Method 1623: Cryptosporidium and Giardia in Water by Filtration/ IMS/ FA June 2003 Draft for Comment Office of Water ( 4607) EPA 815 R 03 XXX http:// www. epa. gov/ microbes/ June 2003 Printed on Recycled Paper i 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 by DynCorp under a U. S. EPA contract, 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 and Prevention, 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, 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 Mary Ann Feige, Technical Support Center, Office of Ground Water and Drinking Water, U. S. Environmental Protection Agency, 26 W. Martin Luther King Drive, Cincinnati, OH 45268 1320, USA Colin Fricker, Thames Water Utilities, Manor Farm Road, Reading, Berkshire, RG2 0JN, England Carrie Hancock, CH Diagnostic & Consulting Service, Inc., 214 S. E. Nineteenth Street, Loveland, CO 80537, USA Stephanie Harris, Manchester Laboratory, U. S. Environmental Protection Agency, Region 10, 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 Cryptosporidium cover photo courtesy of the U. S. Centers for Disease Control Giardia cover photo courtesy of CH Diagnostic & Consulting Service, Inc. ii 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.

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

Laboratory Quality Assurance Program for the Analysis of Cryptosporidium in Water Coordinator Office of Ground Water and Drinking Water Technical Support Center U. S. EPA Office of Water 26 West Martin Luther King Drive Cincinnati, OH 45268 1320 William A. Telliard U. S. EPA Office of Water Analytical Methods Staff Mail Code 4303 Washington, DC 20460 Email: telliard. william@ epa. gov iii 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 assess Cryptosporidium and Giardia occurrence in raw surface waters used as source waters for drinking water treatment plants. EPA Method 1623 was developed to support this assessment.

Method Development and Validation 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 only 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 and developed quality control ( QC) acceptance criteria for the method based on this validation study. To avoid confusion with Method 1622, which already had been validated and was in use both domestically and internationally as a stand alone Cryptosporidium only detection method, EPA designated the new combined procedure EPA Method 1623.

The interlaboratory validated versions of Method 1622 ( January 1999; EPA 821 R 99 001) and Method 1623 ( April 1999; EPA 821 R 99 006) were used to analyze approximately 3,000 field and QC samples during the Information Collection Rule Supplemental Surveys ( ICRSS) between March 1999 and February 2000. Method 1622 was used to analyze samples from March 1999 to mid July 1999; Method 1623 was used from mid July 1999 to February 2000. The April 2001 revision of both methods include updated QC acceptance criteria based on analysis of the QC samples analyzed during the ICRSS.

Changes in the April 2001 Versions of the Methods Both methods were revised in April 2001, after completion of the ICRSS and multiple meetings with researchers and experienced laboratory staff to discuss potential method updates. Changes incorporated in the April 2001 revisions of the methods ( EPA 821 R 01 025 and EPA 821 R 01 026) included the following:

C Nationwide approval of modified versions of the methods using the following components: ( a) Whatman Nuclepore CrypTest   filter ( b) IDEXX Filta Max   filter ( c) Waterborne Aqua Glo   G/ C Direct FL antibody stain ( d) Waterborne Crypt a Glo   and Giardi a Glo   antibody stains C Clarified sample acceptance criteria C Modified capsule filter elution procedure C Modified concentrate aspiration procedure iv C Modified IMS acid dissociation procedure C Updated QC acceptance criteria for IPR and OPR tests C Addition of a troubleshooting section for QC failures C Modified holding times C Inclusion of flow cytometry   sorted spiking suspensions Changes in the June 2003 Versions of the Methods Both methods were revised again in June 2003 to support proposal of EPA's Long Term 2 Enhanced Surface Water Treatment Rule. Changes incorporated into the December 2002 versions include:

° Nationwide approval of a modified version of the methods using the Pall Gelman Envirochek   HV filter ° Removal of Whatman Nuclepore CrypTest   filter from the methods as a result of discontinuation of the product by the manufacturer ° Nationwide approval of the use of BTF EasySeed   irradiated oocysts and cysts for use in routine quality control ( QC) samples ° Minor clarifications and corrections ° Rejection criteria for sample condition upon receipt ° Guidance on measuring sample temperatures ° Clarification of QC sample requirements and use of QC sample results ° Guidance on minimizing carry over debris onto microscope slides after IMS Performance Based Method Concept and Modifications Approved for Nationwide Use EPA Method 1623 is a performance based method applicable to the determination of Cryptosporidium and Giardia in aqueous matrices. EPA Method 1623 requires filtration, immunomagnetic separation of the oocysts and cysts from the material captured, and enumeration of the target organisms based on the results of immunofluorescence assay, 4', 6 diamidino 2 phenylindole ( DAPI) staining results, and differential interference contrast microscopy.

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

Since the interlaboratory validation of EPA Method 1623, interlaboratory validation studies have been performed to demonstrate the equivalency of modified versions of the method using the following components: ° Whatman Nuclepore CryptTest   filter ( no longer available) ° IDEXX Filta Max   filter ° Pall Gelman Envirochek   HV filter ° Waterborne Aqua Glo   G/ C Direct FL antibody stain ° Waterborne Crypt a Glo   and Giardi a Glo   antibody stains ° BTF EasySeed   irradiated oocysts and cysts for use in routine QC samples v The validation studies for these modified versions of the method met EPA's performance based measurement system Tier 2 validation for nationwide use ( see Section 9.1.2 for details), and have been accepted by EPA as equivalent in performance to the original version of the method validated by EPA. The equipment and reagents used in these modified versions of the method are noted in Sections 6 and 7 of the method; the procedures for using these equipment and reagent options are available from the manufacturers.

Because this is a performance based method, other alternative components not listed in the method may be available for evaluation and use by the laboratory. Confirming the acceptable performance of a modified version of the method using alternate components in a single laboratory does not require that an interlaboratory validation study be conducted. However, method modifications validated only in a single laboratory have not undergone sufficient testing to merit inclusion in the method. Only those modified versions of the method that have been demonstrated as equivalent at multiple laboratories on multiple water sources through a Tier 2 interlaboratory study will be cited in the method. vi Table of Contents 1.0 Scope and Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2.0 Summary of Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3.0 Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 4.0 Contamination, Interferences, and Organism Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 5.0 Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 6.0 Equipment and Supplies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 7.0 Reagents and Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 8.0 Sample Collection and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 9.0 Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 10.0 Microscope Calibration and Analyst Verification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 11.0 Oocyst and Cyst Suspension Enumeration and Spiking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 12.0 Sample Filtration and Elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 13.0 Sample Concentration and Separation ( Purification) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 14.0 Sample Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 15.0 Examination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 16.0 Analysis of Complex Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 17.0 Method Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 18.0 Pollution Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 19.0 Waste Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 20.0 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 21.0 Tables and Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 22.0 Glossary of Definitions and Purposes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 June 2003 1 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 water by filtration concentration, 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 ( DIC) microscopy. The method has been validated in surface water, but may be used in other waters, provided the laboratory demonstrates that the method's performance acceptance criteria are met.

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 22.2 as analysts ( and principal analysts, in applicable programs). 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 DIC 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 water sample is filtered and the oocysts, cysts, and extraneous materials are retained on the filter. Although EPA has only validated the method using laboratory filtration of bulk water samples shipped from the field, field filtration also can may be used.

2.2 Elution and separation 2.2.1 Materials on the filter are eluted and 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 ( DIC) 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 cysts. Potential oocysts or cysts are confirmed through DAPI staining characteristics and Method 1623  Cryptosporidium and Giardia June 2003 2 DIC 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). Cryptosporidium is defined in this method as those determined by brilliant apple green fluorescence under UV light, size ( 4 to 6 µ m), and shape ( round to oval), excluding atypical organisms specifically identified as other microbial organisms by FITC and DIC ( for example, those possessing spikes, stalks, appendages, pores, one or two large nuclei filling the cell, red fluorescing chloroplasts, crystals, spores, etc.).

3.2 Giardia is 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). Giardia is defined in this method as those determined by brilliant apple green fluorescence under UV light, size ( 8 to 18 µ m long by 5 to 15 µ m wide), and shape ( oval), excluding atypical organisms specifically identified as other microbial organisms by FITC and DIC ( for example, those possessing spikes, stalks, appendages, pores, one or two large nuclei filling the cell, red fluorescing chloroplasts, crystals, spores, etc.).

3.3 Definitions for other terms used in this method are given in the glossary ( Section 22.0).

4.0 Contamination, Interferences, and Organism 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 immunofluorescence assay ( FA) ( Reference 20.1).

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 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). Method 1623  Cryptosporidium and Giardia June 2003 3 4.4 Freezing samples, filters, eluates, concentrates, or slides may interfere with the detection and/ or identification of oocysts and cysts.

4.5 All equipment should be cleaned according to manufacturers' instructions. Disposable supplies should be used wherever possible.

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, laboratory staff 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 carcinogenicity 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 laboratory staff 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.

5.5 Centers for Disease Control ( CDC) regulations ( 42 CFR 72) prohibit interstate shipment of more than 4 L of solution known to contain infectious materials ( see http:// www. cdc. gov/ od/ ohs/ biosfty/ shipregs. htm for details). State regulations may contain similar regulations for intrastate commerce. 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 CDC or state regulations, the sample should be shipped in accordance with these regulations.

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. Method 1623  Cryptosporidium and Giardia June 2003 4 6.1 Sample collection equipment for shipment of bulk water samples for laboratory filtration. Collapsible LDPE cubitainer for collection of 10 L bulk sample( s)   Cole Parmer cat. no. U 06100 30 or equivalent. Fill completely to ensure collection of a full 10 L sample. Discard after one use.

6.2 Equipment for sample filtration. Three options have been demonstrated to be acceptable for use with Method 1623. Other options may be used if their acceptability is demonstrated according to the procedures outlined in Section 9.1.2.

6.2.1 Cubitainer spigot to facilitate laboratory filtration of sample ( for use with any filtration option)   Cole Parmer cat. no. U 06061 01, or equivalent.

6.2.2 Original Envirochek   sampling capsule or Envirochek   HV sampling capsule equipment requirements ( for use with the procedure described in Section 12.2). The versions of the method using this filter was these filters were validated using 10 L and 50 L sample volumes, respectively. Alternate sample volumes may be used, provided the laboratory demonstrates acceptable performance on initial and ongoing spiked reagent water and source water samples ( Section 9.1.2).

<IP_ADDRESS> Sampling capsule <IP_ADDRESS>.1 Envirochek   , Pall Gelman Laboratory, Ann Arbor, MI, product no. 12110 ( individual filter) and or product no. 12107 ( box of 25 filters) ( www. pall. com/ gelman or ( 800) 521 1520 ext. 2)

<IP_ADDRESS>.2 Envirochek   HV, Pall Gelman Laboratory, Ann Arbor, MI, product no. 12099 ( individual filter) or product no. 12098 ( box of 25 filters) ( www. pall. com/ gelman or ( 800) 521 1520 ext. 2)

<IP_ADDRESS> Laboratory shaker with arms for agitation of sampling capsules <IP_ADDRESS>.1 Laboratory shaker   Lab Line model 3589, VWR Scientific cat. no. 57039 055, Fisher cat. no. 14260 11, or equivalent <IP_ADDRESS>.2 Side arms for laboratory shaker   Lab Line Model 3587 4, VWR Scientific cat. no. 57039 045, Fisher cat. no. 14260 13, or equivalent 6.2.3 CrypTest   capsule filter equipment requirements. Follow the manufacturer's instructions when using this filtration option. The version of the method using this filter was validated using 10 L sample volumes; alternate sample volumes may be used, provided the laboratory demonstrates acceptable performance on initial and ongoing spiked reagent water and matrix samples ( Section 9.1.2).

<IP_ADDRESS> Capsule filter   CrypTest   , Whatman Inc, Clifton, NJ, product no. 610064 <IP_ADDRESS> Cartridge housing   Ametek 5 in. clear polycarbonate, Whatman cat. no. 71503, or equivalent <IP_ADDRESS> Ultrasonic bath   VWR Model 75T# 21811 808, or equivalent <IP_ADDRESS> Laboratory tubing   Tygon formula R 3603, or equivalent 6.2.3 Filta Max   foam filter equipment requirements ( for use with the procedure described in Section 12.3). The version of the method using this filter was validated using 50 L Method 1623  Cryptosporidium and Giardia June 2003 5 sample volumes; alternate sample volumes may be used, provided the laboratory demonstrates acceptable performance on initial and ongoing spiked reagent water and matrix samples ( Section 9.1.2).

<IP_ADDRESS> Foam filter   Filta Max   , IDEXX, Westbrook, ME. Filter module and membrane: product code FMC 10601; filter membranes ( 100 pack), product code FMC 10800 NOTE: Check at least one filter per batch to ensure that the filters have not been affected by improper storage or other factors that could result in brittleness or other problems. At a minimum confirm that the test filter expands properly in water before using the batch or shipping filters to the field.

<IP_ADDRESS> Filter processing equipment   Filta Max starter kit, IDEXX, Westbrook, ME, cat. no. FMC 11002. Includes all equipment required to run and process Filta Max filter modules ( manual wash station ( FMC 10102) including plunger head ( FMC 12001), elution tubing set ( FMC 10301), vacuum set ( FMC 10401), filter housing ( FMC 10501), and magnetic stirrer ( FMC 10901). 6.3 Ancillary sampling equipment 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 autoclaved, thoroughly rinsed with detergent solution, followed by repeated rinsing with reagent water to minimize sample contamination. Alternately, decontaminate using hypochlorite solution, sodium thiosulfate, and multiple reagent water rinses; dispose of tubing when wear is evident. Dispose of tubing after one use whenever possible. 6.3.2 Flow control valve   0.5 gpm ( 0.03 L/ s), Bertram Controls, Plast O Matic cat. no. FC050B ½  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 Simer, model number, M40, or equivalent. 6.3.4 Flow meter   Sameco cold water totalizer, E. Clark and Associates, Northboro, MA, product no. WFU 10.110, or equivalent 6.4 Equipment for spiking samples in the laboratory 6.4.1 10 L carboy with bottom delivery port ( ½ " )   Cole Palmer cat. no. 06080 42, or equivalent; calibrate to 10.0 L and mark level with waterproof marker 6.4.2 Stir bar   Fisher cat. no. 14 511 93, or equivalent 6.4.3 Stir plate   Fisher cat. no. 14 493 120S, or equivalent 6.4.4 Hemacytometer   Neubauer type, Hauser Scientific, Horsham, PA, cat. no. 3200 or 1475, or equivalent 6.4.5 Hemacytometer coverslip   Hauser Scientific, cat. no. 5000 ( for hemacytometer cat. no. 3200) or 1461 ( for hemacytometer cat. no 1475), or equivalent 6.4.6 Lens paper without silicone   Fisher cat. no. 11 995, or equivalent 6.4.7 Polystyrene or polypropylene conical tubes with screw caps   15 and 50 mL Method 1623  Cryptosporidium and Giardia June 2003 6 6.4.8 Equipment required for enumeration of spiking suspensions using membrane filters <IP_ADDRESS> Glass microanalysis filter holder   25 mm diameter, with fritted glass support, Fisher cat. no. 09 753E, or equivalent. Replace stopper with size 8, one hole rubber stopper, Fisher Cat. No. 14 135M, or equivalent. <IP_ADDRESS> Three port vacuum filtration manifold and vacuum source   Fisher Cat. No. 09 753 39A, or equivalent <IP_ADDRESS> Cellulose acetate support membrane   1.2 µ m pore size, 25 mmdiameter Fisher cat. no. A12SP02500, or equivalent <IP_ADDRESS> Polycarbonate track etch hydrophilic membrane filter   1 µ m pore size, 25 mm diameter, Fisher cat. no. K10CP02500, or equivalent <IP_ADDRESS> 100 × 15 mm polystyrene petri dishes ( bottoms only) <IP_ADDRESS> 60 × 15 mm polystyrene petri dishes <IP_ADDRESS> Glass microscope slides   1 in. × 3 in or 2 in. × 3 in. <IP_ADDRESS> Coverslips   25 mm2 6.5 Immunomagnetic separation ( IMS) apparatus 6.5.1 Sample mixer   Dynal Inc., Lake Success, NY, cat. no. 947.01, or equivalent 6.5.2 Magnetic particle concentrator for 10 mL test tubes   Dynal MPC 1 ® , cat. no. 120.01, or equivalent 6.5.3 Magnetic particle concentrator for microcentrifuge tubes   Dynal MPC M ® , cat. no. 120.09, or equivalent 6.5.4 Flat sided sample tubes   16 × 125 mm Leighton type tubes with 60 × 10 mm flat sided magnetic capture area, Dynal L10, cat. no. 740.03, or equivalent 6.6 Powder free latex gloves   Fisher cat no. 113945B, or equivalent 6.7 Graduated cylinders, autoclavable   10, 100, and 1000 mL 6.8 Centrifuges 6.8.1 Centrifuge capable of accepting 15 to 250 mL conical centrifuge tubes and achieving 1500 × G   International Equipment Company, Needham Heights, MA, Centrifuge Size 2, Model K with swinging bucket, or equivalent 6.8.2 Centrifuge tubes   Conical, graduated, 1.5, 50, and 250 mL 6.9 Microscope 6.9.1 Epifluorescence/ differential interference contrast ( DIC) with stage and ocular micrometers and 20X ( N. A.= 0.4) to 100X ( N. A.= 1.3) objectives   Zeiss   Axioskop, Olympus   BH, or equivalent 6.9.2 Excitation/ band pass filters for immunofluorescence 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 Method 1623  Cryptosporidium and Giardia June 2003 7 6.9.3 Excitation/ band pass filters for DAPI   Filters cited below ( Chroma Technology, Brattleboro, VT), or equivalent Microscope model Fluoro chrome Excitation filter ( nm) Dichroic beamsplitting mirror ( nm) Barrier or suppression filter ( nm) Chroma catalog number Zeiss    Axioskop DAPI ( UV) 340 380 400 420 CZ902 Zeiss    IM35 DAPI ( UV) 340 380 400 420 CZ702 Olympus   BH DAPI ( UV) 340 380 400 420 11000 Filter holder 91002 Olympus   BX DAPI ( UV) 340 380 400 420 11000 Filter holder 91008 Olympus   IMT2 DAPI ( UV) 340 380 400 420 11000 Filter holder 91003 6.10 Ancillary equipment for microscopy 6.10.1 Well slides   Spot On well slides, Dynal cat. no. 740.04; treated, 12 mm diameter well slides, Meridian Diagnostics Inc., Cincinnati, OH, cat. no. R2206; or equivalent 6.10.2 Glass coverslips   22 × 50 mm 6.10.3 Nonfluorescing immersion oil   Type FF, Cargille cat. no. 16212, or equivalent 6.10.4 Micropipette, adjustable: 0 to 10 µ L with 0 to 10 µ L tips 10 to 100 µ L, with 10 to 200 µ L tips 100 to 1000 µ L with 100 to 1000 µ L tips 6.10.5 Forceps   Splinter, fine tip 6.10.6 Forceps   Blunt end 6.10.7 Desiccant   Drierite   Absorbent, Fisher cat. no. 07 577 1A, or equivalent 6.10.8 Humid chamber   A tightly sealed plastic container containing damp paper towels on top of which the slides are placed 6.11 Pipettes   Glass or plastic 6.11.1 5, 10, and 25 mL 6.11.2 Pasteur, disposable 6.12 Balances 6.12.1 Analytical   Capable of weighing 0.1 mg 6.12.2 Top loading   Capable of weighing 10 mg 6.13 pH meter 6.14 Incubator   Fisher Scientific Isotemp   , or equivalent 6.15 Vortex mixer   Fisons Whirlmixer, or equivalent 6.16 Vacuum source   Capable of maintaining 25 in. Hg, equipped with shutoff valve and vacuum gauge 6.17 Miscellaneous labware and supplies 6.17.1 Test tubes and rack 6.17.2 Flasks   Suction, Erlenmeyer, and volumetric, various sizes 6.17.3 Beakers   Glass or plastic, 5, 10, 50, 100, 500, 1000, and 2000 mL 6.17.4 Lint free tissues Method 1623  Cryptosporidium and Giardia June 2003 8 6.18 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 6.19 Filters for filter sterilizing reagents   Sterile Acrodisc, 0.45 µ m, Gelman Sciences cat no. 4184, 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 ( HCl)   ACS reagent grade, 6.0 N, 1.0 N, and 0.1 N in reagent water NOTE: Due to the low volumes of pH adjusting reagents used in this method, and the impact that changes in pH have on the immunofluorescence assay, the laboratory should purchase standards at the required normality directly from a vendor. Normality should not be adjusted by the laboratory.

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 filters NOTE: Laboratories should store prepared eluting solution for no more than 1 week or when noticeably turbid, whichever comes sooner.

7.4.1 Reagents for eluting Envirochek   sampling capsules ( Section 6.2.2) <IP_ADDRESS> 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. <IP_ADDRESS> 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 HCl or NaOH. Dilute to a final 1000 mL with reagent water and adjust the final pH. Filtersterilize through a 0.2 µ m membrane into a sterile plastic container and store at room temperature. <IP_ADDRESS> 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 of reagent water and adjust pH to 8.0 with 6.0 N HCl or NaOH. Dilute to a final volume of 1000 mL with reagent water and adjust to pH 8.0 with 1.0 N HCl or NaOH. <IP_ADDRESS> Antifoam A   Sigma Chemical Co. cat. no. A5758, or equivalent <IP_ADDRESS> Preparation of elution buffer solution   Add the contents of a preprepared Laureth 12 vial ( Section <IP_ADDRESS>) 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 <IP_ADDRESS>), 2 mL of EDTA solution ( Section <IP_ADDRESS>), and 150 µ L Antifoam A ( Section <IP_ADDRESS>). Dilute to 1000 mL with reagent water. 7.4.2 Reagents for eluting CrypTest   capsule filters ( Section 6.2.3). To 900 mL of reagent water add 8.0 g NaCl, 0.2 g KH2PO4, 2.9 g Na2HPO4 ( 12H2O) 0.2 g KCl, 0.2 g sodium lauryl sulfate ( SDS), 0.2 mL Tween 80, and 0.02 mL Antifoam A ( Sigma Chemical Co. Method 1623  Cryptosporidium and Giardia June 2003 9 cat. no. A5758, or equivalent). Adjust volume to 1 L with reagent water and adjust pH to 7.4 with 1 N NaOH or HCl. 7.4.2 Reagents for eluting Filta Max   foam filters ( Section 6.2.3) <IP_ADDRESS> Phosphate buffered saline ( PBS), pH 7.4   Sigma Chemical Co. cat. no. P 3813, or equivalent. Alternately, prepare PBS by adding the following to 1 L of reagent water: 8 g NaCl; 0.2 g KCl; 1.15 g Na2HPO4, anhydrous; and 0.2 g KH2PO4. <IP_ADDRESS> Tween 20   Sigma Chemical Co. cat. no. P 7949, or equivalent <IP_ADDRESS> High vacuum grease   BDH/ Merck. cat. no. 636082B, or equivalent <IP_ADDRESS> Preparation of PBST elution buffer. Add 100 µ L of Tween 20 to prepared PBS ( Section <IP_ADDRESS> <IP_ADDRESS>). Alternatively, add the contents of one sachet packet of PBS to 1.0 L of reagent water. Dissolve by stirring for 30 minutes. Add 100 µ L of Tween 20. Mix by stirring for 5 minutes. 7.5 Reagents for immunomagnetic separation ( IMS)   Dynabeads ® GC Combo, Dynal cat. nos. 730.02, 730.12, or equivalent 7.6 Direct antibody labeling reagents for detection of oocysts and cysts. Store reagents at 0 ° C to 8 ° C and return promptly to this temperature after each use. Do not allow any of the reagents to freeze. The reagents should be protected from exposure to light. Diluted, unused working reagents should be discarded after 48 hours. Discard reagents after the expiration date is reached. The labeling reagents in Sections 7.6.1 7.6.3 have been approved for use with this method. 7.6.1 Merifluor Cryptosporidium/ Giardia, Meridian Diagnostics cat. no. 250050, Cincinnati, OH, or equivalent 7.6.2 Aqua Glo   G/ C Direct FL, Waterborne cat. no. A100FLR, New Orleans, LA, or equivalent 7.6.3 Crypt a Glo   and Giardi a Glo   , Waterborne cat. nos. A400FLR and A300FLR, respectively, New Orleans, LA, or equivalent NOTE: If a laboratory will use multiple types of labeling reagents, the laboratory must demonstrate acceptable performance through an initial precision and recovery test ( Section 9.4) for each type, and must perform positive and negative staining controls for each batch of slides stained using each product. However, the laboratory is not required to analyze additional ongoing precision and recovery samples or method blank samples for each type. The performance of each labeling reagent used also should be monitored in each source water type.

7.6.4 Diluent for labeling reagents   Phosphate buffered saline ( PBS) ( Section 7.4.1). , pH 7.4   Sigma Chemical Co. cat. no. P 3813, or equivalent. Alternately, prepare PBS by adding the following to 1 L of reagent water: 8 g NaCl; 0.2 g KCl; 1.15 g Na2HPO4, anhydrous; and 0.2 g KH2PO4. Filter sterilize ( Section 6.19) or autoclave. Discard if growth is detected or after 6 months, whichever comes first. 7.7 4', 6 diamidino 2 phenylindole ( DAPI) stain   Sigma Chemical Co. cat. no. D9542, or equivalent 7.7.1 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. Do not allow to freeze. Discard unused solution when positive staining control fails. 7.7.2 Staining solution ( 1/ 5000 dilution in PBS [ Section 7.6.4])   Add 10 µ 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 µ g/ 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. Method 1623  Cryptosporidium and Giardia June 2003 10 7.8 Mounting medium 7.8.1 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 7.6.4]). After the DABCO has dissolved completely, adjust the solution volume to 100 mL by adding an appropriate volume of glycerol/ PBS solution. Alternately, dissolve the DABCO in 40 mL of PBS, then add azide ( 1 mL of 100X, or 10% solution), then 60 mL of glycerol. 7.8.2 Mounting medium supplied with Merifluor direct labeling kit ( Section 7.6.1) 7.9 Clear fingernail polish or clear fixative, PGC Scientifics, Gaithersburg, MD, cat. no. 60 4890, or equivalent 7.10 Oocyst and cyst suspensions for spiking 7.10.1 Enumerated spiking suspensions prepared by flow cytometer   not heat fixed or formalin fixed: Wisconsin State Laboratory of Hygiene Flow Cytometry Unit or equivalent. <IP_ADDRESS> Live, flow cytometer   sorted oocysts and cysts   Wisconsin State Laboratory of Hygiene Flow Cytometry Unit ([ 608] 224 6260), or equivalent <IP_ADDRESS> Irradiated, flow cytometer   sorted oocysts and cysts   flow cytometer   sorted oocysts and cysts   BTF EasySeed   ( www. biotechnologyfrontiers. com), or equivalent 7.10.2 Materials for manual enumeration of spiking suspensions <IP_ADDRESS> Purified Cryptosporidium oocyst stock suspension for manual enumeration   not heat fixed or formalin fixed: Sterling Parasitology Laboratory, University of Arizona, Tucson, or equivalent <IP_ADDRESS> Purified Giardia cyst stock suspension for manual enumeration   not heat fixed or formalin fixed: Waterborne, Inc., New Orleans, LA; Hyperion Research, Medicine Hat, Alberta, Canada; or equivalent <IP_ADDRESS> Tween 20, 0.01%   Dissolve 1.0 mL of a 10% solution of Tween 20 in 1 L of reagent water 7.10.3 Storage procedure   Store oocyst and cyst suspensions at 0 ° C to 810 ° C, until ready to use; do not allow to freeze 7.11 Additional reagents for enumeration of spiking suspensions using membrane filtration ( Section 11.3.6)   Sigmacote ® Sigma Company Product No. SL 2, or equivalent 8.0 Sample Collection and Storage 8.1 Sample collection, shipment, and receipt 8.1.1 Sample collection. Samples are collected as bulk samples and shipped to the laboratory for processing through the entire method, or are filtered in the field and shipped to the laboratory for processing from elution ( Section 12.2.6) onward. 8.1.2 Sample shipment. Ambient water samples are dynamic environments and, depending on sample constituents and environmental conditions, Cryptosporidium oocysts or Giardia cysts present in a sample can degrade, potentially biasing analytical results. Samples that are not analyzed the same day they are collected should be chilled to reduce biological activity, and preserve the state of source water samples between collection and analysis. Samples must analyzed by an off site laboratory should be shipped via overnight service on the day they are collected. Overnight service may not be necessary if the samples are maintained below 10 ° C ( but not frozen) and holding times are met. Method 1623  Cryptosporidium and Giardia June 2003 11 NOTE: See transportation precautions in Section 5.5.

<IP_ADDRESS> If samples are collected early in the day, chill samples as much as possible between collection and shipment by storing in a refrigerator or pre icing the sample in a cooler. If the sample is pre iced before shipping, replace with fresh ice immediately before shipment. <IP_ADDRESS> If samples are collected later in the day, these samples may be held overnight in a refrigerator. This should be considered for bulk water samples that will be shipped off site, as this minimizes the potential for water samples collected during the summer to melt the ice in which they are packed and arrive at the laboratory at > 10 ° C. <IP_ADDRESS> Samples should be shipped at 0 ° C to < 10 ° C 8 ° C and not frozen, unless the time required to chill the sample to 8 ° C 10 ° C would prevent the sample from being shipped overnight for receipt at the laboratory the day after collection. Samples must not be allowed to freeze. <IP_ADDRESS> Public water systems shipping samples to off site laboratories for analysis should include in the shipping container a means for monitoring the temperature of the sample during shipping to verify that the sample did not freeze or exceed 10 ° C. Suggested approaches for monitoring sample temperature during shipping are discussed in Section 8.1.4. 8.1.3 Sample receipt. Upon receipt, the laboratory must record the temperature. Samples that were not collected the same day they were received, and that are received at > 10 ° C or frozen, or samples that the laboratory has determined exceeded > 10 ° C or froze during shipment, must be rejected. After receipt, samples must be stored at the laboratory at < 10 ° C, and not frozen, until processed. Results from samples shipped overnight to the laboratory and received at > 8 ° C should be qualified by the laboratory.. 8.1.4 Suggestions on measuring sample temperature. Given the importance of maintaining sample temperatures for Cryptosporidium and Giardia determination, laboratories performing analyses using this method must establish acceptance criteria for receipt of samples transported to their laboratory. Several options are available to measure sample temperature upon receipt at the laboratory and, in some cases, during shipment: <IP_ADDRESS> Temperature sample. One option, for filtered samples only ( not for 10 L bulk samples), is for the sampler to fill a small, inexpensive sample bottle with water and pack this " temperature sample" next to the filtered sample. The temperature of this extra sample volume is measured upon receipt to estimate the temperature of the filter. Temperature sample bottles are not appropriate for use with bulk samples because of the potential effect that the difference in sample volume may have in temperature equilibration in the sample cooler. Example product: Cole Parmer cat. no. U 06252 20. <IP_ADDRESS> Thermometer vial. A similar option is to use a thermometer that is securely housed in a liquid filled vial. Unlike temperature samples, the laboratory does not need to perform an additional step to monitor the temperature of the vial upon receipt, but instead just needs to read the thermometer. Example product: Eagle Picher Sentry Temperature Vial 3TR 40CS F or 3TR 40CS. <IP_ADDRESS> iButton. Another option for measuring the sample temperature during shipment and upon receipt is a Thermocron ® iButton. An iButton is a small, waterproof device that contains a computer chip that can be programmed to record temperature at different time intervals. The information is then downloaded from the iButton onto a computer. The Method 1623  Cryptosporidium and Giardia June 2003 12 iButton should be placed in a temperature sample ( Section <IP_ADDRESS>), rather than placed loose in the cooler. Information on Thermocron ® iButtons is available from http:// www. ibutton. com/. Distributors include http:// www. pointsix. com/, http:// www. rdsdistributing. com, and http:// www. scigiene. com/. <IP_ADDRESS> Stick on temperature strips. Another option is for the laboratory to apply a stick on temperature strip to the outside of the sample container upon receipt at the laboratory. This option does not measure temperature as precisely as the other options, but still mitigates the risk of sample contamination while providing an indication of sample temperature to verify that the sample temperature is acceptable. Example product: Cole Parmer cat. no. U 90316 00. <IP_ADDRESS> Infrared thermometers. A final option is to measure the temperature of the surface of the sample container or filter using an infrared thermometer. The thermometer is pointed at the sample, and measures the temperature without coming in contact with the sample volume. Example product: Cole Parmer cat. no. EW 39641 00. As with other laboratory equipment, all temperature measurement devices must be calibrated routinely to ensure accurate measurements. See the EPA Manual for the Certification of Laboratories Analyzing Drinking Water ( Reference 20.6) for more information. 8.2 Sample holding times. Samples must be processed or examined within each of the holding times specified in Sections 8.2.1  8.2.4. Sample processing should be completed as soon as possible by the laboratory. The laboratory should complete sample filtration, elution, concentration, purification, and staining the day the sample is received wherever possible. However, the laboratory is permitted to split up the sample processing steps if processing a sample completely in one day is not possible. If this is necessary, sample processing can be halted after filtration, application of the purified sample onto the slide, or staining. Table 1, in Section 21.0 provides a breakdown of the holding times for each set of steps. Sections 8.2.1 through 8.2.4 provide descriptions of these holding times. 8.2.1 Sample collection and filtration. Sample elution must be initiated within 96 hours of sample collection ( if shipped to the laboratory as a bulk sample) or filtration ( if filtered in the field). 8.2.2 Sample elution, concentration, and purification. The laboratory must complete the elution, concentration, and purification ( Sections 12.2.6 through <IP_ADDRESS>) in one work day. It is critical that these steps be completed in one work day to minimize the time that any target organisms present in the sample sit in eluate or concentrated matrix. This process ends with the application of the purified sample on the slide for drying. 8.2.3 Staining. The sample must be stained within 72 hours of application of the purified sample to the slide. 8.2.4 Examination. Although immunofluorescence assay ( FA) and 4', 6 diamidino 2 phenylindole ( DAPI) and differential interference contrast ( DIC) microscopy examination and confirmation should be performed immediately after staining is complete, laboratories have up to 7 days from completion of sample staining to complete the examination and confirmation of samples. However, if fading/ diffusion of FITC or DAPI staining is noticed, the laboratory must reduce this holding time. In addition the laboratory may adjust the concentration of the DAPI staining solution ( Sections 7.7.2) so that fading/ diffusion does not occur. 8.5 Spiking suspension enumeration holding times. Flow cytometer sorted spiking suspensions ( Sections 7.10.1 and 11.2) used for spiked quality control ( QC) samples ( Section 9) must be used within the expiration date noted on the suspension. Laboratories should use flow cytometer Method 1623  Cryptosporidium and Giardia June 2003 13 sorted spiking suspensions containing live organisms within two weeks of preparation at the flow cytometry laboratory. Manually enumerated spiking suspensions must be used within 24 hours of enumeration of the spiking suspension if the hemacytometer chamber technique is used ( Section 11.3.4); or within 24 hours of application of the spiking suspension to the slides if the well slide or membrane filter enumeration technique is used ( Sections 11.3.5 and 11.3.6). Oocyst and cyst suspensions must be stored at 0 ° C to 10 ° C, until ready to use; do not allow to freeze 9.0 Quality Control 9.1 Each laboratory that uses this method is required to operate a formal quality assurance ( QA) program ( Reference 20.7). The minimum requirements of this program consist of an initial demonstration of laboratory capability through performance of the initial precision and recovery ( IPR) 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 required quality control ( QC) tests are performed and all QC acceptance criteria are met. 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 to replace immunofluorescence assay in this method ( the use of different determinative techniques are considered to be different methods, rather than modified version of this method). However, the laboratory is permitted to modify the immunofluorescence assay procedure, provided that all required QC tests are performed ( Section <IP_ADDRESS>) and all QC acceptance criteria are met ( see guidance on the use of multiple labeling reagents in Section 7.6).

NOTE: Method modifications should be considered only to improve method performance, reduce cost, or reduce sample processing time. Method modifications that reduce cost or sample processing time, but that result in poorer method performance should not be used.

<IP_ADDRESS> Method modification validation/ equivalency demonstration requirements <IP_ADDRESS>.1 Method modifications at a single laboratory. Each time a modification is made to this method for use in single laboratory, the laboratory must, at a minimum, validate the modification according to Tier 1 of EPA's performance based measurement system ( PBMS) ( Table 2 and Reference 20.8) to demonstrate that the modification produces results equivalent or superior to results produced by this method as written. Briefly, each time a modification is made to this method, the laboratory is required to demonstrate acceptable modified method performance through the IPR test ( Section 9.4). IPR results must meet the QC acceptance criteria in Tables 3 and 4 in Section 21.0, and should be comparable to previous results using the unmodified procedure. Although not required, the laboratory also should perform a matrix spike/ matrix spike duplicate ( MS/ MSD) test to demonstrate the performance of the modified method in at least one real world matrix before Method 1623  Cryptosporidium and Giardia June 2003 14 analyzing field samples using the modified method. The laboratory is required to perform MS samples using the modified method at the frequency noted in Section 9.1.8. If the modified method involves changes that cannot be adequately evaluated through these tests, additional tests may be required to demonstrate acceptability. <IP_ADDRESS>.2 Method modifications for nationwide approval. If the laboratory or a manufacturer seeks EPA approval of a method modification for nationwide use, the laboratory or manufacturer must, at a minimum, validate the modification according to Tier 2 of EPA's PBMS ( Table 2 and Reference 20.8). Briefly, at least three laboratories must perform IPR tests ( Section 9.4) and MS/ MSD ( Section 9.5) tests using the modified method, and all tests must meet the QC acceptance criteria specified in Tables 3 and 4 in Section 21.0. Upon nationwide approval, laboratories electing to use the modified method still must demonstrate acceptable performance in their own laboratory according to the requirements in Section <IP_ADDRESS>.1. If the modified method involves changes that cannot be adequately evaluated through these tests, additional tests may be required to demonstrate acceptability. <IP_ADDRESS> The laboratory is required to maintain records of modifications made to this method. These records include the following, at a minimum: <IP_ADDRESS>.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. <IP_ADDRESS>.2 A listing of the analyte( s) measured ( Cryptosporidium and Giardia). <IP_ADDRESS>.3 A narrative stating reason( s) for the modification. <IP_ADDRESS>.4 Results from all QC tests comparing the modified method to this method, including: ( a) IPR ( Section 9.4) ( b) MS/ MSD ( Section 9.5) ( c) Analysis of method blanks ( Section 9.6) <IP_ADDRESS>.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 7.10) ( 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 <IP_ADDRESS>) ( g) Filtration and elution dates and times ( h) Pellet volume, resuspended concentrate volume, resuspended concentrate volume transferred to Method 1623  Cryptosporidium and Giardia June 2003 15 IMS, and all calculations required to verify the percent of concentrate examined ( Section 13.2) ( i) Purification completion dates and times ( Section <IP_ADDRESS>) ( j) Staining completion dates and times ( Section 14.10) ( k) Staining control results ( Section 15.2.1) ( l) All required examination information ( Section 15.2.2) ( m) Examination completion dates and times ( Section 15.2.4) ( n) Analysis sequence/ run chronology ( o) Lot numbers of elution, IMS, and staining reagents ( p) Copies of bench sheets, logbooks, and other recordings of raw data ( q) 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 in Sections <IP_ADDRESS> 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 MS samples ( Section 9.5.1) at a minimum frequency of 1 MS sample per 20 field samples and at least 12 months must elapse between the first and last MS sample. The laboratory should analyze an MS sample when samples are first received from a PWS for which the laboratory has never before analyzed samples to identify potential method performance issues with the matrix ( Section 9.5.1; Tables 3 and 4). If an MS sample cannot be analyzed on the first sampling event, the first MS sample should be analyzed as soon as possible to identify potential method performance issues with the matrix. 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, or the calibration can be performed by the laboratory, provided the laboratory maintains a detailed procedure that can be evaluated by an independent auditor. 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 Method 1623  Cryptosporidium and Giardia June 2003 16 dispensing capacity of the micropipette, 50% of the capacity, and 10% of the capacity. Ten replicates should be performed at each weight. Record the weight of the water ( assume that 1.00 mL of reagent water weighs 1.00 g) and calculate the relative standard deviation ( RSD) for each. If the weight of the reagent water is within 1% of the desired weight ( mL) and the RSD of the replicates at each weight is within 1%, 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 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 11.4 and enumerated spiking suspensions ( Section 7.10.1 or Section 11.3), spike, filter, elute, concentrate, separate ( purify), stain, and examine four reagent water samples spiked with 100 to 500 oocysts and 100 to 500 cysts. <IP_ADDRESS> The laboratory is permitted to analyze the four spiked reagent samples on the same day or on as many as four different days ( provided that the spiked reagent samples are analyzed consecutively), and also may use different analysts and/ or reagent lots for each sample ( however, the procedures used for all analyses must be identical). Laboratories should note that the variability of four measurements performed on multiple days or using multiple analysts or reagent lots may be greater than the variability of measurements performed on the same day with the same analysts and reagent lots. As a result, the laboratory is at a greater risk of generating unacceptably variable IPR results if the test is performed across multiple days, analysts, and / or reagent lots. <IP_ADDRESS> 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. <IP_ADDRESS> The set of four IPR tests samples must be accompanied by analysis of a an acceptable method blank ( Section 9.6). 9.4.2 Using results of the four analyses, calculate the average percent recovery and the relative standard deviation ( RSD) of the recoveries for Cryptosporidium and for Giardia. The RSD is the standard deviation divided by the mean, times 100. 9.4.3 Compare the RSD and the mean with the corresponding limits for initial precision and recovery in Tables 3 and 4 in Section 21.0. If the RSD and the mean meet the acceptance criteria, system performance is acceptable and analysis of blanks and samples may begin. If the RSD or the mean falls outside the range for recovery, system performance is unacceptable. In this event, trouble shoot the problem by starting at the end of the method ( see guidance in Section 7.9.5 9.7.5), 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 and field sample must be collected from the same sampling location as split samples or as samples sequentially collected immediately after one another. The MS sample volume Method 1623  Cryptosporidium and Giardia June 2003 17 analyzed must be within 10% of the field sample volume. The MS shall be analyzed according to the frequency in Section 9.1.8. <IP_ADDRESS> Analyze an unspiked field sample according to the procedures in Sections 12.0 to 15.0. Using the spiking procedure in Section 11.4 and enumerated spiking suspensions ( Section 7.10.1 or Section 11.3), spike, filter, elute, concentrate, separate ( purify), stain, and examine a second field sample aliquot with a similar the number of organisms as that used in the IPR or OPR tests ( Sections 9.4 and 9.7). <IP_ADDRESS> For each organism, calculate the percent recovery ( R) using the following equation.

R = 100 x Nsp  Ns T 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 T is the true value of the oocysts or cysts spiked <IP_ADDRESS> Compare the recovery for each organism with the corresponding limits in Tables 3 and 4 in Section 21.0.

NOTE: Some sample matrices may prevent the acceptance criteria in Tables 3 and 4 from being met. An assessment of the distribution of MS recoveries across 430 MS samples from 87 sites during the ICR Supplemental Surveys is provided in Table 5.

<IP_ADDRESS> 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 each organism passes the tests in Section <IP_ADDRESS>, the laboratory should calculate 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 regularly across all MS samples and stratified by MS samples for each source. 9.5.2 Matrix spike duplicate   MSD analysis is required as part of nationwide approval of a modified version of this method to demonstrate that the modified version of this method produces results equal or superior to results produced by the method as written ( Section <IP_ADDRESS>.2). At the same time the laboratory spikes and analyzes the second field sample aliquot in Section <IP_ADDRESS>, the laboratory shall spike and analyze a third, identical field sample aliquot.

NOTE: Matrix spike duplicate samples are only required for Tier 2 validation studies. They are recommended for Tier 1 validation, but not required.

<IP_ADDRESS> For each organism, calculate the percent recovery ( R) using the equation in Section <IP_ADDRESS>. <IP_ADDRESS> Calculate the mean of the number of oocysts or cysts in the MS and MSD ( Xmean) (= [ MS+ MSD]/ 2). Method 1623  Cryptosporidium and Giardia June 2003 18 <IP_ADDRESS> Calculate the relative percent difference ( RPD) of the recoveries using the following equation:

RPD = 100 x | NMS  NMSD |

XMEAN where RPD is the relative percent difference NMS is the number of oocysts or cysts detected in the MS NMSD is the number of oocysts or cysts detected in the MSD Xmean is the mean number of oocysts or cysts detected in the MS and MSD <IP_ADDRESS> Compare the mean MS/ MSD recovery and RPD with the corresponding limits in Tables 3 and 4 in Section 21.0 for each organism. 9.6 Method blank ( negative control sample, laboratory blank): Reagent water blanks are analyzed to demonstrate freedom from contamination. Analyze the blank immediately prior to after analysis of the IPR test ( 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 Actions <IP_ADDRESS> If Cryptosporidium oocysts, Giardia cysts, or potentially interfering organisms or materials that may be misidentified as oocysts or cysts are not found in the method blank, the method blank test is acceptable and analysis of samples may proceed Any method blank in which oocysts or cysts are not detected is assumed to be uncontaminated and may be reported. <IP_ADDRESS> If Cryptosporidium oocysts, or Giardia cysts ( as defined in Section 3) or any potentially interfering organism or materials that may be misidentified as oocysts or cysts are found in the method blank, the method blank test is unacceptable. Any field sample in a batch associated with an unacceptable method blank 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. Analysis of additional samples is halted until the source of contamination is eliminated, the method blank test is performed again, and no evidence of contamination is detected. 9.7 Ongoing precision and recovery ( OPR; positive control sample; laboratory control sample): Using the spiking procedure in Section 11.4 and enumerated spiking suspensions ( Section 7.10.1 or Section 11.3), filter, elute, concentrate, separate ( purify), stain, and examine at least one reagent water sample spiked with 100 to 500 oocysts and 100 to 500 cysts each 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. If multiple method variations are used, separate OPR samples must be prepared for each method variation. 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 should samples be analyzed. 9.7.1 Examine the slide from the OPR prior to analysis of samples from the same batch. <IP_ADDRESS> Using 200X to 400X magnification, more than 50% of the oocysts or cysts must appear undamaged and morphologically intact; otherwise, the Method 1623  Cryptosporidium and Giardia June 2003 19 organisms in the spiking suspension may be of unacceptable quality or the analytical process is may be damaging the organisms. Examine the spiking suspension organisms directly ( by centrifuging, if possible, to concentrate the organisms in a volume that can be applied directly to a slide). If the organisms appear undamaged and morphologically intact under DIC, determine the step or reagent that is causing damage to the organisms. Correct the problem and repeat the OPR test. <IP_ADDRESS> Identify and enumerate each organism using epifluorescence microscopy. The first three presumptive Cryptosporidium oocysts and three Giardia cysts identified in the OPR sample must be examined using FITC, DAPI, and DIC, as per Section 15.2, and the detailed characteristics ( size, shape, DAPI category, and DIC category) reported on the Cryptosporidium and Giardia report form, as well as any additional comments on organism appearance, if notable. 9.7.2 For each organism, calculate the percent recovery ( R) using the following equation:

R = 100 x N T where: R = the percent recovery N = the number of oocysts or cysts detected T = the number of oocysts or cysts spiked 9.7.3 Compare the recovery with the limits for ongoing precision and recovery in Tables 3 and 4 in Section 21.0. 9.7.4 Actions <IP_ADDRESS> If the recoveries for Cryptosporidium and Giardia meet the acceptance criteria, system performance is acceptable and analysis of blanks and samples may proceed. <IP_ADDRESS> If, however, the recovery for Cryptosporidium or Giardia falls outside of the criteria range given, system performance is unacceptable. Any sample in a batch associated with an unacceptable OPR sample is unacceptable. Analysis of additional samples is halted until the analytical system is brought under control. In this event, there may be a problem with the microscope or with the filtration or separation systems. Troubleshoot the problem using the procedures at Section 9.7.5 as a guide. After assessing the issue, perform another OPR test and verify that Cryptosporidium and Giardia recoveries meet the acceptance criteria. All samples must be associated with an OPR that passes the criteria in Section 21.0. Samples that are not associated with an acceptable OPR must be flagged accordingly. 9.7.5 Troubleshooting. If an OPR sample has failed, and the cause of the failure is not known, the laboratory generally should identify the problem working backward in the analytical process from the microscopic examination to filtration. <IP_ADDRESS> Quality of spiked organisms. Examine the spiking suspension organisms directly ( by centrifuging, if possible, to concentrate the organisms in a volume that can be applied directly to a slide). If the organisms appear damaged under DIC, obtain fresh spiking materials. If the organisms appear undamaged and morphologically intact, determined whether the problem is associated with the microscope system or antibody stain ( Section <IP_ADDRESS>). Method 1623  Cryptosporidium and Giardia June 2003 20 <IP_ADDRESS> Microscope system and antibody stain: To determine if the failure of the OPR test is due to changes in the microscope or problems with the antibody stain, re examine the positive staining control ( Section 15.2.1), check Köhler illumination, and check the fluorescence of the fluoresceinlabeled monoclonal antibodies ( Mabs) and 4', 6 diamidino 2 phenylindole ( DAPI). If results are unacceptable, re examine the previously prepared positive staining control to determine whether the problem is associated with the microscope or the antibody stain. <IP_ADDRESS> Separation ( purification) system: To determine if the failure of the OPR test is attributable to the separation system, check system performance by spiking a 10 mL volume of reagent water with 100  500 oocysts and cysts and processing the sample through the IMS, staining, and examination procedures in Sections 13.3 through 15.0. Recoveries should be greater than 70%. <IP_ADDRESS> Filtration/ elution/ concentration system: If the failure of the OPR test is attributable to the filtration/ elution/ concentration system, check system performance by processing spiked reagent water according to the procedures in Section 12.2 through <IP_ADDRESS> 13.2.2, and filter, stain, and examine the sample concentrate according to Section 11.3.6. 9.7.6 The laboratory should add results that pass the specifications in Section 9.7.3 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, all analysts must familiarize themselves with operation of the microscope. 10.3 Microscope adjustment and calibration ( adapted from Reference 20.7) 10.3.1 Preparations for adjustment <IP_ADDRESS> 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 Method 1623  Cryptosporidium and Giardia June 2003 21 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. <IP_ADDRESS> 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. <IP_ADDRESS> 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 Köhler illumination. <IP_ADDRESS> 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. <IP_ADDRESS> Remove the diffuser lens between the lamp and microscope or swing it out of the transmitted light path. <IP_ADDRESS> Using a prepared microscope slide, adjust the focus so the image in the oculars is sharply defined. <IP_ADDRESS> Replace the slide with a business card or a piece of lens paper. <IP_ADDRESS> 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. <IP_ADDRESS> Mount the mercury lamp house on the microscope without the UV diffuser lens in place and turn on the mercury bulb. <IP_ADDRESS> 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. <IP_ADDRESS> 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. <IP_ADDRESS> Reattach the objective to the nosepiece. <IP_ADDRESS> Insert the diffuser lens into the light path between the mercury lamp house and the microscope. <IP_ADDRESS> Turn off the transmitted light and replace the card with a slide of fluorescent material. Check the field for even fluorescent illumination. Method 1623  Cryptosporidium and Giardia June 2003 22 Adjustment of the diffuser lens probably will be required. Additional slight adjustments as in Section <IP_ADDRESS> above may be required. <IP_ADDRESS> 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. <IP_ADDRESS> Remove the diffuser lens between the lamp and microscope or swing it out of the transmitted light path. <IP_ADDRESS> Using a prepared microscope slide and a 40X ( or similar) objective, adjust the focus so the image in the oculars is sharply defined. <IP_ADDRESS> Without the ocular or Bertrand optics in place, view the pupil and filament image at the bottom of the tube. <IP_ADDRESS> Focus the lamp filament image with the appropriate adjustment on the lamp house. <IP_ADDRESS> Similarly, center the lamp filament image within the pupil with the appropriate adjustment( s) on the lamp house. <IP_ADDRESS> 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 <IP_ADDRESS> assumes use of a microscope with both oculars adjustable; Section <IP_ADDRESS> assumes use of a microscope with a single adjustable ocular. The procedure must be followed each time an analyst uses the microscope. <IP_ADDRESS> Interpupillary distance <IP_ADDRESS>.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. <IP_ADDRESS>.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. <IP_ADDRESS> Ocular adjustment for microscopes capable of viewing a photographic frame through the viewing binoculars: This procedure assumes both oculars are adjustable. <IP_ADDRESS>.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 interpupillary distance. Bring an image located in the center of the field of view into as sharp a focus as possible. <IP_ADDRESS>.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. Method 1623  Cryptosporidium and Giardia June 2003 23 <IP_ADDRESS> 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. <IP_ADDRESS>.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. <IP_ADDRESS>.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. <IP_ADDRESS> 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. <IP_ADDRESS> 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. <IP_ADDRESS> Without changing the stage adjustment, find a point as distant as possible from the two 0 lines where two other lines are exactly superimposed. <IP_ADDRESS> 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. <IP_ADDRESS> Calculate the number of mm/ ocular micrometer space. For example:

0.6 mm = 0.0125 mm 48 ocular micrometer spaces ocular micrometer space <IP_ADDRESS> Because most measurements of microorganisms are given in µ m rather than mm, the value calculated above must be converted to µ m by multiplying it by 1000 µ m/ mm. For example:

0.0125 mm x 1,000 µ m = 12.5 µ m ocular micrometer space mm ocular micrometer space Method 1623  Cryptosporidium and Giardia June 2003 24 <IP_ADDRESS> 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. Objective power Description No. of ocular micrometer spaces No. of stage micrometer mm1 µ m/ ocular micrometer space2 1 10X N. A. 3=

2 20X N. A.=

3 40X N. A.=

4 100X N. A.=

11000 µ m/ mm 2( Stage micrometer length in mm × ( 1000 µ m/ mm)) ÷ no. ocular micrometer spaces 3N. A. refers to numerical aperature. The numerical aperature value is engraved on the barrel of the objective.

10.3.6 Köhler illumination: This section assumes that Köhler illumination will be established for only the 100X oil DIC 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 DIC, then each time the objective is changed, Köhler 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 Köhler illumination is not properly established, then DIC 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 time the objective is changed. <IP_ADDRESS> 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. <IP_ADDRESS> 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. <IP_ADDRESS> 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. <IP_ADDRESS> 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. <IP_ADDRESS> The aperture diaphragm of the condenser should is now be 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. Method 1623  Cryptosporidium and Giardia June 2003 25 <IP_ADDRESS> 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 DIC. 10.4 Microscope cleaning procedure 10.4.1 Use canned air to remove dust from the lenses, filters, and microscope body. 10.4.2 Use a Kimwipe dampened with a microscope cleaning solution ( MCS) ( consisting of 2 parts 90% isoproponal and 1 part acetone) to wipe down all surfaces of the microscope body. Dry off with a clean, dry Kimwipe. 10.4.3 Protocol for cleaning oculars and condenser <IP_ADDRESS> Use a new, clean Q tip dampened with MCS to clean each lense. Start at the center of the lens and spiral the Q tip outward using little to no pressure. Rotate the Q tip head while spiraling to ensure a clean surface is always contacting the lens. <IP_ADDRESS> Repeat the procedure using a new, dry Q tip. <IP_ADDRESS> Repeat Sections <IP_ADDRESS> and <IP_ADDRESS>. <IP_ADDRESS> Remove the ocular and repeat the cleaning procedure on the bottom lens of the ocular. 10.4.4 Protocol for cleaning objective lenses <IP_ADDRESS> Wipe 100X oil objective with lense paper to remove the bulk of the oil from the objective. <IP_ADDRESS> Hold a new Q tip dampened with MCS at a 45 ° angle on the objective and twirl. <IP_ADDRESS> Repeat Sections <IP_ADDRESS> with a new, dry Q tip. <IP_ADDRESS> Repeat Sections <IP_ADDRESS> and <IP_ADDRESS>. <IP_ADDRESS> Clean all objectives whether they are used or not. 10.4.5 Protocol for cleaning light source lens and filters <IP_ADDRESS> Using a Kimwipe dampened with microscope cleaning solution, wipe off the surface of each lens and filter. <IP_ADDRESS> Repeat the procedure using a dry Kimwipe. <IP_ADDRESS> Repeat Sections <IP_ADDRESS> and <IP_ADDRESS>. 10.4.6 Protocol for cleaning microscope stage <IP_ADDRESS> Using a Kimwipe dampened with microscope cleaning solution, wipe off the stage and stage clip. Be sure to clean off any residual immersion oil or fingernail polish. Remove the stage clip if necessary to ensure that it is thoroughly cleaned. 10.4.7 Use 409 and a paper towel to clean the bench top surrounding the microscope. 10.4.8 Frequency <IP_ADDRESS> Perform Sections 10.4.2, 10.4.3, 10.4.4, 10.4.5 and 10.4.7 after each microscope session. <IP_ADDRESS> Perform complete cleaning each week. 10.5 Protozoa libraries: Each laboratory is encouraged to develop libraries of photographs and drawings for identification of protozoa. 10.5.1 Take color photographs of Cryptosporidium oocysts and Giardia cysts by FA, and 4', 6 diamidino 2 phenylindole ( DAPI), and DIC that the analysts ( Section 22.2) determine are accurate ( Section 15.2). Method 1623  Cryptosporidium and Giardia June 2003 26 10.5.2 Similarly, take color photographs of interfering organisms and materials by FA, and DAPI , and DIC that the analysts believe 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 accurate positive identification of DAPI positive or negative organisms. 10.6 Verification of analyst 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 positive or negative oocysts and cysts, this method shall rely upon the ability of the analyst for identification and enumeration of oocysts and cysts. 10.6.1 At least monthly when microscopic examinations are being performed, the laboratory 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 positive and undamaged under DIC. 10.6.2 Each analyst shall determine the total number of oocysts and cysts and the number that are DAPI positive and DAPI negative for the entire slide. For 10 oocysts and 10 cysts, each analyst shall determine using the slide prepared in Section 10.5.1. the number of nuclei by DAPI and the DIC category ( empty, containing amorphous structures, or containing identifiable internal structures) of each. 10.6.3 Requirements for laboratories with multiple analysts <IP_ADDRESS> The total number and the number of DAPI positive or and DAPI negative oocysts and cysts determined by each analyst ( Section 10.5.2.) must be within ± 10% of each other. If the number is not within this range, the analysts must identify the source of any variability between analysts' examination criteria, prepare a new slide, and repeat the performance verification ( Sections 10.5.1 to 10.5.2). Differences in the number of nuclei by DAPI and in DIC categorizations among analysts must be discussed and resolved, and these resolutions must be documented. <IP_ADDRESS> Document the date, name( s) of analyst( s), number of total, DAPI positive or negative oocysts and cysts determined by the analyst( s), whether the test was passed/ failed and the results of attempts before the test was passed. <IP_ADDRESS> Only after an analyst has passed the criteria in Section 10.6.3, may oocysts and cysts in QC samples and field samples be identified and enumerated. 10.6.4 Laboratories with only one analyst should maintain a protozoa library ( Section 10.5) and compare the results of the examinations performed in Sections 10.6.1 and 10.6.2 to photographs of oocysts and cysts and interfering organisms to verify that examination results are consistent with these references. These laboratories also should perform repetitive counts of a single verification slide for FITC and DAPI. These laboratories should also coordinate with other laboratories to share slides and compare counts.

11.0 Oocyst and Cyst Suspension Enumeration and Spiking 11.1 This method requires routine analysis of spiked QC samples to demonstrate acceptable initial and ongoing laboratory and method performance ( initial precision and recovery samples [ Section 9.4], matrix spike and matrix spike duplicate samples [ Section 9.5], and ongoing precision and recovery samples [ Section 9.7]). The organisms used for these samples must be enumerated to calculate recoveries and precision. EPA recommends that flow cytometry be used for this enumeration, rather than manual techniques. Flow cytometer   sorted spikes generally are characterized by a relative standard deviation of # 2.5%, versus greater variability for manual enumeration techniques ( Reference 20.8). Guidance on preparing spiking suspensions using a flow cytometer is provided in Section 11.2. Manual enumeration procedures are provided in Method 1623  Cryptosporidium and Giardia June 2003 27 Section 11.3. The procedure for spiking bulk samples in the laboratory is provided in Section 11.4. 11.2 Flow cytometry enumeration guidelines. Although it is unlikely that many laboratories performing Method 1623 will have direct access to a flow cytometer for preparing spiking suspensions, flow sorted suspensions are available from commercial vendors and other sources ( Section 7.10.1). The information provided in Sections 11.2.1 through 11.2.4 is simply meant as a guideline for preparing spiking suspensions using a flow cytometer. Laboratories performing flow cytometry must develop and implement detailed standardized protocols for calibration and operation of the flow cytometer. 11.2.1 Spiking suspensions should be prepared using unstained organisms that have not been heat fixed or formalin fixed. 11.2.2 Spiking suspensions should be prepared using Cryptosporidium parvum oocysts < 3 months old, and Giardia intestinalis cysts < 2 weeks old. 11.2.3 Initial calibration. Immediately before sorting spiking suspensions, an initial calibration of the flow cytometer should be performed by conducting 10 sequential sorts directly onto membranes or well slides. The oocyst and cyst levels used for the initial calibration should be the same as the levels used for the spiking suspensions. Each initial calibration sample should be stained and manually counted microscopically and the manual counts used to verify the accuracy of the system. The relative standard deviation ( RSD) of the 10 counts should be # 2.5%. If the RSD is > 2.5%, the laboratory should perform the initial calibration again, until the RSD of the 10 counts is # 2.5%. In addition to counting the organisms, the laboratory also should evaluate the quality of the organisms using DAPI and DIC to confirm that the organisms are in good condition. 11.2.4 Ongoing calibration. When sorting the spiking suspensions for use in QC samples, the laboratory should perform ongoing calibration samples at a 10% frequency, at a minimum. The laboratory should sort the first run and every eleventh sample directly onto a membrane or well slide. Each ongoing calibration sample should be stained and manually counted microscopically and the manual counts used to verify the accuracy of the system. The mean of the ongoing calibration counts also should be used as the estimated spike dose, if the relative standard deviation ( RSD) of the ongoing calibration counts is # 2.5%. If the RSD is > 2.5%, the laboratory should discard the batch. 11.2.5 Method blanks. Depending on the operation of the flow cytometer, method blanks should be prepared and examined at the same frequency as the ongoing calibration samples ( Section 11.2.4). 11.2.6 Holding time criteria. Flow cytometer sorted spiking suspensions ( Sections 7.10.1 and 11.2) used for spiked quality control ( QC) samples ( Section 9) must be used within the expiration date noted on the suspension. The holding time specified by the flow cytometry laboratory should be determined based on a holding time study. Laboratories should use flow cytometer sorted spiking suspensions containing live organisms within two weeks of preparation at the flow cytometry laboratory. 11.3 Manual enumeration procedures. Two sets of manual enumerations are required per organism before purified Cryptosporidium oocyst and Giardia cyst stock suspensions ( Sections <IP_ADDRESS> and <IP_ADDRESS> Section 7.10.2) received from suppliers can be used to spike samples in the laboratory. First, the stock suspension must be diluted and enumerated ( Section 11.3.3) 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 hemacytometer chamber counting ( Section 11.3.4), well slide counting ( Section 11.3.5), or membrane filter counting ( Section 11.3.6). 11.3.1 Precision criteria. The relative standard deviation ( RSD) of the calculated mean spike dose for manually enumerated spiking suspensions must be # 16% for Cryptosporidium and # 19% for Giardia before proceeding ( these criteria are based on the pooled RSDs of Method 1623  Cryptosporidium and Giardia June 2003 28 105 manual Cryptosporidium enumerations and 104 manual Giardia enumerations submitted by 20 different laboratories under the EPA Protozoa Performance Evaluation Program). 11.3.2 Holding time criteria. Manually enumerated spiking suspensions must be used within 24 hours of enumeration of the spiking suspension if the hemacytometer chamber technique is used ( Section 11.3.4); or within 24 hours of application of the spiking suspension or membrane filter to the slides if the well slide or membrane filter enumeration technique is used ( Sections 11.3.5 and 11.3.6). 11.3.3 Enumerating and diluting stock suspensions <IP_ADDRESS> Purified, concentrated stock suspensions ( Sections <IP_ADDRESS> and <IP_ADDRESS>) 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 <IP_ADDRESS>), to a concentration of 20 to 50 organisms per large hemacytometer square before proceeding to Section <IP_ADDRESS>. <IP_ADDRESS> Apply a clean hemacytometer coverslip ( Section 6.4.5) to the hemacytometer and load the hemacytometer chamber with 10 µ 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 <IP_ADDRESS>, below, for the hemacytometer cleaning procedure. <IP_ADDRESS> 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. <IP_ADDRESS> Use 200X magnification. <IP_ADDRESS> Move the chamber so the ruled area is centered underneath it the objective. <IP_ADDRESS> Move the objective close to the coverslip while watching it from the side of the microscope, rather than through the microscope. <IP_ADDRESS> Focus up from the coverslip until the hemacytometer ruling appears. <IP_ADDRESS> 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. <IP_ADDRESS> Use the following formula to determine the number of organisms per mL of suspension:

number of organisms counted × 10 × dilution factor × 1000 mm2 = number of organisms number of mm2 counted 1 mm 1 1 µ LmL µ LmL <IP_ADDRESS> Record the result on a hemacytometer data sheet. Method 1623  Cryptosporidium and Giardia June 2003 29 <IP_ADDRESS> A total of six different hemacytometer chambers must be loaded, counted, and averaged for each suspension to achieve optimal counting accuracy. <IP_ADDRESS> 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 µ L); however, ranges as great as 5000 to 15,000 organisms per mL ( 50 to 150 organisms per 10 µ L) can be used.

NOTE: If the diluted stock suspensions ( the spiking suspensions) will be enumerated using hemacytometer chamber counts ( Section 11.3.4) or membrane filter counts ( Section 11.3.6), then the stock suspensions should be diluted with 0.01% Tween 20. If the spiking suspensions will be enumerated using well slide counts ( Section 11.3.3 11.3.5), then the stock suspensions should be diluted in reagent water.

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

volume of stock suspension ( µ L) required = required number of organisms x 1000 : L number of organisms/ µ mL of stock suspension If the volume is less than 10 µ 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 µ L, use the following formula:

total volume (: L) = number of organisms required x 10: L predicted number of organisms per 10: L ( 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 (: L) = total volume (: L)  stock suspension volume required (: L)

<IP_ADDRESS> 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. <IP_ADDRESS>.1 Rinse the hemacytometer and cover glass first with tap water, then 70% ethanol, and finally with acetone. <IP_ADDRESS>.2 Dry and polish the hemacytometer chamber and cover glass with lens paper. Store it in a secure place. Method 1623  Cryptosporidium and Giardia June 2003 30 <IP_ADDRESS> Several factors are known to introduce errors into hemacytometer counts, including:

C Inadequate mixing of suspension before flooding the chamber C Irregular filling of the chamber, trapped air bubbles, dust, or oil on the chamber or coverslip C Total number of organisms counted is too low to provide statistical confidence in the result C Error in recording tally C Calculation error; failure to consider dilution factor, or area counted C Inadequate cleaning and removal of organisms from the previous count C Allowing filled chamber to sit too long, so that the chamber suspension dries and concentrates. 11.3.4 Enumerating spiking suspensions using a hemacytometer chamber NOTE: Spiking suspensions enumerated using a hemacytometer chamber must be used within 24 hours of enumeration.

<IP_ADDRESS> Vortex the tube containing the spiking suspension ( diluted stock suspension; Section 11.3.3) for a minimum of 2 minutes. Gently invert the tube three times. <IP_ADDRESS> 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 µ L micropipette without touching the stir bar. Cover the beaker with a watch glass or petri dish to prevent evaporation between sample withdrawals. <IP_ADDRESS> Allow the beaker contents to stir for a minimum of 30 minutes before beginning enumeration. <IP_ADDRESS> While the stir bar is still spinning, remove a 10 µ L aliquot and carefully load one side of the hemacytometer. Count all organisms on the platform, at 200X magnification using phase contrast or darkfield microscopy. The count must include the entire area under the hemacytometer, not just the four outer 1 mm2 squares. Repeat this procedure nine times. This step allows confirmation of the number of organisms per 10 µ L ( Section <IP_ADDRESS>). Based on the 10 counts, calculate the mean, standard deviation, and RSD of the counts. Record the counts and the calculations on a spiking suspension enumeration form. The relative standard deviation ( RSD) of the calculated mean spike dose must be # 16% for Cryptosporidium and # 19% for Giardia before proceeding. If the RSD is unacceptable, or 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 <IP_ADDRESS> for factors that may introduce errors. Method 1623  Cryptosporidium and Giardia June 2003 31 11.3.5 Enumerating spiking suspensions using well slides NOTE: Spiking suspensions enumerated using well slides must be used within 24 hours of application of the spiking suspension to the slides.

<IP_ADDRESS> Prepare well slides for sample screening and label the slides. 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. <IP_ADDRESS> Vortex the tube containing the spiking suspension ( diluted stock suspension; Section 11.3.3) for a minimum of 2 minutes. Gently invert the tube three times. <IP_ADDRESS> Remove a 10 µ L aliquot from the spiking suspension and apply it to the center of a well. <IP_ADDRESS> Before removing subsequent aliquots, cap the tube and gently invert it three times to ensure that the oocysts or cysts are in suspension. <IP_ADDRESS> Ten wells must be prepared and counted, and the counts averaged, to sufficiently enumerate the spike dose. Air dry the well slides. Because temperature and humidity varies from laboratory to laboratory, no minimum time is specified. However, the laboratory must take care to ensure that the sample has dried completely before staining to prevent losses during the rinse steps. A slide warmer set at 35 ° C to 42 ° C also can be used. <IP_ADDRESS> Positive and negative controls must be prepared. <IP_ADDRESS>.1 For the positive control, pipette 10 µ 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. <IP_ADDRESS>.2 For the negative control, pipette 50 µ L of PBS onto the center of a well and spread it over the well area with a pipette tip. <IP_ADDRESS>.3 Air dry the control slides. <IP_ADDRESS> Apply 50 µ L of absolute methanol to each well containing the dried sample and allow to air dry for 3 to 5 minutes. <IP_ADDRESS> Follow the manufacturer's instructions ( Section 7.6) in applying the stain to the slide. <IP_ADDRESS> Place the slides in a humid chamber in the dark and incubate according to manufacturer's directions. 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. <IP_ADDRESS> Apply one drop of wash buffer ( prepared according to the manufacturer's instructions [ Section 7.6]) to each well. Tilt each slide on a clean paper towel, long edge down. Gently aspirate the excess detection reagent from below the well using a clean Pasteur pipette or absorb with a paper towel or other absorbent material. Avoid disturbing the sample.

NOTE: If using the Merifluor stain ( Section 7.6.1), do not allow slides to dry completely.

<IP_ADDRESS> Add mounting medium ( Section 7.8) to each well. <IP_ADDRESS> Apply a cover slip. Use a tissue to remove excess mounting fluid from the edges of the coverslip. Seal the edges of the coverslip onto the slide using clear nail polish. Method 1623  Cryptosporidium and Giardia June 2003 32 <IP_ADDRESS> Record the date and time that staining was completed. If slides will not be read immediately, store in a humid chamber in the dark at 0 ° C to 8 ° C < 10 ° C and not frozen until ready for examination. <IP_ADDRESS> After examination of the 10 wells, calculate the mean, standard deviation, and RSD of the 10 replicates. Record the counts and the calculations on a spiking suspension enumeration form. The relative standard deviation ( RSD) of the calculated mean spike dose must be # 16% for Cryptosporidium and # 19% for Giardia before proceeding. If the RSD is unacceptable, or 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. 11.3.6 Enumeration of spiking suspensions using membrane filters NOTE: Spiking suspensions enumerated using membrane filters must be used within 24 hours of application of the filters to the slides.

<IP_ADDRESS> Precoat the glass funnels with Sigmacote ® by placing the funnel in a large petri dish and applying 5 mL of Sigmacoat ® to the funnel opening using a pipette and allowing it to run down the inside of the funnel. Repeat for all funnels to be used. The pooled Sigmacoat ® may be returned to the bottle for re use. Place the funnels at 35 ° C or 41 ° C for approximately 5 minutes to dry. <IP_ADDRESS> Place foil around the bottoms of the 100 × 15 mm petri dishes. <IP_ADDRESS> Filter sterilize ( Section 6.19) approximately 10 mL of PBS pH 7.2 ( Section 7. 9. 4 7.6.4). Dilute detection reagent ( Section 7.7) as per manufacturer's instructions using sterile PBS. Multiply the anticipated number of filters to be stained by 100 mL to calculate total volume of stain required. Divide the total volume required by 5 to obtain the microliters of antibody necessary. Subtract the volume of antibody from the total stain volume to obtain the required microliters of sterile PBS to add to the antibody. <IP_ADDRESS> Label the tops of foil covered, 60 × 15 mm petri dishes for 10 spiking suspensions plus positive and negative staining controls and multiple filter blanks controls ( one negative control, plus a blank after every five sample filters to control for carry over). Create a humid chamber by laying damp paper towels on the bottom of a stain tray ( the inverted foillined petri dishes will protect filters from light and prevent evaporation during incubation). <IP_ADDRESS> Place a decontaminated and cleaned filter holder base ( Section <IP_ADDRESS>) into each of the three ports of the vacuum manifold ( Section <IP_ADDRESS>). <IP_ADDRESS> Pour approximately 10 mL of 0.01% Tween 20 into a 60 × 15 mm petri dish. <IP_ADDRESS> Using forceps, moisten a 1.2 µ m cellulose acetate support membrane ( Section <IP_ADDRESS>) in the 0.01% Tween 20 and place it on the fritted glass support of one of the filter bases. Moisten a polycarbonate filter ( Section <IP_ADDRESS>) the same way and position it on top of the cellulose acetate support membrane. Carefully clamp the glass funnel to the loaded filter support. Repeat for the other two filters. <IP_ADDRESS> Add 5 mL of 0.01% Tween 20 to each of the three filtration units and allow to stand. Method 1623  Cryptosporidium and Giardia June 2003 33 <IP_ADDRESS> Vortex the tube containing the spiking suspension ( diluted stock suspension; Section 11.3.3) for a minimum of 2 minutes. Gently invert the tube three times. <IP_ADDRESS> Using a micropipettor, sequentially remove two, 10 µ L aliquots from the spiking suspension and pipet into the 5 mL of 0.01% Tween 20 standing in the unit. Rinse the pipet tip twice after each addition. Apply 10 µ L of 0.01% Tween 20 to the third unit to serve as the negative control. Apply vacuum at 2" Hg and allow liquid to drain to miniscus, then close off vacuum. Pipet 10 mL of reagent water into each funnel and drain to miniscus, closing off the vacuum. Repeat the rinse and drain all fluid, close off the vacuum. <IP_ADDRESS> Pipet 100 mL of diluted antibody to the center of the bottom of a 60 × 15 mm petri dish for each sample. <IP_ADDRESS> Unclamp the top funnel and transfer each cellulose acetate support membrane/ polycarbonate filter combination onto the drop of stain using forceps ( apply each membrane/ filter combination to a different petri dish containing stain). Roll the filter into the drop to exclude air. Place the small petri dish containing the filter onto the damp towel and cover with the corresponding labeled foil covered top. Incubate for approximately 45 minutes at room temperature. <IP_ADDRESS> Reclamp the top funnels, apply vacuum and rinse each three times, each time with 20 mL of reagent water. <IP_ADDRESS> Repeat Sections <IP_ADDRESS> through <IP_ADDRESS> for the next three samples ( if that the diluted spiking suspension has sat less than 15 minutes, reduce the suspension vortex time to 60 seconds). Ten, 10 µ L spiking suspension aliquots must be prepared and counted, and the counts averaged, to sufficiently enumerate the spike dose. Include a filter blank sample at a frequency of every five samples; rotate the position of filter blank to eventually include all three filter placements. <IP_ADDRESS> Repeat Sections <IP_ADDRESS> through <IP_ADDRESS> until the 10 µ L spiking suspensions have been filtered. The last batch should include a 10 µ L 0.01 Tween 20 blank control and 20 µ L of positive control antigen as a positive staining control. <IP_ADDRESS> Label slides. After incubation is complete, for each sample, transfer the cellulose acetate filter support and polycarbonate filter from drop of stain and place on fritted glass support. Cycle vacuum on and off briefly to remove excess fluid. Peel the top polycarbonate filter off the supporting filter and place on labeled slide. Discard cellulose acetate filter support. Mount and apply coverslips to the filters immediately to avoid drying. <IP_ADDRESS> To each slide, add 20 µ L of mounting medium ( Section 7.8). <IP_ADDRESS> Apply a coverslip. Seal the edges of the coverslip onto the slide using clear nail polish. ( Sealing may be delayed until cover slips are applied to all slides.) <IP_ADDRESS> Record the date and time that staining was completed. If slides will not be read immediately, store sealed slides in a closed container in the dark at 0 ° C to < 8 ° C ( but not frozen) until ready for examination. <IP_ADDRESS> After examination of the 10 slides, calculate the mean, standard deviation, and RSD of the 10 replicates. Record the counts and the calculations on a spiking suspension enumeration form. The relative standard deviation ( RSD) of the calculated mean spike dose must be Method 1623  Cryptosporidium and Giardia June 2003 34 # 16% for Cryptosporidium and # 19% for Giardia before proceeding. If the RSD is unacceptable, or 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. <IP_ADDRESS> If oocysts or cysts are detected on the filter blanks, modify the rinse procedure to ensure that no carryover occurs and repeat enumeration. 11.4 Procedure for spiking samples in the laboratory with enumerated spiking suspensions. 11.4.1 Arrange a bottom dispensing container to feed the filter or insert the influent end of the tube connected to the filter through the top of a carboy to allow siphoning of the sample. 11.4.2 For initial precision and recovery ( Section 9.4) and ongoing precision and recovery ( Section 9.7) samples, fill the container with 10 L of reagent water or a volume of reagent water equal to the volume of the field samples analyzed in the analytical batch. For matrix spike samples ( Section 9.5), fill the container with the field sample to be spiked. Continuously mix the sample ( using a stir bar and stir plate for smaller volume samples and alternate means for larger volume samples). 11.4.3 Follow the procedures in Section <IP_ADDRESS> for flow cytometer   enumerated suspensions and the procedures in Section <IP_ADDRESS> for manually enumerated suspensions. Vortex the spiking suspension( s) ( Section 11.2 or Section 11.3) for a minimum of 2 minutes. <IP_ADDRESS> For flow cytometer   enumerated suspensions ( where the entire volume of a spiking suspension tube will be used): <IP_ADDRESS>.1 Add 400 µ L of Antifoam A to 100 mL of reagent water, and mix well to emulsify. <IP_ADDRESS>.2 Add 500 µ L of the diluted antifoam to the tube containing the spiking suspension and vortex for 30 seconds 2 minutes. <IP_ADDRESS>.3 Pour the suspension into the sample container. <IP_ADDRESS>.4 Add 20 mL of reagent water to the empty tube, cap, vortex 10 seconds to rinse, and add the rinsate to the carboy. <IP_ADDRESS>.5 Repeat this rinse using another 20 mL of reagent water. <IP_ADDRESS>.6 Record the estimated number of organisms spiked, the date and time the sample was spiked, and the sample volume spiked on a bench sheet. Proceed to Section 11.4.4. <IP_ADDRESS> For manually enumerated spiking suspensions: <IP_ADDRESS>.1 Vortex the spiking suspension( s) ( Section 11.2 or Section 11.3) for a minimum of 30 seconds. <IP_ADDRESS>.2 Rinse a pipette tip with 0.01% Tween 20 once, then rinse with the well mixed spiking suspension a minimum of five times before pulling an aliquot to be used to spike the container. <IP_ADDRESS>.3 Add the spiking suspension( s) to the carboy, delivering the aliquot below the surface of the water. <IP_ADDRESS>.4 Record the estimated number of organisms spiked, the date and time the sample was spiked, and the sample volume spiked on a bench sheet. Proceed to Section 11.4.4 Method 1623  Cryptosporidium and Giardia June 2003 35 11.4.4 Allow the spiking suspensions to mix for approximately 1 minute in the container. 11.4.5 Turn on the pump and allow the flow rate to stabilize. Set flow at the rate designated for the filter being used. As the carboy is depleted, check the flow rate and adjust if necessary. 11.4.6 When the water level approaches the discharge port of the carboy, tilt the container so that it is completely emptied. At that time, turn off the pump and add sufficient reagent water to the container to rinse. Swirl the contents to rinse down the sides. Additional rinses may be performed. 11.4.7 Turn on the pump. Allow all of the water to flow through the filter and turn off the pump.

12.0 Sample Filtration and Elution 12.1 A water sample is filtered according to the procedures in Section 12.2 or Section 12.3. Alternate procedures may be used if the laboratory first demonstrates that the alternate procedure provides equivalent or superior performance per Section 9.1.2.

NOTE: Sample elution must be initiated within 96 hours of sample collection ( if shipped to the laboratory as a bulk sample) or filtration ( if filtered in the field).

12.2 Capsule filtration ( adapted from Reference 20.9). This procedure was validated using 10 L sample volumes ( for the original Envirochek   filter) and 50 L sample volumes ( for the Envirochek   HV filter). Alternate sample volumes may be used, provided the laboratory demonstrates acceptable performance on initial and ongoing spiked reagent water and source water samples ( Section 9.1.2).

NOTE: The filtration procedures specified in Section 12.2.1  <IP_ADDRESS> are specific to laboratory filtration of a bulk sample, and reflect the procedures used during the interlaboratory validation of this method ( Reference 20.10). These procedures may require modification if samples will be filtered in the field.

12.2.1 Flow rate adjustment <IP_ADDRESS> Connect the sampling system, minus the capsule, to a carboy filled with reagent water ( Figure 3). <IP_ADDRESS> Turn on the pump and adjust the flow rate to 2.0 L/ min. <IP_ADDRESS> 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), sample type, and sample filtration start date and time on a bench sheet. 12.2.4 Filtration <IP_ADDRESS> Mix the sample well by shaking and connect the sampling system to the field carboy of sample water, or transfer the sample water to the laboratory carboy used in Section <IP_ADDRESS>. If the sample will be filtered from a field carboy, a spigot ( Section 6.2.1) can be used with the carboy to facilitate sample filtration.

NOTE: If the bulk field sample is transferred to a laboratory carboy, the laboratory carboy must be cleaned and disinfected before it is used with another field sample. Method 1623  Cryptosporidium and Giardia June 2003 36 <IP_ADDRESS> 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 6.18). This container will be used to determine the sample volume filtered. Alternately, connect a flow meter ( Section 6.3.4) downstream of the filter, and record the initial meter reading. <IP_ADDRESS> Allow the carboy discharge tube and capsule to fill with sample water by gravity. Vent residual air using the bleed valve/ vent port, gently shaking or tapping the capsule, if necessary. Turn on the pump to start water flowing through the filter. Verify that the flow rate is 2 L/ min. <IP_ADDRESS> After all of the sample has passed through the filter, turn off the pump. Allow the pressure to decrease until flow stops. ( If the sample was filtered in the field, and excess sample remains in the filter capsule upon receipt in the laboratory, pull the remaining sample volume through the filter before eluting the filter [ Section 12.2.6].) 12.2.5 Disassembly <IP_ADDRESS> 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. <IP_ADDRESS> Based on the water level in the graduated container and ½  L hash marks or meter reading, record the volume filtered on the bench sheet to the nearest quarter liter. Discard the contents of the graduated container. <IP_ADDRESS> Loosen the outlet fitting, then cap the inlet and outlet fittings. 12.2.6 Elution NOTE: The laboratory must complete the elution, concentration, and purification ( Sections 12.2.6 through <IP_ADDRESS>) in one work day. It is critical that these steps be completed in one work day to minimize the time that any target organisms present in the sample sit in eluate or concentrated matrix. This process ends with the application of the purified sample on the slide for drying.