Viability staining of Cryptosporidium oocysts and Giardia cysts combined with flow cytometry

The incorporation of flow cytometry as an additional purification step has improved the detection method for Cryptosporidium oocysts and Giardia cysts in water. Flow cytometry allows separation of (oo)cysts from interfering debris particles present in water concentrates and thus enables the application of fluorogenic vital dyes such as propidium iodide (PI) to assess (oo)cyst viability. Staining with DAPI (4',6-diamidine-2-phenylindole-dihydrochloride) and PI, followed by cell sorting with the Becton Dickinson FACSort flow cytometer, resulted in microscopic preparations in which the readability of vital staining was excellent. Sorting had no effect on oocyst viability and both PI positive and negative oocysts were sorted equally efficiently. Sample processing steps (centrifugation, sonification, flotation, washing) did not affect oocyst viability. Clarification of membrane filters on to which oocyst were sorted, by dehydration, in order to enable the use of DIC microscopy , had a strong negative effect on oocyst viability. All sample processing steps caused a considerable decrease in Giardia cyst viability, indicating that the current detection method can only detect the presence of Giardia cysts it cannot give information on their viability. Extension of the detection method with the DAPI/PI stain provides information on Cryptosporidium oocyst viability. The information is limited, since DIC microscopy cannot be used to examine whether DAPI and PI negative oocysts have contents and should be considered viable or dead. Due to the limitation of vital staining assays, other methods to assess oocyst viability are still needed. Cell culture techniques may prove valuable tools in the near future