1National Exposure Research Laboratory, Office of Research and Development, US Environmental Protection Agency, 2Shaw Environmental & Infrastructure, 3Office of Ground Water and Drinking Water, US Environmental Protection Agency
Rhodes, E. R., Villegas, L. F., Shaw, N. J., Miller, C., Villegas, E. N. A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.. J. Vis. Exp. (65), e4177, doi:10.3791/4177 (2012).
Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies 12. The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.
Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocysts and Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously1-3,5-8,10,11. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters4. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices9,13 .
This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.
1. Tangential Flow Hollow-fiber Ultrafiltration Procedure
2. Immunomagnetic Separation Procedure
3. Staining and Examination
Note: Additional information about the original procedure can be found in the December 2005 version of EPA Method 162312. The tangential flow hollow-fiber ultrafiltration procedure described is used in place of Section 12.0 of EPA Method 1623. The heat dissociation modifies Section 13.3.3 of EPA Method 1623. The procedure also describes an additional PBS rinse during the IMS process which can be inserted into the December 2005 version of Method 1623 after section 18.104.22.168. The complete list of consumables, reagents and equipment used for EPA Method 1623 including these modifications is listed in the equipment list.
4. Representative Results
Cryptosporidium oocysts and Giardia cysts recovered through the processes of filtration and immunomagnetic separation are detected by microscopic analysis. At 200X total magnification, each organism exhibiting a typical staining pattern, size, and shape as shown in Figure 2 should be further observed using oil immersion at 1000X total magnification. This will allow for measurement and identification of either typical defining features or atypical features that would rule out positive identification. Cryptosporidium is an ovoid to spherical object 4 to 6 μm in diameter which exhibits brilliant apple-green FITC fluorescence with brightly highlighted edges (Figure 3A). With DAPI UV, an oocyst will exhibit one of the following typical feature categories: light blue internal staining with a green rim and no distinct nuclei (DAPI negative), intense blue internal staining, or up to four distinct, sky-blue nuclei (DAPI positive - Figure 3B). Atypical features include deviations in color, structure, or DAPI fluorescence (e.g., too many stained nuclei, red fluorescing internal structures). If the fluorescent object has met criteria for typical FITC and DAPI staining, it is examined using differential interference contrast (DIC). The object is examined for atypical external or internal morphological characteristics such as cell wall ornamentation, or one or two large nuclei filling the cell. If atypical structures are not observed, the object is recorded in the total IFA count and categorized as an empty amorphous structure or with one to four sporozoites present (Figure 3C). Similarly, Giardia-like objects are examined with regard to FITC and DAPI staining as well as DIC characteristics, like axonemes, median bodies, and nuclei. Giardia cysts are round to ovoid brilliant apple-green objects, 8 - 18 μm long by 5 - 15 μm wide with brightly highlighted edges (Figure 3D). With DAPI UV, the Giardia cyst will exhibit DAPI-negative staining, or DAPI-positive characteristics (Figure 3E). The fluorescent object is examined by DIC for typical and atypical features in the same manner as described for Cryptosporidium. If atypical features are not observed, the object is recorded in the total IFA count and categorized as empty containing amorphous structure, or with one or more type of internal structures present (Figure 3F).
Any organism that is observed to have atypical features should not be counted as an (oo)cyst. Microscopic analysis of environmental samples can be challenging as there are organisms that may auto-fluoresce or cross-react with the FITC-conjugated anti-Cryptosporidium and/or anti-Giardia antibodies 1. It is recommended that an analyst be familiar with aquatic microbes and review dozens of slides to gain experience identifying Cryptosporidium and Giardia. At least three (oo)cysts on the positive staining control slide should be characterized prior to every session at the microscope.
Quality control samples may be spiked with (oo)cysts to determine the percent recovery for each protozoan using the calculation:
(Oo)cyst Percent Recovery = ((QC Sample Count - Count from Unspiked Sample) / Spike) x 100.
Figure 1. Graphic representation of the tangential flow hollow-fiber ultrafiltration system. The tubing is color coded to aid is assembly of the system.
Figure 2. Representative fluorescence image of Cryptosporidium and Giardia (oo)cysts. Cryptosporidium oocysts and Giardia cysts were stained with FITC labeled anti-Cryptosporidium/Giardia antibodies. Arrows, Giardia cysts; arrowheads, Cryptosporidium oocysts. A total of four Cryptosporidium oocysts and six Giardia cysts were found in the plane of focus. Samples observed under 200X magnification.
Figure 3. Representative microscopic images of Cryptosporidium oocysts and Giaridia cysts used for characterization. Cryptosporidium oocysts (A - C). Brilliant apple-green FITC fluorescence of spherical objects 4 to 6 μm in diameter with brightly highlighted edges (A) containing up to four distinct, sky-blue DAPI nuclei (B) and one to four sporozoites (S) per oocyst (C). Giardia cysts (D - F). Brilliant apple-green FITC fluorescence of round to ovoid objects 8 - 18 μm long by 5 - 15 μm wide with brightly highlighted edges (D) containing up to four sky-blue DAPI nuclei (E) and with one or more discernable internal structure such as nuclei (N), median body (M) and or axonemes(A) (F). White arrows, brilliant apple green fluorescence staining Cryptosporidium oocysts and Giardia cysts walls; white arrowheads, DAPI positive nuclei. Samples observed under 1000X magnification.
Tangential flow hollow-fiber ultrafiltration is an alternative and effective technique for the initial concentration of Cryptosporidium oocysts and Giardia cysts from water. Hollow-fiber ultrafiltration is less expensive than traditional filters. Since it has the ability to concentrate Cryptosporidium oocysts and Giardia cysts from a variety of different water matrices it is a useful alternative to the current filtration techniques used for EPA Method 1623. As with most other filtration methods, hollow-fiber ultrafiltration is prone to fouling with extremely turbid samples. High water pressure would result from the filter fouling; therefore it is recommended to monitor the pressure during the filtration run. In addition to Cryptosporidium oocysts and Giardia cysts, hollow-fiber ultrafiltration has been shown to be capable of concentrating bacteria and viruses1-3,5,8 . Hollow-fiber ultrafiltration outlined in this method can be used to concentrate multiple organisms in a single sample. It is noteworthy that obtaining a final volume between 200 and 250 ml is the critical final step in the concentration procedure so that extra centrifugation steps, that may result in (oo)cyst loss, are avoided (step 2.2). However, allowing the volume in the bottle to drop too low can have unfavorable effects on the recoveries since there will not be enough liquid volume to force all the oocysts or cysts into the retentate bottle. Therefore it is recommended to maintain a final volume between 200 and 250 ml.
Heat dissociation is an alternative to the acid dissociation step in Method 1623. This alternative step has been shown to improve Cryptosporidium oocyst recovery and reduce the method variation when isolated from either river or reagent water9. A side-by-side comparison of acid and heat dissociation methods demonstrated that using heat to dissociate the organisms from the immunomagnetic beads produced higher mean recoveries for both Cryptosporidium and Giardia. In addition, the precision of Cryptosporidium and Giardia recoveries was better in samples processed with heat dissociation compared with acid dissociation9.
The incorporation of HFUF as the concentration step allows more flexibility by providing the ability to concentrate multiple organisms. In addition it is a less expensive alternative to current Method 1623 filtration options.
The United States Environmental Protection Agency through its Office of Research and Development in collaboration with the Office of Ground Water and Drinking Water's Technical Support Center funded the research described here. All work was supported on-site at the US EPA, Cincinnati, Ohio. Although the information described in this article has been funded wholly or in part by the United States Environmental Protection Agency under contract (Contract EP-C-06-031) to Shaw Environmental and Infrastructure, Inc, it does not necessarily reflect the views of the Agency and no official endorsement should be inferred. It has been subjected to Agency review and approved for publication.
We would like to thank Ann Grimm and Michael Zimmerman for critical review of this manuscript and Doug Hamilton for his technical support.
|Asahi Kasei Rexeed 25 S/R wet hollow-fiber ultrafilters||Dial Medical||REXEED25S/R|
|I/P 73 (Masterflex R-3603), or equivalent||Cole-Parmer||EW-06408-73|
|L/S 24 (Masterflex Platinum-Cured), or equivalent||Cole-Parmer||EW-96410-24|
|L/S 15 (Masterflex Platinum-Cured), or equivalent||Cole-Parmer||EW-96410-15|
|L/S 36 (Masterflex Platinum-Cured), or equivalent||Cole-Parmer||EW-96410-36|
|I/P Precision Brushless Drive||Cole-Parmer||EW-77410-10|
|I/P Easy Load Pump Head||Cole-Parmer||EW-77601-10|
|Black HDPE Tee, 1/4"x 3/8" x 3/8"||US Plastics||62064|
|Masterflex T-connector L/S 15-25||Cole-Parmer||EG-30613-12|
|Nalgene heavy-duty pp 1 L bottle||Cole-Parmer||EW-06257-10|
|10 ml pipettes||Fisher Scientific||13-678-11C|
|Nalgene filling/venting cap for 1/4" tubing, 53B||Cole-Parmer||EW-06258-10|
|Straight coupling, NPT(F), 1/4"||Cole-Parmer||EW-06469-18|
|NPT branch tee, natural pp||Cole-Parmer||A-30610-75|
|Pinch clamps, 1/2"||Cole-Parmer||EW-06833-00|
|Custom fit DIN adapters||Molded Products Corp||MPC-855NS.250|
|Ring stand||Fisher Scientific||14-670B|
|Ring stand clamps||Fisher Scientific||05-769-6Q|
|Keck ramp clamp, 14mm||Cole-Parmer||EW-06835-10|
|Sodium thiosulfate pentahydrate||Sigma-Aldrich||72050|
|Antifoam Y-30 emulsion||Sigma-Aldrich||A5758|
|10 L Collapsible high-density polyethylene cubitainer||VWR international||IR314-0025|
|Centrifuge bottle rack||Fisher Scientific||05-663-103|
|250 ml conical centrifuge tubes||Corning||430776|
|Centrifuge||Beckman Coulter Inc.||Allegra X-15R|
|Swinging bucket rotor||Beckman Coulter Inc.||ARIES SX4750|
|Centrifuge bucket adapters for 250 ml conical tubes||Beckman Coulter Inc.||349849|
|200 μl large bore pipette tips||Fisher Scientific||02-707-134|
|5 ml pipettes||Fisher Scientific||13-678-11D|
|Dynabeads: Cryptosporidium/Giardia combo kit||IDEXX||73002|
|50 ml conical centrifuge tubes||Falcon BD||352098|
|Dynal L10 flat sided tubes||IDEXX||74003|
|Dynal MPC-6 magnet||IDEXX||12002D|
|1 ml pipettes||VWR international||53283-700|
|1.5 ml low adhesion microcentrifuge tubes||Fisher Scientific||02-681-320|
|1000 μl pipette & corresponding barrier tips||Gilson, Inc.||P1000/DF1000ST|
|100 μl pipette & corresponding barrier tips||Gilson, Inc.||P100/DF100ST|
|9 inch Pasteur pipettes||VWR international||14672-412|
|Dynal MPC-S magnet||IDEXX||12020D|
|Dynabeads rotator mixer||IDEXX||94701|
|Heat block||Fisher Scientific||11-718-2|
|Lab Armor Beads||Lab Armor||42370-750|
|Digital thermometer||Fisher Scientific||15-077-60|
|Phosphate-buffer saline 1X pH 7.4 (1X PBS)||Sigma-Aldrich||P4417|
|Single Spot slides||IDEXX||30201|
|EasyStain direct kit||BTF||-|
|10 μl pipette & corresponding barrier tips||Gilson, Inc.||P10 & DF10ST|
|4’,6’-Diamidino-2-phenyl indole dihydrochloride (DAPI)||Sigma-Aldrich||D9542|
|Clear nail polish||Fisher Scientific||S30697|
|slide warmer||Fisher Scientific||11-474-521|
|Immersion oil, Type A ND= 1.515||Nikon Instruments||MXA20234|
|Nikon 90i microscope with DIC capabilities||Nikon Instruments||MBA 77000|
|Plan APO 100X oil objective||Nikon Instruments||MRD01901|
|Plan Achro 20X||Nikon Instruments||MRL00202|
|FITC filter||Nikon Instruments||96302|
|DAPI filter||Nikon Instruments||96301|
|X-cite fluorescence illuminator||Nikon Instruments||87540|
|Lens paper||Nikon Instruments||76997|
|Biohazard disposable bag||Fisher Scientific||01-829D|
|Biohazard sharps container||Fisher Scientific||14-827-117|
|3 % hydrogen peroxide||VWR international||BDH3540-2|