&EPA
United States
Environmental Protection
Agency
Method 1622: Cryptosporidium in Water
by Filtration/I MS/FA
December 2005
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Office of Water (4607)
EPA815-R-05-001
http ://www. epa. gov/microbes/
December 2005
Printed on Recycled Paper
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Acknowledgments
This method was prepared under the direction of William A. Telliard of the Engineering and Analysis
Division within the U.S. Environmental Protection Agency (U.S. EPA) Office of Water. This document
was prepared by CSC 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 Cornell, CSC, 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 (retired), 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 OJN, England
Carrie Moulton (Hancock), 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
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 HI, 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
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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:
Carrie Moulton
Coordinator, Laboratory Quality Assurance Program for the Analysis of Cryptosporidium
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
Technical Support Center, MCI40
26 West Martin Luther King Drive
Cincinnati, OH 45268-1320
(513)569-7919
(513)569-7191 (fax)
moulton.carrie@epa.gov
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Introduction
To support future regulation of protozoa in drinking water, the Safe Drinking Water Act Amendments of
1996 require the U.S. Environmental Protection Agency (EPA) to evaluate the risk to public health posed
by drinking water contaminants, including waterborne parasites, such as Cryptosporidium. To implement
these requirements, EPA must assess Cryptosporidium occurrence in raw surface waters used as source
waters for drinking water treatment plants. EPA Method 1622 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.
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.
Changes in the April 2001 Version of the Method
The method was 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 revision of the method (EPA-821-R-01-025) included the following:
• 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
• Clarified sample acceptance criteria
• Modified capsule filter elution procedure
• Modified concentrate aspiration procedure
• Modified IMS acid dissociation procedure
• Updated QC acceptance criteria for IPR and OPR tests
• Addition of a troubleshooting section for QC failures
• Modified holding times
• Inclusion of flow cytometry-sorted spiking suspensions
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Changes in the June 2003 Version of the Method
The method was 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 version 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 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
Changes in the December 2005 Version of the Method
The method was revised again in 2005 to support promulgation of EPA's Long Term 2 Enhanced Surface
Water Treatment Rule. Changes incorporated into the June 2003 version include:
• Nationwide approval of the use of portable continuous-flow centrifugation as a modified version
of the method. The product met all method acceptance criteria for Cryptosporidium using 50-L
source water samples.
• Addition of BTF EasyStain™ monoclonal antibody stain as an acceptable reagent for staining in
Methods 1622. The product was validated through an interlaboratory validation study using the
Pall Envirochek™ HV filter.
• Clarification of the analyst verification procedure
• Clarification of sample condition criteria upon receipt
Performance-Based Method Concept and Modifications Approved for Nationwide Use
EPA Method 1622 is a performance-based method applicable to the determination of Cryptosporidium in
aqueous matrices. EPA Method 1622 requires filtration, immunomagnetic separation of the oocysts 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 1622 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.
IV
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Since the interlaboratory validation of EPA Method 1622, 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
• Portable Continuous-Flow Centrifugation (PCFC)
• Waterborne Aqua-Glo™ G/C Direct FL antibody stain
• Waterborne Crypt-a-Glo™ and Giardi-a-Glo™ antibody stains
• BTF EasyStain™ antibody stain
• BTF EasySeed™ irradiated oocysts for use in routine QC samples
The validation studies for the 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.
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.
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Table of Contents
1.0 Scope and Application 1
2.0 Summary of Method 1
3.0 Definitions 2
4.0 Contamination, Interferences, and 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 11
9.0 Quality Control 13
10.0 Microscope Calibration and Analyst Verification 21
11.0 Oocyst Suspension Enumeration and Sample Spiking 28
12.0 Sample Filtration and Elution 36
13.0 Sample Concentration and Separation (Purification) 45
14.0 Sample Staining 51
15.0 Examination 52
16.0 Analysis of Complex Samples 53
17.0 Method Performance 54
18.0 Pollution Prevention 54
19.0 Waste Management 54
20.0 References 54
21.0 Tables and Figures 56
22.0 Glossary of Definitions and Purposes 65
VI
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Method 1622: Cryptosporidium in Water
by Filtration/IMS/FA
1.0 Scope and Application
•| •] This method is for the detection of Cryptosporidium (CAS Registry number 137259-50-8) and in
water by concentration, immunomagnetic separation (IMS), and immunofluorescence assay (FA)
microscopy. Cryptosporidium may be verified using 4',6-diamidino-2-phenylindole (DAPI)
staining and differential interference contrast (DIG) 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.
•j 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 identifies the genera, Cryptosporidium, but not the species. The method cannot
determine the host species of origin, nor can it determine the viability or infectivity of detected
oocysts.
•j 4 This method is for use only by persons experienced in the determination of Cryptosporidium by
filtration, IMS, and FA. Experienced persons are defined in Section 22.2 as analysts or principal
analysts. 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 DIG microscopy.
•| 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"| A water sample is filtered and the oocysts 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 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
the supernatant fluid is aspirated.
2.2.2 The oocysts are magnetized by attachment of magnetic beads conjugated to anti-
Cryptosporidium antibodies. The magnetized oocysts 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.
2 3 Enumeration
2.3.1 The oocysts are stained on well slides with fluorescently labeled monoclonal antibodies
and 4',6-diamidino-2-phenylindole (DAPI). The stained sample is examined using
fluorescence and differential interference contrast (DIG) 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.
December 2005
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Method 1622 - Cryptosporidium
2.3.3 Quantitative analysis is performed by counting the total number of objects on the slide
confirmed as oocysts.
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 a genus of protozoan parasites potentially found in water and other media.
The recent taxonomy of the genus Cryptosporidium includes the following species and their
potential hosts: C. hominis (humans; formerly C. parvum genotype I; Reference 20.1); C. parvum
(bovine and other mammals including humans; formerly genotype II;); C. baileyi and C.
meleagridis (birds); C. muris (rodents); C. canis (dogs); C.felis (cats); C. serpentis (reptiles); and
C. nasorum (fish). Cryptosporidium oocysts are defined in this method as objects exhibiting
brilliant apple green fluorescence under UV light (FA-positive), typical size (4 to 6 (im) and shape
(round to oval), and no atypical characteristics by FA, DAPI fluorescence, or DIG microscopy.
Examination and characterization using fluorescence (FITC and DAPI stain) and DIG microscopy
are required for exclusion of atypical organisms (e.g., those possessing spikes, stalks, appendages,
pores, one or two large nuclei filling the cell, red fluorescing chloroplasts, crystals, spores, etc.).
3 2 Definitions for other terms used in this method are given in the glossary (Section 22.0).
4.0 Contamination, Interferences, and Organism Degradation
4 •] Turbidity caused by inorganic and organic debris can interfere with the concentration, separation,
and examination of the sample for Cryptosporidium oocysts. In addition to naturally-occurring
debris, e.g. clays and algae, chemicals, e.g. iron, alum coagulants and polymers added to source
waters during the treatment process may result in additional interference.
4 2 Organisms and debris that autofluoresce or demonstrate non-specific immunofluorescence, such
as algal and yeast cells, when examined by epifluorescent microscopy, may interfere with the
detection of oocysts and contribute to false positives by immunofluorescence assay (FA)
(Reference 20.3).
4 3 Solvents, reagents, labware, and other sample-processing hardware may yield artifacts that may
cause misinterpretation of microscopic examinations for oocysts. All materials used must 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 Freezing samples, filters, eluates, concentrates, or slides may interfere with the detection and/or
identification of oocysts.
4 5 All equipment should be cleaned according to manufacturers' instructions. Disposable supplies
should be used wherever possible.
December 2005
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Method 1622 - Cryptosporidium
5.0 Safety
5 •] The biohazard associated with, and the risk of infection from, oocysts 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 current knowledge 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 20.4
through 20.7.
5 3 Samples may contain high concentrations of biohazards and toxic compounds, and must be
handled with gloves. Reference materials and standards containing oocysts 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. 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 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.
g •] Sample collection equipment for shipment of bulk water samples for laboratory filtration.
Collapsible LDPE cubitainer for collection of 10-L bulk sample(s)—Cole Farmer cat. no. U-
06100-30 or equivalent. Fill completely to ensure collection of a full 10-L sample. Discard after
one use.
g 2 Equipment for sample filtration. Four options have been demonstrated to be acceptable for use
with Method 1622. Other options may be used if their acceptability is demonstrated according to
the procedures outlined in Section 9.1.2.
3 December 2005
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Method 1622 - Cryptosporidium
6.2.1 Cubitainer spigot to facilitate laboratory filtration of sample (for use with any filtration
option)—Cole Farmer 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 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).
6.2.2.1 Sampling capsule
6.2.2.1.1 Envirochek™, Pall Corporation, Ann Arbor, MI, part no.
12110 (individual filter) and or part no. 12107 (box of 25
filters) (www.Dall.com or (800) 521-1520 ext. 2)
6.2.2.1.2 Envirochek™ HV, Pall Corporation, Ann Arbor, MI, part
no. 12099 (individual filter) or part no. 12098 (box of 25
filters) (www.pall.com or (800) 521-1520 ext. 2)
6.2.2.2 Laboratory shaker with arms for agitation of sampling capsules
6.2.2.2.1 Laboratory shaker—Lab-Line model 3589 (available
through VWR Scientific cat. no. 57039-055), Pall
Corporation part no. 4821, Fisher cat. no. 14260-11, or
equivalent
6.2.2.2.2 Side arms for laboratory shaker—Lab-Line Model 3587-
4 (available through VWR Scientific cat. no. 57039-045),
Fisher cat. no. 14260-13, 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
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).
6.2.3.1 Foam filter—Filta-Max®, IDEXX, Westbrook, ME. Filter module cat.
no. FMC 10603
NOTE: Check at least one/liter 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.
6.2.3.2 Filter processing equipment—Filta-Max® starter kit, IDEXX,
Westbrook, ME, cat. no. FMC 11002. Starter kit includes manual wash
station with clamp set (FMC 10101 or 10106) including plunger head
(FMC 12001), tubing set (FMC 10307), vacuum set (FMC 10401), MKII
filter housing with hose-tail fittings (FMC 10504) and green housing
tools (FMC 10506). In addition, processing requires magnetic stirrer
(FMC 10901) and filter membranes, 100 pk, (FMC 10800).
6.2.4 Portable Continuous-Flow Centrifuge (PCFC) requirements (for use with procedures
described in Section 12.4). The version of the method using this technique was validated
for Cryptosporidium in 50-L sample volumes; alternate sample volumes may be used,
December 2005 4
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Method 1622 - Cryptosporidium
provided the laboratory demonstrates acceptable performance on initial and ongoing
spiked reagent water and matrix samples (Section 9.1.2). The technique is based on
technology from Haemonetics Corporation, Braintree, MA.
g 3 Ancillary sampling equipment
6.3.1 Tubing—Glass, polytetrafluoroethylene (PTFE), high-density polyethylene (HDPE), or
other tubing to which oocysts 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 after one use whenever possible or when wear is
evident.
6.3.2 Flow control valve—0.5 gpm (0.03 L/s), Bertram Controls, Plast-O-Matic cat. no.
FC050BV2-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 Pump— peristaltic, centrifugal, impeller, or diaphragm pump; MasterFlex I/P®
EasyLoad® peristaltic pump (Cole-Parmer cat. No. EW-77963-10) with 77601-10
pumphead, 77410-00 drive unit, and 06429-73 Tygon LFL tubing; Dayton, model
number 3YU61 (available through Grainger), Jabsco Flexible Impeller Pump (Cole-
Parmer cat. No. EW-75202-00); Simer, model number M40; or equivalent. It is
recommended that the pump be placed on the effluent side of the filter, when possible, to
reduce the risk of contamination and the amount of tubing replaced or cleaned.
6.3.4 Flow meter—SaMeCo cold water totalizer, E. Clark and Associates, Northboro, MA,
product no. WFU 10.110; Omega flow meter, Stamford, CT, model FTB4105; or
equivalent. Alternatively, use a graduated carboy(s) (See Section 6.18)
g 4 Equipment for spiking samples in the laboratory
6.4.1 Collapsible 10-L LDPE cubitainer with cubitainer spigot—Cole Farmer cat. no. U-
06100-30 or equivalent and Cole Farmer cat. no. U-06061-01, or equivalent. Discard
after one use to eliminate possible contamination. Alternatively, use clean, 10-L carboy
with bottom delivery port (Yz"), 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. 14-513-66, or equivalent
6.4.3 Stir plate—Fisher cat. no. 11-51049S, S50461HP, or equivalent
6.4.4 Hemacytometer—Neubauer type, Hausser Scientific, Horsham, PA, product no. 3200 or
1475, or equivalent
6.4.5 Hemacytometer coverslip—Hausser Scientific, product 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
6.4.8 Equipment required for enumeration of spiking suspensions using membrane filters
6.4.8.1 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.
6.4.8.2 Three-port vacuum filtration manifold and vacuum source—Fisher Cat.
No. 09-753-39A, or equivalent
December 2005
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Method 1622 - Cryptosporidium
6.4.8.3 Cellulose acetate support membrane—1.2-um-pore-size, 25-mm-
diameter, Fisher cat. no. A12SP02500, or equivalent
6.4.8.4 Polycarbonate track-etch hydrophilic membrane filter—1 -^m-pore-size,
25-mm-diameter, Fisher cat. no. K10CP02500, or equivalent
6.4.8.5 100 x 15 mm polystyrene petri dishes (bottoms only)
6.4.8.6 60 x 15 mm polystyrene petri dishes
6.4.8.7 Glass microscope slides—1 in. x 3 in or 2 in. x 3 in.
6.4.8.8 Coverslips—25 mm2
g 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
MPC®-6, cat. No 120.02, or equivalent
6.5.3 Magnetic particle concentrator for microcentrifuge tubes—Dynal MPC®-M, cat. no.
120.09 (no longer available); Dynal MPC®-S, cat. no. 120.20, or equivalent
6.5.4 Flat-sided sample tubes—16 x 125 mm Leighton-type tubes with 60 x 10 mm flat-sided
magnetic capture area, Dynal L10, cat. no. 740.03, or equivalent
g g Powder-free latex gloves—Fisher cat no. 113945B, or equivalent
g 7 Graduated cylinders, autoclavable—10-, 100-, and 1000-mL
g_g Centrifuges
6.8.1 Centrifuge capable of accepting 15- to 250-mL conical centrifuge tubes and achieving
1500 x 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
g g Microscope
6.9.1 Epifluorescence/differential interference contrast (DIG) with stage and ocular
micrometers and 20X (N.A.=0.4) to 100X (N.A.=1.3) objectives—Zeiss™ Axioskop,
Olympus™ BH, or equivalent. Hoffman Modulation Contrast optics may be 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
December 2005
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Method 1622 - Cryptosporidium
6.9.3 Excitation/band-pass filters for DAPI—Filters cited below (Chroma Technology,
Brattleboro, VT), or equivalent
6.11
6.12
6.13
6.14
6.15
6.16
6.17
Microscope
model
Zeiss™ -
Axioskop
Zeiss™ -IM35
Olympus™ BH
Olympus™ BX
Olympus™
IMT2
Fluoro-chrome
DAP I (UV)
DAP I (UV)
DAP I (UV)
Excitation
filter (nm)
340-380
340-380
340-380
Dichroic
beam- splitting
mirror (nm)
400
400
400
Barrier or
suppression
filter (nm)
420
420
420
Filter holder
DAP I (UV)
340-380
400
420
Filter holder
DAP I (UV)
340-380
400
420
Filter holder
Chroma
catalog
number
CZ902
CZ702
11000
91002
11000
91008
11000
91003
610 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 x 50 mm
6.10.3 Nonfluorescing immersion oil—Type FF, Cargille cat. no. 16212, or equivalent
6.10.4 Micropipette, adjustable: 0- to 10-^iL with 0- to lO-^iLtips
10- to 100-fiL, with 10- to 200-^L tips
100- to 1000-uL with 100- to lOOO-^L tips
6.10.5 Forceps—Splinter, fine tip
6.10.6 Forceps—Blunt-end
6.10.7 Desiccant—Drierite™ Absorbent, Fisher cat. no. 07-577-1 A, or equivalent
6.10.8 Humid chamber—A tightly sealed plastic container containing damp paper towels on top
of which the slides are placed
Pipettes—Glass or plastic
6.11.1 5-, 10-, and25-mL
6.11.2 Pasteur, disposable
Balances
6.12.1 Analytical—Capable of weighing 0.1 mg
6.12.2 Top loading—Capable of weighing 10 mg
pH meter
Incubator—Fisher Scientific Isotemp™, or equivalent
Vortex mixer—Fisons Whirlmixer, or equivalent
Vacuum source—Capable of maintaining 25 in. Hg, equipped with shutoff valve and vacuum
gauge
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 orplastic, 5-, 10-, 50-, 100-, 500-, 1000-, and 2000-mL
December 2005
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Method 1622 - Cryptosporidium
6.17.4 Lint-free tissues
g 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
g 19 Filters for filter-sterilizing reagents—Sterile Acrodisc, 0.45 urn, Pall Corporation, 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 (HC1)—ACS reagent grade, 6.ON, l.ON, andO.l N in reagent water
NOTE: Due to the low volumes ofpH-adjusting reagents used in this method, and the
impact that changes in pH have on the immunofluorescence assay, the laboratory must
purchase standards at the required normality directly from a vendor. Normality must not
be adjusted by the laboratory.
-j 2 Solvents—Acetone, glycerol, ethanol, and methanol, ACS reagent grade
7 3 Reagent water—Water in which oocysts and interfering materials and substances, including
magnetic minerals, are not detected by this method. See Reference 20.8 (Section 9020) for reagent
water requirements.
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™ and Envirochek™ HV sampling capsules (Section
6.2.2)
7.4.1.1 Laureth-12—PPG Industries, Gurnee, IL, cat. no. 06194, or equivalent.
Store Laureth-12 as a 10% solution in reagent water. Weigh 10 g of
Laureth-12 and dissolve using a microwave or hot plate in 90 mL of
reagent water. Dispense 10-mL aliquots into sterile vials and store at
room temperature for up to 2 months, or in the freezer for up to a year.
7.4.1.2 1 M Tris, pH 7.4—Dissolve 121.1 g Tris (Fisher cat. no. BP152) in 700
mL of reagent water and adjust pH to 7.4 with 1 N HC1 or NaOH. Dilute
to a final 1000 mL with reagent water and adjust the final pH. Filter-
sterilize through a 0.2-um membrane into a sterile plastic container and
store at room temperature. Alternatively, use prepared TRIS, Sigma
T6066 or equivalent.
7.4.1.3 0.5 M EDTA, 2 Na, pH 8.0—Dissolve 186.1 g ethylenediamine
tetraacetic acid, disodium salt dihydrate (Fisher cat. no. S311) in 800 mL
of reagent water and adjust pH to 8.0 with 6.0 N HC1 or NaOH. Dilute to
a final volume of 1000 mL with reagent water and adjust to pH 8.0 with
1.0 N HC1 or NaOH. Alternatively, use prepared EDTA, Sigma E5134 or
equivalent.
7.4.1.4 Antifoam A—Sigma Chemical Co. cat. no. A5758, or equivalent
December 2005
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Method 1622 - Cryptosporidium
7.4.1.5 Preparation of elution buffer solution—Add the contents of a pre-
prepared Laureth-12 vial (Section 7.4.1.1) to a 1000-mL graduated
cylinder. Rinse the vial several times to ensure the transfer of the
detergent to the cylinder. Add 10 mL of Tris solution (Section 7.4.1.2), 2
mL of EDTA solution (Section 7.4.1.3), and 150 uL Antifoam A (Section
7.4.1.4). Dilute to 1000 mL with reagent water.
7.4.2 Reagents for eluting Filta-Max® foam filters (Section 6.2.3)
7.4.2.1 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 KC1; 1.15 g Na2HPO4, anhydrous;
and 0.2 g KH2PO4.
7.4.2.2 Tween® 20 —Sigma Chemical Co. cat. no. P-7949, or equivalent
7.4.2.3 High-vacuum grease—BDH/Merck. cat. no. 636082B, or equivalent
7.4.2.4 Preparation of PBST elution buffer. Add 100 ^L of Tween® 20 to
prepared PBS (Section 7.4.2.1). Alternatively, add the contents of one
packet of PBS to 1.0 L of reagent water. Dissolve by stirring for 30
minutes. Add 100 uL of Tween® 20 . Mix by stirring for 5 minutes.
7.4.3 Reagents for Portable Continuous-Flow Centrifuge (Section 6.2.4)
7.4.3.1 Sodium dodecyl sulfate—Sigma Chemical Co. cat. no. 71730 or
equivalent
7.4.3.2 TWEEN 80— Sigma Chemical Co. cat. no. P1754 or equivalent
7.4.3.3 Antifoam A—Sigma Chemical Co. cat. no. A5758, or equivalent
7.4.3.4 Preparation of concentrated elution buffer. Add above reagents to obtain
a final concentration of 1% sodium dodecyl sulfate, 0.01% TWEEN 80,
and 0.001% Antifoam A in concentrated sample volume of ~250mL
7 5 Reagents for immunomagnetic separation (IMS)—Dynabeads® anti-Cryptosporidium beads,
Dynal cat. nos. 730.01/730.11, or equivalent
7 g Direct antibody labeling reagents for detection of oocysts. Store reagents between 1°C and 10°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. AlOOFLR, New Orleans, LA, or
equivalent
7.6.3 Crypt-a-Glo™, Waterborne cat. no. A400FLR, New Orleans, LA, or equivalent
7.6.4 EasyStain™C&G, BTF Pty Limited, Sydney, Australia 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.
7.6.5 Diluent for labeling reagents—Phosphate buffered saline (PBS) (Section 7.4.2).
9 December 2005
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Method 1622 - Cryptosporidium
1 i 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 between 1°C and 10°C in the dark. Do not allow to
freeze. Discard unused solution when positive staining control fails or after specified
time determined by laboratory.
7.7.2 Staining solution—Follow antibody kit manufacturer's instructions. Add 10 (iL of 2
mg/mL DAPI stock solution to 50 mL of PBS for use with Aqua-Glo™ G/C Direct FL or
MeriFluor® Cryptosporidium/Giardia. Add 50 f^L of 2 mg/mL DAPI stock solution to
50 mL of PBS for use with EasyStain™. Prepare working solution daily and store
between 1°C and 10°C (do not allow to freeze). DAPI is light sensitive; therefore, store
in the dark except when staining. The DAPI concentration may be increased if
fading/diffusion of DAPI staining is encountered, but the staining solution must be tested
first on expendable environmental samples to confirm that staining intensity is
appropriate.
7 g 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). 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® Cryptosporidium/Giardia, Meridian
Diagnostics cat. no. 250050, or equivalent (Section 7.6.1)
7.8.3 Mounting medium supplied with Aqua-Glo™ G/C Direct FL kit, Waterborne cat. no.
A100FLR, cat. no. M101, or equivalent (Section 7.6.2)
7.8.4 Mounting medium supplied with EasyStain™C&G, BTF Pty Limited or equivalent
(Section 7.6.4)
7.8.5 Elvanol or equivalent permanent, non-fade archiving mounting medium
7 g Clear fingernail polish or clear fixative, PGC Scientifics, Gaithersburg, MD, cat. no. 60-4890-00,
or equivalent
y-|0 Oocyst suspensions for spiking
7.10.1 Enumerated spiking suspensions prepared by flow cytometer—not formalin fixed.
7.10.1.1 Live, flow cytometer-sorted oocysts —Wisconsin State Laboratory of
Hygiene Flow Cytometry Unit ([608] 224-6260), or equivalent
7.10.1.2 Irradiated, flow cytometer-sorted oocysts —flow cytometer-sorted
oocysts —BTF EasySeed™ (contact@btfbio.com). or equivalent
7.10.2 Materials for manual enumeration of spiking suspensions
7.10.2.1 Purified Cryptosporidium oocyst stock suspension for manual
enumeration—not formalin-fixed: Sterling Parasitology Laboratory,
University of Arizona, Tucson, or equivalent
7.10.2.2 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 suspensions between 1°C and 10°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
December 2005 10
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Method 1622 - Cryptosporidium
8.0 Sample Collection and Storage
g 1 Sample collection, shipment, and receipt
8.1.1 Sample collection. Samples are collected as bulk samples and shipped to the laboratory
on ice for processing through the entire method, or are filtered in the field and shipped to
the laboratory on ice 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 present in a
sample can degrade, potentially biasing analytical results. Samples should be chilled to
reduce biological activity, and preserve the state of source water samples between
collection and analysis. Samples analyzed by an off-site laboratory should be shipped on
ice via overnight service on the day they are collected.
NOTE: See transportation precautions in Section 5.5.
8.1.2.1 If samples are collected early in the day, chill samples by storing in a
refrigerator between 1°C and 10°C or pre-icing the sample in a cooler. If
the sample is pre-iced before shipping, replace with fresh ice immediately
before shipment.
8.1.2.2 If samples are collected later in the day, these samples may be chilled
overnight in a refrigerator between 1°C and 10°C. 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
>20°C.
8.1.2.3 If samples are shipped after collection at >20°C with no chilling, the
sample will not maintain the temperature during shipment at <20°C.
8.1.2.4 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 20°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 sample temperature.
Samples that were not collected the same day they were received, and that are received at
>20°C or frozen, or samples that the laboratory has determined exceeded >20°C or froze
during shipment, must be rejected. After receipt, samples must be stored at the laboratory
between 1°C and 10°C, and not frozen, until processed.
8.1.4 Suggestions on measuring sample temperature. Given the importance of maintaining
sample temperatures for Cryptosporidium 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:
8.1.4.1 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
Farmer cat. no. U-06252-20.
11 December 2005
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Method 1622 - Cryptosporidium
8.1.4.2 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. The thermometer vial is appropriate for use with filtered
samples not bulk samples. Example product: Eagle-Picher Sentry
Temperature Vial 3TR-40CS-F or 3TR-40CS.
8.1.4.3 iButton. Measures the sample temperature during shipment and upon
receipt. 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 iButton should be placed in a temperature sample,
rather than placed loose in the cooler, or attached to the sample container.
This option is appropriate for use with both filtered and bulk samples.
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/.
8.1.4.4 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 provides an indication of sample
temperature to verify that the sample temperature is acceptable. This
option is appropriate for use with both filtered and bulk samples. Example
product: Cole Farmer cat. no. U-90316-00.
8.1.4.5 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. This
option is appropriate for use with both filtered and bulk samples. Example
product: Cole Farmer 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 20.9) for more
information.
g 2 Sample holding times. Samples must be processed or examined within each of the holding times
specified in Sections 8.2.1 through 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 abreakdown 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
elution, concentration, and purification (Sections 12.2.6 through 13.3.3.11) 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.
December 2005 12
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Method 1622 - Cryptosporidium
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 (DIG) microscopy
examination and characterization should be performed immediately after staining is
complete, laboratories have up to 168 hours (7 days) from the completion of sample
staining to perform the examination and verification 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.
g 3 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. 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 suspensions must be stored between l°Cand 10°C, until
ready to use; do not allow to freeze.
9.0 Quality Control
g •] Each laboratory that uses this method is required to operate a formal quality assurance (QA)
program that addresses and documents data quality, instrument and equipment maintenance and
performance, reagent quality and performance, analyst training and certification, and records
storage and retrieval. General requirements and recommendations for QA and quality control
(QC) procedures for microbiology laboratories are provided in References 20.8, 20.9, 20.10. The
minimum analytical requirements of this program consist of an initial demonstration of laboratory
capability (IDC) through performance of the initial precision and recovery (IPR) test (Section
9.4), and ongoing demonstration of laboratory capability and method performance through the
matrix spike (MS) test (Section 9.5.1), the method blank test (Section 9.6), the ongoing precision
and recovery (OPR) test (Section 9.7), staining controls (Section 14.1 and 15.2.1), and analyst
verification tests (Section 10.6). 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 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 9.1.2.1) and all QC
acceptance criteria are met (see guidance on the use of multiple labeling reagents in
Section 7.6).
13 December 2005
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Method 1622 - Cryptosporidium
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.
9.1.2.1 Method modification validation/equivalency demonstration requirements
9.1.2.1.1 Method modifications at a single laboratory. Each
time a modification is made to this method for use in a
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) 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 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.
9.1.2.1.2 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). 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 9.1.2.1.1. If the modified
method involves changes that cannot be adequately
evaluated through these tests, additional tests may be
required to demonstrate acceptability.
9.1.2.2 The laboratory is required to maintain records of modifications made to
this method. These records include the following, at a minimum:
9.1.2.2.1 The names, titles, addresses, and telephone numbers of
the analyst(s) who performed the analyses and
December 2005 14
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Method 1622 - Cryptosporidium
modification, and of the quality control officer who
witnessed and will verify the analyses and modification.
9.1.2.2.2 A listing of the analyte(s) measured (Cryptosporidium).
9.1.2.2.3 A narrative stating reason(s) for the modification.
9.1.2.2A 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)
9.1.2.2.5 Data that will allow an independent reviewer to validate
each determination by tracing the following processing
and analysis steps leading to the final result:
(a) Sample numbers and other identifiers
(b) Source of spiking suspensions, as well as lot
number and date received (Section 7.10)
(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 12.2.5.2)
(g) Filtration and elution dates and times
(h) Pellet volume, resuspended concentrate volume,
resuspended concentrate volume transferred to
IMS, and all calculations required to verify the
percent of concentrate examined (Section 13.2)
(i) Purification completion dates and times (Section
13.3.3.11)
(j) Staining completion dates and times (Section
14.10)
(k) Staining control results (Section 15.2.1)
(1) 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. The frequency of the MS test is described in Section 9.1.8 and the
procedures are described in Section 9.5.1.
9.1.4 Analysis of method blanks is required to demonstrate freedom from contamination. The
frequency of the analysis of method blanks is described in Section 9.1.7 and 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. Frequency of
OPR samples is described in Section 9.1.7 and the procedures are described in Section
9.7.
15 December 2005
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Method 1622 - Cryptosporidium
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 9.5.1.4 and 9.7.6.
9.1.7 The laboratory shall analyze one method blank (Section 9.6) and one OPR sample
(Section 9.7) each week (7 day or 168 hours time period which begins with processing
the OPR) in 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 one week (7 day or 168
hours) period.
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 from each source analyzed. 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.
g 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
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.
g 3 Microscope adjustment and calibration —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, method blanks, OPRs,
field samples, and MS/MSDs.
g 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 the four reagent water samples spiked with -100-500 oocysts.
9.4.1.1 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
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Method 1622 - Cryptosporidium
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 unacceptable IPR results if the test is performed across
multiple days, analysts, and /or reagent lots.
9.4.1.2 If more than one modification will be used for filtration and/or separation
of samples, a separate set of IPR samples must be prepared for each
modification.
9.4.1.3 The set of four IPR samples must be accompanied by analysis of an
acceptable method blank (Section 9.6).
9.4.2 Calculate the percent recovery (R) using the following equation:
N
R= 100x
T
where:
R = the percent recovery
N = the number of oocysts counted
T = the number of oocysts spiked
This calculation assumes that the total volume spiked was processed and examined.
9.4.3 Using percent recovery (R) of the four analyses, calculate the mean percent recovery and
the relative standard deviation (RSD) of the recoveries for Cryptosporidium. The RSD is
the standard deviation divided by the mean, times 100.
9.4.4 Compare the mean and RSD to the corresponding method performance acceptance
criteria for initial precision and recovery in Table 3 in Section 21.0. If the mean and RSD
for recovery meet the acceptance criteria, system performance is acceptable and analysis
of blanks and samples may begin. If the mean or RSD 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 9.7.5), correct the problem
and repeat the IPR test (Section 9.4.1).
9.4.5 Examine and document the IPR slides following the procedure in Section 15.0. The first
three Cryptosporidium oocysts identified in each IPR sample must be characterized (size,
shape, DAPI category, and DIG category) and documented on the examination form, as
well as any additional comments on organisms appearance, if notable.
9.4.6 Using 200X to 400X magnification, more than 50% of the oocysts must appear
undamaged and morphologically intact; otherwise, the organisms in the spiking
suspension maybe of unacceptable quality or the analytical process maybe damaging
the organisms. If the quality of the organisms on the IPR test slides is unacceptable,
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
unprocessed organisms appear undamaged and morphologically intact under DIG,
determine the step or reagent that is causing damage to the organisms. Correct the
problem (see Section 9.7.5) and repeat the IPR test.
g 5 Matrix spike (MS) and matrix spike duplicate (MSD)
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Method 1622 - Cryptosporidium
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 recovery. The MS and field
sample must be that was collected from the same sampling location as split samples or as
samples sequentially collected immediately after one another. The MS sample volume
analyzed must be within 10% of the field sample volume. The MS shall be analyzed
according to the frequency in Section 9.1.8.
9.5.1.1 Analyze an unspiked field sample according to the procedures in Sections
12.0 to 15.0. Using the spiking procedure in Section 11.4 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 number of organisms as that used in the IPR or
OPR tests (Sections 9.4 and 9.7).
9.5.1.2 Calculate the percent recovery (R) using the following equation.
N5P - N5
R= 100x
where
R is the percent recovery
Nsp is the number of oocysts counted in the spiked sample
Ns is the number of oocysts counted in the unspiked sample
T is the true value of the oocysts spiked
9.5.1.3 Compare the recovery with the acceptance criteria in Table 3 in Section
21.0.
NOTE: Some sample matrices may prevent the acceptance criteria in Tables 3 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.
9.5.1.4 As part of the QA program forthe laboratory, method precision for
samples should be assessed and records maintained. After the analysis of
five samples, the laboratory should calculate the mean 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 Tier 2 or 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 9.1.2.1.2). At the same time the laboratory spikes and analyzes the
second field sample aliquot in Section 9.5.1.1, the laboratory shall spike and analyze 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.
9.5.2.1 Calculate the percent recovery (R) using the equation in Section 9.5.1.2.
December 2005 18
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Method 1622 - Cryptosporidium
9.5.2.2 Calculate the mean of the number of oocysts in the MS and MSD
(= [MS+MSD]/2).
9.5.2.3 Calculate the relative percent difference (RPD) of the recoveries using the
following equation:
RPD= 100 x
NMS - NMSD
where
RPD is the relative percent difference
NMS is the number of oocysts counted in the MS
NMSD is the number of oocysts counted in the MSD
Xmean is the mean number of oocysts counted in the MS and MSD
9.5.2.4 Compare the mean MS/MSD recovery and RPD with the acceptance
criteria in Table 3 in Section 21.0.
g g Method blank (negative control sample, laboratory blank)—Reagent water blanks are routinely
analyzed to demonstrate freedom from contamination. Analyze the blank immediately 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
method blank per week (Section 9.1.7) according to the procedures in Sections 12.0 to
15.0. A method blank must be analyzed each week (7 day or 168 hours time period that
begins with processing the OPR) in which samples are analyzed if 20 or fewer field
samples are analyzed during this period. If more than 20 samples are analyzed in a week
(7 days or 168 hours), process and analyze one reagent water method blank for every 20
samples.
9.6.2 Actions
9.6.2.1 If Cryptosporidium oocysts or potentially interfering organisms or
materials that may be misidentified as oocysts are not found in the
method blank, the method blank test is acceptable and analysis of samples
may proceed.
9.6.2.2 If Cryptosporidium oocysts (as defined in Section 3) or any potentially
interfering organism or materials that may be misidentified as oocysts are
found in the method blank, the method blank test is unacceptable. Any
field sample in a batch associated with an unacceptable method blank is
assumed to be contaminated and should be recollected. 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.
g 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 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 should samples be analyzed.
9.7.1 Examine the slide from the OPR prior to analysis of samples from the same batch.
19 December 2005
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Method 1622 - Cryptosporidium
9.7.1.1 Using 200X to 400X magnification, more than 50% of the oocysts must
appear undamaged and morphologically intact; otherwise, the organisms
in the spiking suspension maybe of unacceptable quality or the analytical
process 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 DIG,
determine the step or reagent that is causing damage to the organisms.
Correct the problem and repeat the OPR test.
9.7.1.2 Identify and enumerate each organism using epifluorescence microscopy.
The first three Cryptosporidium oocysts identified in the OPR sample
must be examined using FITC, DAPI, and DIG, as per Section 15.2, and
the detailed characteristics (size, shape, DAPI category, and DIG
category) reported on the Cryptosporidium report form, as well as any
additional comments on organism appearance, if notable.
9.7.2 Calculate the percent recovery (R) using the following equation:
N
R= 100x
T
where:
R = the percent recovery
N = the number of oocysts detected
T = the number of oocysts spiked
9.7.3 Compare the recovery with the acceptance criteria for ongoing precision and recovery in
Table 3 in Section 21.0.
9.7.4 Actions
9.7.4.1 If the recoveries for Cryptosporidium meet the acceptance criteria, system
performance is acceptable and analysis of samples may proceed.
9.7.4.2 If the recovery for Cryptosporidium falls outside of the criteria, 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.
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 recoveries meet the acceptance criteria.
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.
9.7.5.1 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 DIG, 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 9.7.5.2).
9.7.5.2 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),
December 2005 20
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Method 1622 - Cryptosporidium
check Kohler illumination, and check the fluorescence of the fluorescein-
labeled monoclonal antibodies (Mabs) and 4',6-diamidino-2-phenylindole
(DAPI). If results are unacceptable, re-examine a previously-prepared
positive staining control to determine whether the problem is associated
with the microscope or the antibody stain.
9.7.5.3 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 processing the sample through the IMS, staining, and
examination procedures in Sections 13.3 through 15.0. Recoveries should
be greater than 70%.
9.7.5.4 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 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 mean 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%.
g g 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.
g 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 by this method.
910 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
•101 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.
102 Using the manuals provided with the microscope, all analysts must familiarize themselves with
operation of the microscope.
103 Microscope adjustment and calibration (adapted from Reference 20.10)
10.3.1 Preparations for adjustment
10.3.1.1 The microscopy portion of this procedure depends upon proper alignment
and adjustment of very sophisticated optics. Without proper alignment
and adjustment, the microscope will not function at maximal efficiency,
and reliable identification and enumeration of oocysts will not be
21 December 2005
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Method 1622 - Cryptosporidium
possible. Consequently, it is imperative that all portions of the
microscope from the light sources to the oculars are properly adjusted.
10.3.1.2 While microscopes from various vendors are configured somewhat
differently, they all operate on the same general physical principles.
Therefore, slight deviations or adjustments may be required to make the
procedures below work for a particular instrument.
10.3.1.3 The sections below assume that the mercury bulb has not exceeded time
limits of operation, that the lamp socket is connected to the lamp house,
and that the condenser is adjusted to produce Kohler illumination.
10.3.1.4 Persons with astigmatism should always wear contact lenses or glasses
when using the microscope.
CAUTION: In the procedures below, do not touch the quartz portion of the mercury
bulb with your bare fingers. Finger oils can cause rapid degradation of the quartz and
premature failure of the bulb.
WARNING: Never look at the ultraviolet (UV) light from the mercury lamp, lamp
house, or the UV image without a barrier filter in place. UV radiation can cause serious
eye damage.
10.3.2 Epifluorescent mercury bulb adjustment: The purpose of this procedure is to ensure even
field illumination. This procedure must be followed when the microscope is first used,
when replacing bulbs, and if problems such as diminished fluorescence or uneven field
illumination are experienced.
10.3.2.1 Remove the diffuser lens between the lamp and microscope or swing it
out of the transmitted light path.
10.3.2.2 Using a prepared microscope slide, adjust the focus so the image in the
oculars is sharply defined.
10.3.2.3 Replace the slide with a business card or a piece of lens paper.
10.3.2.4 Close the field diaphragm (iris diaphragm in the microscope base) so only
a small point of light is visible on the card. This dot of light indicates the
location of the center of the field of view.
10.3.2.5 Mount the mercury lamp house on the microscope without the UV
diffuser lens in place and turn on the mercury bulb.
10.3.2.6 Remove the objective in the light path from the nosepiece. A primary
(brighter) and secondary image (dimmer) of the mercury bulb arc should
appear on the card after focusing the image with the appropriate
adjustment.
10.3.2.7 Using the lamp house adjustments, adjust the primary and secondary
mercury bulb images so they are side by side (parallel to each other) with
the transmitted light dot in between them.
10.3.2.8 Reattach the objective to the nosepiece.
10.3.2.9 Insert the diffuser lens into the light path between the mercury lamp
house and the microscope.
10.3.2.10 Turn off the transmitted light and replace the card with a slide of
fluorescent material. Check the field for even fluorescent illumination.
Adjustment of the diffuser lens probably will be required. Additional
slight adjustments as in Section 10.3.2.7 above may be required.
December 2005 22
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Method 1622 - Cryptosporidium
10.3.2.11 Maintain a log of the number of hours the UV bulb has been used. Never
use the bulb for longer than it has been rated. Fifty-watt bulbs should not
be used longer than 100 hours; 100-watt bulbs should not be used longer
than 200 hours.
10.3.3 Transmitted bulb adjustment: The purpose of this procedure is to center the filament and
ensure even field illumination. This procedure must be followed when the bulb is
changed.
10.3.3.1 Remove the diffuser lens between the lamp and microscope or swing it
out of the transmitted light path.
10.3.3.2 Using a prepared microscope slide and a 40X (or similar) objective,
adjust the focus so the image in the oculars is sharply defined.
10.3.3.3 Without the ocular or Bertrand optics in place, view the pupil and
filament image at the bottom of the tube.
10.3.3.4 Focus the lamp filament image with the appropriate adjustment on the
lamp house.
10.3.3.5 Similarly, center the lamp filament image within the pupil with the
appropriate adjustment(s) on the lamp house.
10.3.3.6 Insert the diffuser lens into the light path between the transmitted lamp
house and the microscope.
10.3.4 Adjustment of the interpupillary distance and oculars for each eye: These adjustments
are necessary so that eye strain is reduced to a minimum, and must be made for each
individual using the microscope. Section 10.3.4.2 assumes use of a microscope with both
oculars adjustable; Section 10.3.4.3 assumes use of a microscope with a single adjustable
ocular. The procedure must be followed each time an analyst uses the microscope.
10.3.4.1 Interpupillary distance
10.3.4.1.1 Place a prepared slide on the microscope stage, turn on
the transmitted light, and focus the specimen image using
the coarse and fine adjustment knobs.
10.3.4.1.2 Using both hands, move the oculars closer together or
farther apart until a single circle of light is observed
while looking through the oculars with both eyes. Note
interpupillary distance.
10.3.4.2 Ocular adjustment for microscopes capable of viewing a photographic
frame through the viewing binoculars: This procedure assumes both
oculars are adjustable.
10.3.4.2.1 Place a card between the right ocular and eye keeping
both eyes open. Adjust the correction (focusing) collar on
the left ocular by focusing the left ocular until it reads the
same as the interpupillary distance. Bring an image
located in the center of the field of view into as sharp a
focus as possible.
10.3.4.2.2 Transfer the card to between the left eye and ocular.
Again keeping both eyes open, bring the same image into
as sharp a focus for the right eye as possible by adjusting
the ocular correction (focusing) collar at the top of the
right ocular.
23 December 2005
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Method 1622 - Cryptosporidium
10.3.4.3 Ocular adjustment for microscopes without binocular capability: This
procedure assumes a single focusing ocular. The following procedure
assumes that only the right ocular is capable of adjustment.
10.3.4.3.1 Place a card between the right ocular and eye keeping
both eyes open. Using the fine adjustment, focus the
image for the left eye to its sharpest point.
10.3.4.3.2 Transfer the card to between the left eye and ocular.
Keeping both eyes open, bring the image for the right eye
into sharp focus by adjusting the ocular collar at the top
of the ocular without touching the coarse or fine
adjustment.
10.3.5 Calibration of an ocular micrometer: This section assumes that a reticle has been
installed in one of the oculars by a microscopy specialist and that a stage micrometer is
available for calibrating the ocular micrometer (reticle). Once installed, the ocular reticle
should be left in place. The more an ocular is manipulated the greater the probability is
for it to become contaminated with dust particles. This calibration should be done for
each objective in use on the microscope. If there is a top lens on the microscope, the
calibration procedure must be done for the respective objective at each top lens setting.
The procedure must be followed when the microscope is first used and each time the
objective is changed.
10.3.5.1 Place the stage micrometer on the microscope stage, turn on the
transmitted light, and focus the micrometer image using the coarse and
fine adjustment knobs for the objective to be calibrated. Continue
adjusting the focus on the stage micrometer so you can distinguish
between the large (0.1 mm) and the small (0.01 mm) divisions.
10.3.5.2 Adjust the stage and ocular with the micrometer so the "0" line on the
ocular micrometer is exactly superimposed on the "0" line on the stage
micrometer.
10.3.5.3 Without changing the stage adjustment, find a point as distant as possible
from the two 0 lines where two other lines are exactly superimposed.
10.3.5.4 Determine the number of ocular micrometer spaces as well as the number
of millimeters on the stage micrometer between the two points of
superimposition. For example: Suppose 48 ocular micrometer spaces
equal 0.6 mm.
10.3.5.5 Calculate the number of mm/ocular micrometer space. For example:
0.6mm 0.0125mm
48 ocular micrometer spaces ocular micrometer space
10.3.5.6 Because most measurements of microorganisms are given in u,m rather
than mm, the value calculated above must be converted to u.m by
multiplying it by 1000 u.m/mm. For example:
0.0125mm 1,000 urn 12.5 urn
x =
ocular micrometer space mm ocular micrometer space
December 2005 24
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Method 1622 - Cryptosporidium
10.3.5.7
Follow the procedure below for each objective. Record the information as
shown in the example below and keep the information available at the
microscope.
Item
no.
1
2
3
4
Objective
power
10X
20X
40X
100X
Description
N.A.3=
N.A.=
N.A.=
N.A.=
No. of ocular
micrometer
spaces
No. of stage
micrometer
mm1
|jm/ocular
micrometer
space2
11000 |jm/mm
2(Stage micrometer length in mm x (1000 |jm/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 Kohler illumination: This section assumes that Kohler illumination will be established
for only the 100X oil DIG objective that will be used to identify internal morphological
characteristics in Cryptosporidium oocysts. If more than one objective is to be used for
DIG, then each time the objective is changed, Kohler illumination must be reestablished
for the new objective lens. Previous sections have adjusted oculars and light sources.
This section aligns and focuses the light going through the condenser underneath the
stage at the specimen to be observed. If Kohler illumination is not properly established,
then DIG 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.
10.3.6.1 Place a prepared slide on the microscope stage, place oil on the slide,
move the 100X oil objective into place, turn on the transmitted light, and
focus the specimen image using the coarse and fine adjustment knobs.
10.3.6.2 At this point both the radiant field diaphragm in the microscope base and
the aperture diaphragm in the condenser should be wide open. Now close
down the radiant field diaphragm in the microscope base until the lighted
field is reduced to a small opening.
10.3.6.3 Using the condenser centering screws on the front right and left of the
condenser, move the small lighted portion of the field to the center of the
visual field.
10.3.6.4 Now look to see whether the leaves of the iris field diaphragm are sharply
defined (focused) or not. If they are not sharply defined, then they can be
focused distinctly by changing the height of the condenser up and down
with the condenser focusing knob while you are looking through the
binoculars. Once you have accomplished the precise focusing of the
radiant field diaphragm leaves, open the radiant field diaphragm until the
leaves just disappear from view.
10.3.6.5 The aperture diaphragm of the condenser should 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.
25
December 2005
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Method 1622 - Cryptosporidium
10.3.6.6 After completing the adjustment of the aperture diaphragm in the
condenser, return the ocular to its tube and proceed with the adjustments
required to establish DIG.
104 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
10.4.3.1 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.
10.4.3.2 Repeat the procedure using a new, dry Q-tip.
10.4.3.3 Repeat Sections 10.4.3.1 and 10.4.3.2.
10.4.3.4 Remove the ocular and repeat the cleaning procedure on the bottom lens
of the ocular.
10.4.4 Protocol for cleaning objective lenses
10.4.4.1 Wipe 100X oil objective with lens paper to remove the bulk of the oil
from the objective.
10.4.4.2 Hold a new Q-tip dampened with MCS at a 45° angle on the objective
and twirl.
10.4.4.3 Repeat Sections 10.4.4.2 with a new, dry Q-tip.
10.4.4.4 Repeat Sections 10.4.4.2 and 10.4.4.3.
10.4.4.5 Clean all objectives whether they are used or not.
10.4.5 Protocol for cleaning light source lens and filters
10.4.5.1 Using a Kimwipe dampened with microscope cleaning solution, wipe off
the surface of each lens and filter.
10.4.5.2 Repeat the procedure using a dry Kimwipe.
10.4.5.3 Repeat Sections 10.4.5.1 and 10.4.5.2.
10.4.6 Protocol for cleaning microscope stage
10.4.6.1 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
10.4.8.1 Perform Sections 10.4.2, 10.4.3, 10.4.4, 10.4.5 and 10.4.7 after each
microscope session.
10.4.8.2 Perform complete cleaning each week.
105 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 by FA, 4',6-diamidino-2-
phenylindole (DAPI), and DIG that the analysts (Section 22.2) determine are accurate
(Section 15.2).
December 2005 26
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Method 1622 - Cryptosporidium
10.5.2 Similarly, take color photographs of interfering organisms and materials by FA, DAPI,
and DIG that the analysts believe are not Cryptosporidium oocysts. Quantify the size,
shape, microscope settings, and other characteristics that can be used to differentiate
oocysts from interfering debris and that will result in accurate identification of positive
or negative organisms.
106 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, this method shall rely upon the ability of the analyst
for identification and enumeration of oocysts. The goal of analyst verification is to encourage
comparison and discussion among analysts to continually refine the consistency of
characterizations between analysts.
10.6.1 At least monthly when microscopic examinations are being performed, the laboratory
shall prepare a slide containing 40 to 200 oocysts. More than 50% of the oocysts must be
DAPI positive and undamaged under DIG.
10.6.2 Each analyst shall determine the total number of oocysts detected by FITC on the entire
slide meeting the criteria in 10.6.1. For the same 10 oocysts, each analyst shall determine
the DAPI category (DAPI negative, DAPI positive internal intense blue and DAPI
positive number of nuclei) and the DIG category (empty, containing amorphous
structures, or containing identifiable internal structures) of each. The DAPI/DIC
comparisons may be performed on the slide prepared in 10.6.1, OPR slide, MS slide, or a
positive staining control slide.
10.6.3 Requirements for laboratories with multiple analysts
10.6.3.1 The total number of oocysts determined by each analyst (Section 10.6.2.)
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 10.6.1 to 10.6.2). It is recommended that the DAPI
and DIG categorization of the same 10 oocysts occur with all analysts at
the same time, i.e. each analyst determines the categorizations
independently, then the differences in the DAPI and DIG categorizations
among analysts are discussed and resolved, and these resolutions
documented. Alternatively, organism coordinates may be recorded for
each analyst to locate and categorize the organisms at different times.
Differences among analysts must be discussed and resolved.
10.6.3.2 Document the date, name(s) of analyst(s), number of total oocysts, and
DAPI and DIG categories determined by the analyst(s), whether the test
was passed/failed and the results of attempts before the test was passed.
10.6.3.3 Only after an analyst has passed the criteria in Section 10.6.3, may
oocysts 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 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. These laboratories should also coordinate with
other laboratories to share slides and compare counts.
27 December 2005
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Method 1622 - Cryptosporidium
11.0 Oocyst Suspension Enumeration and Sample Spiking
•j 1 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) and monitor method performance. 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 20.11). Guidance on preparing spiking
suspensions using a flow cytometer is provided in Section 11.2. Manual enumeration procedures
are provided in Section 11.3. The procedure for spiking bulk samples in the laboratory is provided
in Section 11.4.
•j 1 2 Flow cytometry enumeration guidelines. Although it is unlikely that many laboratories
performing Method 1622 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
formalin-fixed.
11.2.2 Spiking suspensions should be prepared using Cryptosporidium parvum oocysts <3
months 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 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
fluorescence and DIG 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.
•j 1 3 Manual enumeration procedures. Two sets of manual enumerations are required per organism
before purified Cryptosporidium oocyst (Section 7.10.2) received from suppliers can be used to
December 2005 28
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Method 1622 - Cryptosporidium
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 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
before proceeding (these criteria are based on the pooled RSDs of 105 manual
Cryptosporidium enumerations 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
11.3.3.1 Purified, concentrated stock suspensions (Sections 7.10.2.1 and 7.10.2.2)
must be diluted and enumerated before the diluted suspensions are used
to spike samples in the laboratory. Stock suspensions should be diluted
with reagent water/Tween® 20 , 0.01% (Section 7.10.2.3), to a
concentration of 20 to 50 organisms per large hemacytometer square
before proceeding to Section 11.3.3.2.
11.3.3.2 Apply a clean hemacytometer coverslip (Section 6.4.5) to the
hemacytometer and load the hemacytometer chamber with 10 uL of
vortexed suspension per chamber. If this operation has been properly
executed, the liquid should amply fill the entire chamber without bubbles
or overflowing into the surrounding moats. Repeat this step with a clean,
dry hemacytometer and coverslip if loading has been incorrectly
performed. See Section 11.3.3.13, below, for the hemacytometer cleaning
procedure.
11.3.3.3 Place the hemacytometer on the microscope stage and allow the oocysts
to settle for 2 minutes. Do not attempt to adjust the coverslip, apply clips,
or in any way disturb the chamber after it has been filled.
11.3.3.4 Use 200X magnification.
11.3.3.5 Move the chamber so the ruled area is centered underneath the objective.
11.3.3.6 Move the objective close to the coverslip while watching it from the side
of the microscope, rather than through the microscope.
11.3.3.7 Focus up from the coverslip until the hemacytometer ruling appears.
11.3.3.8 At each of the four corners of the chamber is a 1-square-mm area divided
into 16 squares in which organisms are to be counted (Figure 1).
Beginning with the top row of four squares, count with a hand-tally
counter in the directions indicated in Figure 2. Avoid counting organisms
twice by counting only those touching the top and left boundary lines.
Count each square millimeter in this fashion.
29 December 2005
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Method 1622 - Cryptosporidium
11.3.3.9 Use the following formula to determine the number of organisms per uL
of suspension:
number of ._ dilution . 3 number of
organisms counted factor organisms
number of mm2 „ „ „ , ,
counted 1mm 1 1^L ^L
11.3.3.10 Record the result on a hemacytometer data sheet.
11.3.3.11 A total of six different hemacytometer chambers must be loaded, counted,
and averaged for each suspension to achieve optimal counting accuracy.
11.3.3.12 Based on the hemacytometer counts, the stock suspension should be
diluted to a final concentration of between 8 to 12 organisms per uL;
however, ranges as great as 5 to 15 organisms per uL 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 11.3.5), then the
stock suspensions should be diluted in reagent water.
To calculate the volume (in uL) of stock suspension required per uL of
reagent water (or reagent water/Tween® 20 , 0.01%), use the following
formula:
required number of organisms
volume of stock suspension (uL) required =
number of organisms/ uL of stock suspension
If the volume is less than 10 u.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 u.L, use the following formula:
number of organisms required x 10uL
total volume (uL) =
predicted number of organisms per 10uL (8 to 12)
To calculate the volume of reagent water (or reagent water/Tween® 20 ,
0.01%) needed, use the following formula:
reagent water volume (uL) = total volume (uL) - stock suspension volume required (uL)
11.3.3.13 After each use, the hemacytometer and coverslip must be cleaned
immediately to prevent the organisms and debris from drying on it. Since
this apparatus is precisely machined, abrasives cannot be used to clean it,
as they will disturb the flooding and volume relationships.
11.3.3.13.1 Rinse the hemacytometer and cover glass first with tap
water, then 70% ethanol, and finally with acetone.
December 2005 30
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Method 1622 - Cryptosporidium
11.3.3.13.2 Dry and polish the hemacytometer chamber and cover
glass with lens paper. Store it in a secure place.
11.3.3.14 Several factors are known to introduce errors into hemacytometer counts,
including:
• Inadequate mixing of suspension before flooding the chamber
• Irregular filling of the chamber, trapped air bubbles, dust, or oil
on the chamber or coverslip
• Total number of organisms counted is too low to provide
statistical confidence in the result
• Error in recording tally
• Calculation error; failure to consider dilution factor, or area
counted
• Inadequate cleaning and removal of organisms from the previous
count
• Allowing filled chamber to sit too long, so that the chamber
suspension dries and concentrates.
11.3.4 Enumerating spiking suspensions using a hemacytometer chamber
NOTE: Spiking suspensions enumerated using a hemacytometer chamber must be used
within 24 hours of enumeration.
11.3.4.1 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.
11.3.4.2 To an appropriate-size beaker containing a stir bar, add enough spiking
suspension to perform all spike testing and the enumeration as described.
The liquid volume and beaker relationship should be such that a spinning
stir bar does not splash the sides of the beaker, the stir bar has unimpeded
rotation, and there is enough room to draw sample from the beaker with a
10-(iL micropipette without touching the stir bar. Cover the beaker with a
watch glass or petri dish to prevent evaporation between sample
withdrawals.
11.3.4.3 Allow the beaker contents to stir for a minimum of 30 minutes before
beginning enumeration.
11.3.4.4 While the stir bar is still spinning, remove a 10-(iL 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 uL (Section
11.3.3.12). 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 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
11.3.3.14 for factors that may introduce errors.
31 December 2005
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Method 1622 - Cryptosporidium
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.
11.3.5.1 Prepare well slides for sample screening and label the slides.
11.3.5.2 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.
11.3.5.3 Remove a 10-^iL aliquot from the spiking suspension and apply it to the
center of a well.
11.3.5.4 Before removing subsequent aliquots, cap the tube and gently invert it
three times to ensure that the oocysts are in suspension.
11.3.5.5 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.
11.3.5.6 Positive and negative controls must be prepared.
11.3.5.6.1 For the positive control, pipette 10 (iL of positive antigen
or 200 to 400 intact oocysts to the center of a well and
distribute evenly over the well area.
11.3.5.6.2 For the negative control, pipette 50 (iL of PBS onto the
center of a well and spread it over the well area with a
pipette tip.
11.3.5.6.3 Air-dry the control slides.
11.3.5.7 Follow the manufacturer's instructions (Section 7.6) in applying the stain
to the slide.
11.3.5.8 Place the slides in a humid chamber in the dark and incubate according to
manufacturer's directions. The humid chamber consists of a tightly sealed
plastic container containing damp paper towels on top of which the slides
are placed.
11.3.5.9 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® Cryptosporidium/Giardia stain (Section 7.6.1), do not
allow slides to dry completely.
11.3.5.10 Add mounting medium (Section 7.8) to each well.
11.3.5.11 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.
December 2005 32
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Method 1622 - Cryptosporidium
11.3.5.12 Record the date and time that staining was completed. If slides will not be
read immediately, store in a humid chamber in the dark between 1°C and
10°C until ready for examination.
11.3.5.13 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 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.
11.3.6.1 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.
11.3.6.2 Place foil around the bottoms of the 100 x 15 mm petri dishes.
11.3.6.3 Filter-sterilize (Section 6.19) approximately 10 mL of PBS (Section
7.4.2.1). Dilute detection reagent (Section 7.6) 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.
11.3.6.4 Label the tops of foil-covered, 60 x 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 foil-
lined petri dishes will protect filters from light and prevent evaporation
during incubation).
11.3.6.5 Place a decontaminated and cleaned filter holder base (Section 6.4.8.1)
into each of the three ports of the vacuum manifold (Section 6.4.8.2).
11.3.6.6 Pour approximately 10 mL of 0.01% Tween® 20 into a 60 x 15 mm petri
dish.
11.3.6.7 Using forceps, moisten a 1.2-[im cellulose-acetate support membrane
(Section 6.4.8.3) 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 6.4.8.4) 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.
11.3.6.8 Add 5 mL of 0.01% Tween® 20 to each of the three filtration units and
allow to stand.
33 December 2005
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Method 1622 - Cryptosporidium
11.3.6.9 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.
11.3.6.10 Using a micropipettor, sequentially remove two, 10-uL 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 (iL 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.
11.3.6.11 Pipet 100 mL of diluted antibody to the center of the bottom of a 60 x 15
mm petri dish for each sample.
11.3.6.12 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.
11.3.6.13 Reclamp the top funnels, apply vacuum and rinse each three times, each
time with 20 mL of reagent water.
11.3.6.14 Repeat Sections 11.3.6.4 through 11.3.6.10 forthe 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-^iL 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.
11.3.6.15 Repeat Sections 11.3.6.4 through 11.3.6.10 until the 10-|j,L spiking
suspensions have been filtered. The last batch should include a 10-^iL
0.01 Tween® 20 blank control and 20 \aL of positive control antigen as a
positive staining control.
11.3.6.16 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.
11.3.6.17 To each slide, add 20 \aL of mounting medium (Section 7.8).
11.3.6.18 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.)
11.3.6.19 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
between 1°C and 10°C until ready for examination.
11.3.6.20 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
December 2005 34
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Method 1622 - Cryptosporidium
standard deviation (RSD) of the calculated mean spike dose must be
< 16% for Cryptosporidium 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.
11.3.6.21 If oocysts are detected on the filter blanks, modify the rinse procedure to
ensure that no carryover occurs and repeat enumeration.
•| •] 4 Procedure for spiking samples in the laboratory with enumerated spiking suspensions.
11.4.1 Arrange a disposable cubitainer or 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 11.4.3.1 or manufacturer's instructions for flow
cytometer-enumerated suspensions and the procedures in Section 11.4.3.2 for manually
enumerated suspensions.
11.4.3.1 For flow cytometer-enumerated suspensions (where the entire volume of
a spiking suspension tube will be used):
11.4.3.1.1 Add 400 uL of Antifoam A to 100 mL of reagent water,
and mix well to emulsify.
11.4.3.1.2 Add 500 uL of the diluted antifoam to the tube
containing the spiking suspension and vortex for 30
seconds.
11.4.3.1.3 Pour the suspension into the sample container.
11.4.3.1.4 Add 20 mL of reagent water to the empty tube, cap,
vortex 10 seconds to rinse, and add the rinsate to the
carboy.
11.4.3.1.5 Repeat this rinse using another 20 mL of reagent water.
11.4.3.1.6 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.
11.4.3.2 For manually enumerated spiking suspensions:
11.4.3.2.1 Vortex the spiking suspension(s) (Section 11.2 or Section
11.3) for a minimum of 30 seconds.
11.4.3.2.2 Rinse a pipette tip with 0.01% Tween® 20 once, then
repeatedly pipette the well-mixed spiking suspension a
minimum of five times before withdrawing an aliquot to
spike the sample.
11.4.3.2.3 Add the spiking suspension(s) to the carboy, delivering
the aliquot below the surface of the sample.
11.4.3.2.4 Record the estimated number of organisms spiked, the
date and time the sample was spiked, and the sample
35 December 2005
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Method 1622 - Cryptosporidium
volume spiked on a bench sheet. Proceed to Section
11.4.4
11.4.4 Allow the spiked sample 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 1-L PBST or
reagent water to the 10-L carboy to rinse (5 L PBST or reagent water rinse to 50-L
carboy). 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.
11.4.8 Proceed to filter disassembly.
12.0 Sample Filtration and Elution
121 A water sample is filtered according to the procedures in Section 12.2, 12.3, or 12.4. 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 (iffiltered in the field).
122 Capsule filtration (adapted from Reference 20.12). 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).
12.2.1 Flow rate adjustment
12.2.1.1 Connect the sampling system, minus the capsule, to a carboy filled with
reagent water (Figure 3).
12.2.1.2 Turn on the pump and adjust the flow rate to 2.0 L/min.
12.2.1.3 Allow 2 to 10 L of reagent water to flush the system. Adjust the pump
speed as required during this period. Turn off the pump when the flow
rate has been adjusted.
12.2.2 Install the capsule filter in the line, securing the inlet and outlet ends with the appropriate
clamps/fittings.
12.2.3 Record the sample number, sample turbidity (if not provided with the field sample),
sample type, and sample filtration start date and time on a bench sheet.
12.2.4 Filtration
12.2.4.1 Mix the sample well by shaking, add stir bar and place on stir plate. Turn
on stir plate to lowest setting needed to keep sample thoroughly mixed.
Connect the sampling system to the field carboy of sample water, or
transfer the sample water to the laboratory carboy used in Section
12.2.1.1. 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.
December 2005 36
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Method 1622 - Cryptosporidium
12.2.4.2
12.2.4.3
12.2.4.4
12.2.5
12.2.6
12.2.4.5
12.2.4.6
Disassembly
12.2.5.1
12.2.5.2
12.2.5.3
Elution
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.
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.
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].)
Turn off stir plate; add 1 L PBST or reagent water rinse (to 10-L carboy)
or 5 L PBST or reagent water rinse (to 50-L carboy). Swirl or shake the
carboy to rinse down the side walls.
Reconnect to pump, turn on pump and allow pump to pull all water
through filter; turn off pump.
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 from the filter. Restart the pump and
allow as much water to drain as possible. Turn off the pump.
Based on the water level in the graduated container and Vz-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.
Loosen the outlet fitting, then cap the inlet and outlet fittings.
NOTE: The laboratory must complete the elution, concentration, and purification
(Sections 12.2.6 through 13.3.3.11) in oneworkday. 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.
12.2.6.1
Setup
12.2.6.1.1
12.2.6.1.2
12.2.6.1.3
Assemble the laboratory shaker with the clamps aligned
vertically so that the filters will be aligned horizontally.
Extend the clamp arms to their maximum distance from
the horizontal shaker rods to maximize the shaking
action.
Prepare sufficient quantity of elution buffer to elute all
samples that are associated with the OPR/MB which used
that batch of elution buffer. Elution may require up to
275 mL of buffer per sample.
Designate at least one 250-mL conical centrifuge tube for
each sample and label with the sample number.
37
December 2005
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Method 1622 - Cryptosporidium
12.2.6.2
Elution
12.2.6.2.1
12.2.6.2.2
12.2.6.2.3
12.2.6.2.4
12.2.6.2.5
12.2.6.2.6
12.2.6.2.7
Record the elution date and time on the bench sheet.
Using a ring stand or other means, clamp each capsule in
a vertical position with the inlet end up.
Remove the inlet cap, pour elution buffer through the
inlet fitting, and allow the liquid level to stabilize.
Sufficient elution buffer must be added to cover the
pleated white membrane with buffer solution or elution
buffer may be measured to ensure the use of one 250-mL
centrifuge tube. Replace the inlet cap.
Securely clamp the capsule in one of the clamps on the
laboratory shaker with the bleed valve positioned at the
top on a vertical axis (in the 12 o'clock position). Turn on
the shaker and set the speed to maximum (approximately
900 rpm or per manufacturer's instructions). Agitate the
capsule for approximately 5 minutes. Time the agitation
using a lab timer, rather than the timer on the shaker to
ensure accurate time measurement.
Remove the filter from the shaker, remove the inlet cap,
and pour the contents of the capsule into the 250-mL
conical centrifuge tube.
Clamp the capsule vertically with the inlet end up and
add sufficient volume of elution buffer through the inlet
fitting to cover the pleated membrane. Replace the inlet
cap.
Return the capsule to the shaker with the bleed valve
positioned at the 4 o'clock position. Turn on the shaker
and agitate the capsule for approximately 5 minutes.
Remove the filter from the shaker, but leave the elution
12.3
buffer in the capsule. Re-clamp the capsule to the shaker
at the 8 o'clock position. Turn on the shaker and agitate
the capsule for a final 5 minutes.
12.2.6.2.8 Remove the filter from the shaker and pour the contents
into the 250-mL centrifuge tube. Rinse down the inside
of the capsule filter walls with reagent water or elution
buffer using a squirt bottle inserted in the inlet end of the
capsule. Invert the capsule filter over the centrifuge tube
and ensure that as much of the eluate as possible has been
transferred.
12.2.7 Proceed to Section 13.0 for concentration and separation (purification).
Sample filtration using the Filta-Max® foam filter. This procedure was validated using 50-L
sample volumes. 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 Sections 12.3.1.2 - 12.3.1.6.3 are specific
to laboratory filtration of a bulk sample. These procedures may require modification if
samples will be filtered in the field.
December 2005
38
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Method 1622 - Cryptosporidium
12.3.1 Filtration
12.3.1.1 Flow rate adjustment
12.3.1.1.1 Connect the sampling system, minus the filter housing, to
a carboy filled with reagent water.
12.3.1.1.2 Place the peristaltic pump upstream of the filter housing.
12.3.1.1.3 Turn on the pump and adjust the flow rate to 1 to 4 L per
minute.
NOTE: A head pressure of 0.5 bar (7.5 psi) is required to create flow through the filter,
and the recommended pressure of 5 bar (75 psi) should produce the flow rate of 3 to 4 L
per minute. The maximum operating pressure of 8 bar (120 psi) should not be exceeded.
12.3.1.1.4 Allow 2 to 10 L of reagent water to flush the system.
Adjust the pump speed as necessary during this period.
Turn off the pump when the flow rate has been adjusted.
12.3.1.2 Place filter module into the filter housing bolt head down and secure lid,
hand tighten housings, apply gentle pressure to create the seal between
the module and the 'O' rings in the base and the lid of the housing.
Excessive tightening is not necessary, and may shorten the life of the 'O'
rings. Tools may be used to tighten housing to the alignment marks (refer
to manufacturer's instructions). 'O' rings should be lightly greased before
use (refer to manufacturer's instructions).
12.3.1.3 Install the filter housing in the line, securing the inlet and outlet ends with
the appropriate clamps/fittings. Verify that the filter housing is installed
so that the end closest to the screw top cap is the inlet and the opposite
end is the outlet.
12.3.1.4 Record the sample number, sample turbidity (if not provided with the
field sample), and the name of the analyst filtering the sample on a bench
sheet.
12.3.1.5 Filtration
12.3.1.5.1 Connect the sampling system to the field carboy of
sample water, or transfer the sample water to the
laboratory carboy used in Section 12.3.1.1.1. If the
sample will be filtered from a field carboy, a spigot 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.
12.3.1.5.2 Place the drain end of the sampling system tubing into an
empty graduated container with a capacity greater than or
equal to the volume to be filtered. This container will be
used to determine the sample volume filtered.
Alternately, connect a flow meter downstream of the
filter, and record the initial meter reading.
12.3.1.5.3 Allow the carboy discharge tube and filter housing to fill
with sample water. Turn on the pump to start water
flowing through the filter. Verify that the flow rate is
between 1 and 4 L per min.
39 December 2005
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Method 1622 - Cryptosporidium
12.3.1.5.4 After all of the sample has passed through the filter, turn
off the pump. Allow the pressure to decrease until flow
stops.
12.3.1.6 Disassembly
12.3.1.6.1 Disconnect the inlet end of the filter housing assembly
while maintaining the level of the inlet fitting above the
level of the outlet fitting to prevent backwashing and the
loss of oocysts from the filter. Restart the pump and
allow as much water to drain as possible. Turn off the
pump.
12.3.1.6.2 Based on the water level in the graduated container or the
meter reading, record the volume filtered on a bench
sheet to the nearest quarter liter.
12.3.1.6.3 Loosen the outlet fitting, the filter housing should be
sealed with rubber plugs.
NOTE: Filters should be prevented from drying out, as this can impair their ability to
expand when decompressed.
12.3.2 Elution
12.3.2.1 The filter is eluted to wash the oocysts from the filter. This can be
accomplished using the Filta-Max® wash station, which moves a plunger
up and down a tube containing the filter and eluting solution (Section
12.3.2.2), or a stomacher, which uses paddles to agitate the stomacher bag
containing the foam filter in the eluting solution (Section 12.3.2.3). If the
Filta-Max® automatic wash station is used please see the manufacturer's
operator's guide for instructions on its use. If Filta-Max® Quick Connect
kit is used please follow manufacturer's instructions.
12.3.2.2 Filta-Max® wash station elution procedure
12.3.2.2.1 First wash
(a) Detach the removable plunger head using the tool
provided, and remove the splash guard.
(b) Place the filter membrane flat in the concentrator
base with the rough side up. Locate the concentrator
base in the jaws of the wash station and screw on the
concentrator tube (the longer of the two tubes),
creating a tight seal at the membrane. Take the
assembled concentrator out of the jaws and place on
the bench.
(c) Replace the splash guard and temporarily secure it at
least 15 cm above the end of the rack. Secure the
plunger head with the tool provided ensuring that the
lever is fully locked down.
(d) Remove the filter module from the filter housing or
transportation container. Pour excess liquid into the
assembled concentrator, then rinse the housing or
container with PBST and add the rinse to the
concentrator tube. Screw the filter module onto the
base of the plunger. Locate the elution tube base in
December 2005 40
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Method 1622 - Cryptosporidium
the jaws of the wash station and screw the elution
tube (the shorter of the two tubes) firmly in place.
(e) Pull the plunger down until the filter module sits at
the bottom of the elution tube; the locking pin (at the
top left of the wash station) should "click" to lock the
plunger in position.
(f) Remove the filter module bolt by turning the adapted
alien key (provided) in a clockwise direction (as seen
from above). Attach the steel tube to the elution tube
base.
(g) Add 600 mL of PBST to the assembled concentrator.
If more than 50 mL of liquid has been recovered from
the shipped filter module, reduce the volume of
PBST accordingly. Screw the concentrator tube onto
the base beneath the elution tube. Release the locking
pin.
NOTE: Gentle pressure on the lever, coupled with a pulling action on the locking pin
should enable the pin to be easily released.
(h) Wash the foam disks by moving the plunger up and
down 20 times. Gentle movements of the plunger are
recommended to avoid generating excess foam.
NO TE: The plunger has an upper movement limit during the wash process to prevent it
popping out of the top of the chamber.
(i) Detach the concentrator and hold it such that the
stainless steel tube is just above the level of the
liquid. Purge the remaining liquid from the elution
tube by moving the plunger up and down 5 times,
then lock the plunger in place. To prevent drips,
place the plug provided in the end of the steel tube.
(j) Prior to the second wash the eluate from the first
wash can be concentrated using the Filta-Max®
apparatus according to Section 12.3.3.2.1 or the
eluate can be decanted into a 2-L pooling beaker and
set aside.
12.3.2.2.2 Second wash
(a) Add an additional 600 mL of PBST to the
concentrator module, remove the plug from the end
of the steel tube and screw the concentrator tube back
onto the elution module base. Release the locking
pin.
(b) Wash the foam disks by moving the plunger up and
down 10 times. Gentle movements of the plunger are
recommended to avoid generating excess foam.
(c) The eluate can be concentrated using the Filta-Max®
apparatus according to Section 12.3.3.2.2 or the
eluate can be decanted into the 2-L pooling beaker
containing the eluate from the first wash and
41 December 2005
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Method 1622 - Cryptosporidium
concentrated using centrifugation, as described in
Section 12.3.3.3.
12.3.2.3 Stomacher elution procedure
12.3.2.3.1 First wash
(a) Place the filter module in the stomacher bag then use
the alien key to remove the bolt from the filter
module, allowing the rings to expand. Remove the
end caps from the stomacher bag and rinse with
PBST into the stomacher bag.
(b) Add 600 mL of PBST to stomacher bag containing
the filter pads. Place bag in stomacher and wash for 5
minutes on a normal setting.
(c) Remove the bag from the stomacher and decant the
eluate into a 2-L pooling beaker.
12.3.2.3.2 Second wash
(a) Add a second 600-mL aliquot of PBST to the
stomacher bag. Place bag in stomacher and wash for
5 minutes on a normal setting. Remove the bag from
the stomacher and decant the eluate from the
stomacher bag into the 2-L pooling beaker. Wring the
stomacher bag by hand to remove eluate from the
foam filter and add to the pooling beaker. Remove
the foam filter from the bag and using a squirt bottle,
rinse the stomacher bag with reagent water and add
the rinse to the pooling beaker.
(b) Proceed to concentration (Section 12.3.3).
12.3.3 Concentration
12.3.3.1 The eluate can be concentrated using the Filta-Max® concentrator
apparatus, which pulls most of the eluate through a membrane filter
leaving the oocysts concentrated in a small volume of the remaining
eluting solution (Section 12.3..2), or by directly centrifuging all of the
eluting solution used to wash the filter (Section 12.3.2.3).
12.3.3.2 The Filta-Max® concentrator procedure
12.3.3.2.1 Concentration of first wash
(a) If the stomacher was used to elute the sample
(Section 12.3.2.3), transfer 600 mL of eluate from the
pooling beaker to the concentrator tube. Otherwise
proceed to Step (b).
(b) Stand the concentrator tube on a magnetic stirring
plate and attach the lid (with magnetic stirrer bar).
Connect the waste bottle trap and hand or electric
vacuum pump to the valve on the concentrator base.
Begin stirring and open the tap. Increase the vacuum
using the hand pump.
NOTE: The force of the vacuum should not exceed 30 cmHg.
(c) Allow the liquid to drain until it is approximately
level with the middle of the stirrer bar then close the
valve. Remove the magnetic stirrer, and rinse it with
December 2005 42
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Method 1622 - Cryptosporidium
PBST or distilled water to recover all oocysts. Decant
the concentrate into a 50-mL tube, then rinse the
sides of the concentration tube and add the rinsate to
the 50-mL tube.
12.3.3.2.2 Concentration of second wash
(a) If the stomacher was used to elute the sample
(Section 12.3.2.3), transfer the remaining 600 mL of
eluate from the pooling beaker to the concentrator
tube. Otherwise proceed to Step (b).
(b) Add the concentrate, in the 50-mL tube, retained
from the first concentration (Section 12.3.3.2.1 (c)) to
the 600 mL of eluate from the second wash, then
repeat concentration steps from Sections 12.3.3.2.1
(b) and 12.3.3.2.1 (c). The final sample can be poured
into the same 50-mL tube used to retain the first
concentrate. Rinse the sides of the concentrator tube
with PBST and add the rinse to the 50-mL tube.
(c) Remove the magnetic stirrer. Insert the empty
concentrator module into the jaws of the wash station
and twist off the concentrator tube.
(d) Transfer the membrane from the concentrator base to
the bag provided using membrane forceps.
12.3.3.2.3 Membrane elution. The membrane can be washed
manually or using a stomacher:
• Manual wash. Add 5 mL of PBST to the bag
containing the membrane. Rub the surface of the
membrane through the bag until the membrane
appears clean. Using a pipette, transfer the eluate to a
50-mL tube. Repeat the membrane wash with another
5 mL of PBST and transfer the eluate to the 50-mL
tube. (Optional: Perform a third wash using another 5
mL of PBST, by hand-kneading an additional minute
or placing the bag on a flat-headed vortexer and
vortexing for one minute. Transfer the eluate to the
50-mL tube.)
NOTE: Mark the bag with an "X" to note which side of the membrane has the oocysts to
encourage the hand-kneading to focus on the appropriate side of the membrane.
• Stomacher wash. Add 5 mL of PBST to the bag
containing the membrane. Place the bag containing
the membrane into a small stomacher and stomach
for 3 minutes. Using a pipette transfer the eluate to a
50-mL tube. Repeat the wash two times using the
stomacher and 5-mL aliquots of PBST. (Optional:
Perform a fourth wash using another 5 mL of PBST,
by hand-kneading an additional minute or placing the
bag on a flat-headed vortexer and vortexing for one
minute. Transfer the eluate to the 50-mL tube.)
12.3.3.2.4 If the membrane filter clogs before concentration is
complete, there are two possible options for completion
43 December 2005
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Method 1622 - Cryptosporidium
of concentration. One option is replacing the membrane
as often as necessary. Filter membranes may be placed
smooth side up during the second concentration step.
Another option is concentrating the remaining eluate
using centrifugation. Both options are provided below.
• Using multiple membranes. Disassemble the
concentrator tube and pour any remaining eluate back
into the pooling beaker. Remove the membrane using
membrane forceps, placing it in the bag provided.
Place a new membrane in the concentrator tube
smooth side up, reassemble, return the eluate to the
concentrator tube, rinse the pooling beaker and add
rinse to the eluate, and continue the concentration.
Replace the membrane as often as necessary.
• Centrifuging remaining volume. Decant the
remaining eluate into a 2-L pooling beaker. Rinse the
sides of the concentrator tube and add to the pooling
beaker. Remove the filter membrane and place it in
the bag provided. Wash the membrane as described
in Section 12.3.3.2.3, then concentrate the sample as
described in Section 12.3.3.3.1.
12.3.3.3 If the Filta-Max® concentrator is not used for sample concentration, or if
the membrane filter clogs before sample concentration is complete, then
the procedures described in Section 12.3.3.3.1 should be used to
concentrate the sample. If less than 50 mL of concentrate has been
generated, the sample can be further concentrated, as described in Section
12.3.3.3.2, to reduce the volume of sample to be processed through IMS.
NOTE: The volume must not be reduced to less than 5 mL above the packed pellet. The
maximum amount of pellet that should be processed through IMS is 0.5 mL. If the packed
pellet is greater than 0.5 mL then the pellet may be subsampled as described in Section
13.2.4.
12.3.3.3.1 Centrifugation of greater than 50 mL of eluate
(a) Decant the eluate from the 2-L pooling beaker into
250-mL conical centrifuge tubes. Make sure that the
centrifuge tubes are balanced.
(b) Centrifuge the 250-mL centrifuge tubes containing
the eluate at 1500 x G for 15 minutes. Allow the
centrifuge to coast to a stop.
(c) Using a Pasteur pipette, carefully aspirate off the
supernatant to 5 mL above the pellet. If the sample is
reagent water (e.g. initial or ongoing precision and
recovery sample) extra care must be taken to avoid
aspirating oocysts during this step.
(d) Vortex each 250-mL tube vigorously until pellet is
completely resuspended. Swirl the centrifuge tube
gently to reduce any foaming after vortexing.
Combine the contents of each 250-mL centrifuge
tube into a 50-mL centrifuge tube. Rinse each of the
December 2005 44
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Method 1622 - Cryptosporidium
250-mL centrifuge tubes with PBST and add the
rinse to the 50-mL tube.
(e) Proceed to Section 12.3.3.3.2.
12.3.3.3.2 Centrifugation of less than 50 mL of eluate
(a) Centrifuge the 50-mL centrifuge tube containing the
combined concentrate at 1500 x G for 15 minutes.
Allow the centrifuge to coast to a stop. Record the
initial pellet volume (volume of solids) and the date
and time that concentration was completed on a
bench sheet.
(b) Proceed to Section 13.0 for concentration and
separation (purification).
12.3.4 Maintenance and cleaning
12.3.4.1 Maintenance of O-rings
12.3.4.1.1 Check all rubber O-rings for wear or deterioration prior
to each use and replace as necessary.
12.3.4.1.2 Lubricate the plunger head O-ring inside and out with
silicon before each use.
12.3.4.1.3 Lubricate all other O-rings (concentrator tube set, filter
housing) regularly in order to preserve their condition.
12.3.4.2 Cleaning
12.3.4.2.1 All components of the Filta-Max® system can be cleaned
using warm water and laboratory detergent. After
washing, rinse all components with oocyst and cyst free
reagent water and dry them. All O-rings should be re-
lubricated. Alternatively a mild (40°C) dishwasher cycle
without bleach or rinse aid can be used.
12.3.4.2.2 To wash the detachable plunger head slide the locking
pin out and wash the plunger head and locking pin in
warm water and laboratory detergent. Rinse the plunger
head and locking pin with oocyst and cyst free reagent
water and dry. Lightly lubricate the locking pin and re-
assemble the plunger head.
•|24 Sample collection (filtration and concentration) using portable continuous-flow centrifugation.
Please follow manufacturer's instructions. This procedure was validated for the detection of
Cryptosporidium using 50-L sample volumes. Alternate sample volumes maybe used, provided
the laboratory demonstrates acceptable performance on initial and ongoing spiked reagent water
and source water samples (Section 9.1.2).
13.0 Sample Concentration and Separation (Purification)
•j 3 1 During concentration and separation, the filter eluate is concentrated through centrifugation, and
the oocysts in the sample are separated from other particulates through immunomagnetic
separation (IMS). Alternate procedures and products maybe used if the laboratory first
demonstrates equivalent or superior performance as per Section 9.1.2.
132 Adjustment of pellet volume
13.2.1 Centrifuge the 250-mL centrifuge tube containing the capsule filter eluate at 1500 x G
for 15 minutes. Allow the centrifuge to coast to a stop—do not use the brake. Record the
pellet volume (volume of solids) on the bench sheet.
45 December 2005
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Method 1622 - Cryptosporidium
NOTE: Recoveries may be improved if centrifugation force is increased to 2000 x G.
However, do not use this higher force if the sample contains sand or other gritty material
that may degrade the condition of any oocysts in the sample.
13.2.2 Using a Pasteur pipette, carefully aspirate the supernatant to 5 mL above the pellet. Extra
care must be taken to avoid aspirating oocysts during this step, particularly if the sample
is reagent water (e.g. initial or ongoing precision and recovery sample).
13.2.3 If the packed pellet volume is < 0.5 mL, vortex the tube vigorously until pellet is
completely resuspended. Swirl the centrifuge tube gently to reduce any foaming after
vortexing. Record the resuspended pellet volume on the bench sheet. Proceed to Section
13.3.
NOTE: Extra care must be taken with samples containing sand or other gritty material
when vortexing to ensure that the condition of any oocysts in the sample is not
compromised.
13.2.4 If the packed pellet volume is > 0.5 mL, the concentrate must be separated into
multiple subsamples (a subsample is equivalent to no greater than 0.5 mL of packed
pellet material, the recommended maximum amount of particulate material to process
through the subsequent purification and examination steps in the method). Use the
following formula to determine the total volume required in the centrifuge tube before
separating the concentrate into two or more subsamples:
pellet volume
total volume (mL) required = x 5 mL
0.5 mL
(For example, if the packed pellet volume is 1.2 mL, the total volume required is 12 mL.)
Add reagent water to the centrifuge tube to bring the total volume to the level calculated
above.
NOTE: Extra care must be taken with samples containing sand or other gritty material
when vortexing to ensure that the condition of any oocysts in the sample is not
compromised.
13.2.4.1 Analysis of entire sample. If analysis of the entire sample is required,
determine the number of subsamples to be processed independently
through the remainder of the method:
13.2.4.1.1 Calculate number of subsamples: Divide the total
volume in the centrifuge tube by 5 mL and round up to
the nearest integer (for example, if the resuspended
volume in Section 13.2.4 is 12 mL, then the number of
subsamples would be 12 mL / 5 mL = 2.4, rounded = 3
subsamples).
13.2.4.1.2 Determine volume of resuspended concentrate per
subsample. Divide the total volume in the centrifuge
tube by the calculated number of subsamples (for
example, if the resuspended volume in Section 13.2.4 is
12 mL, then the volume to use for each subsample = 12
mL / 3 subsamples = 4 mL).
13.2.4.1.3 Process subsamples through IMS. Vortex the tube
vigorously for 10 to 15 seconds to completely resuspend
the pellet. Record the resuspended pellet volume on the
December 2005 46
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Method 1622 - Cryptosporidium
bench sheet. Proceed immediately to Section 13.3, and
transfer aliquots of the resuspended concentrate
equivalent to the volume in the previous step to multiple,
flat-sided sample tubes in Section 13.3.2.1. Process the
sample as multiple, independent subsamples from Section
13.3 onward, including the preparation and examination
of separate slides for each aliquot. Record the volume of
resuspended concentrate transferred to IMS on the bench
sheet (this will be equal to the volume recorded in
Section 13.2.4). Also record the number of subsamples
processed independently through the method on the
bench sheet.
13.2.4.2 Analysis of partial sample. If not all of the concentrate will be
examined, vortex the tube vigorously for 10 to 15 seconds to completely
resuspend the pellet. Record the resuspended pellet volume on the bench
sheet. Proceed immediately to Section 13.3, and transfer one or more 5-
mL aliquots of the resuspended concentrate to one or more flat-sided
sample tubes in Section 13.3.2.1. Record the volume of resuspended
concentrate transferred to IMS on the bench sheet. To determine the
volume analyzed, calculate the percent of the concentrate examined using
the following formula:
total volume of resuspended concentrate transferred to IMS
percent examined = x100%
total volume of resuspended concentrate in Section 13.2.4
Then multiply the volume filtered (Section 12.2.5.2) by this percentage to
determine the volume analyzed.
133 IMS procedure (adapted from Reference 20.13)
NOTE: The IMS procedure should be performed on a bench top with all materials at
room temperature, ranging from 15°C to 25°C.
13.3.1 Preparation and addition of reagents
13.3.1.1 Prepare a IX dilution of SL-buffer-A from the 10X SL-buffer-A (clear,
colorless solution) supplied. Use reagent water (demineralized; Section
7.3) as the diluent. For every 1 mL of IX SL-buffer-A required, mix 100
u,L of 10X SL-buffer-A and 0.9 mL diluent water. A volume of 1.5 mL of
IX SL-buffer-A will be required per sample or subsample on which the
Dynal IMS procedure is performed.
13.3.1.2 For each lOmL sample or subsample (Section 13.2) to be processed
through IMS, add 1 mL of the 10X SL-buffer-A (supplied—not the
diluted IX SL-buffer-A) to a flat-sided tube (Section 6.5.4).
13.3.1.3 For each subsample, add 1 mL of the 10X SL-buffer-B (supplied—
magenta solution) to the flat-sided tube containing the 1 OX SL-buffer-A.
13.3.2 Oocyst and cyst capture
13.3.2.1 Use a graduated, 10-mL pipette that has been pre-rinsed with elution
buffer to transfer the water sample concentrate from Section 13.2 to the
flat-sided tube(s) containing the SL-buffers. If all of the concentrate is
used, rinse the centrifuge tube twice with reagent water and add the
rinsate to the flat-sided tube containing the concentrate (or to the tube
47 December 2005
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Method 1622 - Cryptosporidium
containing the first subsample, if multiple subsamples will be processed).
Each of the two rinses should be half the volume needed to bring the total
volume in the flat-sided sample tube to 12 mL (including the buffers
added in Sections 13.3.1.2 and 13.3.1.3). (For example, if the tube
contained 1 mL of SL-buffer-A and 1 mL of SL-buffer-B, and 5 mL of
sample was transferred after resuspension of the pellet, for a total of 7
mL, the centrifuge tube would be rinsed twice with 2.5 mL of reagent
water to bring the total volume in the flat-sided tube to 12 mL.) Visually
inspect the centrifuge tube after completing the transfer to ensure that no
concentrate remains. If multiple subsamples will be processed, bring the
volume in the remaining flat-sided tubes to 12 mL with reagent water.
Label the flat-sided tube(s) with the sample number (and subsample
letters).
13.3.2.2 Vortex the Dynabeads® anti-Cryptosporidium beads vial from the IMS
kit for approximately 10 seconds to suspend the beads. Ensure that the
beads are fully resuspended by inverting the sample tube and making sure
that there is no residual pellet at the bottom.
13.3.2.3 Add 100 (iL of the resuspended Dynabeads® anti-Cryptosporidium beads
(Section 13.3.2.2) to the sample tube(s) containing the water sample
concentrate and SL-buffers.
13.3.2.4 Affix the sample tube(s) to a rotating mixer and rotate at approximately
18 rpm for 1 hour at room temperature.
13.3.2.5 After rotating for 1 hour, remove each sample tube from the mixer and
place the tube in the magnetic particle concentrator (MFC®-1 or MPC®-
6) with flat side of the tube toward the magnet.
13.3.2.6 Without removing the sample tube from the MPC®-1, place the magnet
side of the MPC®-1 downwards, so the tube is horizontal and the flat side
of the tube is facing down.
13.3.2.7 Gently rock the sample tube by hand end-to-end through approximately
90°, tilting the cap-end and base-end of the tube up and down in turn.
Continue the tilting action for 2 minutes with approximately one tilt per
second.
13.3.2.8 Ensure that the tilting action is continued throughout this period to
prevent binding of low-mass, magnetic or magnetizable material. If the
sample in the MFC®-1 is allowed to stand motionless for more than 10
seconds, remove the flat-sided tube from the MPC®-1, shake the tube to
resuspend all material, replace the sample tube in the MPC®-1 and repeat
Section 13.3.2.9 before continuing to Section 13.3.2.11.
13.3.2.9 Return the MPC®-1 to the upright position, sample tube vertical, with
cap at top. Immediately remove the cap and, keeping the flat side of the
tube on top, pour off all of the supernatant from the tube held in the
MPC®-1 into a suitable container. Do not shake the tube and do not
remove the tube from MPC®-1 during this step. Allow more supernatant
to settle; aspirate additional supernatant with pipette.
13.3.2.10 Remove the sample tube from the MPC®-1 and resuspend the sample in
0.5 mL IX SL-buffer-A (prepared from 10X SL-buffer-A
stock—supplied). Mix very gently to resuspend all material in the tube.
Do not vortex.
13.3.2.11 Quantitatively transfer (transfer followed by two rinses) all the liquid
from the sample tube to a labeled, 1.5-mL microcentrifuge tube. Use 0.5
December 2005 48
-------
Method 1622 - Cryptosporidium
mL of IX SL-buffer-A to perform the first rinse and 0.5 mL of IX SL-
buffer-A for the second rinse. Allow the flat-sided sample tube to sit for
a minimum of 1 minute after transfer of the second rinse volume, then use
a pipette to collect any residual volume that drips down to the bottom of
the tube to ensure that as much sample volume is recovered as possible.
Ensure that all of the liquid and beads are transferred.
13.3.2.12 Place the microcentrifuge tube into the second magnetic particle
concentrator (MPC®-M or MPC®-S), with its magnetic strip in place.
13.3.2.13 Without removing the microcentrifuge tube from MPCCD-M, gently
rock/roll the tube through 180° by hand. Continue for approximately 1
minute with approximately one 180° roll/rock per second. At the end of
this step, the beads should produce a distinct brown dot at the back of the
tube.
13.3.2.14 Immediately aspirate the supernatant from the tube and cap held in the
MPC®-M. If more than one sample is being processed, conduct three 90°
rock/roll actions before removing the supernatant from each tube. Take
care not to disturb the material attached to the wall of the tube adjacent to
the magnet. Do not shake the tube. Do not remove the tube from MPC®-
M while conducting these steps.
13.3.3 Dissociation of beads/oocyst complex
NOTE: Two acid dissociations are required.
13.3.3.1 Remove the magnetic strip from the MPC®-M.
13.3.3.2 Add 50 (iL of 0.1 N HC1, then vortex at the highest setting for
approximately 50 seconds.
NOTE: The laboratory must use 0.1-N standards purchased directly from a vendor,
rather than adjusting the normality in-house.
13.3.3.3 Place the tube in the MPC®-M without the magnetic strip in place and
allow to stand in a vertical position for at least 10 minutes at room
temperature.
13.3.3.4 Vortex vigorously for approximately 30 seconds.
13.3.3.5 Ensure that all of the sample is at the base of the tube. Place the
microcentrifuge tube in the MPC®-M.
13.3.3.6 Replace magnetic strip in MPC®-M and allow the tube to stand
undisturbed for a minimum of 10 seconds.
13.3.3.7 Prepare a well slide for sample screening and label the slide.
13.3.3.8 Add 5 (iL of 1.0 N NaOH to the sample wells of two well slides (add 10
\\L to the sample well of one well slide if the volume from the two
required dissociations will be added to the same slide).
NOTE: The laboratory must use 1.0-N standards purchased directly from a vendor
rather than adjusting the normality in-house.
13.3.3.9 Without removing the microcentrifuge tube from the MPC®-M, transfer
all of the sample from the microcentrifuge tube in the MPC®-M to the
sample well with the NaOH. Do not disturb the beads at the back wall of
the tube. Ensure that all of the fluid is transferred.
49 December 2005
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Method 1622 - Cryptosporidium
13.3.3.10 Do not discard the beads or microcentrifuge tube after transferring the
volume from the first acid dissociation to the well slide. Perform the steps
in Sections 13.3.3.1 through 13.3.3.9 a second time. The volume from the
second dissociation can be added to the slide containing the volume from
the first dissociation, or can be applied to a second slide.
NOTE: The wells on Dynal Spot-On slides are likely to be too small to accommodate the
volumes from both dissociations.
13.3.3.11 Record the date and time the purified sample was applied to the slide(s).
13.3.3.12 Air-dry the sample on the well slide(s). Because temperature and
humidity vary 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.
13.3.4 Tips for minimizing carry-over of debris onto microscope slides after IMS
• Make sure the resuspended pellet is fully homogenized before placing the tube in the
MPC®-1 or MPC®-M to avoid trapping "clumps" or a dirty layer between the beads
and the side of the tube.
• When using the MPC®-1 magnet, make sure that the tube is snugged flat against the
magnet. Push the tube flat if necessary. Sometimes the magnet is not flush with the
outside of the holder and, therefore, the attraction between the beads and the magnet
is not as strong as it should be. However, it can be difficult to determine this if you do
not have more than one MPC®-1 to make comparisons.
• After the supernatant has been poured off at Section 13.3.2.11, leave the tube in the
MPC®-1 and allow time for any supernatant remaining in the tube to settle down to
the bottom. Then aspirate the settled supernatant and associated particles from the
bottom of the tube. The same can be done at Section 13.3.2.16 with the
microcentrifuge tube.
• An additional rinse can also be performed at Section 13.3.2.11. After the supernatant
has been poured off and any settled material is aspirated off the bottom, leave the tube
in the MPC®-1 and add an additional 10 mL of reagent water or PBS to the tube and
repeat Sections 13.3.2.9 and 13.3.2.11. Although labs have reported successfully
using this technique to reduce carryover, because the attraction between the MPC®-1
and the beads is not as great as the attraction between the MPC®-M and the beads,
the chances would be greater for loss of oocysts doing the rinse at this step instead of
at Section 13.3.2.16.
• After the supernatant has been aspirated from the tube at Section 13.3.2.16, add 0.1
mL of PBS, remove the tube from the MPC®-M, and resuspend. Repeat Sections
13.3.2.15 and 13.3.2.16.
• Use a slide with the largest diameter well available to spread out the sample as much
as possible.
December 2005 50
-------
Method 1622 - Cryptosporidium
14.0 Sample Staining
NOTE: The sample must be stained within 72 hours of application of the purified sample
to the slide.
•j 4 •] Prepare positive and negative controls.
14.1.1 For the positive control, pipette 10 \\L of positive antigen or 200 to 400 intact oocysts to
the center of a well.
14.1.2 For the negative control, pipette 50 \aL of PBS (Section 7.4.2.1) into the center of a well
and spread it over the well area with a pipette tip.
14.1.3 Air-dry the control slides (see Section 13.3.3.12 for guidance).
NOTE: If the laboratory has a large batch of slides that will be examined over several
days, and is concerned that a single positive control may fade, due to multiple
examinations, the laboratory should prepare multiple control slides with the batch of field
slides and alternate between the positive controls when performing the positive control
check.
142 Follow manufacturer's instructions in applying stain to slides.
-| 4 3 Place the slides in a humid chamber in the dark and incubate at room temperature for
approximately 30 minutes. The humid chamber consists of a tightly sealed plastic container
containing damp paper towels on top of which the slides are placed.
-|4 4 Remove slides from humid chamber and allow condensation to evaporate, if present.
•|45 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 paper
towel or other absorbent material placed at edge of slide. Avoid disturbing the sample.
146 Apply 50 f^L of 4',6-diamidino-2-phenylindole (DAPI) staining solution (Section 7.7.2) to each
well. Allow to stand at room temperature for a minimum of 1 minute. (The solution concentration
may be increased up to 1 (ig/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.)
-| 4 7 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 DAPI staining solution from below the well using a clean Pasteur pipette or absorb with
paper towel or other absorbent material placed at edge of slide. Avoid disturbing the sample.
NOTE: If using the MeriFluor® Cryptosporidium/Giardia (Section 7.6.1), do not allow
slides to dry completely.
148 Add mounting medium (Section 7.8) to each well.
149 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.
1410 Record the date and time that staining was completed on the bench sheet. If slides will not be read
immediately, store in a humid chamber in the dark between 1 °C and 10°C until ready for
examination.
51 December 2005
-------
Method 1622 - Cryptosporidium
15.0 Examination
NOTE: Although immunofluorescence assay (FA) and 4',6-diamidino-2-phenylindole
(DAPI) and differential interference contrast (DIC) microscopy examination should be
performed immediately after staining is complete, laboratories have up to 168 hours (7
days) from completion of sample staining to complete the examination and verification of
samples. However, if fading/diffusion ofFITC or DAPI fluorescence 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.
•| 5 •] Scanning technique: Scan each well in a systematic fashion. An up-and-down or a side-to-side
scanning pattern may be used (Figure 4).
•|52 Examination using immunofluorescence assay (FA), 4',6-diamidino-2-phenylindole (DAPI)
staining characteristics, and differential interference contrast (DIC) microscopy. The minimum
magnification requirements for each type of examination are noted below.
NOTE: All characterization (DAPI and DIC) and size measurements must be determined
using IOOOXmagnification and reported to the nearest 0.5 ^m.
Record examination results for Cryptosporidium oocysts on a Cryptosporidium examination form.
All organisms that meet the criteria specified in Sections 15.2.2 and 15.2.3, less atypical
organisms specifically identified as non-target organisms by DIC or DAPI (e.g. possessing spikes,
stalks, appendages, pores, one or two large nuclei filling the cell, red fluorescing chloroplasts,
crystals, spores, etc), must be reported.
15.2.1 Positive and negative staining control. Positive and negative staining controls must be
acceptable before proceeding with examination of field sample slides.
15.2.1.1 Each analyst must characterize a minimum of three Cryptosporidium
oocysts on the positive staining control slide before examining field
sample slides. This characterization must be performed by each analyst
during each microscope examination session. FITC examination must be
conducted at a minimum of 200X total magnification, DAPI examination
must be conducted at a minimum of 400X, and DIC examination and size
measurements must be conducted at a minimum of 1 OOOX. Size, shape,
and DIC and DAPI characteristics of three Cryptosporidium oocysts must
be recorded by the analyst on a microscope log. The analyst also must
indicate on each sample examination form whether the positive staining
control was acceptable.
15.2.1.2 Examine the negative staining control to confirm that it does not contain
any oocysts (Section 14.1). Indicate on each sample examination form
whether the negative staining control was acceptable.
15.2.1.3 If the positive staining control contains oocysts within the expected range
and at the appropriate fluorescence for both FA and DAPI, and the
negative staining control does not contain any oocysts (Section 14.1),
proceed to Sections 15.2.2 and 15.2.3.
15.2.2 Sample examination—Cryptosporidium
15.2.2.1 FITC examination (the analyst must use a minimum of 200X total
magnification). Use epifluorescence to scan the entire well for apple-
green fluorescence of oocyst and cyst shapes. When brilliant apple-green
fluorescing ovoid or spherical objects 4 to 6 urn in diameter are observed
with brightly highlighted edges, increase magnification to 400X and
December 2005 52
-------
Method 1622 - Cryptosporidium
switch the microscope to the UV filter block for DAPI (Section 15.2.2.2),
then to DIG (Section 15.2.2.3) at 1000X.
15.2.2.2 DAPI fluorescence examination (the analyst must use a minimum of
400X total magnification). Using the UV filter block for DAPI, the
object will exhibit one of the following characteristics:
(a) Light blue internal staining (no distinct nuclei) with a green rim
(b) Intense blue internal staining
(c) Up to four distinct, sky-blue nuclei
Look for atypical DAPI fluorescence, e.g., more than four stained nuclei,
size of stained nuclei, and wall structure and color. Record oocysts in
category (a) as DAPI-negative; record oocysts in categories (b) and (c) as
DAPI-positive.
15.2.2.3 DIC examination (the analyst must use a minimum of 1000X total
magnification [oil immersion lens]). Using DIC, look for external or
internal morphological characteristics atypical of Cryptosporidium
oocysts (e.g., spikes, stalks, appendages, pores, one or two large nuclei
filling the cell, red fluorescing chloroplasts, crystals, spores, etc.)
(adapted from Reference 20.10). If atypical structures are not observed,
then categorize each apple-green fluorescing object as:
(a) An empty Cryptosporidium oocyst
(b) A Cryptosporidium oocyst with amorphous structure
(c) A Cryptosporidium oocyst with internal structure (one to four
sporozoites/oocyst)
Using 1000X total magnification, record the shape, measurements (to the
nearest 0.5 urn), and number of sporozoites (if applicable) for each apple-
green fluorescing object meeting the size and shape characteristics.
Although not a defining characteristic, surface oocyst folds may be
observed in some specimens.
15.2.2.4 A positive result is a Cryptosporidium oocyst which exhibits typical IFA
fluorescence, typical size and shape and exhibits nothing atypical on IFA,
DAPI fluorescence, or DIC microscopy. A positive result must be
characterized and assigned to one of the DAPI and DIC categories in
Sections 15.2.2.2 and 15.2.2.3.
15.2.3 Record the date and time that sample examination was completed on the examination
form.
15.2.4 Report Cryptosporidium concentrations as oocysts/L.
15.2.5 Record analyst name.
16.0 Analysis of Complex Samples
•j g 1 Some samples may contain high levels (>1000/L) of oocysts and/or interfering organisms,
substances, or materials. Some samples may clog the filter (Section 12.0); others will not allow
separation of the oocysts from the retentate or eluate; and others may contain materials that
preclude or confuse microscopic examination.
162 If the sample holding time has not been exceeded and a full-volume sample cannot be filtered,
dilute an aliquot of sample with reagent water and filter this smaller aliquot (Section 12.0). This
dilution must be recorded and reported with the results.
53 December 2005
-------
Method 1622 - Cryptosporidium
•| g 3 If the holding times for the sample and for microscopic examination of the cleaned up
retentate/eluate have been exceeded, the site should be re-sampled. If this is not possible, the
results should be qualified accordingly.
-| g 4 Some samples may adhere to the centrifuge tube walls. The use of siliconized or low-adhesion
centrifuge tubes (Fisherbrand siliconized/low retention microcentrifuge tubes, 02-681-320 or
equivalent) may reduce adhesion. Alternately, rinse centrifuge tubes with PBST elution buffer or
Sigmacote® prior to use.
17.0 Method Performance
•| 7 •] Method acceptance criteria are shown in Tables 3 and 4 in Section 21.0. The initial and ongoing
precision and recovery criteria are based on the results of spiked reagent water samples analyzed
during the Information Collection Rule Supplemental Surveys (Reference 20.11). The matrix
spike and matrix spike duplicate criteria are based on spiked source water data generated during
the interlaboratory validation study of Method 1622 involving 11 laboratories and 11 raw surface
water matrices across the U.S. (Reference 20.14).
NOTE: Some sample matrices may prevent the MS acceptance criteria in Tables 3 and 4
to be 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.
18.0 Pollution Prevention
•j g •] The solutions and reagents used in this method pose little threat to the environment when recycled
and managed properly.
182 Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize
the volume of expired materials that need to be discarded.
19.0 Waste Management
•j g •] It is the laboratory's responsibility to comply with all federal, state, and local regulations govern-
ing waste management, particularly the biohazard and hazardous waste identification rules and
land disposal restrictions, and to protect the air, water, and land by minimizing and controlling all
releases from fume hoods and bench operations. Compliance with all sewage discharge permits
and regulations is also required. An overview of these requirements can be found in the
Environmental Management Guide for Small Laboratories (EPA 233-B-98-001).
192 Samples, reference materials, and equipment known or suspected to have viable oocysts attached
or contained must be sterilized prior to disposal.
193 F°r further information on waste management, consult The Waste Management Manual for
Laboratory Personnel and Less is Better: Laboratory Chemical Management for Waste
Reduction, both available from the American Chemical Society's Department of Government
Relations and Science Policy, 1155 16th Street N.W., Washington, D.C. 20036.
20.0 References
20 1 Morgan-Ryan, UM, A. Fall, L.A Ward, N. Hijjawi, I. Sulaiman, R. Payer, R.C.Thompson, M.
Olson, A. Lai, L. Xiao. 2002. Cryptosporidium hominis n. sp. (Apicomplexa: Cryptosporidiidae
from Homo sapiens). Journal Eukaryot Microbiol 49(6):433-450.
20.2 Rodgers, MarkR., Flanigan, Debbie J., and Jakubowski, Walter, 1995. Applied and
Environmental Microbiology 6\_ (10), 3759-3763.
20 3 Fleming, Diane O., et al.(eds.), Laboratory Safety: Principles and Practices, 2nd edition. 1995.
ASM Press, Washington, DC
December 2005 54
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Method 1622 - Cryptosporidium
20.4 "Working with Carcinogens," DREW, PHS, CDC, NIOSH, Publication 77-206, (1977).
20.5 "OSHA Safety and Health Standards, General Industry," OSHA2206, 29 CFR 1910 (1976).
20 6 "Safety in Academic Chemistry Laboratories," ACS Committee on Chemical Safety (1979).
20 7 APHA, AWWA, and WEF. 2005. Standard Methods for the Examination of Water and
Wastewater; 21th Edition. American Public Health Association, American Water Works
Association, Washington, D.C.
20 8 USEPA 2005. Manual for the Certification of Laboratories Analyzing Drinking Water; Criteria
and Procedures; Quality Assurance. Fifth Edition. EPA 815-R-05-004. Office of Ground Water
and Drinking Water, U.S. Environmental Protection Agency, 26 West Martin Luther King Drive,
Cincinnati, OH 45268.
20 9 ICR Microbial Laboratory Manual, EPA/600/R-95/178, National Exposure Research Laboratory,
Office of Research and Development, U.S. Environmental Protection Agency, 26 Martin Luther
King Drive, Cincinnati, OH 45268 (1996).
20.10 Connell K-> c-c- Rodgers, H.L. Shank-Givens, J Scheller, M.L Pope, and K. Miller, 2000.
Building a Better Protozoa Data Set. Journal AWWA, 92:10:30.
20 11 "Envirochek™ Sampling Capsule," PN 32915, Gelman Sciences, 600 South Wagner Road, Ann
Arbor, MI 48103-9019 (1996).
20.12 "Dynabeads® GC-Combo," Dynal Microbiology R&D, P.O. Box 8146 Dep., 0212 Oslo, Norway
(September 1998, Revision no. 01).
20 13 USEPA. Results of the Interlaboratory Method Validation Study for Determination of
Cryptosporidium and Giardia Using USEPA Method 1623, EPA-821-R-01-028. Office of Water,
Office of Science and Technology, Engineering and Analysis Division, Washington, DC (2001).
20 14 USEPA. Implementation and Results of the Information Collection Rule Supplemental Surveys.
EPA-815-R-01-003. Office of Water, Office of Ground Water and Drinking Water, Standards and
Risk Management Division, Washington, DC (2001).
20 15 Connell, K., J. Scheller, K. Miller, and C.C. Rodgers, 2000. Performance of Methods 1622 and
1623 in the ICR Supplemental Surveys. Proceedings, American Water Works Association Water
Quality Technology Conference, November 5-9, 2000, Salt Lake City, UT.
55 December 2005
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Method 1622 - Cryptosporidium
21.0 Tables and Figures
Table 1. Method Holding Times (See Section 8.2 for details)
Sample Processing Step
Maximum Allowable Time between Breaks
(Samples should be processed as soon as possible)
Collection
Filtration
X Up to 96 hours are permitted between sample collection (if shipped to the laboratory as a bulk
sample) or filtration (if filtered in the field) and initiation of elution
Elution
Concentration
Purification
Application of purified sample to slide
These steps must be completed in 1 working day
Drying of sample
X Up to 72 hours are permitted from application of the purified sample to the slide to staining
Staining
X Up to 7 days are permitted between sample staining and examination
Examination
December 2005
56
-------
Method 1622 - Cryptosporidium
Table 2. Tier 1 and Tier 2 Validation/Equivalency Demonstration Requirements
Test
IPR
(Section 9.4)
Method blank
(Section 9.6)
MS
(Section 9.5.1)
MS/MSD
(Section 9.5)
Description
4 replicates of spiked
reagent water
Unspiked reagent
water
Spiked matrix water
2 replicates of spiked
matrix water
Tier 1 modification111
Required. Must be accompanied by a
method blank.
Required
Required on each water to which the
modification will be applied and on every
20th sample of that water thereafter. Must be
accompanied by an unspiked field sample
collected at the same time as the MS sample
Recommended, but not required. Must be
accompanied by an unspiked field sample
collected at the same time as the MS sample
Tier 2 modification'2'
Required per laboratory
Required per laboratory
Not required
Required per laboratory.
Each laboratory must
analyze a different water.
(1) If a modification will be used only in one laboratory, these tests must be performed and the results must meet all
of the QC acceptance criteria in the method (these tests also are required the first time a laboratory uses the
validated version of the method)
(2) If nationwide approval of a modification is sought for one type of water matrix (such as surface water), a
minimum of 3 laboratories must perform the tests and the results from each lab individually must meet all QC
acceptance criteria in the method. If more than 3 laboratories are used in a study, a minimum of 75% of the
laboratories must meet all QC acceptance criteria.
NOTE: The initial precision and recovery and ongoing precision and recovery (OPR)
acceptance criteria listed in Table 3 are based on results from 293 Cryptosporidium OPR
samples analyzed by six laboratories during the Information Collection Rule
Supplemental Surveys (Reference 20.15). The matrix spike acceptance criteria are based
on data generated through interlaboratory validation of Method 1623 (Reference 20.14).
57
December 2005
-------
Method 1622 - Cryptosporidium
Table 3. Quality Control Acceptance Criteria for Cryptosporidium
Performance test
Initial precision and recovery
Mean recovery (percent)
Precision (as maximum relative standard deviation)
Ongoing precision and recovery (percent)
Matrix spike/matrix spike duplicate (for method modifications)
Mean recovery1'2 (as percent)
Precision (as maximum relative percent difference)
Section
9.4
9.4.3
9.4.3
9.7
9.5
9.5.2.2
9.5.2.3
Acceptance criteria
24-100
55
11 - 100
13-143
67
(1) The acceptance criteria for mean MS/MSD recovery serves as the acceptance criteria for MS recovery during
routine use of the method (Section 9.5.1).
(2) Some sample matrices may prevent the acceptance criteria from being met. An assessment of the distribution of
MS recoveries from multiple MS samples from 87 sites during the ICR Supplemental Surveys is provided in Table
4.
December 2005
58
-------
Method 1622 - Cryptosporidium
Table 4. Distribution of Matrix Spike Recoveries from Multiple Samples Collected from 87 Source Waters
During the ICR Supplemental Surveys (Adapted from Reference 20.16)
MS Recovery Range
<10%
>10%-20%
>20% - 30%
>30% - 40%
>40% - 50%
>50% - 60%
>60% - 70%
>70% - 80%
>80% - 90%
>90%
Percent of 430 Cryptosporidium MS Samples in
Recovery Range
6.7%
6.3%
14.9%
14.2%
18.4%
17.4%
11.2%
8.4%
2.3%
0.2%
59
December 2005
-------
Method 1622 - Cryptosporidium
1 mm
A
1/5 mm
D
B
C
Figure 1. Hemacy to meter Platform Ruling. Squares 1, 2, 3, and 4
are used to count stock suspensions of Cryptosporidium
oocysts (after Miale, 1967)
December 2005
60
-------
Method 1622 - Cryptosporidium
o
•
o
*
•
O
-O
0
O
c
6
o
Figure 2. Manner of Counting Oocysts in 1 Square mm.
Dark organisms are counted and light
organisms are omitted (after Miale, 1967).
61
December 2005
-------
Method 1622 - Cryptosporidium
e\
Sampte
Envirodiek71 capsute
tit
Flew rate meter
with valve \j
P iV^lve)
Influent
tubing
9lM CenWugal
tubmg
pump
ORECf ION OF FLOW
Flowtoteizer Effluent
Ouflet6uttng
is-*
Flow rate meter*—
with valve
/
Influent
Effluent tub ing
Pressure Wet tubing
regulator
Envirochek™
capsule
Flow tola!izer
>,, FScw conW
valw may be
used it pte 'A
flow rate i»ttr
OiRECTOM 01- PU3W
Figure 3a. Filtration Systems for Envirochek™ or
Envirochek™HV Capsule (unpressurized source - top,
pressurized source - bottom)
62
December 2005
-------
Method 1622 - Cryptosporidium
\ / .,
, Influent tubing Inlet tubing
Sample
Flowtolaletr
I—(Valve)
Outlet tubing
ORKTtOM Of fUMi
Ftow rate meter"- —
K
InFlyert
tubing
ri
Effluent tubing
I / • k .'
I / ut, to
Pressure In let tubing I
regulator Filta-Max™
filter housing
Rowtottizer
:"\ Ficnv contra
flo«rtt»T«1sf
Figure 3b. Filtration Systems for Filta-Max© filters (unpressurized
source - top, pressurized source - bottom)
December 2005
63
-------
Method 1622 - Cryptosporidium
Til
Figure 4. Methods for Scanning a Well Slide
December 2005
64
-------
Method 1622 - Cryptosporidium
22.0 Glossary of Definitions and Purposes
These definitions and purposes are specific to this method but have been conformed to common
usage as much as possible.
22 1 Units of weight and measure and their abbreviations
22.1.1 Symbols
°C degrees Celsius
(iL microliter
< less than
> greater than
% percent
22.1.2 Alphabetical characters
cm centimeter
g gram
G acceleration due to gravity
hr hour
ID inside diameter
in. inch
L liter
m meter
MCS microscope cleaning solution
mg milligram
mL milliliter
mm millimeter
mM millimolar
N normal; gram molecular weight of solute divided by hydrogen equivalent of
solute, per liter of solution
RSD relative standard deviation
sr standard deviation of recovery
X mean percent recovery
22 2 Definitions, acronyms, and abbreviations (in alphabetical order)
Analyst—The analyst should have at least 2 years of college in microbiology or equivalent or
closely related field. The analyst also should have a minimum of 6 months of continuous bench
experience with Cryptosporidium and IFA microscopy. The analyst should have a minimum of 3
months experience using EPA Method 1622 and/or EPA Method 1623 and should have
successfully analyzed a minimum of 50 samples using EPA Method 1622 and/or EPA Method
1623.
Analyte—A protozoan parasite tested for by this method. The analyte in this method is
Cryptosporidium.
Cyst—A phase or a form of an organism produced either in response to environmental conditions
or as a normal part of the life cycle of the organism. It is characterized by a thick and
environmentally resistant cell wall.
Flow cytometer—A particle-sorting instrument capable of counting protozoa.
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Method 1622 - Cryptosporidium
Immunomagnetic separation (IMS)—A purification procedure that uses microscopic, magnetically
responsive particles coated with an antibodies targeted to react with a specific pathogen in a fluid
stream. Pathogens are selectively removed from other debris using a magnetic field.
Initial precision and recovery (IPR)—Four aliquots of spiking suspension analyzed to establish
the ability to generate acceptable precision and accuracy. An IPR is performed prior to the first
time this method is used and anytime the method or instrumentation is modified.
Laboratory blank—See Method blank
Laboratory control sample (LCS)—See Ongoing precision and recovery (OPR) standard
Matrix spike (MS)—A sample prepared by adding a known quantity of organisms to a specified
amount of sample matrix for which an independent estimate of target analyte concentration is
available. A matrix spike is used to determine the effect of the matrix on a method's recovery
efficiency.
May—This action, activity, or procedural step is neither required nor prohibited.
May not—This action, activity, or procedural step is prohibited.
Method blank—An aliquot of reagent water that is treated exactly as a sample, including exposure
to all glassware, equipment, solvents, and procedures that are used with samples. The method
blank is used to determine if analytes or interferences are present in the laboratory environment,
the reagents, or the apparatus.
Must—This action, activity, or procedural step is required.
Negative control—See Method blank
Nucleus—A membrane-bound organelle containing genetic material. Nuclei are a prominent
internal structure seen both in Cryptosporidium oocysts. In Cryptosporidium oocysts, there is one
nucleus per sporozoite.
Oocyst—The encysted zygote of some sporozoa; e.g., Cryptosporidium. The oocyst is a phase or
form of the organism produced as a normal part of the life cycle of the organism. It is
characterized by a thick and environmentally resistant outer wall.
Ongoing precision and recovery (OPR) standard—A method blank spiked with known quantities
of analytes. The OPR is analyzed exactly like a sample. Its purpose is to assure that the results
produced by the laboratory remain within the limits specified in this method for precision and
recovery.
Oocyst and cyst spiking suspension—See Spiking suspension
Oocyst and cyst stock suspension—See Stock suspension
Positive control—See Ongoing precision and recovery standard
Principal analyst—The principal analyst (may not be applicable to all monitoring programs)
should have a BS/BA in microbiology or closely related field and a minimum of 1 year of
continuous bench experience with Cryptosporidium and IFA microscopy. The principal analyst
also should have a minimum of 6 months experience using EPA Method 1622 and/or EPA
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Method 1622 - Cryptosporidium
Method 1623 and should have analyzed a minimum of 100 samples using EPA Method 1622
and/or EPA Method 1623.
PTFE—Polytetrafluoroethylene
Quantitative transfer—The process of transferring a solution from one container to another using
a pipette in which as much solution as possible is transferred, followed by rinsing of the walls of
the source container with a small volume of rinsing solution (e.g., reagent water, buffer, etc.),
followed by transfer of the rinsing solution, followed by a second rinse and transfer.
Reagent water—Water demonstrated to be free from the analytes of interest and potentially
interfering substances at the method detection limit for the analyte.
Reagent water blank—see Method blank
Relative standard deviation (RSD)—The standard deviation divided by the mean times 100.
RSD—See Relative standard deviation
Should—This action, activity, or procedural step is suggested but not required.
Spiking suspension—Diluted stock suspension containing the organism(s) of interest at a
concentration appropriate for spiking samples.
Sporozoite—A motile, infective stage of certain protozoans; e.g., Cryptosporidium. There are four
sporozoites in each Cryptosporidium oocyst, and they are generally banana-shaped.
Stock suspension—A concentrated suspension containing the organism(s) of interest that is
obtained from a source that will attest to the host source, purity, authenticity, and viability of the
organism(s).
Technician—The technician filters samples, performs centrifugation, elution, concentration, and
purification using IMS, and places purified samples on slides for microscopic examination, but
does not perform microscopic protozoan detection and identification. No minimum education or
experience requirements with Cryptosporidium and IFA microscopy apply to the technician. The
technician should have at least 3 months of experience in filter extraction and processing of
protozoa samples by EPA Method 1622/1623 and should have successfully processed a minimum
of 50 samples using EPA Method 1622/1623.
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