscopic confirmation. The recovery of the entire sample processing procedure may be improved when flow cytometry is coupled with calcium carbonate flocculation. Sorted samples containing (oo)cysts and relatively low levels of debris may be suitable for the application of techniques predicting viability which is a key characteristic in determining the impact of environmental (oo)cysts on human health. Pure suspensions of sorted environmental (oo)cysts may also be used in species-specific PCR assays.<br>