EPA-MICRO: 1622: Cryptosporidium by filtration/IMS/FA microscopy
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Official Method Name
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EPA Method 1622: Cryptosporidium in Water by Filtration/IMS/FA |
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Current Revision
| April 2001 |
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Media
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WATER |
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Instrumentation
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Filtration-immunomagnetic Separation-immunofluorescent Microscopy |
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Method Subcategory
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Microbiological |
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Method Source
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Citation
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Brief Method Summary
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The method is a performance based method--if equivalent or better performance can be shown, modifications of the method may be used. A 10-L or greater sample is put through a 1-micron membrane filter, the materials on the filter are eluted with an aqueous buffered salt and detergent solution, and the eluate is concentrated. Magnetic beads conjugated to an antibody are added to the concentrate, and the magnetized oocysts are separated from the extraneous materials using a magnet. The magnetic bead complex is detached from the oocysts and the purified sample is applied to a well slide. The oocysts are stained on well slides with fluorescently labeled monoclonal antibodies and 4',6-diamidino-2-phenylindole (DAPI). The stained sample is examined using fluorescence and differential interference contrast (DIC) microscopy. |
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Scope and Application
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Ambient, compliance monitoring: ambient surface waters. EPA Fed Reg (Aug 2001) for cryptosporidium, ambient only: fresh, marine, or estuarine surface waters; applicability must be demonstrated for other matrices. USEPA. 1996 (May 14). National primary drinking water regulations: monitoring requirements for public drinking water supplies - Cryptosporidium, Giardia, viruses, disinfection byproducts, water treatment plant data and other information requirements. Fed. Reg. 61(94):24354-24388. USEPA. 2001 (August 30). Guidelines establishing test procedures for the analysis of pollutants; Analytical methods for biological pollutants in ambient water; proposed rule. Fed. Reg. 66(169)45811-45829. Clean Water Act section 401. 40 CFR 136.1(c). (state certification, licenses) for compliance monitoring in programs 303(c), 304(a), and 501(a). 136.3 Identification of test procedures. |
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Applicable Concentration Range
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40 to 500 oocysts/10 L (method); 100 - 10000 oocysts/10 L (Clancy et al 1999) |
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Interferences
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Inorganic and organic debris, clays and algae, and iron and alum coagulants may interfere with concentration, separation, and examination of the sample. Organisms that autofluoresce may contribute to false positives. Solvents, reagents, labware, and other hardware may yield artifacts that interfere with microscopic examinations. Freezing samples, filters, eluates, concentrates, or slides may interfere with detection of oocysts. |
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Quality Control Requirements
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Lab must operate a formal QA program. Method blank (negative control sample) should be run initially and a minimum of once per week or after changes in source of reagent water. Initial precision and recovery test (IPR). Analysis of spiked samples to evaluate and document data quality (matrix spike, MS). Analysis of standards and blanks as tests of continued performance: ongoing precision and recovery tests (OPR) and positive and negative staining control. All equipment should be thoroughly cleaned after use. |
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Sample Handling
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Sample preservation: Samples are chilled to 8oC; must not freeze. Techniques for collection: Collect minimum 10 L of sample in plastic carboy or filtered in the field. Must be shipped to the lab the same day as collected; see Standard Methods, 20th ed., L. Clesceri, A. Greenberg, and A. Eaton (editors). APHA: Washington, DC. 1998. Section 9060A and 9213B.2b Sample processing time 16-20 h. |
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Maximum Holding Time
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96 hours between sample collection/filtration and initiation of elution. Sample to slide preparation must be completed in 1 working day. 72 hours between application of the purified sample to the slide to staining. 7 days between sample staining and examination. |
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Relative Cost
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Greater than $400 |
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Sample Preparation Methods
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Membrane filtration, magnetic separation |
