AWWA WQTC56951

The main objectives of this study were to develop methods to isolate microsporidia from water and, once they were isolated, to determine the most efficient and accurate method to identify the recovered microsporidia. Identification techniques included microscopic morphology, immunological reactions, and molecular assays such as PCR. The only US Environmental Protection Agency (USEPA) approved method for recovery of protozoa from water (Method 1623; USEPA 1999) involves filtration of a 10 L water sample followed by elution from the filter, concentration by centrifugation, immunomagnetic purification of target organisms and visualization by an immunofluorescence assay. Since this laboratory routinely obtains high recovery efficiencies with low coefficients of variation for Cryptosporidium and Giardia using this method, it was used as the basis for developing microsporidia recovery methods. A variety of filtration methods were tested using several different filter technologies with commercially available and custom-made filters. A variety of fluorescent and non-fluorescent stains were tested for morphological identification and many primers and probes were tested for their specificity and affinity to DNA from different species of microsporidia. Includes 8 references, table.