United States           Office of Water        EPA-821-R-99-006
Environmental Protection     Washington, DC 20460 April 1999
Agency


Method 1623:  Cryptosporidium and

Giardia in Water by Filtration/IMS/FA
                                    Printed on Recycled Paper

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                                  Acknowledgments

This method was prepared under the direction of William A. Telliard of the Engineering and Analysis
Division within the U.S. Environmental Protection Agency (U.S. EPA) Office of Water. This document
was prepared under U.S. EPA Contract No. 68-C-98-139 by DynCorp, I&ET, with assistance from its
subcontractor, Interface, Inc.

The U.S. EPA Office of Water gratefully acknowledges the contributions of the following persons and
organizations to the development of this method:

Mike Arrowood, Centers for Disease Control, Division of Parasitic Diseases (MS-F13), 4770 Buford
       Highway, N.E., Atlanta, GA 30341-3724, USA
Phil Berger, Office of Groundwater and Drinking Water, U.S. Environmental Protection Agency, 401 M
       Street, S.W., Washington, DC 20460, USA
Jennifer Clancy, Clancy Environmental Consultants, Inc., P.O. Box 314, St. Albans, VT 05478, USA
Kevin Connell, DynCorp I&ET, 6101 Stevenson Avenue, Alexandria, VA 22314, USA
Ricardo DeLeon, Metropolitan Water District of Southern California, 700 Moreno Avenue, LaVerne, CA
       91760, USA
Shirley Dzogan, EnviroTest Laboratories, 745 Logan Avenue, Winnipeg, Manitoba R3E 3L5, Canada
Colin Fricker, Thames Water Utilities, Manor Farm Road, Reading, Berkshire, RG2 OJN, England
Carrie Hancock, CH Diagnostic &  Consulting Service, Inc., 214 S.E. Nineteenth Street, Loveland, CO
       80537, USA
Stephanie Harris, Region 10 Laboratory, U.S. Environmental Protection Agency, 7411 Beach Drive East,
       Port Orchard, WA 98366, USA
Dale Rushneck, Interface, Inc.,  3194 Worthington Avenue, Fort Collins, CO 80526, USA
Frank Schaefer III, National Exposure Research Laboratory, U.S. Environmental Protection Agency, 26
       W. Martin Luther King Drive, Cincinnati, OH 45268-1320, USA
Steve Schaub, Health and Ecological Criteria Division (4304), Office of Science and Technology, U.S.
       Environmental Protection Agency, 401 M Street, S.W., Washington, DC 20460, USA
Ajaib Singh, City of Milwaukee Health Department, 841 North Broadway, Milwaukee, WI 53202, USA
Huw Smith, Department of Bacteriology, Scottish Parasite Diagnostic Laboratory, Stobhill NHS Trust,
       Springburn, Glasgow, G21 3UW, Scotland
Timothy Straub, Lockheed Martin, 7411 Beach Drive East, Port Orchard, WA 98366, USA
William A. Telliard, Office of Science and Technology, U.S. Environmental Protection Agency, 401 M
       Street, S.W., Washington, DC 20460, USA
Giardia cover photo courtesy of CH Diagnostic & Consulting Service, Inc.
                                       Disclaimer

This method has been reviewed by the U.S. EPA Office of Water and approved for publication. Mention of
trade names or commercial products does not constitute endorsement or recommendation for use.

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Questions regarding this method or its application should be addressed to:

       William A. Telliard
       U.S. EPA Office of Water
       Analytical Methods Staff
       Mail Code 4303
       Washington, DC 20460
       Phone: 202/260-7120
       Fax:   202/260-7185

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                                        Introduction

To support future regulation of protozoa in drinking water, the Safe Drinking Water Act Amendments of
1996 require the U.S. Environmental Protection Agency (EPA) to evaluate the risk to public health posed
by drinking water contaminants, including waterborne parasites, such as Cryptosporidium and Giardia. To
implement these requirements, EPA must accurately assess Cryptosporidium and Giardia occurrence hi
raw surface waters used as source waters for drinking water treatment plants. Method 1623 was developed
to support this assessment.

EPA initiated an effort in 1996 to identify new and innovative technologies for protozoan monitoring and
analysis. After evaluating potential alternatives to the  then-current method through literature searches,
discussions with research and commercial laboratories, and meetings with experts in the field, the
Engineering and Analysis Division within the Office of Science and Technology within EPA's Office of
Water developed draft Method 1622 for Cryptosporidium detection in December 1996. This
Cryptosporidium-Q-rAy method was validated through an interlaboratory study in August 1998, and was
revised as a final, valid method for detecting Cryptosporidium in water in January 1999.

Although development of an acceptable immunomagnetic separation system for Giardia lagged behind
development of an acceptable system for Cryptosporidium, an acceptable system was identified in October
1998, and EPA validated a method for simultaneous detection of Cryptosporidium and Giardia in
February 1999. To avoid confusion with Method 1622, which already had been validated and was in use
both domestically and internationally as a stand-alone Cryptosporidium- detection method, EPA designated
the new combined procedure Method 1623.

Method 1623 is a performance-based method applicable to the determination of Cryptosporidium and
Giardia in aqueous matrices. Method 1623 requires filtration, immunomagnetic separation of the oocysts
and cysts from the material captured, and an immunofluorescence assay for determination of oocyst and
cyst concentrations, with confirmation through vital dye staining and differential interference contrast
microscopy.

The interlaboratory validation of Method 1623 used the Gelman capsule filtration procedure, Dynal
immunomagnetic separation (IMS) procedure, and Meridian sample staining procedure described in this
method. However, alternate procedures are allowed, provided that required quality control tests are
performed and all quality control acceptance criteria in this method are met.

Alternate filtration procedures to the Gelman capsule filter include, but are not limited to, the following:
        Coming membrane disk filtration
•       Corning capsule filtration
•       ImmuCell vortex flow filtration
        Water analyzer developed by the Marshfield Clinic and Molecular Biology Resources
        Continuous-operation water centrifuge developed by the Johns Hopkins University

Alternate IMS procedures to the Dynal IMS kit include, but are not limited to, the following:
        ImmuCell IMS
        Hach IMS

Alternate staining procedures to  the Meridian kit include, but are not limited to, the following:
        Waterborne combined Cryptosporidium/Giardia antibody staining kit
•       CelLabs combined Cryptosporidium/Giardia staining kit

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                                   Table of Contents


1.0     Scope and Application . .       . .           ...         . .       	    .	    1

2.0     Summary of Method      ....        , .	      . .     .	  ....  1

3.0     Definitions  ....      	      	         	       .....  	2

4.0     Contamination, Interferences, and Oocyst Degradation	  	2

5.0     Safety		    ......	  3

6.0     Equipment and Supplies   . .       	         .		    3

7.0     Reagents and Standards     	           ...	      . .   .....    6

8.0     Sample Collection and Storage  ....	 .       ....     ......    .     ,7

9.0     Quality Control . .	      8

10.0   Microscope Calibration and Analyst Verification		  ....  .  14

11.0   Oocyst and Cyst Suspension Enumeration and Spiking	20

12.0   Sample Filtration and Elution   ...    . .	24

13.0   Sample Concentration and Separation (Purification)...        	26

14.0   Sample Staining      	      	      .... 29

15.0   Examination	      	       	30

16.0   Analysis of Complex Samples  . .       	31

17.0   Method Performance	         	       	32

18.0   Pollution Prevention	       .	       . .       . . 32

19.0   Waste Management	     	   32

20.0   References	  32

21.0   Tables and Figures	33
       Table 1.       Quality control acceptance criteria for Cryptosporidium	  33
       Table 2.       Quality control acceptance criteria for Giardia  	  33
       Figure 1.      Hemacytometer platform ruling   	34
       Figure 2.      Manner of counting oocysts and cysts in 1 square mm	  35
       Figure 3.      Laboratory filtration system	  36
       Figure 4.      Methods for scanning a well  slide     	37

22.0   Glossary of Definitions and Purposes	38

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VI

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       Method 1623: Cryptosporidium and  Giardia  in Water
                                by Filtration/IMS/FA

1.0   Scope and Application
1.1    This method is for determination of the identity and concentration of Cryptosporidium (CAS
       Registry number 137259-50-8) and Giardia (CAS Registry number 137259-49-5) in untreated
       surface water and in other waters by filtration, immunomagnetic separation (IMS), and
       immunofluorescence assay (FA) microscopy. Cryptosporidium and Giardia may be confirmed
       using 4',6-diamidino-2-phenylindole (DAPI) staining and differential interference contrast (D.I.C.)
       microscopy.
1.2    This method is designed to meet the survey and monitoring requirements of the U.S. Environmental
       Protection Agency (EPA). It is based on laboratory testing of recommendations by a panel of
       experts convened by EPA. The panel was charged with recommending an improved protocol for
       recovery and detection of protozoa that could be tested and implemented with minimal additional
       research.
1.3    This method will not identify the species of Cryptosporidium or Giardia or the host species of
       origin, nor can it determine the viability or infectivity of detected oocysts and cysts.
1.4    This method is for use only by persons experienced in the determination of Cryptosporidium and
       Giardia by filtration, IMS, and FA. Experienced persons are defined in Section 22.0 at the end of
       this method as the principal analyst/supervisor, analyst, and technician. Laboratories unfamiliar
       with analyses of environmental samples by the techniques in this method should gain experience
       using water filtration techniques, IMS, fluorescent antibody staining with monoclonal antibodies,
       and microscopic examination of biological particulates using bright-field and D.I.C. microscopy.
1.5    Any modification of the method beyond those expressly permitted is subject to the application and
       approval of alternative test procedures under 40 CFR Part  141.27.

2.0   Summary of Method
2.1    A 10-L volume of water is collected in a carboy in the field and shipped to the laboratory. The
       sample is filtered in the laboratory and the oocysts, cysts, and extraneous materials are retained on
       the filter.
2.2    Elution and separation
       2.2.1   Materials on the filter are removed by elution with an aqueous buffered salt and detergent
               solution. The eluate is centrifuged to pellet the oocysts and cysts, and the supernatant
               fluid is aspirated.
       2.2.2   The oocysts and cysts are magnetized by attachment of magnetic beads conjugated to
               anti-Cryptosporidium and anti-Giardia antibodies. The magnetized oocysts and cysts are
               separated from the extraneous materials using a magnet, and the  extraneous materials are
               discarded. The magnetic bead complex is then detached from the oocysts and cysts.
2.3    Enumeration
       2.3.1   The oocysts and cysts 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 (D.I.C.) microscopy.
       2.3.2   Qualitative analysis is performed by scanning each slide well for objects that meet the
               size, shape, and fluorescence characteristics of Cryptosporidium oocysts or Giardia


                                               1                                      April 1999

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Method 1623 - Crvptosooridium and Giardia
                cysts. Potential oocysts or cysts are confirmed through DAPI staining characteristics and
                D.I.C. microscopy. Oocysts and cysts are identified when the size, shape, color, and
                morphology agree with specified criteria and examples in a photographic library.
       2.3.3   Quantitative analysis is performed by counting the total number of objects on the slide
                confirmed as oocysts or cysts.
2.4    Quality is assured through reproducible calibration and testing of the filtration, immunomagnetic
       separation (IMS), staining, and microscopy systems. Detailed information on these tests is
       provided in Section 9.0.

3.0   Definitions
3.1    Cryptosporidium is defined as a protozoan parasite potentially found in water and other media.
       The six species of Cryptosporidium and their potential hosts are C. parvum (mammals, including
       humans); C. baileyi and C. meleagridis (birds); C. muris (rodents); C. serpentis (reptiles); and C.
       nasorum (fish).
3.2    Giardia is defined as a protozoan parasite potentially found in water and other media. The two
       species of Giardia and their potential hosts are G. intestinalis (humans) and G. muris (mice).
3.3    Definitions for other terms used in this method are given in the glossary (Section 22.0).

4.0   Contamination, Interferences, and Oocyst Degradation
4.1    Turbidity caused by inorganic and organic debris can interfere with the concentration, separation,
       and examination of the sample for Cryptosporidium oocysts and Giardia cysts. In addition to
       naturally-occurring debris, such as clays and algae, chemicals, such as iron and alum coagulants
       and polymers, may be added to finished waters during the treatment process, which may result in
       additional interference.
4.2    Organisms and debris that autofluoresce or demonstrate non-specific fluorescence, such as algal
       and yeast cells, when examined by epifluorescent microscopy, may interfere with the detection of
       oocysts and cysts and contribute to false positives by immunofluorescent assay (FA).
4.3    Solvents, reagents, labware, and other sample-processing hardware may yield artifacts that may
       cause misinterpretation of microscopic examinations for oocysts and cysts. All materials used shall
       be demonstrated to be free from interferences under the conditions of analysis by running a method
       blank (negative control sample)  initially and a minimum of every week or after changes in source
       of reagent water.  Specific selection of reagents and purification of solvents and other materials may
       be required.
4.4    Interferences co-extracted from samples will vary considerably from source to source, depending
       on the the water being sampled. Experience suggests that high levels of algae, bacteria, and other
       protozoa can interfere in the identification of oocysts and cysts (Reference 20.1).
4.5    Freezing 10-L samples, filters, eluates, concentrates, or slides may interfere with the detection
       and/or identification of oocysts and cysts.
4.6    All equipment should be autoclaved after use and before washing. Clean equipment by scrubbing
       with warm detergent solution and exposing to hypochlorite solution (minimum of 5%) for at least
       30 minutes at room temperature. Rinse the equipment with reagent water and place in an oocyst-
       and cyst-free environment until dry. Disposable supplies should be used wherever possible.
April 1999

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                                                        Method 1623 - Cryptosporidium and Giardia
5.0   Safety
5.1    The biohazard associated with, and the risk of infection from, oocysts and cysts is high in this
       method because live organisms are handled. This method does not purport to address all of the
       safety problems associated with its use. It is the responsibility of the laboratory to establish
       appropriate safety and health practices prior to use of this method. In particular, the
       analyst/technician must know and observe the safety procedures required in a microbiology
       laboratory that handles pathogenic organisms while preparing, using, and disposing of sample
       concentrates, reagents and materials, and while operating sterilization equipment.
5.2    The toxicity or carcinogeniciry of each compound or reagent used in this method has not been
       precisely determined; however, each chemical compound should be treated as a potential health
       hazard. Exposure to these compounds should be reduced to the lowest possible level.  The
       laboratory is responsible for maintaining a current awareness file of Occupational Safety and
       Health Administration regulations regarding the safe handling of the chemicals specified in this
       method. A reference file of material safety data sheets should be made available to all personnel
       involved in these analyses. Additional information on laboratory safety can be found in References
       20.2 through 20.5.
5.3    Samples may contain high concentrations of biohazards and toxic compounds, and must be handled
       with gloves and opened in a biological safety cabinet to prevent exposure. Reference materials and
       standards containing oocysts and cysts must also be handled with gloves and the analyst/technician
       must never place gloves in or near the face after exposure to solutions known or suspected to
       contain oocysts and cysts. Do not mouth-pipette.
5.4    Laboratory personnel must change gloves after handling filters and other contaminant-prone
       equipment and reagents. Gloves must be removed or changed before touching any other laboratory
       surfaces or equipment.

6.0   Equipment and Supplies

       NOTE: Brand names, suppliers, and part numbers are for illustrative purposes only.
       No endorsement is implied. Equivalent performance may be achieved using apparatus
       and materials other than those specified here, but demonstration of equivalent
       performance that meets the requirements  of this method is the responsibility of the
       laboratory.

6.1    Equipment for spiking samples in the laboratory
       6.1.1   10-L carboy with bottom delivery port ('/2")—Cole-Palmer cat. no. 06080-42, or
                equivalent; calibrate to 10.0 L and mark level with waterproof marker
       6.1.2   Stir bar—Fisher cat. no. 14-511-93, or equivalent
       6.1.3   Stir plate—Fisher cat. no. 14-493-120S, or equivalent
       6.1.4   Hemacytometer—Hauser Scientific, Horsham, PA, cat. no. 3200 or 1475, or equivalent
       6.1.5   Hemacytometer coverslip—Hauser Scientific, cat. no. 5000 (for hemacytometer cat. no.
                3200) or 1461 (for hemacytometer cat. no 1475), or equivalent
       6.1.6   Lens paper without silicone—Fisher cat. no. 11-995, or equivalent
       6.1.7   Polystyrene or polypropylene conical tubes with screw caps—15- and 50-rnL
                                                                                       April 1999

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Method 1623 - Crvptosporidium and Giardia
6.2    Equipment for laboratory filtration of samples
       6.2.1    Capsule filter—Approximately 6-cm diameter x 21-cm long with approximately 1300
                cm2 polyethersulfone filter media and '/2-in. inlet and outlet fittings, Pall Gelman
                Laboratory, Ann Arbor, MI, Envirochek™ Sampling Capsule, product 12110, or
                equivalent
       6.2.2    Laboratory shaker with arms for agitation of sampling capsules
                6.2.2.1        Laboratory shaker—Lab-Line model 3589, VWR Scientific cat. no.
                              57039-055, Fisher cat. no 14260-11, or equivalent
                6.2.2.2       Side arms for laboratory shaker—Lab-Line Model 3587-4, VWR
                              Scientific cat. no. 57039-045, Fisher cat. no. 14260-13, or equivalent
6.3    Ancillary sampling equipment required for using capsule filter
       6.3.1    Tubing—Glass, polytetrafluoroethylene (PTFE), high-density polyethylene (HDPE), or
                other tubing to which oocysts and cysts will not easily adhere—Tygon formula R-3603,
                or equivalent. If rigid tubing (glass, PTFE, HDPE) is used and the sampling system uses
                a peristaltic pump, a minimum length of compressible tubing may be used in the pump.
                Before use, the tubing must be thoroughly rinsed with detergent solution, followed by
                repeated rinsing with reagent water to minimize sample contamination.
       6.3.2    Flow control valve—0.5 gpm (0.03 L/s), Bertram Controls, Plast-O-Matic cat. no.
                FC050B'/2-PV, or equivalent; or 0.4- to 4-Lpm flow meter with valve—Alamo Water
                Treatment, San Antonio, TX, cat.  no. R5310, or equivalent
       6.3.3    Centrifugal pump—Grainger, Springfield, VA, cat. no. 2P613, or equivalent
6.4    Immunomagnetic separation (IMS) apparatus
       6.4.1    Sample  mixer—Dynal Inc., Lake  Success, NY, cat.  no. 947.01, or equivalent
       6.4.2    Magnetic particle concentrator for 10-mL test tubes—Dynal MPC-1® , cat. no. 120.01,
                or equivalent
       6.4.3    Magnetic particle concentrator for microcentrifuge tubes—Dynal MPC-M® , cat. no.
                120.09,  or equivalent
       6.4.4    Flat-sided sample tubes—16 x  125 mm Leighton-type tubes with 60 x 10 mm flat-sided
                magnetic capture area, Dynal L10, cat. no. 740.03, or equivalent
6.5    Powder-free latex gloves—Fisher cat no. 113945B, or equivalent
6.6    Graduated cylinders, autoclavable—10-,  100-, and 1000-mL
6.7    Centrifuges
       6.7.1    Centrifuge capable of accepting 15- to 250-mL conical centrifuge tubes and achieving
                1100 x G—International Equipment Company, Needham Heights, MA, Centrifuge Size
                2, Model K with swinging bucket, or equivalent
       6.7.2    Centrifuge tubes—Conical, graduated, 1.5-, 50-, and 250-mL
6.8    Microscope
       6.8.1    Epifluorescence/differential interference contrast (D.I.C.) with stage and ocular
                micrometers and 20X (N.A.=0.4)  to 100X (N.A.=1.3)  objectives—Zeiss™ Axioskop,
                Olympus™ BH, or equivalent
April 1999

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                                                         Method 1623 - Cryptosporidium and Giardia
       6.8.2    Excitation/band-pass filters for immunofluorescent assay (FA)—Zeiss™ 487909 or
                equivalent, including, 450- to 490-nm exciter filter, 510-nm dicroic beam-splitting mirror,
                and 515- to 520-nm barrier or suppression filter
       6.8.3    Excitation/band-pass filters for DAPI—Filters cited below (Chroma Technology,
                Brattleboro, VT), or equivalent
Microscope
model
Zeiss™ -Axioskop
Zeiss™ -IM35
Olympus™ BH
Olympus™ BX
Olympus™ IMT2
Fluoro-
chrome
DAPI (UV)
DAPI (UV)
DAPI (UV)
Excitation
filter (nm)
340-380
340-380
340-380
Dichroic beam-
splitting mirror (nm)
400
400
400
Barrier or suppression
filter (nm)
420
420
420
Filter holder
DAPI (UV)
340-380
400
420
Filter holder
DAPI (UV)
340-380
400
420
Filter holder
Chroma catalog
number
CZ902
CZ702
11000
91002
11000
91008
11000
91003
6.9    Ancillary equipment for microscopy
       6.9.1    Well slides—Dynal Spot-On well slides cat. no. 740.04, or equivalent
       6.9.2    Glass coverslips—22 * 50 mm
       6.9.3    Fingernail polish—Clear or clear fixative, PGC Scientifics, Gaithersburg, MD, cat. no.
                60-4890, or equivalent
       6.9.4    Nonfluorescing immersion oil
       6.9.5    Micropipette, adjustable:       0- to IQ-^L with 0- to 10-^L tips
                                             10- to 100-AiL, with 10- to 200-^L tips
                                             100- to 1000-AiL with 100- to 1000-,uL tips
       6.9.6    Forceps—Splinter, fine tip
       6.9.7    Forceps—Blunt-end
       6.9.8    Desiccant—Drierite™ Absorbent, Fisher cat. no. 07-577-1A, or equivalent
6.10   Pipettes—Glass or plastic
       6.10.1  5-, 10-, and25-mL
       6.10.2  Pasteur, disposable
6.11   Balances
       6.11.1  Analytical—Capable of weighing 0.1 mg
       6.11.2  Top loading—Capable of weighing 10 mg
6.12   pH meter
6.13   Incubator—Fisher Scientific Isotemp™, or equivalent
6.14   Vortex mixer—Fisons Whirhnixer, or equivalent
6.15   Vacuum source—Capable of maintaining 25 in. Hg, equipped with shutoff valve and vacuum
       gauge
                                                                                        April 1999

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Method 1623 - Cryptosporidium and Giardia
6.16   Miscellaneous labware and supplies
       6.16.1  Test tubes and rack
       6.16.2  Flasks—Suction, Erlenmeyer, and volumetric, various sizes
       6.16.3  Beakers—Glass or plastic, 5-, 10-, 50-, 100-, 500-, 1000-, and 2000-mL
       6.16.4  Lint-free tissues
6.17   10- to 15-L graduated container—Fisher cat. no. 02-961-50B, or equivalent; calibrate to 9.0, 9.5,
       10.0, 10.5, and 11.0 L and mark levels with waterproof marker
6.18   Equipment for field sampling and shipping
       6.18.1   10-L carboy—Cole Farmer cat. no. 06100-33, or equivalent
       6.18.2  Shipping container—Cole Farmer cat. no. 06100-03, or equivalent

7.0   Reagents and  Standards
7.1    Reagents for adjusting pH
       7.1.1   Sodium hydroxide (NaOH)—ACS reagent grade, 6.0 N and 1.0 N in reagent water
       7.1.2   Hydrochloric acid (HC1)—ACS reagent grade, 6.0 N, 1.0 N, and 0.1 N in reagent water
7.2    Solvents—Acetone, glycerol, ethanol, and methanol, ACS reagent grade
7.3    Reagent water—Water in which oocysts and cysts and interfering materials and substances,
       including magnetic minerals, are not detected by this method
7.4    Reagents for eluting capsule filters
       7.4.1   Laureth-12—PPG Industries, Gurnee, IL, cat. no. 06194, or equivalent. Store Laureth-12
               as a 10% solution in reagent water. Weigh 10 g of Laureth-12 and dissolve using a
               microwave or hot plate in 90 mL of reagent water. Dispense 10-mL aliquots into sterile
               vials and store at room temperature for up to 2 months, or in the freezer for up to a year.
       7.4.2    1 M Tris, pH 7.4—Dissolve 121.1 g Tris (Fisher cat. no. BP152)  in 700 mL of reagent
               water and adjust pH to 7.4 with 1 N HC1 or NaOH. Dilute to a final 1000 mL with
               reagent water and adjust the final pH. Filter-sterilize through a 0.2-,um membrane into a
               sterile plastic container and store at room temperature.
       7.4.3   0.5 M EDTA, 2 Na, pH  8.0—Dissolve 186.1  g ethylenediamine tetraacetic acid,
               disodium salt dihydrate (Fisher cat. no. S311) in 800 mL and adjust pH to 8.0 with 6.0 N
               HC1 or NaOH. Dilute to a final volume of 1000 mL with reagent water and adjust to pH
               8.0 with 1.0 N HC1 or NaOH.
       7.4.4   Antifoam A—Sigma Chemical Co. cat. no. A5758, or equivalent
       7.4.5   Preparation of elution buffer solution—Add the contents of a pre-prepared Laureth-12
               vial (Section 7.4.1) to a 1000-mL graduated cylinder. Rinse the vial several times to
               ensure the transfer of the detergent to the cylinder. Add 10 mL of Tris solution (Section
               7.4.2), 2 mL of EDTA solution (Section 7.4.3), and 150 yL Antifoam A (Section 7.4.4).
               Dilute to 1000 mL with reagent water.
7.5    Reagents for immunomagnetic separation (IMS)—Dynabeads® GC-Combo, Dynal cat no. 730.02,
       or equivalent
7.6    DABCO/glycerol mounting medium (2%)—Dissolve 2 g of DABCO (Sigma Chemical Co. cat no.
       D-2522, or equivalent) in 95 mL of warm glycerol/PBS (60% glycerol, 40% PBS [Section 7.9.4]).

April 1999                                      6

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                                                        Method 1623 - Cryptosporidium and Giardia
       After the DABCO has dissolved completely, adjust the solution volume to 100 mL by adding an
       appropriate volume of glycerol/PBS solution.
7.7    Direct labeling kit for detection of oocysts and cysts—Merifluor Cryptosporidium/Giardia,
       Meridian Diagnostics cat. no. 250050, Cincinnati, OH, or equivalent. Store the kit at 0°C to 8°C
       and return it promptly to this temperature after each use. Do not allow any of the reagents in this
       kit to freeze. The labeling reagents should be protected from exposure to light. Diluted, unused
       working reagents  should be discarded after 48 hours. Discard the kit after the expiration date is
       reached.
7.8    4',6-diamidino-2-phenylindole (DAPI) stain
       7.8.1    Stock solution—Dissolve 2 mg/mL DAPI in absolute methanol. Prepare volume
                consistent with minimum use. Store at 0°C to 8°C in the dark. Discard unused solution
                after 2 weeks. Do not allow to freeze.
       7.8.2   Staining solution (1/5000 dilution in PBS [Section 7.9.4])—Add 10 ^L of 2 mg/mL
                DAPI stock solution to 50 mL of PBS. Prepare daily. Store at 0°C to 8°C in the dark
                except when staining. Do not allow to freeze. The solution concentration may be
                increased up to 1 Aig/mL if fading/diffusion of DAPI staining is encountered, but the
                staining  solution must be tested first on expendable environmental samples to confirm that
                staining  intensity is appropriate.
7.9    Oocyst and cyst suspensions for spiking
       7.9.1    Purified, live Cryptosporidium oocyst stock suspension—not heat-fixed, formalin-fixed,
                or treated in any way to reduce viability
       7.9.2   Purified, live Giardia cyst stock suspension—not heat-fixed, formalin-fixed, or treated in
                any way to  reduce viability
       7.9.3   Tween-20, 0.01%—Dissolve 1.0 mL of a 10% solution of Tween-20 in 1 L of reagent
                water
       7.9.4   Phosphate buffered saline (PBS), 150 mM—Add 1.07 g Na2HPO4, 0.39 g
                NaH2PO4.2H2O, and 8.5 g NaCl to 800 mL reagent water.  Dissolve and adjust to 1 L
                volume with reagent water. Adjust pH to 7.2 with NaOH or HC1. Prepare weekly.
       7.9.5   Storage procedure—Store oocyst and cyst suspensions at 0°C to 8°C, until ready to use.
                Do not allow to freeze. Samples must be spiked: (1) within 24 hours of enumeration of
                the oocyst and cyst spiking suspension if the hemacytometer chamber technique is used
                (Section 11.3),  or (2) within 24 hours of application of the spiking suspension to the  10
                well slides if the well-slide enumeration technique is used (Section 11.4).

8.0   Sample Collection and  Storage
8.1    Samples are collected in plastic  10-L carboys and shipped to the  laboratory for filtration, elution,
       concentration, immunomagnetic separation (IMS), staining, and examination. Samples must be
       shipped to the laboratory the day they are collected and must arrive at the laboratory within 24
       hours of sample collection.  Store 10-L carboys at 0°C to 8°C between collection and shipment to
       the laboratory and upon receipt at the laboratory until ready for filtration.  Do not allow to freeze.
                                                                                       April 1999

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Method 1623 - Cryptosporidium and Giardia
        NOTE:  U.S. Department of Transportation (DOT) regulations (49 CFR 172) prohibit
        interstate shipment of more than 4 L of solution known to contain infectious materials.
        State regulations may contain similar regulations for intrastate commerce. This method
        requires a minimum sample volume of 10 L.  Unless the sample is known or suspected to
        contain Cryptosporidium, Giardia. or other infectious agents (e.g., during an outbreak),
        samples should be shipped as noninfectious and should not be marked as infectious. If a
        sample is known or suspected to be infectious, and the sample must be shipped to a
        laboratory by a transportation means affected by DOT or state regulations, it is
        recommended that the sample be filtered in the field, and that the filter be shipped to the
        laboratory to avoid violating transport regulations.

8.2     Sample holding times: Laboratory filtration, elution, and concentration of a sample received in a
        carboy must be completed within 72 hours of sample collection. The concentrate must be stored at
        0°C to 8°C  if not proceeding immediately to IMS. Do not allow to freeze.
8.3     Concentrate holding times: IMS and sample staining must be completed within 24  hours of
        completion of sample concentration. Stained slides must be stored at 0°C to 8°C in the dark. Do
        not allow to freeze.
8.4     Stained sample holding times: Although immunofluorescence assay (FA) and 4',6-diamidino-2-
        phenylindole (DAPI) and differential interference contrast (D.I.C.) microscopy examination and
        confirmation should be performed immediately after staining is complete, laboratories have up to
        72 hours from completion of sample staining to complete the examination and confirmation of
        samples. However, if fading/diffusion of DAPI staining is noticed, the laboratory must reduce this
        holding time and/or adjust the concentration of the DAPI staining solution (Section 7.8.2) so that
        fading/diffusion does not occur.
8.5     Spiking suspension enumeration holding times: Initial and ongoing precision and recovery (IPR and
        OPR) samples and matrix spike (MS) samples must be spiked: (1) within 24 hours of enumeration
        of the spiking suspension if the hemacytometer chamber technique is used (Section 11.3), or (2)
        within 24 hours of application of the spiking suspension to the 10 well slides if the well-slide
        enumeration technique is used (Section 11.4).

9.0    Quality Control
9.1     Each laboratory that uses this method is required to operate a formal quality assurance (QA)
        program (Reference 20.6). The minimum requirements of this program consist of an initial
        demonstration of laboratory capability through performance of the initial precision and recovery
        test (Section 9.4), analysis of spiked samples to evaluate and document data quality, and analysis
        of standards and blanks as tests of continued performance. Laboratory performance is compared to
        established performance criteria to determine  if the results of analyses  meet the performance
        characteristics of the method.
        9.1.1    A test of the microscope used for detection of oocysts and cysts is performed prior to
                examination of slides. This test is described in Section 10.0.
        9.1.2   In recognition of advances that are occurring in analytical technology, the laboratory is
                permitted to modify certain method procedures to improve recovery or lower the costs of
                measurements, provided that all quality control (QC) tests cited in Section 9.1.2.1 are
                performed and all QC acceptance criteria (Tables 1 and 2 in  Section 21.0) are met.

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                                         Method 1623 - Cryptosporidium and Giardia
Method procedures that can be modified include front-end techniques, such as filtration or
immunomagnetic separation (IMS). The laboratory is not permitted to use an alternate
determinative technique (such as polymerase chain reaction) to replace
immunofluorescent assay in this method. However the laboratory is permitted to modify
the immunofluorescent assay procedure, provided that all QC tests cited in Section
9.1.2.1 are performed and all QC acceptance criteria are met.
9.1.2.1       Each time a modification is made to this method, the laboratory is
              required to perform the IPR test (Section 9.4) and the matrix spike/matrix
              spike duplicate (MS/MSD) test (Section 9.5) to demonstrate that the
              modification produces results equivalent or superior to results produced
              by this method.
9.1.2.2       The laboratory is required to maintain records of modifications made to
              this method. These records include the following, at a minimum:
              9.1.2.2.1       The names, titles, addresses, and telephone numbers  of
                              the analyst(s) who performed the analyses and
                              modification, and of the quality control officer who
                              witnessed and will verify the analyses and modification.
              9.1.2.2.2       A listing of the analyte(s) measured (Cryptosporidium).
              9.1.2.2.3       A narrative stating reason(s) for the modification.
              9.1.2.2.4       Results from all  QC tests comparing the modified method
                              to this method, including:
                              (a)     Microscope calibration (Section 10.0)
                              (b)     Calibration verification (Section 10.0)
                              (c)     IPR (Section 9.4)
                              (d)     MS/MSD (Section 9.5)
                              (e)     Analysis of method blanks (Section 9.6)
              9.1.2.2.5       Data that will allow an independent reviewer to validate
                              each determination by tracing the following processing
                              and analysis steps leading to the final result:
                              (a)     Sample  numbers and other identifiers
                              (b)     Source of spiking suspensions, as well as lot
                                     number and date received (Section 7.9)
                              (c)     Spike enumeration  date and time
                              (d)     All spiking suspension enumeration counts and
                                     calculations (Section 11.0)
                              (e)     Sample  spiking dates and times
                              (f)     Volume filtered (Section 12.2.5.2)
                              (g)     Filtration and concentration dates and times
                              (h)     Initial pellet volume and resuspended pellet
                                     volume(s) (Section 13.2)
                              (i)     Staining completion dates and times
                              (j)     Staining control results (Section 15.2.1)
                              (k)     All required examination and confirmation
                                     information (Section 15.2)
                              (1)     Examination and confirmation dates and times

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Method 1623 - Cryptosporidium and Giardia
                                             (m)     Analysis sequence/run chronology
                                             (n)      Lot numbers of elution, IMS, and staining
                                                     reagents
                                             (o)      Copies of bench sheets, logbooks, and other
                                                     recordings of raw data
                                             (p)      Data system outputs, and other data to link the
                                                     raw data to the results reported
        9.1.3   The laboratory shall spike a separate sample aliquot from the same source to monitor
                method performance. This MS test is described in Section 9.5.1.
        9.1.4   Analysis of method blanks is required to demonstrate freedom from contamination. The
                procedures and criteria for analysis of a method blank are described in Section 9.6.
        9.1.5   The laboratory shall, on an ongoing basis, demonstrate through analysis of the ongoing
                precision and recovery (OPR) sample that the analysis system is in control. These
                procedures are described in  Section 9.7.
        9.1.6   The laboratory shall maintain records to define the quality of data that are generated.
                Development of accuracy statements is described hi Sections 9.5.1.4 and 9.7.3.
        9.1.7   The laboratory shall analyze one method blank (Section 9.6) and one OPR sample
                (Section 9.7) each week during which samples are analyzed if 20 or fewer field samples
                are analyzed during this period. The laboratory shall analyze one laboratory blank and
                one OPR sample for every 20 samples if more than 20 samples are analyzed in a week.
        9.1.8   The laboratory shall analyze one MS sample (Section 9.5.1) when samples are first
                received from a utility for which the laboratory has never before analyzed samples. The
                MS analysis is performed on an additional (second) sample sent from the utility. If the
                laboratory routinely analyzes samples from 1 or more utilities, 1 MS analysis must be
                performed per 20 field samples. For example, when a laboratory receives the first sample
                from a given site, the laboratory must obtain a second aliquot of this sample to be used
                for the MS. When the laboratory receives the 21st sample from this site, a separate
                aliquot of this 21st sample must be collected and spiked.
9.2     Micropipette calibration
        9.2.1   Micropipettes must be sent to the manufacturer for calibration annually. Alternately, a
                qualified independent technician specializing in micropipette calibration can be used.
                Documentation on the precision of the recalibrated micropipette must be obtained from
                the manufacturer or technician.
        9.2.2   Internal and external calibration records must be kept on file in the laboratory's QA
                logbook.
        9.2.3   If a micropipette calibration problem is  suspected, the laboratory shall tare an empty
                weighing boat on the analytical balance  and pipette the following volumes of reagent
                water into the weigh boat using the pipette in question: 100% of the maximum dispensing
                capacity of the micropipette, 50% of the capacity, and 10% of the capacity. Record the
                weight of the water (assume that 1.00 mL of reagent water weighs 1.00 g). If the weight
                of the reagent water is within 1% of the  desired weight (mL) then the pipette remains
                acceptable for use.
        9.2.4   If the weight of the reagent water is outside the acceptable limits, consult the
                manufacturer's instruction manual troubleshooting section  and repeat steps described in

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                                                         Method 1623 - Cryptosporidium and Giardia
                Section 9.2.3. If problems with the pipette persist, the laboratory must send the pipette to
                the manufacturer for recalibration.
9.3    Microscope adjustment and certification: Adjust the microscope as specified in Section 10.0. All of
       the requirements in Section 10.0 must be met prior to analysis of IPRs, blanks, OPRs, field
       samples, and MS/MSDs.
9.4    Initial precision and recovery (IPR)—To establish the ability to demonstrate control over the
       analytical system and to generate acceptable precision and recovery, the laboratory shall perform
       the following operations:
       9.4.1    Using the spiking procedure in Section 11.5 and enumerated spiking suspension aliquots,
                each containing 100 to 500 oocysts or cysts (Section 11.3 or 11.4), the laboratory must
                filter, elute, concentrate, separate (purify), stain, and examine four 10-L reagent water
                samples. If more than one process will be used for filtration and/or separation of samples,
                a separate set of IPR samples must be prepared for each process.

       NOTE: IPR tests must be accompanied by analysis of a method blank (Section 9.6).

       9.4.2   Using results of the four analyses, compute the average percent recovery (X) and the
                relative standard deviation of the recovery (sr) for  Cryptosporidium.
       9.4.3   Compare sr and X with the corresponding limits for initial precision and recovery in
                Tables 1  and 2 in Section 21.0. If sr and X meet the  acceptance criteria, system
                performance is acceptable and analysis of blanks and samples may begin. If sr or X falls
                outside the range for recovery, system performance is unacceptable. In this event, correct
                the problem and repeat the test (Section 9.4.1).
9.5    Matrix spike (MS) and matrix spike duplicate (MSD):
       9.5.1    Matrix spike—The laboratory shall spike and analyze a separate field sample aliquot to
                determine the effect of the matrix on the method's oocyst and cyst recovery. The MS shall
                be analyzed according to the frequency in Section 9.1.8.
                9.5.1.1       Analyze an unspiked field sample according to the procedures in Sections
                               12.0 to  15.0. Using the spiking procedure in Section 11.5 and an
                              appropriate volume of enumerated oocyst and cyst spiking suspensions
                              (Section 11.3 or 11.4), spike  a second field sample aliquot to produce five
                              times the number of oocysts and cysts detected in  the unspiked sample or
                              the number used in the IPR or OPR tests (Sections 9.4 and 9.7),
                              whichever is greater.
                9.5.1.2       For each organism, compute the percent recovery  (R) using the following
                              equation.
                                       NSD - Ns
                              where
                                      R is the percent recovery
                                      Nsp is the number of oocysts or cysts detected in the spiked
                                      sample
                                      Ns is the number of oocysts or cysts detected in the unspiked
                                      sample
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Method 1623 - Cryptosporidium and Giardia
                                     T is the true value of the oocysts or cysts spiked
                9.5.1 .3       Compare the recovery for each organism with the corresponding limits in
                              Tables 1 and 2 in Section 21.0. If the recovery for oocysts or cysts falls
                              outside the limit for that organism, method performance is unacceptable
                              for that sample. If the results for the method blank (Section 9.6) and for
                              the OPR sample (Section 9.7) associated with this batch of samples are
                              within their respective control limits, a matrix interference may be causing
                              the poor recovery. See Section 16.0 for instructions for dealing with
                              matrix interferences; however the matrix may not be diluted for MS tests
                              performed to support method modifications. If the results for the blank
                              and OPR are not within their control limits, the laboratory is not in
                              control. The problem must be identified and corrected and a fresh sample
                              should be collected and reanalyzed.
                9.5.1 .4       As part of the QA program for the laboratory, method precision for
                              samples should be assessed and records maintained. After the analysis of
                              five samples for which the spike recovery for Cryptosporidium passes the
                              tests in Section 9.5.1.3, the laboratory should compute the average percent
                              recovery (P) and the standard deviation of the percent recovery (sr).
                              Express the precision assessment as a percent recovery interval from P -
                              2 sr to P + 2 sr for each matrix. For example, if P = 80% and sr = 30%,
                              the accuracy interval is expressed as 20% to 140%. The precision
                              assessment should be updated on a regular basis (e.g., after each 5  to 10
                              new accuracy measurements).
        9.5.2   Matrix spike duplicate — MSB analysis is required to demonstrate that a modified version
                of this method produces results equal or superior to results produced by the method as
                written (Section 9. 1 .2). At the same time the laboratory spikes and analyzes the second
                field sample aliquot in Section 9.5.1.1, the laboratory shall spike and analyze an third,
                identical field sample aliquot.
                9.5.2.1       For each organism, calculate the percent recovery (R) using the equation
                              in Section 9.5.1.2. Calculate the mean of the MS and MSB recoveries
                              (X^) (= [MS+MSB]/2).
                9.5.2.2       Calculate the relative percent difference (RPB) of the recoveries using the
                              following equation:
                              where
                                      RPB is the relative percent difference
                                      RMS is the number of oocysts or cysts detected in the MS
                                      RMSD is the number of oocysts or cysts detected in the MSB
                                      Xmcan is the mean of the recoveries for the MS and MSB
                9.5.2.3       Compare the mean MS/MSB recovery and RPB with the corresponding
                              limits in Tables 1 and 2 in Section 21.0 for each organism.
9.6     Method blank (negative control sample): Reagent water blanks are analyzed to demonstrate
        freedom from contamination. Analyze the blank immediately prior to analysis of the IPR test

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                                                        Method 1623 - Cryptosporidium and Giardia
       (Section 9.4) and OPR test (Section 9.7) and prior to analysis of samples for the week to
       demonstrate freedom from contamination.
       9.6.1   Filter, elute, concentrate, separate (purify), stain, and examine at least one reagent water
               blank per week (Section 9.1.7) according to the procedures in Sections 12.0 to 15.0. If
               more than 20 samples are analyzed in a week, process and analyze one reagent water
               blank for every 20 samples.
       9.6.2   If Cryptosporidium oocysts, Giardia cysts, or any potentially interfering organism or
               material is found in the blank, analysis of additional samples is halted until the source of
               contamination is eliminated and a blank shows no evidence of contamination. Any sample
               in a batch associated with a contaminated blank that shows the presence of one or more
               oocysts or cysts is assumed to be contaminated and should be recollected, if possible. Any
               method blank in which oocysts or cysts are not detected is assumed to be uncontaminated
               and may be reported.
9.7    Ongoing precision and recovery ([OPR]; positive control sample; laboratory control sample):
       Using the spiking procedure in Section 11.5 and enumerated spiking suspension  aliquots, each
       containing 100 to 500 oocysts or cysts (Section 11.3 or 11.4), filter, elute, concentrate, separate
       (purify), stain, and examine at least one spiked reagent water sample per week to verify all
       performance criteria. The laboratory must analyze one OPR sample for every 20 samples if more
       than 20 samples are analyzed in a week. Adjustment and/or recalibration of the analytical system
       shall be performed until all  performance criteria are met. Only after all performance criteria are
       met may samples be analyzed.
       9.7.1   Examine the slide  from the OPR prior to analysis of samples from the same batch.
               9.7.1.1        More than 50% of the oocysts or cysts must appear undamaged and
                              morphologically intact; otherwise, the analytical process is damaging the
                              organisms. Determine the step or reagent that is causing damage to the
                              organisms. Correct the problem and repeat the OPR test.
               9.7.1.2       Identify and enumerate each organism using epifluorescence microscopy.
                              Each organism must meet the identification criteria in Section 15.2.
       9.7.2  For each organism, compute the percent recovery using the following equation:

                       P=100x  —

                              where
                      N = the number of oocysts( or cysts detected
                      T = the number of oocysts or cysts spiked
               9.7.2.1        Compare the recovery with the limits for ongoing precision and recovery
                              in Tables 1 and 2 in Section 21.0. If the recovery meets the acceptance
                              criteria, system performance is acceptable and analysis of blanks and
                              samples may proceed. If, however, the recovery falls outside of the range
                              given, system performance is unacceptable. In this event, there may be a
                              problem with the microscope or with the filtration or separation systems.
                              Reanalyze the OPR sample and recollect and reanalyze samples. All
                              samples must be associated with an OPR  that passes the criteria in
                              Section 21.0.
                                                13                                      April 1999

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Method 1623 - Cryptosporidium and Giardia
                9.7.2.2       Microscope system: To determine if the failure of the OPR test (Section
                              9.7.2.1) is due to changes in the microscope, examine a slide containing a
                              known number of freshly prepared oocysts and cysts, check Kohler
                              illumination, and check the fluorescence of the fluorescein-labeled
                              monoclonal antibodies (Mabs) and 4',6-diamidino-2-phenylindole (DAPI).
                9.7.2.3       Filtration/elution/concentration system: If the failure of the OPR test
                              (Section 9.7.2.1) is attributable to the filtration/elution/concentration
                              system, these systems may not be in control. Check filtration/elution/
                              concentration system performance using spiked reagent water, eluting the
                              filter, and analyzing the concentrated sample without separation
                              (purification) using FA.
                9.7.2.4       Separation (purification) system: If the failure of the OPR test (Section
                              9.7.2.1) is attributable to the separation system, this system may not be in
                              control. Check separation system performance by processing a spiked 10-
                              mL volume of reagent water through IMS and analyzing the purified
                              sample using FA (Sections 13.3 through 15.0).
        9.7.3   The laboratory should add results that pass the specifications in Section 9.7.2.1 to initial
                and previous ongoing data and update the QC chart to form a graphic representation of
                continued laboratory performance. The laboratory should develop a statement of
                laboratory accuracy (reagent water, raw surface water) by calculating the average percent
                recovery (R) and the standard deviation of percent recovery (sr). Express the accuracy as
                a recovery interval from R    2 sr to R + 2 sr. For example, if R = 95% and sr = 25%, the
                accuracy is 45% to 145%.
9.8     The laboratory should periodically analyze an external QC sample, such as a performance
        evaluation or standard reference material, when available. The laboratory also should periodically
        participate in interlaboratory comparison studies using the method.
9.9     The specifications contained in this method can be met if the analytical system is under control.
        The standards used for initial (Section 9.4) and ongoing (Section 9.7) precision and recovery
        should be identical, so that the most precise results will be  obtained. The microscope in particular
        will provide the most reproducible results if dedicated to the settings and conditions required for the
        determination of Cryptosporidium and Giardia by this method.
9.10   Depending on specific program requirements, field replicates may be collected to determine the
        precision of the sampling technique, and duplicate spiked samples may be required to determine the
        precision of the analysis.

10.0  Microscope  Calibration and Analyst Verification
10.1   In a room capable of being darkened to near-complete darkness, assemble the microscope, all
        filters, and attachments. The microscope should be placed on a solid surface free from vibration.
        Adequate workspace should be provided on either side of the microscope for taking notes and
        placement of slides and ancillary materials.
10.2   Using the manuals provided with the microscope, the principal analyst/supervisor and all analysts
        must familiarize themselves with operation of the microscope.
10.3   Microscope adjustment and calibration (adapted from Reference 20.6)
        10.3.1  Preparations for adjustment

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                                                 Method 1623 - Cryptosporidium and Giardia
         10.3.1.1       The microscopy portion of this procedure depends upon proper alignment
                       and adjustment of very sophisticated optics. Without proper alignment and
                       adjustment, the microscope will not function at maximal efficiency, and
                       reliable identification and enumeration of oocysts and cysts will not be
                       possible. Consequently, it is imperative that all portions of the microscope
                       from the light sources to the oculars are properly adjusted,
         10.3.1.2      While microscopes from various vendors are configured somewhat
                       differently, they all operate on the same general physical principles.
                       Therefore, slight deviations or adjustments may be required to make the
                       procedures below work for a particular instrument.
         10.3.1.3      The sections below assume that the mercury bulb has not exceeded time
                       limits of operation, that the lamp socket is connected to the lamp house,
                       and that the condenser is adjusted to produce Kohler illumination.
         10.3.1.4      Persons with astigmatism should always wear contact lenses or glasses
                       when using the microscope.

CAUTION:    In the procedures below, do not touch the quartz portion of the mercury
bulb with your bare fingers. Finger oils can cause rapid degradation of the quartz and
premature failure of the bulb.


WARNING:   Never look at the ultraviolet (UV) light from the mercury lamp, lamp
house, or the UV image without a barrier filter in place. UV radiation can cause serious
eye damage.

10.3.2  Epifluorescent mercury bulb adjustment: The purpose of this procedure is  to ensure even
         field illumination. This procedure must be followed when the microscope is first used,
         when replacing bulbs, and if problems such as diminished fluorescence or uneven field
         illumination are experienced.
         10.3.2.1       Remove the diffuser lens between the lamp and microscope or swing it out
                       of the transmitted light path.
         10.3.2.2      Using a prepared microscope  slide, adjust the focus so the image in the
                       oculars is sharply defined.
         10.3.2.3      Replace the slide with a business card or a piece of lens paper.
         10.3.2.4      Close the field diaphragm (iris diaphragm in the microscope base)  so only
                       a  small point of light is visible on the card.  This dot of light indicates the
                       location of the center of the field of view.
         10.3.2.5      Mount the mercury lamp house  on the microscope without the UV diffuser
                       lens in place and turn  on the mercury bulb.
         10.3.2.6      Remove the objective in the light path from the nosepiece.  A primary
                       (brighter) and secondary image  (dimmer) of the mercury bulb arc should
                       appear on the card after focusing the image with the appropriate
                       adjustment.
                                        15                                      April 1999

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Method 1623 - Cryptosporidium and Giardia
                10.3.2.7      Using the lamp house adjustments, adjust the primary and secondary
                              mercury bulb images so they are side by side (parallel to each other) with
                              the transmitted light dot in between them.
                10.3.2.8      Reattach the objective to the nosepiece.
                10.3.2.9      Insert the diffuser lens into the light path between the mercury lamp house
                              and the microscope.
                10.3.2.10     Turn off the transmitted light and replace the card with a slide of
                              fluorescent material. Check the field for even fluorescent illumination.
                              Adjustment of the diffuser lens probably will be required. Additional
                              slight adjustments as in Section 10.3.2.7 above may be required.
                10.3.2.11     Maintain a log of the number of hours the UV bulb has been used. Never
                              use the bulb for longer than it has been rated. Fifty-watt bulbs should not
                              be used longer than 100 hours; 100-watt bulbs should not be used longer
                              than 200 hours.
        10.3.3  Transmitted bulb adjustment: The purpose of this procedure is to center the filament and
                ensure even field illumination. This procedure must be followed when the bulb is changed.
                10.3.3.1      Remove  the diffuser lens between the lamp and microscope or swing it out
                              of the transmitted light path.
                10.3.3.2      Using a prepared microscope slide and a 40X (or similar) objective, adjust
                              the focus so the image in the oculars is sharply defined.
                10.3.3.3      Without  the ocular or Bertrand optics in place, view the pupil and
                              filament  image at the bottom of the tube.
                10.3.3.4      Focus the lamp filament image with the appropriate adjustment on the
                              lamp house.
                10.3.3.5      Similarly, center the lamp filament image within the pupil with the
                              appropriate adjustment(s) on the lamp house.
                10.3.3.6      Insert the diffuser lens into the light path between the transmitted lamp
                              house and the microscope.
        10.3.4  Adjustment of the interpupillary distance and oculars for each eye:  These adjustments are
                necessary so that eye strain is reduced to a minimum, and must be made for each
                individual using the microscope. Section 10.3.4.2 assumes use of a microscope with both
                oculars adjustable; Section 10.3.4.3 assumes use of a microscope with a single adjustable
                ocular. The procedure must be followed each time an analyst uses the microscope.
                10.3.4.1      Interpupillary distance
                              10.3.4.1.1     Place a prepared slide on the microscope stage, turn on
                                             the transmitted light, and focus the specimen image using
                                             the coarse and fine adjustment knobs.
                              10.3.4.1.2    Using both hands, move  the oculars closer together or
                                             farther apart until a single circle of light is observed while
                                             looking through the oculars  with both eyes. Note
                                             interpupillary distance.
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                                                  Method 1623 - Cryptosporidium and Giardia
        10.3.4.2      Ocular adjustment for microscopes capable of viewing a photographic
                      frame through the viewing binoculars: This procedure assumes both
                      oculars are adjustable.
                      10.3.4.2.1      Place a card between the right ocular and eye keeping
                                      both eyes open. Adjust the correction (focusing) collar on
                                      the left ocular by focusing the left ocular  until it reads the
                                      same as the mterpupillary distance. Bring an image
                                      located in the center of the field of view into as sharp a
                                      focus as possible.
                      10.3.4.2.2     Transfer the card to between the left eye and ocular.
                                      Again keeping both eyes open, bring the same image into
                                      as sharp a focus for the right eye as possible by adjusting
                                      the ocular correction (focusing) collar at the top of the
                                      right ocular.
        10.3.4.3      Ocular adjustment for microscopes without binocular capability: This
                      procedure assumes a single focusing ocular. The following procedure
                      assumes that only the right ocular is capable  of adjustment.
                      10.3.4.3.1      Place a card between the right ocular and  eye keeping
                                      both eyes open. Using the fine adjustment, focus the
                                      image for the left eye to its sharpest point.
                      10.3.4.3.2     Transfer the card to between the left eye and ocular.
                                      Keeping both eyes open, bring the image  for the right eye
                                      into sharp focus by adjusting the ocular collar at the top
                                      of the ocular without touching the coarse  or fine
                                      adjustment.
10.3.5  Calibration of an ocular micrometer: This section assumes that a reticle has been installed
        in one of the oculars by a microscopy specialist and that a stage micrometer is available
        for calibrating the ocular micrometer (reticle). Once installed, the ocular reticle should be
        left in place.  The more an ocular is manipulated the greater the probability is for it to
        become contaminated with dust particles. This  calibration should be done for each
        objective in use on the microscope. If there is a top lens on the microscope, the calibration
        procedure must be done for the respective objective at each top lens setting.  The
        procedure must be followed when the microscope is first used and each time the objective
        is  changed.
        10.3.5.1      Place the stage micrometer on the microscope stage, turn on the
                      transmitted light, and focus the  micrometer image using the coarse and
                      fine adjustment knobs for the objective to be calibrated. Continue
                      adjusting the focus on the stage micrometer so you can distinguish
                      between the large (0.1 mm) and the small (0.01 mm) divisions.
        10.3.5.2      Adjust the stage and ocular with the micrometer so the 0 line on the ocular
                      micrometer is exactly superimposed on the 0 line on the stage micrometer.
        10.3.5.3      Without changing the stage adjustment, find a point as distant as possible
                      from the two 0 lines where two other lines are exactly superimposed.
                                         17                                      April 1999

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Method 1623 - Cryptosporidium and Giardia
                10.3.5.4      Determine the number of ocular micrometer spaces as well as the number
                              of millimeters on the stage micrometer between the two points of
                              superimposition. For example: Suppose 48 ocular micrometer spaces
                              equal 0.6 mm.
                10.3.5.5      Calculate the number of mm/ocular micrometer space. For example:
                                        0.6 mm
                                      0.0125 mm
                              48  ocular micrometer spaces    ocular micrometer space
                10.3.5.6      Because most measurements of microorganisms are given in ^m rather
                              than mm, the value calculated above must be converted to ^m by
                              multiplying it by 1000 Mm/mm. For example:
                                     0.0125 mm
                           1,000
                               ocular micrometer space
12.5 /urn
                                                            mm
                                       ocular micrometer space
                10.3.5.7
Follow the procedure below for each objective. Record the information as
shown in the example below and keep the information available at the
microscope.
Item
no.
1
2
3
4
Objective
power
10X
20X
40X
100X
Description
N.A.3=
N.A.=
N.A.=
N.A.=
No. of ocular
micrometer
spaces




No. of stage
micrometer
mm1




j/m/ocular
micrometer
space2




                                             no. ocular micrometer
                              1
                              2(Stage micrometer length in mm * (1000 ^m/mm))
                              spaces
                              3N.A. refers to numerical aperature. The numerical aperature value is engraved
                               on the barrel of the objective.
        1 0.3.6  Kohler illumination: This section assumes that Kohler illumination will be established for
                only the 100X oil D.I.C. objective that will be used to identify internal morphological
                characteristics in Cryptosporidium oocysts and Giardia cysts. If more than one objective
                is to be used for D.I.C., then each time the objective is changed, Kohler illumination must
                be reestablished for the new objective lens. Previous sections have adjusted oculars and
                light sources. This section aligns and focuses the light going through the condenser
                underneath the stage at the specimen to be observed. If Kohler illumination is not properly
                established, then D.I.C, will not work to its maximal potential. These steps need to
                become second nature and must be practiced regularly until they are a matter of reflex
                rather than a chore. The procedure must be followed each time an analyst uses the
                microscope and each tune the objective is changed.
                10.3.6.1      Place a prepared slide on the microscope stage, place oil on the slide,
                              move the  100X oil objective into place, turn on the transmitted light, and
                              focus the specimen image using the coarse and fine adjustment knobs.
April 1999
                  18

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                                                         Method 1623 - Cryptosporidium and Giardia
                10.3.6.2      At this point both the radiant field diaphragm in the microscope base and
                              the aperture diaphragm in the condenser should be wide open. Now close
                              down the radiant field diaphragm in the microscope base until the lighted
                              field is reduced to a small opening.
                10.3.6.3      Using the condenser centering screws on the front right and left of the
                              condenser, move the small lighted portion of the field to the center of the
                              visual field.
                10.3.6.4      Now look to see whether the leaves of the iris field diaphragm are sharply
                              defined (focused) or not. If they are not sharply defined, then they can be
                              focused distinctly by changing the height of the condenser up and down
                              with the condenser  focusing knob while you are  looking through the
                              binoculars. Once you have accomplished the precise focusing of the
                              radiant field diaphragm leaves, open the radiant  field diaphragm until the
                              leaves just disappear from view.
                10.3.6.5      The aperture diaphragm of the condenser is now adjusted to make it
                              compatible with the total numerical aperture of the optical system. This is
                              done by removing an ocular, looking into the tube at the rear focal plane
                              of the objective, and stopping down the aperture diaphragm iris leaves
                              until they are visible just inside the rear plane of the objective.
                10.3.6.6      After completing the adjustment of the aperture  diaphragm in the
                              condenser, return the ocular to its tube and proceed with the adjustments
                              required to establish D.I.C.
10.4   Protozoa libraries: Each laboratory is encouraged to develop libraries of photographs and drawings
       for identification of protozoa.
       10.4.1   Take color photographs of Cryptosporidium oocysts and Giardia cysts by FA and 4',6-
                diamidino-2-phenylindole (DAPI)  that the principal analyst/supervisor (Section 22.2)
                determines are accurate (Section 15.2).
       10.4.2   Similarly, take color photographs of interfering organisms and materials by FA and DAPI
                that the principal analyst/supervisor believes are not Cryptosporidium oocysts or Giardia
                cysts. Quantify the size, shape, microscope settings, and other characteristics that can be
                used to differentiate oocysts and cysts from interfering debris and that will result in
                positive identification of DAPI + or - organisms.
10.5   Verification of performance: Until standard  reference materials, such as National Institute of
       Standards and Technology standard reference materials, are available that contain a reliable
       number of DAPI + or - oocysts and cysts, this method shall rely upon the ability of the principal
       analyst/supervisor for identification and enumeration of oocysts and cysts.
       10.5.1   At least monthly when microscopic examinations are being performed, the principal
                analyst/supervisor shall prepare a slide containing 40 to 100 oocysts and 40 to 100 cysts.
                More than 50% of the oocysts and cysts must be DAPI +. The principal
                analyst/supervisor shall determine  the numbers of total oocysts  and cysts by FA and
                number of oocysts and cysts that are DAPI + or -, using the procedures in this method,
                and these numbers shall be known  only to the principal analyst/supervisor.
                                                19                                      April 1999

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Method 1623 - Cryptosporidium and Giardia
       10.5.2  Each analyst shall determine the total number of oocysts and cysts and the number that
                are DAPI + or -, using the slide provided by the principal analyst/supervisor (Section
                10.5.1).
       10.5.3  The total number and the number of DAPI + or - oocysts and cysts determined by each
                analyst (Section 10.5.2.) must be within ±10% of the number determined by the principal
                analyst/supervisor. If the number is not within this range, the principal analyst/ supervisor
                and the analyst shall resolve how to identify and enumerate DAPI + or - oocysts and
                cysts, and the principal analyst/supervisor shall prepare a new slide and the performance
                verification (Sections 10.5.1  to 10.5.2) shall be repeated.

       NOTE:  If the laboratory has only a principal analyst/supervisor,  the principal analyst/
       supervisor shall perform the identification and enumeration of total and DAPI + and -
       oocysts and cysts on a monthly basis, at a minimum, and the reputation of the laboratory
       shall rest with the principal analyst/supervisor.

       10.5.4  Document the date, name of principal analyst/supervisor, name(s) of analyst(s), number
                of total, DAPI + or - oocysts and cysts placed on the slide, number determined by the
                principal analyst/supervisor,  number determined by the analyst(s), whether the test was
                passed/failed for each analyst, and the number and results  of attempts prior to passage.
       10.5.5  Only after an analyst has passed the criteria in Section 10.5.3, may oocysts and cysts in
                QC samples and field samples be identified and enumerated.

11.0 Oocyst and Cyst Suspension  Enumeration and Spiking
11.1   Two sets of enumerations are required per organism before purified Cryptosporidium oocyst and
       Giardia cyst stock suspensions (Section 7.9) received from suppliers can be used to spike samples
       in the laboratory. First, the stock suspension must be diluted and enumerated to yield a suspension
       at the appropriate oocyst or cyst concentration for spiking (spiking suspension). Then, 10 aliquots
       of spiking suspension must be enumerated to calculate a mean spike dose. Spiking suspensions can
       be enumerated using either hemacytometer chamber counting or well-slide counting. The procedure
       for diluting and enumerating purified stock suspensions is provided in Section  11.2. The two
       procedures for enumerating spiking suspensions are provided in Sections 11.3 and 11.4. The
       procedure for spiking 10-L carboys in the laboratory is provided in  Section 11.5.
11.2   Enumerating and diluting stock suspensions
       11.2.1  Purified, concentrated stock  suspensions (Sections 7.9.1 and 7.9.2) must be diluted and
                enumerated before the diluted suspensions are used to spike samples in the laboratory.
                Stock suspensions should be diluted with reagent water/Tween-20, 0.01% (Section 7.9.3),
                to a concentration of 20 to 50 organisms  per large hemacytometer square before
                proceeding to Section 11.2.2,
       11.2.2  Apply a clean hemacytometer coverslip to the hemacytometer and load the hemacytometer
                chamber with 10 ^L of vortexed suspension per chamber. If this operation has been
                properly executed, the liquid should amply fill the entire chamber without bubbles or
                overflowing into the surrounding moats. Repeat this step with a clean, dry hemacytometer
                and coverslip if loading has been incorrectly performed. See Section 11.2.13, below, for
                the hemacytometer cleaning procedure.
April 1999                                      20

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                                                Method 1623 - Cryptosporidium and Giardia
11.2.3  Place the hemacytometer on the microscope stage and allow the oocysts or cysts to settle
        for 2 minutes Do not attempt to adjust the coverslip, apply clips, or in any way disturb
        the chamber after it has been filled.
11.2.4  Use a magnification of 400X to 500X.
11.2.5  Move the chamber so the ruled area is centered underneath it.
11.2.6  Move the objective close to the coverslip while watching it from the side of the
        microscope, rather than through the microscope.
11.2.7  Focus up from the coverslip until the hemacytometer ruling appears.
11.2.8  At each of the four corners of the chamber is a 1-square-mm area divided into 16 squares
        in which organisms are to be counted (Figure 1). Beginning with the top row of four
        squares, count with a hand-tally counter in the directions indicated in Figure 2. Avoid
        counting organisms twice by counting only those touching the top and left boundary lines.
        Count each square millimeter in this fashion.
11.2.9  Use the following formula to determine the number of organisms per mL of suspension:

        number of organisms counted   10   dilution factor  1000mm3
        	»	—— x	x	x —-—-— = number of organisms mL
           number of mm counted     1mm        1          1 mL

11.2.10 Record the result on a  hemacytometer data sheet.
11.2.11 A total of six different hemacytometer chambers must be loaded, counted, and averaged
        for each suspension to achieve optimal counting accuracy.
11.2.12 Based on the hemacytometer counts, the stock suspension should be diluted to a final
        concentration of between 8000 and 12,000 organisms per mL (80 to 120 organisms per
        10 fjL); however, ranges as  great as 5000 to 15,000 organisms per mL (50 to 150
        organisms per 10 //L) can be used.

NOTE: If the diluted stock suspensions (the spiking suspensions) will be enumerated
using hemacytometer chamber  counts (Section 11.3), then the stock suspensions should
be diluted with reagent water/Tween-20, 0.01%. If the spiking suspensions will be
enumerated using well-slide counts (Section 11.4),  or if both hemacytometer chamber
counts and well-slide counts will be used to enumerate the spiking suspension, then the
stock suspensions should be diluted using reagent water only.

        To calculate the volume (in  #L) of stock suspension required per mL of reagent water (or
        reagent water/Tween-20, 0.01%), use the following formula:

                                                 required number of organisms  x  1000/j.L
        volume of stock suspension  (\iL) required =	:	;—;———•	:—
                          r                    number of organisms I mL of stock suspension


        If the volume is less than 10 /*L, an additional dilution of the stock suspension is
        recommended before proceeding.
        To calculate the dilution factor needed to achieve the required number of organisms per
        10 fjL, use the following formula:
                                        21                                     April 1999

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Method 1623 - Crvotosporidium and Giardia
                                        number of organisms required x 10uL
                total volume (nL) = -         	
                                  predicted number of organisms per 10jiL (80 to 120)


                To calculate the volume of reagent water (or reagent water/Tween-20, 0.01%) needed,
                use the following formula:
                           reagent water volume (\iL) -
                             total volume (fiL) - stock suspension volume  required (fiL)

        11.2.13 After each use, the hemacytometer and coverslip must be cleaned immediately to prevent
                the organisms and debris from drying on it.  Since this apparatus is precisely machined,
                abrasives cannot be used to clean it, as they  will disturb the flooding and volume
                relationships.
                11.2.13.1     Rinse the hemacytometer and cover glass first with tap water, then 70%
                              ethanol, and finally with acetone.
                11.2.13.2    Dry and polish the hemacytometer chamber and cover glass with lens
                              paper. Store it in a secure place.
        11.2.14 Several factors are known to introduce errors into hemacytometer counts, including:
                •      Inadequate mixing of suspension before flooding the chamber
                •      Irregular filling of the chamber, trapped air bubbles, dust, or oil on the chamber or
                       coverslip
                •      Total number of organisms counted is too low to provide statistical confidence in
                       the result
                       Error in recording tally
                •      Calculation error; failure to consider dilution factor, or area counted
                •      Inadequate cleaning and removal of organisms from the previous count
                •      Allowing filled chamber to sit too long, so that the chamber suspension dries and
                       concentrates.
11.3   Enumerating spiking suspensions using a hemacytometer chamber
        11.3.1  Vortex the tube containing the spiking suspension (diluted stock suspension; Section
                11.2) for a minimum of 2 minutes. Gently invert the tube three times.
        11.3.2  To an appropriate-size beaker containing a stir bar, add enough spiking suspension to
                perform all spike testing and the enumeration as described. The liquid volume and beaker
                relationship should be such that a spinning stir bar does not splash the sides of the beaker,
                the stir bar has unimpeded rotation, and there is enough room to draw sample from the
                beaker with a 10-,uL micropipette without touching the stir bar. Cover the beaker with a
                watch glass or petri dish to prevent evaporation between sample withdrawals.
        11.3.3  Allow the beaker contents to stir for a minimum of 30 minutes before beginning
                enumeration.
        11.3.3  While the stir bar is still spinning, remove a 10-^L aliquot and carefully load one side of
                the hemacytometer. Count all organisms on the platform, at 200X magnification using

April 1999                                       22

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                                                         Method 1623 - Cryptosporidium and Giardia
                phase-contrast or darkfield microscopy. The count must include the entire area under the
                hemacytometer, not just the four outer 1-square-mm squares. Repeat this procedure nine
                times. This step allows confirmation of the number of organisms per 10 fjL (Section
                11.2.12). If the mean number is outside the expected range, add additional oocysts from
                stock suspension or dilute the contents of the beaker appropriately with reagent water.
                Repeat the process to confirm counts. Refer to Section  11.2.14 for factors that may
                introduce errors.
11.4   Enumerating spiking suspensions using well slides
       11.4.1  Remove well slides from cold storage and lay the slides on a flat surface for 15  minutes to
                allow them to warm to room temperature.
       11.4.2  Vortex the tube containing the spiking suspension (diluted stock suspension; Section
                11.2) for a minimum of 2 minutes. Gently invert the tube three times.
       11.4.3  Remove a 10-/uL aliquot from the spiking suspension and apply it to the center of a well.
       11.4.4  Before removing subsequent aliquots, cap the tube and gently  invert it three times to
                ensure that the oocysts or cysts are in suspension.
       11.4.5  Ten wells must be counted, and the counts averaged, to sufficiently enumerate the spike
                dose.
       11.4.5  Positive and negative controls must be prepared.
                11.4.5.1      For the positive control, pipette  10 ^L of positive antigen or 200 to 400
                              intact oocysts or cysts to the center of a well and distribute evenly over the
                              well area.
                11.4.5.2     For the negative control, pipette 50 nL of PBS onto the center of a well
                              and spread it over the well area with a pipette tip.
                11.4.5.3     Air-dry the control slides.
       11.4.6  Apply one drop of Detection Reagent (from direct labeling kit, Section 7.7) to each well.
       11.4.7  Apply one drop of Counterstain (from direct labeling kit, Section 7.7) to each well.
       11.4.8  Spread over entire well with applicator stick, if necessary. Do not allow the stick to
                scratch the treated surface of the slide. Use a different applicator stick for each  slide.
       11.4.9  Place the slides in a humid chamber in the dark and incubate at room temperature for
                approximately 30 minutes. The humid chamber consists of a tightly sealed plastic
                container containing  damp paper towels on top of which the slides are placed.
       11.4.10 Apply one drop of IX Wash Buffer (made from 20X concentrate in direct labeling kit,
                Section 7.7) to each well. Tilt each slide on a clean paper towel, long edge down. Gently
                aspirate the excess Detection Reagent and Counterstain from below the well using a clean
                Pasteur pipette. Avoid disturbing the sample.

       NOTE:  Do not allow slides to dry.

       11.4.11 Add one  drop of Mounting Medium (from direct labeling kit, Section 7.7) to each well.
       11.4.12 Apply a cover slip. Use a tissue to remove excess mounting fluid from the edges of the
                coverslip.
                                                23                                       April 1999

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Method 1623 - Cryptosporidium and Giardia
       11.4.13 Record the date and time that staining was completed on the bench sheet. If slides will not
                be read immediately, store in a humid chamber in the dark at 0°C to 8°C until ready for
                examination.
April 1999                                      24

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                                                         Method 1623 - Cryptosporidium and Giardia
11.5   Procedure for spiking samples in the laboratory with enumerated spiking suspensions
       11.5.1   Arrange a bottom-dispensing 10-L carboy to gravity-feed a capsule filter so the outlet will
                feed self-priming centrifugal pump (Figure 3).
       11.5.2  Place a large, sterile stir bar in the carboy. Fill the carboy with 10.0 L of reagent water
                (for initial precision and recovery [Section 9.4] and ongoing precision and recovery
                [Section 9.7] samples) or with the 10-L field sample (for matrix spike samples [Section
                9.5]). Place the carboy on the stir plate. Turn the stirrer on so that the bar creates a
                vortex.
       11.5.3  Vortex the tube containing the Cryptosporidium oocyst spiking suspension and the tube
                containing the Giardia cyst spiking suspension (Section 11.3 or Section 11.4) for a
                minimum of 2 minutes. For each spiking suspension, rinse a pipette tip with Tween-20
                0.01% once, then with the well-mixed spiking suspension a minimum of five times before
                pulling an aliquot to be used to spike the carboy.
       11.5.4  Add one spiking suspension to the carboy, delivering the aliquot below the surface of the
                water. Add the other spiking suspension, again delivering the aliquot below the  surface of
                the water. Allow the spiking suspensions to mix for approximately 1 minute in the
                carboy.
       11.5.5  Turn on the pump and allow the flow rate to stabilize. Set flow at the rate designated for
                the filter under test. As the carboy is depleted, check the flow rate and adjust if necessary.
       11.5.6  When the water level approaches the discharge port of the carboy, turn off the stirrer and
                tilt the carboy so that it is completely emptied. At that time, turn off the pump and add 1
                L of reagent water to the carboy. Swirl the contents to rinse down the sides.
       11.5.7  Turn on the pump. Allow the pump to pull all the water through the filter and turn off the
                pump.

 12.0 Sample Filtration  and Elution
 12.1   A capsule filter is used to filter the  10-L water sample received by the laboratory, according to the
       procedures in Section 12.2. Alternate procedures may be used if the laboratory first demonstrates
       that the quality control acceptance criteria listed in Tables  1 and 2 in Section 21.0 are met.
 12.2   Capsule filtration (adapted from Reference 20.7)
       12.2.1   Flow rate adjustment
                12.2.1.1      Connect the sampling system, minus the capsule,  to a carboy filled with
                              reagent water (Figure 3).
                12.2.1.2      Turn on the pump and adjust the flow rate to 2.0 L/min.
                12.2.1.3      Allow  2 to 10 L of reagent water to flush the system. Adjust the pump
                              speed as required during this period. Turn off the pump when the flow
                              rate has been  adjusted.
       12.2.2  Install the capsule filter in the line, securing the inlet and outlet ends with the appropriate
                clamps/fittings.
       12.2.3  Record the sample number, sample turbidity (if not provided with the field sample), and
                the name of analyst filtering the sample on a bench sheet and on the capsule filter.
                                                25                                      April 1999

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Method 1623 - Cryptosporidium and Giardia
        12.2.4  Filtration
                12.2.4.1
              Connect the sampling system to the field carboy of sample water, or
              transfer the sample water to the laboratory carboy used in Section
              12.2.1.1.
        NOTE: If the field sample is transferred to a laboratory carboy, the laboratory carboy
        must be cleaned and disinfected before it is used with another field sample.
        12.2.5
        12.2.6
12.2.4.2



12.2.4.3


12.2.4.4

Disassembly
12.2.5.1
12.2.5.2


12.2.5.3
Elution
12.2.6.1
                12.2.6.2
Place the drain end of the sampling system tubing into an empty graduated
container with a capacity of 10 to 15 L, calibrated at 9.0, 9.5, 10.0, 10.5,
and 11.0 L (Section 6.17). This container will be used to determine the
sample volume filtered.
Allow the carboy discharge tube and capsule to fill with sample water.
Vent residual air using the bleed valve/vent port. Turn on the pump to
start water flowing through the filter. Verify that the flow rate is 2 L/min.
After the sample has passed through the filter, turn off the pump. Allow
the pressure to decay until flow stops.


Disconnect the inlet end of the capsule filter assembly while maintaining
the level of the inlet fitting above the level of the outlet fitting to prevent
backwashing and the loss of oocysts and cysts from the filter. Restart the
pump and allow as much water to drain as possible. Turn off the pump.
Based on the water level in the graduated container (Section 12.2.4.2),
record the volume filtered on a bench sheet and the capsule filter label to
the nearest quarter liter. Discard the contents of the graduated container.
Loosen the outlet fitting, then cap the inlet and outlet fittings.
                              Setup
                              12.2.6.1.1

                              12.2.6.1.2
              12.2.6.1.3

              Elution
              12.2.6.2.1
                               12.2.6.2.2
               Assemble the laboratory shaker with the clamps aligned
               vertically so that the filters will be aligned horizontally.
               Prepare sufficient elution buffer so that all samples to be
               eluted that day can be eluted with the same batch of
               buffer. Elution may require up to 275 mL of buffer per
               sample.
               Designate at least one 250-mL conical centrifuge tube for
               each sample and label with the sample number.


               Record the name of the analyst performing the elution on
               the bench sheet. Using a ring stand or other means, clamp
               each capsule in a vertical position with the inlet end up.
               Remove the inlet cap and allow the liquid level to
               stabilize.
               Pour elution buffer through the inlet fitting. Sufficient
               elution buffer must be added to cover the pleated white
               membrane with buffer solution. Replace the inlet cap and
               clamp the cap in place.
April 1999
                                                26

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                                                        Method 1623 - Crvptosporidium and Giardia
                             12.2.6.2.3     Securely clamp the capsule in one of the clamps on the
                                            laboratory shaker with the bleed valve positioned at the
                                            top on a vertical axis (in the 12 o'clock position). Turn on
                                            the shaker and set the speed to 80% of maximum
                                            (approximately 600 rpm). Agitate the capsule for
                                            approximately 5 minutes.
                             12.2.6.2.4     Remove the filter from the shaker, remove the inlet cap,
                                            and pour the contents of the capsule into the 250-mL
                                            conical centrifuge tube.
                             12.2.6.2.5     Clamp the capsule vertically with the inlet end up and add
                                            sufficient volume of elution buffer through the inlet fitting
                                            to cover the pleated membrane. Replace the inlet cap and
                                            clamp in place.
                             12.2.6.2.6     Return the capsule to its clamp on the  shaker with the
                                            bleed valve positioned on a horizontal axis (3 or 9 o'clock
                                            position). Turn on the shaker and agitate the capsule for
                                            approximately 5 minutes. Add the contents of the capsule
                                            to the centrifuge tube.
       12.2.7   Proceed to Section 13.0 for concentration and separation (purification).

13.0  Sample Concentration and Separation (Purification)
13.1   During concentration and separation, the filter eluate is concentrated through centrifugation, and
       the oocysts and cysts in the sample are separated from other particulates through immunomagnetic
       separation (IMS). Alternate procedures and products may be used if the laboratory first
       demonstrates that the quality control acceptance criteria listed in Tables 1 and 2 in Section 21.0 are
       met.
13.2   Adjustment of pellet volume
       13.2.1   Centrifuge the 250-mL centrifuge tube containing the capsule filter eluate at 1100 x G for
                15 minutes. Allow the centrifuge to coast to a stop. Record the initial pellet volume
                (volume of solids) and the date and time that concentration was completed on a bench
                sheet.
       13.2.2   Using a Pasteur pipette, carefully aspirate off the supernatant 1 mL above the pellet.  If
                the sample is reagent water (e.g. initial or ongoing precision and recovery sample) extra
                care must be taken to avoid aspirating oocysts and cysts during this step.
                13.2.2.1     If the packed pellet volume is less than or equal to 0.5 mL, add reagent
                             water to the centrifuge tube to bring the total volume to 10 mL. Vortex the
                             tube for 10 to 15 seconds to resuspend the pellet. Proceed to Section 13.3.
                13.2.2.2     If the packed pellet volume is greater than 0.5 mL, use the following
                             formula to determine the total volume required in the centrifuge tube:

                                                  .  ,   pelletvolume  _„  ,
                             totalvolume(mL) required =	——;	x 10mL
                                                          0.5 mL

                             (For example, if the packed pellet volume is 0.8 mL, the total  volume
                             required is  16 mL.) Add reagent water to the centrifuge tube to bring the
                             total volume to the level calculated above. Vortex the tube for 10 to 15
                             seconds to resuspend the pellet. Record this resuspended volume on a
                             bench sheet.
                                               27                                      April 1999

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Method 1623 - Cryptosporidium and Giardia
                              13.2.2.2.1     If the packed pellet volume is greater than 0.5 mL, and
                                            only one slide will be prepared as a representative
                                            portion of the sample,  proceed to Section 13.3, and use a
                                            Pasteur pipette to transfer 10 mL of the resuspended
                                            sample (which will contain 0.5 mL of solids) to the flat-
                                            sided sample tube in Section 13.3.2.1.
                              13.2.2.2.2     If the packed pellet volume is greater than 0.5 mL, and
                                            analysis of the entire sample is required:
                                            (a)     Add additional reagent water to the centrifuge
                                                   tube to bring the volume to an amount evenly
                                                   divisible by 10, then vortex the tube for 10 to  15
                                                   seconds to resuspend. (For example, if the
                                                   resuspended volume measured in Section
                                                    13.2.2.2 is 16 mL, add 4 mL of reagent water to
                                                   bring the volume to 20 mL.) Record this final
                                                   resuspended volume on a bench sheet.
                                            (b)     Proceed to Section 13.3, and process the sample
                                                   as multiple, independent 10-mL subsamples from
                                                   Section 13.3.2 onward, including the preparation
                                                   and  examination of separate slides for each
                                                   aliquot.
13.3   IMS procedure (adapted from Reference 20.8)

        NOTE:  The IMS procedure should be performed on a bench top with all materials at
        room temperature, ranging from 15°C to 25°C.

        13.3.1  Preparation of reagents
                13.3.1.1      Prepare a IX dilution of SL-buffer-A from the 10X SL-buffer-A (clear,
                              colorless solution) supplied. Use reagent water (demineralized;  Section
                              7.3) as the diluent. For every 1  mL of IX SL-buffer-A required, take 100
                              /^L of 10X SL-buffer-A and make up to 1 mL with the diluent water. A
                              volume of 1.5 mL of IX SL-buffer-A will be required per sample or
                              subsample on which the Dynal IMS procedure is performed.
                13.3.1.2      To a flat-sided sample tube (Section 6.4.4), add 1 mL of the 10X SL-
                              buffer-A (supplied—not the diluted IX SL-buffer-A).
                13.3.1.3      Add 1 mL of the 10X SL-buffer-B (supplied—magenta solution) to the
                              sample tube containing the 10X SL-buffer-A.
        13.3.2  Oocyst and cyst capture
                13.3.2.1      Quantitatively transfer the water sample concentrate from Section 13.2.2
                              to the flat-sided sample tube  containing the SL-buffer. Label the tube with
                              the sample number.
                13.3.2.2      Vortex the Dynabeads®Crypto-Combo vial from the IMS kit for
                              approximately 10 seconds to suspend the beads. Ensure that the beads  are
                              fully resuspended by inverting the sample tube and making sure that there
                              is no residual pellet at the bottom.
                13.3.2.3      Add 100 ijL of the resuspended Dynabeads®Crypto-Combo (Section
                              13.3.2.2) to the sample tube containing the water sample concentrate and
                              SL-buffer.
April 1999                                     28

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                                        Method 1623 - Cryptosporidium and Giardia
13.3.2.4      Vortex the Dynabeads®Giardia-Combo vial from the IMS kit for
              approximately 10 seconds to suspend the beads. Ensure that the beads are
              fully resuspended by inverting the tube and making sure that there is no
              residual pellet at the bottom,
13.3.2.5      Add 100 ^L of the resuspended Dynabeads®Giardia-Combo (Section
              13.3.2.4) to the sample tube containing the water sample concentrate,
              Dynabeads®Crypto-Combo, and SL-buffer.
13.3.2.6      Affix the sample tube to a rotating mixer and rotate at approximately 18
              rpm for 1 hour at room temperature.
13.3.2.7      After rotating for 1 hour, remove the sample tube from mixer and place
              the tube in the magnetic particle concentrator (MPC-1) with flat side of
              the tube toward the magnet.
13.3.2.8      Without removing the sample tube from the MFC-1, place the magnet side
              of the MPC-1 downwards, so the tube is horizontal and the flat side of the
              tube is facing down.
13.3.2.9      Gently rock the sample tube by hand end-to-end through approximately
              90°, tilting the cap-end and base-end of the tube up and down in turn.
              Continue the tilting action for 2 minutes with approximately one tilt per
              second.
13.3.2.10     Ensure that the tilting action is continued throughout this period to prevent
              binding of low-mass, magnetic or magnetizable material. If the sample in
              the MPC-1 is allowed to stand motionless for more than 10 seconds,
              repeat Section 13.3.2.9 before continuing to Section 13.3.2.11.
13.3.2.11     Return the MPC-1 to the upright position, sample tube vertical, with cap
              at top. Immediately remove the cap and pour off all of the supernatant
              from the tube held in the MPC-1 into a suitable container. Do not shake
              the tube and do not remove the tube from MPC-1 during this step.
13.3.2.12     Remove the sample tube from the MPC-1 and resuspend the sample in  1-
              mL IX SL-buffer-A (prepared from 10X SL-buffer-A stock—supplied).
              Mix very gently to resuspend all material in the tube. Do not vortex.
13.3.2.13     Quantitatively transfer all the liquid from the sample tube to a labeled,
              1.5-mL microcentrifuge tube. Ensure that all of the liquid and beads are
              transferred.
13.3.2.14     Place the microcentrifuge tube into the second magnetic particle
              concentrator  (MPC-M), with its magnetic strip in place.
13.3.2.15     Without  removing the microcentrifuge tube from MPC-M, gently rock/roll
              the tube through 180° by hand. Continue for approximately 1 minute with
              approximately one 180° roll/rock per second. At the end of this step, the
              beads should produce a distinct brown dot  at the back of the tube.
13.3.2.16     Immediately aspirate the supernatant from  the tube and cap held in the
              MPC-M. If more than one sample is being  processed, conduct three 90°
              rock/roll actions before removing the supernatant from each tube. Take
              care not to disturb the material attached to  the wall of the tube adjacent to
              the magnet. Do not shake the tube. Do not remove the tube from MPC-M
              while conducting these steps.
                               29                                     April 1999

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Method 1623 - Cryptosporidium and Giardia
        13.3.3  Dissociation of beads/oocyst/cyst complex
                13.3.3.1      Remove the magnetic strip from the MPC-M.
                13.3.3.2      Add 50 uL of 0.1 N HC1, then vortex vigorously for 10 to 15 seconds.
                13.3.3.3      Place the tube in the MPC-M without the magnetic strip in place and
                              allow to stand in a vertical position for at least 10 minutes at room
                              temperature.
                13.3.3.4      Vortex vigorously for 5 to 10 seconds.
                13.3.3.5      Ensure that all of the sample is at the base of the tube. Place the
                              microcentrifuge tube in the MPC-M.
                13.3.3.6      Replace magnetic strip in MPC-M and allow the tube to stand undisturbed
                              for approximately 10 seconds.
                13.3.3.7      Prepare a well slide for sample screening and label the slide.
                13.3.3.8      Add 5 ML of 1.0 N NaOH to a sample well.
                13.3.3.9      Without removing the microcentrifuge tube from the MPC-M,  transfer all
                              of the sample from the microcentrifuge tube in the MPC-M to the sample
                              well with the NaOH. Do not disturb the beads at the back wall  of the tube.
                              Ensure that all of the fluid is transferred.
                13.3.3.10    Air-dry the sample onto the well slide.
                13.3.3.11     Proceed to Section 14.0 for sample staining unless a second dissociation is
                              required. If a second dissociation is required (this may enhance recovery
                              of oocysts and cysts in some cases), do not discard the beads or
                              microcentrifuge tube after transferring the sample to a well slide. Perform
                              the steps in Sections 13.3.3.1 through 13.3.3.9 a second time.

 14.0  Sample Staining
 14.1    Prepare positive and negative controls.
        14.1.1  For the positive control, pipette 10 //L of positive antigen or 200 to 400 intact oocysts and
                200 to 400 cysts to the center of a well.
        14.1.2  For the negative control, pipette 50 ^L of 150 mM PBS  (Section 7.9.4) into the center of
                a well and spread it over the well area with a pipette tip.
        14.1.3  Air-dry the control slides.
 14.2    Apply one drop of Detection Reagent (from direct labeling kit, Section 7.7) to each well.
 14.3    Apply one drop of Counterstain (from direct labeling kit, Section  7.7) to each well.
 14.4    Spread over entire well with applicator stick, if necessary. Do not allow the stick to scratch the
        treated surface of the slide. Use a different applicator stick for each slide.
 14.5    Place the slides in a humid chamber in the dark and incubate at room temperature for
        approximately 30 minutes.  The humid chamber consists of a tightly sealed plastic container
        containing damp paper towels on top of which the slides are placed.
 14.6    Apply one drop of IX Wash Buffer (made from 20X concentrate  in direct labeling kit, Section 7.7)
        to each well. Tilt each slide on a clean paper towel, long edge down. Gently aspirate the  excess
        Detection Reagent and Counterstain from below the well using a clean Pasteur pipette. Avoid
        disturbing the sample.

        NOTE:  Do not allow slides to dry.


April 1999                                      30

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                                                         Method 1623 - Cryptosporidium and Giardia
14.7   Apply 50 ,uL of 4',6-diamidino-2-phenylindole (DAPI) staining solution (Section 7.8.2) to each
       well. Allow to stand at room temperature for approximately 1 minute.
14.8   Apply one drop of IX Wash Buffer (from direct labeling kit, Section 7.7) to each well. Tilt each
       slide on a clean paper towel, long edge down. Gently aspirate the excess DAPI staining solution
       from below the well using a clean Pasteur pipette. Avoid disturbing the sample.

       NOTE: Do not allow slides to dry.

14.9   Add one drop of Mounting Medium (from direct labeling kit, Section 7.7) to each well.
14.10 Apply a cover slip. Use a tissue to remove excess mounting fluid from the edges of the coverslip.
14.11 Record the date and time that staining was completed on the bench sheet. If slides will not be read
       immediately, store in a humid chamber in the dark at 0°C to 8°C until ready for examination.

15.0 Examination
15.1   Scanning technique: Scan each well in a systematic fashion. An up-and-down or a side-to-side
       scanning pattern may be used (Figure 4).
15.2   Examination and confirmation using irnmunofluorescence assay  (FA), 4',6-diamidino-2-
       phenylindole (DAPI) staining characteristics, and differential interference contrast (D.I.C.)
       microscopy. Record examination and confirmation results for Cryptosporidium oocysts on a
       Cryptosporidium report form; record examination and confirmation results for Giardia cysts on a
       Giardia report form. All confirmed oocysts and cysts must be reported.
       15.2.1  If the positive staining control contains oocysts and cysts within the expected range and at
                the appropriate fluorescence  for both FA and DAPI, and the negative  staining control
                does not contain any oocysts or cysts (Section 14.1), use epifluorescence to scan the
                entire well for each sample at not less than 200X total magnification for apple-green
                fluorescence of oocyst and cyst shapes.
       15.2.2  When brilliant apple-green fluorescing ovoid or spherical objects 4 to 6 ^m in diameter
                are observed with brightly highlighted edges,  switch the microscope to the UV filter block
                for DAPI, then to D.I.C.
                15.2.2.1      Using the UV filter block for DAPI, the object will exhibit one of the
                              following characteristics:
                              (a)     Up to four distinct, sky-blue nuclei
                              (b)     Intense blue internal staining
                              (c)     Light blue internal staining (no distinct nuclei)
                              (a) and (b) are recorded  as DAPI +; (c) is recorded as DAPI -.
                15.2.2.2     Using D.I.C., look for external or internal morphological characteristics
                              atypical of Cryptosporidium oocysts (e.g., spikes, stalks, appendages,
                              pores, one or two large nuclei filling the cell, red fluorescing chloroplasts,
                              crystals, spores, etc.) (adapted from Reference 20.6).
                              15.2.2.2.1     If atypical structures are not observed, then categorize
                                              each apple-green fluorescing object as:
                                              (a)     An empty Cryptosporidium oocyst
                                              (b)     A Cryptosporidium oocyst with amorphous
                                                     structure
                                              (c)     A Cryptosporidium oocyst with internal
                                                     structure (one to four sporozoites/oocyst)
                                              Record  the shape and measurements to the nearest 0.5 ,um
                                              at 1000X total magnification for each such object.

                                                31                                       April 1999

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Method 1623 - Cryptosporidium and Giardia
                                            Although not a defining characteristic, surface oocyst
                                            folds may be observed in some specimens.
                              15.2.2.2.2    For each oocyst, record the number of sporozoites
                                            observed. Cryptosporidium oocysts with sporozoites
                                            must be confirmed by a principal analyst/supervisor.
                                            Record the date and time that sample examination and
                                            confirmation was completed on the report form.
        15.2.3  When brilliant apple-green fluorescing round to oval objects  (8-18 ,um long by 5 - 15 //m
                wide) are observed, switch the microscope to the UV filter block for DAPI then to D.I.C.
                15.2.3.1      Using the UV filter block for DAPI, the object will exhibit one or more of
                              the following characteristics:
                              (a)     Two to four sky-blue nuclei
                              (b)     Intense blue internal staining
                              (c)     Light blue internal staining (no distinct nuclei) and a green rim
                              (a) and (b) are recorded as DAPI positive; (c) is recorded as DAPI
                              negative.
                15.2.3.2      Using D.I.C., look for external or internal morphological characteristics
                              atypical of Giardia cysts (e.g., spikes, stalks, appendages, pores, one or
                              two large nuclei filling the cell, red fluorescing chloroplasts, crystals,
                              spores, etc.).
                              15.2.3.2.1    If these atypical strucutures are not observed, then
                                            identify such apple-green fluorescing objects of the
                                            aforementioned size and shape as presumptive Giardia
                                            cysts. Record the shape and measurements (to the nearest
                                            0.5 Aon at 1000X) for each such object as part of the
                                            presumptive count.
                              15.2.3.2.2    If two or more internal morphological structures are
                                            observed at this point, record this as a confirmed Giardia
                                            cysts as well. Counts with internal structures must be
                                            confirmed by the principal analyst/supervisor.
16.0  Analysis of Complex Samples
16.1    Some samples may contain high levels (>1000/L) of oocysts and cysts and/or interfering
        organisms, substances, or materials. Some samples may clog the filter (Section 12.0); others will
        not allow separation of the oocysts and cysts from the retentate or eluate; and others may contain
        materials that preclude or confuse microscopic examination. In these cases, dilute the original
        sample and filter and analyze 10 L of the diluted sample. If the sample is diluted at any step during
        analysis, the laboratory must record the original and final volumes and the volume analyzed.
16.2    If the sample holding time has not been exceeded and a full 10-L sample cannot be filtered, dilute
        an aliquot of sample to 10 L with reagent water and filter this smaller aliquot (Section 12.0). This
        dilution must be recorded and reported with the results.
16.3    If the holding times for the sample and for microscopic examination of the cleaned up
        retentate/eluate have been exceeded, the site must be re-sampled.

17.0  Method Performance
April 1999                                      32

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                                                      Method 1623 - Cryptosporidium and Giardia
17.1   Required method performance data are shown in Tables 1 and 2 in Section 21.0. These data are
       based on an interlaboratory validation study of Method 1623 involving 11 laboratories and 11 raw
       surface water matrices across the U.S.

18.0  Pollution Prevention
18.1   The solutions and reagents used in this method pose little threat to the environment when recycled
       and managed properly.
18.2   Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize
       the volume of expired materials to be disposed.

19.0  Waste Management
19.1   It is the laboratory's responsibility to comply with all federal, state, and local regulations governing
       waste management, particularly the biohazard and hazardous waste identification rules and land
       disposal restrictions, and to protect the air, water, and land by minimizing and controlling all
       releases from fume hoods and bench operations. Compliance with all sewage discharge permits and
       regulations is also required. An overview of these requirements can be found in the Environmental
       Management Guide for Small Laboratories (EPA 233-B-98-001).
19.2   Samples, reference materials, and equipment known or suspected to have viable oocysts or cysts
       attached or contained must be  sterilized prior to disposal.
19.3   For further information on waste management, consult The Waste Management Manual for
       Laboratory Personnel and Less is Better: Laboratory Chemical Management for Waste
       Reduction, both available from the American Chemical Society's Department of Government
       Relations and Science Policy,  1155 16th Street N.W., Washington, D.C. 20036.

20.0  References
20.1   Rodgers, Mark R., Flanigan, Debbie J., and Jakubowski, Walter, Applied and Environmental
       Microbiology 61(10), 3759-3763 (October 1995).
20.2   Fleming, Diane O.,  et al. (eds.), Laboratory Safety: Principles and Practices, 2nd edition. 1995.
       ASM Press, Washington, DC
20.3   "Working  with Carcinogens,"  DHEW, PHS, CDC, NIOSH, Publication 77-206, (Aug 1977).
20.4   "OSHA Safety and  Health Standards, General Industry," OSHA 2206, 29 CFR 1910 (Jan 1976).
20.5   "Safety in  Academic Chemistry Laboratories," ACS Committee on Chemical Safety (1979).
20.6   ICR Microbial Laboratory Manual, EPA/600/R-95/178, National Exposure Research Laboratory,
       Office of Research and Development, U.S. Environmental Protection Agency, 26 Martin Luther
       King Drive, Cincinnati, OH 45268,
20.7   "Envirochek™ Sampling Capsule," PN 32915, Gelman Sciences, 600 South Wagner Road, Ann
       Arbor, MI 48103-9019 (September 1996).
20.8   "Dynabeads® GC-Combo," Dynal Microbiology R&D, P.O. Box 8146 Dep., 0212 Oslo, Norway
       (September 1998, Revision no. 01).
                                             33                                    April 1999

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Method 1623 - Cryptosporidium and Giardia
21.0  Tables and  Figures
       NOTE: The acceptance criteria listed in Tables 1 and 2 are based on data generated
       through interlaboratory validation of Method 1623 involving 11 laboratories and 11 raw
       surface water matrices.
Table!
Quality control acceptance criteria for Cryptosporidium
Performance test
Initial precision and recovery
Mean recovery (percent)
Precision (as maximum relative standard deviation)
Ongoing precision and recovery (percent)
Matrix spike/matrix spike duplicate (for method modifications)
Mean recovery* (as percent)
Precision (as maximum relative percent difference)
Section
9.4
9.4.2
9.4.2
9.7
9.5
9.5.2
9.5.2
Acceptance criteria

21 -100
40
19-100

13-111
61
*The acceptance criteria for mean MS/MSD recovery serves as the acceptance criteria for MS recovery during
routine use of the method (Section 9.5.1)
Table 2.
Quality control acceptance criteria for Giardia
Performance test
Initial precision and recovery
Mean recovery (percent)
Precision (as maximum relative standard deviation)
Ongoing precision and recovery (percent)
Matrix spike/matrix spike duplicate (for method modifications)
Mean recovery* (as percent)
Precision (as maximum relative percent difference)
Section
9.4
9.4.2
9.4.2
9.7
9.5
9.5.2
9.5.2
Acceptance criteria

17-100
41
16-100

15-118
30
 *The acceptance criteria for mean MS/MSD recovery serves as the acceptance criteria for MS recovery during
 routine use of the method (Section 9.5.1)
April 1999
                               34

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                                        Method 1623 - Cryptosporidium and Giardia
                                1 mm
         1/5 mm
                                          B
Figure 1.   Hemacytometer platform ruling. Squares 1, 2, 3, and 4 are
           used to count stock suspensions of Cryptosporidium
           oocysts and Giardia cysts (after Miale, 1967)
                                 35
April 1999

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Method 1623 - Cryptosporidium and Giardia
     i
     •
     o
                   O
               I
:^a
   o	o  o
                    v
                        o
                         c
                     TJ   C
                         0
                         0
Figure 2. Manner of counting oocysts and cysts in 1 square mm.
     Dark organisms are counted and light organisms are
     omitted (after Miale, 1967).
April 1999
               36

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                                           Method 1623 - Cryptosporidium and Giardia
   sample
                         capsule
                            filter
                                              flow
                                           controller
                                                                   drain
Figure 3.   Laboratory filtration system
                                    37
April 1999

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Method 1623 - Cryptosporidium and Giardia
Figure 4.    Methods for scanning a well slide
April 1999
38

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                                                        Method 1623 - Cryptosporidium and Giardia
22.0  Glossary of Definitions and Purposes

       These definitions and purposes are specific to this method but have been conformed to common
       usage as much as possible.

22.1   Units of weight and measure and their abbreviations
       22.1.1  Symbols
               °C    degrees Celsius
               /jL    microliter
               <     less than
               >     greater than
               %     percent
       22.1.2  Alphabetical characters
               cm    centimeter
               g     gram
               G     acceleration due to gravity
               hr     hour
               ID    inside diameter
               in.     inch
               L     liter
               m     meter
               mg    milligram
               mL    milliliter
               mm   millimeter
               mM   millimolar
               N     normal; gram molecular weight of solute divided by hydrogen equivalent of solute,
                      per liter of solution
               sr     standard deviation of recovery
               X     average percent recovery

22.2   Definitions, acronyms, and abbreviations (in alphabetical order)

       Analyst—The analyst must have 2 years of college lecture and laboratory course work in
       microbiology or a closely related field. The analyst also must have at least 6 months bench
       experience, must have at least  3 months experience with FA techniques, and must have
       successfully analyzed at least 50 water and/or wastewater samples for Cryptosporidium and
       Giardia. Six months of additional experience in the above  areas may be substituted for two years
       of college.

       Analyte—A protozoan parasite tested for by this method. The analytes  in this method are
       Cryptosporidium and Giardia.

       Axoneme—An  internal flagellar structure that occurs in some protozoa, such as Giardia,
       Spironucleous, and  Trichonmonas.

       Cyst—A phase or a  form of an organism produced either in response to environmental conditions
       or as a normal part of the life cycle of the organism. It is characterized  by a thick and
       environmentally resistant cell wall.
                                               39                                     April 1999

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 Method 1623 - Cryptosporidium and Giardia
        Immunomagnetic separation (IMS)—A purification method that uses microscopic, magnetically
        responsive particles coated with an antibodies targeted to react with a specific pathogen in a fluid
        stream. Pathogens are selectively removed from other debris using a magnetic field.

        Initial precision and recovery (IPR)—Four aliquots of spiking suspension analyzed to establish the
        ability to generate acceptable precision and accuracy. An IPR is performed prior to the first time
        this method is used and any time the method or instrumentation is modified.

        Laboratory blank—See Method blank

        Laboratory control sample (LCS)—See Ongoing precision and recovery (OPR) standard

        Matrix spike (MS)—A  sample prepared by adding a known quantity of organisms to a specified
        amount of sample matrix for which an independent estimate of target analyte concentration is
        available. A matrix spike is used to determine the effect of the matrix on a method's  recovery
        efficiency.

        May—This action, activity, or procedural step is neither required nor prohibited.

        May not—This action, activity, or procedural step is prohibited.

        Median bodies—Prominent, dark-staining, paired organelles consisting of microtubules and found
        in the posterior half of Giardia. In G. intestinalis (from humans), these structures often have a
        claw-hammer shape, while in G. muris (from mice), the median bodies  are round.

        Method blank—An aliquot of reagent water that is treated exactly as a sample including exposure
        to all glassware, equipment, solvents, and procedures that are used with samples. The method
        blank is used to determine if analytes or interferences are present in the laboratory environment, the
        reagents, or the apparatus.

        Must—This  action, activity, or procedural step is required.

        Negative control—See Method blank

        Nucleus—A membrane-bound organelle containing genetic material. Nuclei are a prominent
        internal structure seen both in Cryptosporidium oocysts and Giardia cysts. In Cryptosporidium
        oocysts, there is one nucleus per sporozoite. One to four nuclei can be seen in Giardia cysts.

        Oocyst—The encysted zygote of some  sporozoa; e.g., Cryptosporidium. The oocyst  is a phase or
        form of the organism produced as a normal part of the life cycle of the organism. It is characterized
        by a thick and environmentally resistant outer wall.

        Ongoing precision and recovery (OPR) standard—A method blank spiked with known quantities of
        analytes.  The OPR is analyzed exactly  like a sample. Its purpose is to assure that the results
        produced by the laboratory remain within the limits specified in this method for precision and
        recovery.

        Oocyst and cyst spiking suspension—See Spiking suspension

        Oocyst and cyst stock suspension—See Stock suspension
April 1999

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                                                  Method 1623 - Cryptosporidium and Giardia
Positive control—See Ongoing precision and recovery standard

Principal analyst/supervisor—The principal analyst/supervisor must be an experienced
microbiologist with at least a B.A./B.S. in microbiology or a closely related field. The principal
analyst also must have at least 1 year of continuous bench expenence with immunofluorescent
antibody (FA) techniques and microscopic identification and have analyzed at least 100 water
and/or wastewater samples for Cryptosporidium and Giardia.

PTFE—Polytetrafluoroethylene

Quantitative transfer—The process of transferring a solution from one container to another using a
pipette in which as much solution as possible is transferred, followed by rinsing of the walls of the
source container with a small volume of nnsing  solution (e.g., reagent water, buffer, etc.), followed
by transfer of the rinsing solution, followed by a second rinse and transfer.

Reagent water—Water demonstrated to be free from the analytes of interest and potentially
interfering substances at the method detection limit for the analyte.

Reagent water blank—see Method blank

Relative standard deviation (RSD)—The standard deviation times 100 divided by the mean.

RSD—See Relative standard deviation

Should—This action, activity, or procedural step is suggested but not required.

Spiking suspension—Diluted stock suspension containing the organism(s) of interest at a
concentration appropriate for spiking samples.

Sporozoite—A motile, infective stage of certain protozoans; e.g., Cryptosporidium. There are four
sporozoites in each Cryptosporidium oocyst, and they are generally banana-shaped.

Stock suspension—A concentrated suspension containing the organism(s) of interest that is
obtained from a source that will attest to the host source, purity,  authenticity, and viability of the
organism(s).

Technician—The technician filters samples, performs centrifugation, elution, concentration, and
purification using IMS, and places purified samples on slides for microscopic examination, but
does not perform microscopic protozoan detection and identification. The technician must have at
least 3 months of experience in filter extraction and processing of protozoa samples.
                                         41                                       April 1999

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