EPA Science Inventory



Ware, M. W., L. Wymer, H. A. Lindquist, AND F. W. Schaefer III. EVALUATION OF CRYPTOSPORIDIUM OOCYST RECOVERY IN WATER BY EPA METHOD 1623 WITH A MODIFIED IMS DISSOCIATION PROCEDURE. Presented at International Symposium on Waterborne Pathogens, Lisbon, Portugal, September 22-25, 2002.


EPA Methods 1622 and 1623 are the benchmarks for detection of Cryptosporidium spp. oocysts in water. 5-7 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).5-7 Studies evaluating the recovery of Methods 1622 and 1623 have demonstrated that recovery of oocysts is low and highly variable in real water samples, with the average oocyst recoveries all below 20% 1,3,4 Lindquist, et al. reported that only 30% of the recovered oocysts could be confirmed with DAPI stained nuclei.1 Reynolds, et al. developed a procedure which increased the rate of DAPI confirmation using a solid phase cytometer.2

Study Objectives:

Modifying treatment during and after IMS was evaluated to determine the effect on overall recovery and on the ability to confirm oocysts using DAPI staining.
Experimental Conditions:

Cryptosporidium parvum oocysts (Harley Moon strain) were purified by sieving, step gradients, and cesium chloride and used within 3 months of purification. All oocysts were stained with 1 x Crypt-a-glo or Aqua-glo (Waterborne, Inc.) and counterstained with DAPI (Sigma Chemical) at 0.4 ?g/ml. Briefly, stained oocysts were identified using the criteria in Method 1623.7

A FACS VantageSE (Beckton Dickinson) equipped with clone-cyt software was used for all flow cytometry analysis. Briefly during flow cytometry, a primary gate was drawn around the oocysts using forward (FSC) and side scatter (SSC). Stained oocysts were analyzed by FL1 (fluorescein isothiocyanate (FITC)) and FL5 (DAPI). Unstained, purified oocysts were prepared for experimental manipulations by sorting using the primary (FSC/SSC) gate into 1.5 ml tubes containing 1 ml reagent water.

Cryptosporidium parvum oocysts suspended in reagent water were used to compare DAPI stain reactions using two protocols. Control oocysts were suspended in reagent water. Heat processed oocysts were suspended in reagent water and heated for 10 minutes at 80 C, based on that described by Reynolds.2 The oocysts were then stained in suspension and examined by both flow cytometry and microscopy.

IMS recovery experiments were performed in both reagent and river water to compare the IMS dissociation methods. Grab samples (>300 L) were taken from the Ohio River, filtered with Pall-Gelman Envirochek? filters, and eluted as described in Method 1623. These samples were pelleted, pooled into a packed pellet, and suspended in reagent water 5.

Each IMS reaction was comprised of a 1 ml suspension containing either 150, 100, 50, 20, or 0 flow cytometry enumerated C. parvum oocysts in reagent water, 1 ml of reagent water was used to rinse the suspension tube, and either 8 ml of reagent water was added, or 8 ml river water suspension was added, representing approximately 0.4 ml of packed pellet. The IMS reactions were paired. IMS reagents were added and processed as described in Method 1623 5 through the point of aspiration of the supernatant after the IMS separation in the 1.5 ml tube.

One set of sample was processed with acid dissociation. As described in Method 1623 5, 50 ?l of 0.1 N HCl was added to the oocyst-bead complex. This mixture was incubated for 10 minutes, and the oocysts were separated from the paramagnetic beads by exposure to a magnetic strip. The oocysts in solution were transferred to another 1.5 ml tube containing 5 ?l 1 N NaOH and 50 ?l reagent water. This set of samples was then heated for 10 min 80 C.

One hundred ?l of reagent water was added to each tube in the other set. These oocysts were heated for 10 min at 80 C, separated from the paramagnetic beads by exposure to a magnetic strip, and transferred to another 1.5 ml tube.

Both sets were then stained in solution, filtered through a 0.8 ?m porosity, 13 mm diameter polycarbonate filter, and enumerated by microscopy.


The unheated oocysts had a very different DAPI profile than the heated oocysts when examined by flow cytometry. Over 99% of the nuclei from heat treated oocysts were stained by DAPI. Fewer than 4% of the unheated oocysts were stained. The microscopy results were similar. Exposure to 80 C did not effect either the antibody staining or the DIC appearance of the oocysts.

Seeded reagent and river water samples were analyzed in parallel with either acid dissociation followed by heating or heat only dissociation. In both water types, heat dissociation improves mean oocyst recovery and DAPI confirmation. The mean oocyst recovery by acid dissociation in 48 reagent and river water samples was 28% ? 56 ( ? 2SD). The DAPI confirmation rate for these samples was 48% ? 64 In contrast, the mean oocyst recovery of 48 heat dissociated samples was 63% ? 28. The DAPI confirmation rate in these samples was 85% ? 26. The trends were similar in both water types, although the average oocyst recovery and DAPI confirmation rates were higher in reagent water samples. Relative differences in recovery between the two dissociation methods did not differ significantly by the number of oocysts present.


Water matrix particulates such as algae and cyanobacteria require that Cryptosporidium oocyst-like objects detected by fluorescent antibody be confirmed to prevent false positives. Method 1623 applies two confirmation methods: by demonstration of 1-4 sporozoites or by demonstration of 1-4 DAPI stained nuclei. Rarely are sporozoites visible by DIC microscopy. DAPI staining improves the confirmation rate, however, it remains quite low. This and other studies confirm that heating increases the DAPI confirmation rate.1,2 Furthermore, these DAPI stained oocysts are detectable by flow cytometry and they agree with microscopy results. This study demonstrates the utility of flow cytometry for the automated enumeration and confirmation of Cryptosporidium oocysts.

For both river and reagent water samples, the fraction of oocysts recovered was significantly greater in the samples processed with heat dissociation than by acid dissociation. 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. Since both samples were heated they should have similar DAPI confirmation rates, but this difference may be caused by the poor oocyst recovery of the acid dissociated samples. The performance of Method 1622/3 could be improved by changing the IMS bead-oocyst dissociation procedure from acid to heat which would allow for greater oocyst recovery and confirmation.


1) Refine new, practical methods for the detection of CCL-related and emerging waterborne human protozoa.

2) Perform field tests of devices or methods that have been developed under this task.

3) Evaluate these methods or devices in a variety of water matrices and parasite concentrations.

This work in this task supports CCL2 and 3 and is expected to be completed by 9/07.

Record Details:

Start Date: 09/22/2002
Completion Date: 09/22/2002
Record Last Revised: 06/21/2006
Record Created: 09/26/2003
Record Released: 09/26/2003
Record ID: 63880