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Cryptosporidium Oocyst Recovery in Water By EPA Method 1623: Evaluation of a Modified Ims Dissociation

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Abstract:EPA Methods 1622 and 1623 are the benchmarks for detection of Cryptosporidium spp. oocysts in water. These methods consist of filtration, elution, purification by immunomagnetic separation (IMS), and microscopic analysis after staining with a fluorescein isothiocyanate conjugated monoclonal antibody and counterstaining with 4',6-diamidino-2-phenyl indole (DAPI). Studies evaluating Methods 1622 and 1623 have demonstrated that oocyst recovery is low and highly variable in real water samples. In addition, the confirmation of oocysts is rare. Confirmation is vital to prevent false positive results because some algae can appear to be oocysts.

This study developed an alternative IMS dissociation procedure using heat instead of acid. This modification was evaluated for recovery and DAPI confirmation by performing IMS experiments of seeded reagent and Ohio River water samples which were analyzed in parallel with either acid or heat dissociation.

In both water types, heat dissociation improved mean oocyst recovery and DAPI confirmation. The mean oocyst recovery by acid dissociation in 48 reagent and river water samples was 28% � 28 ( � SD). The DAPI confirmation rate for these samples was 48% � 32 In contrast, the mean oocyst recovery of 48 heat dissociated samples was 63% � 14. The DAPI confirmation rate in these samples was 85% � 13. The trends were similar in both water types, although the average oocyst recovery and DAPI confirmation rates were higher in reagent water samples. The mean oocyst recovery was nearly twice that of acid dissociation (p<0.0001, t-test), and the amount of variation decreased by nearly half (p<0.0001, f-test). The heat dissociated samples had nearly two-fold greater confirmation rate by DAPI. The performance of Method 1622/3 could be improved by changing the IMS dissociation procedure from acid to heat which would allow for greater oocyst recovery and confirmation.
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Citation:Ware, M. W., F. W. Schaefer III, H. A. Lindquist, and L. J. Wymer. Cryptosporidium Oocyst Recovery in Water By EPA Method 1623: Evaluation of a Modified Ims Dissociation. Presented at Ohio River Basin Consortium for Research and Education, Highland Heights, KY, November 6-7, 2002.
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Contact: Mary P. O'Bryant - (919)-541-4871 or obriant.mary@epa.gov
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Division: Microbiological & Chemical Exposure Assessment Division
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Branch: Biohazard Assessment Research Branch
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Product Type: Abstrct/Oral
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Presented: 11/06/2002
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Related Entries:
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Bullet Item Detecting Ccl-Related, Emerging and Regulated Waterborne Human Protozoa for Exposure Assessment
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