United States
Environmental Protection
Agency
Off ice of Water
4603
EPA-821-R-01-026
April 2001
v/EPA Method 1622: Cryptosporidium in
Water by Filtration/IMS/FA
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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 DynCorp I&ET 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, Division of Parasitic Diseases (MS-F13), 4770 Buford
Highway, N.E., Atlanta, GA 30341-3724, USA
Phil Berger, Office of Groundwater and Drinking Water, U.S. Environmental Protection Agency, 401 M
Street, S.W., Washington, DC 20460, USA
Jennifer Clancy, Clancy Environmental Consultants, Inc., P.O. Box 314, St. Albans, VT 05478, USA
Kevin Connell, DynCorp I&ET, 6101 Stevenson Avenue, Alexandria, VA 22314, USA
Ricardo DeLeon, Metropolitan Water District of Southern California, 700 Moreno Avenue, LaVerne, CA
91760, USA
Shirley Dzogan, EnviroTest Laboratories, 745 Logan Avenue, Winnipeg, Manitoba R3E 3L5, Canada
Mary Ann Feige, Technical Support Center, Office of Ground Water and Drinking Water, U.S.
Environmental Protection Agency, 26 W. Martin Luther King Drive, Cincinnati, OH 45268-1320,
USA
Colin Fricker, Thames Water Utilities, Manor Farm Road, Reading, Berkshire, RG2 OJN, England
Carrie Hancock, CH Diagnostic & Consulting Service, Inc., 214 S.E. Nineteenth Street, Loveland, CO
80537, USA
Stephanie Harris, Region 10 Laboratory, U.S. Environmental Protection Agency, 7411 Beach Drive East,
Port Orchard, WA 98366, USA
Dale Rushneck, Interface, Inc., 3194 Worthington Avenue, Fort Collins, CO 80526, USA
Frank Schaefer III, National Exposure Research Laboratory, U.S. Environmental Protection Agency, 26
W. Martin Luther King Drive, Cincinnati, OH 45268-1320, USA
Steve Schaub, Health and Ecological Criteria Division (4304), Office of Science and Technology, U.S.
Environmental Protection Agency, 401 M Street, S.W., Washington, DC 20460, USA
Ajaib Singh, City of Milwaukee Health Department, 841 North Broadway, Milwaukee, WI 53202, USA
Huw Smith, Department of Bacteriology, Scottish Parasite Diagnostic Laboratory, Stobhill NHS Trust,
Springburn, Glasgow, G21 3UW, Scotland
Timothy Straub, Lockheed Martin, 7411 Beach Drive East, Port Orchard, WA 98366, USA
William A. Telliard, Office of Science and Technology, U.S. Environmental Protection Agency, 401 M
Street, S.W., Washington, DC 20460, USA
Cryptosporidium cover photo courtesy of the U.S. Centers for Disease Control
Disclaimer
This method has been reviewed by the U.S. EPA Office of Water and approved for publication. Mention
of trade names or commercial products does not constitute endorsement or recommendation for use.
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Questions regarding this method or its application should be addressed to:
William A. Telliard
U.S. EPA Office of Water
Analytical Methods Staff
Mail Code 4303
Washington, DC 20460
Email: telliard.william@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.
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-on\y 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. Quality control
(QC) acceptance criteria for the method were developed from the interlaboratory study data.
The interlaboratory validated versions of both Method 1622 (January 1999; EPA-821-R-99-001) and the
subsequent combined Cryptosporidium/Giardia version of the method, Method 1623 (April 1999; EPA-
821-R-99-006), were used to analyze approximately 3,000 field and QC samples during the Information
Collection Rule Supplemental Surveys (ICRSS) between March 1999 and February 2000. Method 1622
was used to analyze samples from March 1999 to mid-July 1999; Method 1623 was used from mid-July
1999 to February 2000. The April 2001 revision of both methods include updated QC acceptance criteria
based on analysis of the QC samples analyzed during the ICRSS.
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 an immunofluorescence assay for determination of oocyst concentrations,
with confirmation through vital dye staining 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 Waterborne Crypt-a-
Glo™ sample staining procedure described in this document. Alternate procedures are allowed,
provided that required quality control tests are performed and all quality control acceptance criteria in
this method are met.
Since the interlaboratory validation of EPA Method 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
IDEXX Filta-Max™ filter
Waterborne Aqua-Glo™ G/C Direct FL antibody stain
• Meridian Diagnostics Merifluor Cryptosporidium/Giardia
The validation studies for these modified versions of the method met EPA's performance-based
measurement system Tier 2 validation for nationwide use (see Section 9.1.2 for details), and have been
accepted by EPA as equivalent in performance to the original version of the method validated by EPA.
The equipment and reagents used in these modified versions of the method are noted in Sections 6 and 7
of the method; the procedures for using these equipment and reagent options are available from the
manufacturers.
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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.
IV
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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 2
6.0 Equipment and Supplies 3
7.0 Reagents and Standards 7
8.0 Sample Collection and Storage 10
9.0 Quality Control 11
10.0 Microscope Calibration and Analyst Verification 17
11.0 Oocyst Suspension Enumeration and Spiking 22
12.0 Sample Filtration and Elution 31
13.0 Sample Concentration and Separation (Purification) 33
14.0 Sample Staining 38
15.0 Examination 39
16.0 Analysis of Complex Samples 40
17.0 Method Performance 40
18.0 Pollution Prevention 41
19.0 Waste Management 41
20.0 References 42
21.0 Tables and Figures 43
Table 1. Method Holding Times 43
Table 2. Tier 1 and Tier 2 Validation/Equivalency Demonstration Requirements .... 43
Table 3. Quality Control Acceptance Criteria for Cryptosporidium 44
Table 4. Distribution of Matrix Spike Recoveries from Multiple Samples
Collected from 87 Source Waters During the ICR Supplemental Surveys ... 44
Figure 1. Hemacytometer Platform Ruling 45
Figure 2. Manner of Counting Oocysts in 1 Square mm 46
Figure 3. Laboratory Filtration System 47
Figure 4. Methods for Scanning a Well Slide 48
22.0 Glossary of Definitions and Purposes 49
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Method 1622: Cryptosporidium in Water
by Filtration/IMS/FA
1.0 Scope and Application
1.1 This method is for determination of the identity and concentration of Cryptosporidium (CAS
Registry number 137259-50-8) in water by filtration, immunomagnetic separation (IMS), and
immunofluorescence assay (FA) microscopy. Cryptosporidium may be confirmed 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.
1.2 This method is designed to meet the survey and monitoring requirements of the U.S.
Environmental Protection Agency (EPA). It is based on laboratory testing of recommendations
by a panel of experts convened by EPA. The panel was charged with recommending an
improved protocol for recovery and detection of protozoa that could be tested and implemented
with minimal additional research.
1.3 This method will not identify the species of Cryptosporidium or the host species of origin, nor
can it determine the viability or infectivity of detected oocysts.
1.4 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.
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.
1.5 Any modification of the method beyond those expressly permitted is subject to the application
and approval of alternative test procedures under 40 CFR Part 141.27.
2.0 Summary of Method
2.1 A water sample is filtered and the oocysts and extraneous materials are retained on the filter.
Although EPA has only validated the method using laboratory filtration of bulk water samples
shipped from the field, field-filtration also can 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. Potential
oocysts are confirmed through DAPI staining characteristics and DIG microscopy.
1 April 2001
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Method 1622 - Cryptosporidium
Oocysts are identified when the size, shape, color, and morphology agree with specified
criteria and examples in a photographic library.
2.3.3 Quantitative analysis is performed by counting the total number of objects on the slide
confirmed as oocysts.
2.4 Quality is assured through reproducible calibration and testing of the filtration, immunomagnetic
separation (IMS), staining, and microscopy systems. Detailed information on these tests is
provided in Section 9.0.
3.0 Definitions
3.1 Cryptosporidium is defined as a protozoan parasite potentially found in water and other media.
The six species of Cryptosporidium and their potential hosts are C. parvum (mammals,
including humans); C. baileyi and C. meleagridis (birds); C. muris (rodents); C. serpentis
(reptiles); and C. nasorum (fish).
3.2 Definitions for other terms used in this method are given in the glossary (Section 22.0).
4.0 Contamination, Interferences, and Organism Degradation
4.1 Turbidity caused by inorganic and organic debris can interfere with the concentration,
separation, and examination of the sample for Cryptosporidium oocysts. In addition to naturally-
occurring debris, such as clays and algae, chemicals, such as iron and alum coagulants and
polymers, may be added to finished waters during the treatment process, which may result in
additional interference.
4.2 Organisms and debris that autofluoresce or demonstrate non-specific fluorescence, such as algal
and yeast cells, when examined by epifluorescent microscopy, may interfere with the detection
of oocysts and contribute to false positives by immunofluorescence assay (FA).
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 shall be
demonstrated to be free from interferences under the conditions of analysis by running a method
blank (negative control sample) initially and a minimum of every week or after changes in
source of reagent water. Specific selection of reagents and purification of solvents and other
materials may be required.
4.4 Interferences co-extracted from samples will vary considerably from source to source,
depending on the water being sampled. Experience suggests that high levels of algae, bacteria,
and other protozoa can interfere in the identification of oocysts (Reference 20.1).
4.5 Freezing samples, filters, eluates, concentrates, or slides may interfere with the detection and/or
identification of oocysts.
4.6 All equipment should be cleaned according to manufacturers' instructions. Disposable supplies
should be used wherever possible.
5.0 Safety
5.1 The biohazard associated with, and the risk of infection from, oocysts 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
April 2001 2
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Method 1622 - Cryptosporidium
and observe the safety procedures required in a microbiology laboratory that handles pathogenic
organisms while preparing, using, and disposing of sample concentrates, reagents and materials,
and while operating sterilization equipment.
5.2 The toxicity or carcinogenicity of each compound or reagent used in this method has not been
precisely determined; however, each chemical compound should be treated as a potential health
hazard. Exposure to these compounds should be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file of Occupational Safety and
Health Administration regulations regarding the safe handling of the chemicals specified in this
method. A reference file of material safety data sheets should be made available to all personnel
involved in these analyses. Additional information on laboratory safety can be found in
References 20.2 through 20.5.
5.3 Samples may contain high concentrations of biohazards and toxic compounds, and must be
handled with gloves and opened in a biological safety cabinet to prevent exposure. Reference
materials and standards containing oocysts 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. 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.
6.1 Sample collection equipment for shipment of bulk water samples for laboratory filtration.
Collapsible LDPE cubitainer for collection of 10-L bulk sample(s)—Cole Farmer cat. no. U-
06100-30 or equivalent. Fill completely to ensure collection of a full 10-L sample. Discard after
one use.
6.2 Equipment for sample filtration. Three options have been demonstrated to be acceptable for use
with Method 1622. Other options may be used if their acceptability is demonstrated according to
the procedures outlined in Section 9.1.2.
6.2.1 Cubitainer spigot to facilitate laboratory filtration of sample (for use with any filtration
option)—Cole Farmer cat. no. U-06061-01, or equivalent
April 2001
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Method 1622 - Cryptosporidium
6.2.2 Envirochek™ sampling capsule equipment requirements for use with the procedure
described in Section 12.0. The version of the method using this filter was validated
using 10-L sample volumes; alternate sample volumes may be used, provided the
laboratory demonstrates acceptable performance on initial and ongoing spiked reagent
water and source water samples (Section 9.1.2).
6.2.2.1 Sampling capsule—Envirochek™, Pall German Laboratory, Ann Arbor,
MI, product 12110
6.2.2.2 Laboratory shaker with arms for agitation of sampling capsules
6.2.2.2.1 Laboratory shaker—Lab-Line model 3589, VWR
Scientific cat. no. 57039-055, Fisher cat. no. 14260-11,
or equivalent
6.2.2.2.2 Side arms for laboratory shaker—Lab-Line Model 3587-
4, VWR Scientific cat. no. 57039-045, Fisher cat. no.
14260-13, or equivalent
6.2.3 CrypTest™ capsule filter equipment requirements. Follow the manufacturer's
instructions when using this filtration option. The version of the method using this filter
was validated using 10-L sample volumes; alternate sample volumes may be used,
provided the laboratory demonstrates acceptable performance on initial and ongoing
spiked reagent water and matrix samples (Section 9.1.2).
6.2.3.1 Capsule filter—CrypTest™, Whatman Inc, Clifton, NJ, product no.
610064
6.2.3.2 Cartridge housing—Ametek 5-in. clear polycarbonate, Whatman cat. no.
71503, or equivalent
6.2.3.3 Ultrasonic bath—VWR Model 75T#21811-808, or equivalent
6.2.3.4 Laboratory tubing—Tygon formula R-3603, or equivalent
6.2.4 Filta-Max™ foam filter equipment requirements. Follow the manufacturer's
instructions when using this filtration option. 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).
6.2.4.1 Foam filter—Filta-Max™, IDEXX, Westbrook, ME. Filter module and
membrane: product code FMC 10601; filter membranes (100 pack),
product code FMC 10800
NOTE: Check at least one filter per batch to ensure that the filters have not been
affected by improper storage or other factors that could result in brittleness or other
problems. At a minimum confirm that the test filter expands properly in water before
using the batch or shipping filters to the field.
6.2.4.2 Filter processing equipment—Filta-Max starter kit, IDEXX, Westbrook,
ME, cat. no. FMC 11002. Includes all equipment required to run and
process Filta-Max filter modules (manual wash station (FMC 10102)
including plunger head (FMC 12001), elution tubing set (FMC 10301),
vacuum set (FMC 10401), filter housing (FMC 10501), and magnetic
stirrer(FMC 10901).
April 2001 4
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Method 1622 - Cryptosporidium
6.3 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 when wear is evident. Dispose of
tubing after one use whenever possible.
6.3.2 Flow control valve—0.5 gpm (0.03 L/s), Bertram Controls, Plast-O-Matic cat. no.
FC050B!/2-PV, or equivalent; or 0.4- to 4-Lpm flow meter with valve—Alamo Water
Treatment, San Antonio, TX, cat. no. R5310, or equivalent
6.3.3 Centrifugal pump—Grainger, Springfield, VA, cat. no. 2P613, or equivalent
6.3.4 Flow meter—Sameco cold water totalizer, E. Clark and Associates, Northboro, MA,
product no. WFU 10.110, or equivalent.
6.4 Equipment for spiking samples in the laboratory
6.4.1 10-L carboy with bottom delivery port (!/2")—Cole-Palmer cat. no. 06080-42, or
equivalent; calibrate to 10.0 L and mark level with waterproof marker
6.4.2 Stir bar—Fisher cat. no. 14-511-93, or equivalent
6.4.3 Stir plate—Fisher cat. no. 14-493-120S, or equivalent
6.4.4 Hemacytometer—Neubauer type, Hauser Scientific, Horsham, PA, cat. no. 3200 or
1475, or equivalent
6.4.5 Hemacytometer coverslip—Hauser Scientific, cat. no. 5000 (for hemacytometer cat. no.
3200) or 1461 (for hemacytometer cat. no 1475), or equivalent
6.4.6 Lens paper without silicone—Fisher cat. no. 11-995, or equivalent
6.4.7 Polystyrene or polypropylene conical tubes with screw caps—15- and 50-mL
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
6.4.8.3 Cellulose acetate support membrane—1.2-|jm-pore-size, 25-mm-
diameter, Fisher cat. no. A12SP02500, or equivalent
6.4.8.4 Polycarbonate track-etch hydrophilic membrane filter—l-|jm-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
6.5 Immunomagnetic separation (IMS) apparatus
6.5.1 Sample mixer—Dynal Inc., Lake Success, NY, cat. no. 947.01, or equivalent
April 2001
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Method 1622 - Cryptosporidium
6.5.2 Magnetic particle concentrator for 10-mL test tubes—Dynal MPC-1® , cat. no. 120.01,
or equivalent
6.5.3 Magnetic particle concentrator for microcentrifuge tubes—Dynal MPC-M® , cat. no.
120.09, or equivalent
6.5.4 Flat-sided sample tubes—16 x 125 mm Leighton-type tubes with 60 x 10 mm flat-sided
magnetic capture area, Dynal L10, cat. no. 740.03, or equivalent
Powder-free latex gloves—Fisher cat no. 113945B, or equivalent
Graduated cylinders, autoclavable—10-, 100-, and 1000-mL
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
Microscope
6.6
6.7
6.8
6.9
6.9.1
6.9.2
6.9.3
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
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
Excitation/band-pass filters for DAPI—Filters cited below (Chroma Technology,
Brattleboro, VT), or equivalent
Microscope
model
Zeiss™ -
Axioskop
Zeiss™ -IM35
Olympus™ BH
Olympus™ BX
Olympus™
IMT2
Fluoro-chrome
DAPI (UV)
DAPI (UV)
DAPI (UV)
Excitation
filter (nm)
340-380
340-380
340-380
Dichroic beam-
splitting mirror
(nm)
400
400
400
Barrier or
suppression
filter (nm)
420
420
420
Filter holder
DAPI (UV)
340-380
400
420
Filter holder
DAPI (UV)
340-380
400
420
Filter holder
Chroma
catalog
number
CZ902
CZ702
11000
91002
11000
91008
11000
91003
6.10 Ancillary equipment for microscopy
6.10.1 Well slides—Treated, 12-mm diameter well slides, Meridian Diagnostics Inc.,
Cincinnati, OH, cat. no. R2206; Spot-On well slides, Dynal cat. no. 740.04; or
equivalent
6.10.2 Glass coverslips—22 x 50 mm
6.10.3 Nonfluorescing immersion oil
6.10.4 Micropipette, adjustable: 0- to 10-|jL with 0- to 10-|jL tips
10- to 100-jL, with 10- to 200-|jL tips
100- to 1000-|aL with 100- to 1000-|jL tips
April 2001
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Method 1622 - Cryptosporidium
6.10.5 Forceps—Splinter, fine tip
6.10.6 Forceps—Blunt-end
6.10.7 Desiccant—Drierite™ Absorbent, Fisher cat. no. 07-577-1A, or equivalent
6.10.8 Humid chamber—A tightly sealed plastic container containing damp paper towels on
top of which the slides are placed
6.11 Pipettes—Glass or plastic
6.11.1 5-, 10-, and 25-mL
6.11.2 Pasteur, disposable
6.12 Balances
6.12.1 Analytical—Capable of weighing 0.1 mg
6.12.2 Top loading—Capable of weighing 10 mg
6.13 pH meter
6.14 Incubator—Fisher Scientific Isotemp™, or equivalent
6.15 Vortex mixer—Fisons Whirlmixer, or equivalent
6.16 Vacuum source—Capable of maintaining 25 in. Hg, equipped with shutoff valve and vacuum
gauge
6.17 Miscellaneous labware and supplies
6.17.1 Test tubes and rack
6.17.2 Flasks—Suction, Erlenmeyer, and volumetric, various sizes
6.17.3 Beakers—Glass or plastic, 5-, 10-, 50-, 100-, 500-, 1000-, and 2000-mL
6.17.4 Lint-free tissues
6.18 10- to 15-L graduated container—Fisher cat. no. 02-961-50B, or equivalent; calibrate to 9.0, 9.5,
10.0, 10.5, and 11.0 L and mark levels with waterproof marker
6.19 Filters for filter-sterilizing reagents—Sterile Acrodisc, 0.45 jjm, Gelman Sciences cat no. 4184, or
equivalent
7.0 Reagents and Standards
7.1 Reagents for adjusting pH
7.1.1 Sodium hydroxide (NaOH)—ACS reagent grade, 6.0 N and 1.0 N in reagent water
7.1.2 Hydrochloric acid (HC1)—ACS reagent grade, 6.0 N, 1.0 N, and 0.1 N in reagent water
NOTE: Due to the low volumes of pH-adjusting reagents used in this method, and the
impact that changes in pH have on the immunofluorescence assay, the laboratory
should purchase standards at the required normality directly from a vendor. Normality
should not be adjusted by the laboratory.
7.2 Solvents—Acetone, glycerol, ethanol, and methanol, ACS reagent grade
7.3 Reagent water—Water in which oocysts and interfering materials and substances, including
magnetic minerals, are not detected by this method
7.4 Reagents for eluting filters
7.4.1 Reagents for eluting Envirochek™ 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
7 April 2001
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Method 1622 - Cryptosporidium
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-|jm membrane into a sterile plastic container and
store at room temperature.
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
and adjust pH to 8.0 with 6.0 N HC1 or NaOH. Dilute to a final volume
of 1000 mL with reagent water and adjust to pH 8.0 with 1.0 N HC1 or
NaOH.
7.4.1.4 Antifoam A—Sigma Chemical Co. cat. no. A5758, or equivalent
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 |jL Antifoam A
(Section 7.4.1.4). Dilute to 1000 mL with reagent water.
7.4.2 Reagents for eluting CrypTest™ capsule filters (Section 6.2.3). To 900 mL of reagent
water add 8.0 g NaCl, 0.2 g KH2PO4, 2.9 g Na2HPO4 (12H2O) 0.2 g KC1, 0.2 g sodium
lauryl sulfate (SDS), 0.2 mL Tween 80, and 0.02 mL Antifoam A (Sigma Chemical Co.
cat. no. A5758, or equivalent). Adjust volume to 1 L with reagent water and adjust pH
to 7.4 with 1 N NaOH or HC1.
7.4.3 Reagents for eluting Filta-Max™ foam filters (Section 6.2.4)
7.4.3.1 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.
7.4.3.2 Tween 20—Sigma Chemical Co. cat. no. P-7949, or equivalent
7.4.3.3 High-vacuum grease—BDH/Merck. cat. no. 636082B, or equivalent
7.4.3.4 Preparation of PBST elution buffer. Add the contents of one sachet of
PBS to 1.0 L of reagent water. Dissolve by stirring for 30 minutes. Add
100 joL of Tween 20. Mix by stirring for 5 minutes.
7.5 Reagents for immunomagnetic separation (IMS)—Dynabeads® anti-Cryptosporidium kit, Dynal
cat. nos. 730.01, 730.11, or equivalent
7.6 Direct antibody labeling reagents for detection of oocysts. Store reagents at 0 °C to 8 °C and
return promptly to this temperature after each use. Do not allow any of the reagents to freeze.
The reagents should be protected from exposure to light. Diluted, unused working reagents
should be discarded after 48 hours. Discard reagents after the expiration date is reached. The
labeling reagents in Sections 7.6.1-7.6.3 have been approved for use with this method.
7.6.1 Crypt-a-Glo™, Waterborne cat. no. A400FLR, New Orleans, LA, or equivalent
7.6.2 Merifluor Cryptosporidium/Gictrdia, Meridian Diagnostics cat. no. 250050, Cincinnati,
OH, or equivalent
7.6.3 Aqua-Glo™ G/C Direct FL, Waterborne cat. no. A100FLR, New Orleans, LA, or
equivalent
April 2001 8
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Method 1622 - Cryptosporidium
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.
7.6.4 Diluent for labeling reagents—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. Filter-sterilize (Section 6.19) or autoclave. Discard if growth is detected
or after 6 months, whichever comes first.
7.7 4',6-diamidino-2-phenylindole (DAPI) stain—Sigma Chemical Co. cat. no. A5758, or equivalent
7.7.1 Stock solution—Dissolve 2 mg/mL DAPI in absolute methanol. Prepare volume
consistent with minimum use. Store at 0 °C to 8 °C in the dark. Do not allow to freeze.
Discard unused solution when positive staining control fails.
7.7.2 Staining solution (1/5000 dilution in PBS [Section 7.6.4])—Add 10 |jL of 2 mg/mL
DAPI stock solution to 50 mL of PBS. Prepare daily. Store at 0 °C to 8 °C in the dark
except when staining. Do not allow to freeze. The solution concentration may be
increased up to 1 |jg/mL if fading/diffusion of DAPI staining is encountered, but the
staining solution must be tested first on expendable environmental samples to confirm
that staining intensity is appropriate.
7.8 Mounting medium
7.8.1 DABCO/glycerol mounting medium (2%)—Dissolve 2 g of DABCO (Sigma Chemical
Co. cat no. D-2522, or equivalent) in 95 mL of warm glycerol/PBS (60% glycerol, 40%
PBS [Section 7.6.4]). After the DABCO has dissolved completely, adjust the solution
volume to 100 mL by adding an appropriate volume of glycerol/PBS solution.
Alternately, dissolve the DABCO in 40 mL of PBS, then add azide (1 mL of 100X, or
10% solution), then 60 mL of glycerol.
7.8.2 Mounting medium supplied with Merifluor direct labeling kit (Section 7.6.2)
7.9 Clear fingernail polish or clear fixative, PGC Scientifics, Gaithersburg, MD, cat. no. 60-4890, or
equivalent
7.10 Oocyst suspensions for spiking
7.10.1 Enumerated spiking suspensions prepared by flow cytometer—not heat-fixed or
formalin fixed: Wisconsin State Laboratory of Hygiene Flow Cytometry Unit or
equivalent
7.10.2 Materials for manual enumeration of spiking suspensions
7.10.2.1 Purified Cryptosporidium oocyst stock suspension for manual
enumeration—not heat-fixed or 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
7.10.2.3 Storage procedure—Store oocyst suspensions at 0 °C to 8 °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
April 2001
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Method 1622 - Cryptosporidium
8.0 Sample Collection and Storage
8.1 Samples are collected as bulk samples and shipped to the laboratory for processing through the
entire method, or are filtered in the field and shipped to the laboratory for processing from
elution (Section 12.2.6) onward. Samples must be shipped via overnight service on the day they
are collected. Chill samples as much as possible between collection and shipment by storing in a
refrigerator or pre-icing the sample in a cooler. If the sample is pre-iced before shipping, replace
with fresh ice immediately before shipment. Samples should be shipped at 0 °C to 8 °C, unless the
time required to chill the sample to 8 ° C would prevent the sample from being shipped overnight
for receipt at the laboratory the day after collection. Samples must not be allowed to freeze.
Upon receipt, the laboratory should record the temperature of the samples and store them
refrigerated at 0 °C to 8 °C until processed. Results from samples shipped overnight to the
laboratory and received at >8 ° C should be qualified by the laboratory.
NOTE: See transportation precautions in Section 5.5.
8.2 Sample holding times. Sample processing should be completed as soon as possible by the
laboratory. The laboratory should complete sample filtration, elution, concentration, purification,
and staining the day the sample is received wherever possible. However, the laboratory is
permitted to split up the sample processing steps if processing a sample completely in one day is
not possible. If this is necessary, sample processing can be halted after filtration, application of
the purified sample onto the slide, or staining. Table 1, in Section 21.0 provides a breakdown of
the holding times for each set of steps. Sections 8.2.1 through 8.2.4 provide descriptions of these
holding times.
8.2.1 Sample collection and filtration. Sample elution must be initiated within 96 hours of
sample collection (if shipped to the laboratory as a bulk sample) or filtration (if filtered
in the field).
8.2.2 Sample elution, concentration, and purification. The laboratory must complete the
elution, concentration, and purification (Sections 12.2.6 through 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.
8.2.3 Staining. The sample must be stained within 72 hours of application of the purified
sample to the slide.
8.2.4 Examination. Although immunofluorescence assay (FA) and 4',6-diamidino-2-
phenylindole (DAPI) and differential interference contrast (DIC) microscopy
examination and confirmation should be performed immediately after staining is
complete, laboratories have up to 7 days from completion of sample staining to
complete the examination and confirmation of samples. However, if fading/diffusion of
FITC or DAPI staining is noticed, the laboratory must reduce this holding time. In
addition the laboratory may adjust the concentration of the DAPI staining solution
(Sections 7.7.2) so that fading/diffusion does not occur.
8.5 Spiking suspension enumeration holding times. Flow-cytometer-sorted spiking suspensions
(Sections 7.10.1 and 11.2) used for spiked quality control (QC) samples (Section 9) must be used
within the expiration date noted on the suspension. Laboratories should use flow-cytometer-
sorted spiking suspensions containing live organisms within two weeks of preparation at the flow
cytometry laboratory. Manually enumerated spiking suspensions must be used within 24 hours of
enumeration of the spiking suspension if the hemacytometer chamber technique is used (Section
11.3.4); or within 24 hours of application of the spiking suspension to the slides if the well slide
or membrane filter enumeration technique is used (Sections 11.3.5 and 11.3.6).
April 2001 10
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Method 1622 - Cryptosporidium
9.0 Quality Control
9.1 Each laboratory that uses this method is required to operate a formal quality assurance (QA)
program (Reference 20.6). The minimum requirements of this program consist of an initial
demonstration of laboratory capability through performance of the initial precision and recovery
(IPR) test (Section 9.4), analysis of spiked samples to evaluate and document data quality, and
analysis of standards and blanks as tests of continued performance. Laboratory performance is
compared to established performance criteria to determine if the results of analyses meet the
performance characteristics of the method.
9.1.1 A test of the microscope used for detection of oocysts 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).
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 is required to validate
the modification according to Tier 1 of EPA's
performance-based measurement system (PBMS) (Table
2 and Reference 20.7) 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 Table 3 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.
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 validate the modification
11 April 2001
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Method 1622 - Cryptosporidium
according to Tier 2 of EPA's PBMS (Table 2 and
Reference 20.7). 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 Table 3 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.
9.1.2.2 The laboratory is required to maintain records of modifications made to
this method. These records include the following, at a minimum:
9.1.2.2.1 The names, titles, addresses, and telephone numbers of
the analyst(s) who performed the analyses and
modification, and of the quality control officer who
witnessed and will verify the analyses and modification.
9.1.2.2.2 A listing of the analyte(s) measured (Cryptosporidium).
9.1.2.2.3 A narrative stating reason(s) for the modification.
9.1.2.2.4 Results from all QC tests comparing the modified
method to this method, including:
(a) 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
3.3.3.11)
(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
April 2001 12
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Method 1622 - Cryptosporidium
(o) Lot numbers of elution, IMS, and staining
reagents
(p) Copies of bench sheets, logbooks, and other
recordings of raw data
(q) Data system outputs, and other data to link the
raw data to the results reported
9.1.3 The laboratory shall spike a separate sample aliquot from the same source to monitor
method performance. This MS test is described in Section 9.5.1.
9.1.4 Analysis of method blanks is required to demonstrate freedom from contamination.
The procedures and criteria for analysis of a method blank are described in Section 9.6.
9.1.5 The laboratory shall, on an ongoing basis, demonstrate through analysis of the ongoing
precision and recovery (OPR) sample that the analysis system is in control. These
procedures are described in Section 9.7.
9.1.6 The laboratory shall maintain records to define the quality of data that are generated.
Development of accuracy statements is described in Sections 9.5.1.4 and 9.7.3.
9.1.7 The laboratory shall analyze one method blank (Section 9.6) and one OPR sample
(Section 9.7) each week during which samples are analyzed if 20 or fewer field samples
are analyzed during this period. The laboratory shall analyze one laboratory blank and
one OPR sample for every 20 samples if more than 20 samples are analyzed in a week.
9.1.8 The laboratory shall analyze one MS sample (Section 9.5.1) when samples are first
received from a utility for which the laboratory has never before analyzed samples. The
MS analysis is performed on an additional (second) sample sent from the utility. If the
laboratory routinely analyzes samples from 1 or more utilities, 1 MS analysis must be
performed per 20 field samples. For example, when a laboratory receives the first
sample from a given site, the laboratory must obtain a second aliquot of this sample to
be used for the MS. When the laboratory receives the 21st sample from this site, a
separate aliquot of this 21st sample must be collected and spiked.
9.2 Micropipette calibration
9.2.1 Micropipettes must be sent to the manufacturer for calibration annually. Alternately, a
qualified independent technician specializing in micropipette calibration can be used.
Documentation on the precision of the recalibrated micropipette must be obtained from
the manufacturer or technician.
9.2.2 Internal and external calibration records must be kept on file in the laboratory's QA
logbook.
9.2.3 If a micropipette calibration problem is suspected, the laboratory shall tare an empty
weighing boat on the analytical balance and pipette the following volumes of reagent
water into the weigh boat using the pipette in question: 100% of the maximum
dispensing capacity of the micropipette, 50% of the capacity, and 10% of the capacity.
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.
13 April 2001
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Method 1622 - Cryptosporidium
9.3 Microscope adjustment and certification: Adjust the microscope as specified in Section 10.0. All
of the requirements in Section 10.0 must be met prior to analysis of IPRs, blanks, OPRs, field
samples, and MS/MSDs.
9.4 Initial precision and recovery (IPR) — To establish the ability to demonstrate control over the
analytical system and to generate acceptable precision and recovery, the laboratory shall perform
the following operations:
9.4.1 Using the spiking procedure in Section 1 1.4 and enumerated spiking suspensions
(Section 7.10.1 or Section 11.3), spike, filter, elute, concentrate, separate (purify), stain,
and examine four reagent water samples spiked with 100 to 500 oocysts. If more than
one process will be used for filtration and/or separation of samples, a separate set of
_ IPR samples must be prepared for each process. _
NOTE: IPR tests must be accompanied by analysis of a method blank (Section 9. 6).
9.4.2 Using results of the four analyses, calculate the average 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.
9.4.3 Compare RSD and the mean with the corresponding limits for initial precision and
recovery in Table 3 in Section 21.0. If the RSD and the mean meet the acceptance
criteria, system performance is acceptable and analysis of blanks and samples may
begin. If the RSD or the mean falls outside the range for recovery, system performance
is unacceptable. In this event, correct the problem and repeat the test (Section 9.4. 1).
9.5 Matrix spike (MS) and matrix spike duplicate (MSD):
9.5.1 Matrix spike — The laboratory shall spike and analyze a separate field sample aliquot to
determine the effect of the matrix on the method's oocyst recovery. The MS shall be
analyzed according to the frequency in Section 9.1.8.
9.5.1 .1 Analyze an unspiked field sample according to the procedures in
Sections 12.0 to 15.0. Using the spiking procedure in Section 1 1.4 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 the number of organisms 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.
A/,n - A/,
where
R is the percent recovery
Nsp is the number of oocysts detected in the spiked sample
Ns is the number of oocysts detected in the unspiked sample
T is the true value of the oocysts spiked
9.5.1 .3 Compare the recovery with the corresponding limits in Table 3 in
Section 21.0.
NOTE: Some sample matrices may prevent the acceptance criteria in Table 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 for the laboratory, method precision for
samples should be assessed and records maintained. After the analysis
April 2001 14
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Method 1622 - Cryptosporidium
of five samples for which the spike recovery passes the tests in Section
9.5.1.3, the laboratory should calculate the average percent recovery (P)
and the standard deviation of the percent recovery (sr). Express the
precision assessment as a percent recovery interval from P - 2 sr to P +
2 sr for each matrix. For example, if P = 80% and sr = 30%, the accuracy
interval is expressed as 20% to 140%. The precision assessment should
be updated regularly across all MS samples and stratified by MS samples
for each source.
9.5.2 Matrix spike duplicate — MSB analysis is required as part of nationwide approval of a
modified version of this method to demonstrate that the modified version of this
method produces results equal or superior to results produced by the method as written
(Section 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 athird,
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.
9.5.2.2 Calculate the mean of the number of oocysts in the MS and MSB (Xmem)
(= [MS+MSB]/2).
9.5.2.3 Calculate the relative percent difference (RPB) of the recoveries using
the following equation:
Xmsan
where
RPB is the relative percent difference
NMS is the number of oocysts detected in the MS
NMSD is the number of oocysts detected in the MSB
Xmem is the mean number of oocysts detected in the MS and
MSB
9.5.2.4 Compare the mean MS/MSB recovery and RPB with the corresponding
limits in Table 3 in Section 21.0.
9.6 Method blank (negative control sample, laboratory blank): Reagent water blanks are analyzed to
demonstrate freedom from contamination. Analyze the blank immediately prior to analysis of the
IPRtest (Section 9.4) and OPRtest (Section 9.7) and prior to analysis of samples for the week to
demonstrate freedom from contamination.
9.6.1 Filter, elute, concentrate, separate (purify), stain, and examine at least one reagent water
blank per week (Section 9.1.7) according to the procedures in Sections 12.0 to 15.0. If
more than 20 samples are analyzed in a week, process and analyze one reagent water
blank for every 20 samples.
9.6.2 If Cryptosporidium oocysts or any potentially interfering organism or material is found
in the blank, analysis of additional samples is halted until the source of contamination
is eliminated and a blank shows no evidence of contamination. Any sample in a batch
associated with a contaminated blank that shows the presence of one or more oocysts is
assumed to be contaminated and should be recollected, if possible. Any method blank
in which oocysts are not detected is assumed to be uncontaminated and may be
reported.
15 April 2001
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Method 1622 - Cryptosporidium
9.7 Ongoing precision and recovery ([OPR]; positive control sample; laboratory control sample):
Using the spiking procedure in Section 11.4 and enumerated spiking suspensions (Section
7.10.1 or Section 11.3), filter, elute, concentrate, separate (purify), stain, and examine at least one
reagent water sample spiked with 100 to 500 oocysts each week to verify all performance
criteria. The laboratory must analyze one OPR sample for every 20 samples if more than 20
samples are analyzed in a week. If multiple method variations are used, separate OPR samples
must be prepared for each method variation. Adjustment and/or recalibration of the analytical
system shall be performed until all performance criteria are met. Only after all performance
criteria are met may samples be analyzed.
9.7.1 Examine the slide from the OPR prior to analysis of samples from the same batch.
9.7.1.1 Using 200X to 400X magnification, more than 50% of the oocysts must
appear undamaged and morphologically intact; otherwise, the analytical
process is damaging the organisms. Determine the step or reagent that is
causing damage to the organisms. Correct the problem and repeat the
OPR test.
9.7.1.2 Identify and enumerate each organism using epifluorescence
microscopy. The first three presumptive 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= lOOx —
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 limits for ongoing precision and recovery in Table 3 in
Section 21.0. If the recovery meets the acceptance criteria, system performance is
acceptable and analysis of blanks and samples may proceed. If, however, the recovery
falls outside of the range given, system performance is unacceptable. In this event,
there may be a problem with the microscope or with the filtration or separation
systems. Troubleshoot the problem using the procedures at Section 9.7.4 as a guide.
After assessing the issue, reanalyze the OPR sample. All samples must be associated
with an OPR that passes the criteria in Section 21.0. Samples that are not associated
with an acceptable OPR must be flagged accordingly.
9.7.4 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.
9.7.4.1 Microscope system and antibody stain: To determine if the failure of
the OPR test is due to changes in the microscope or problems with the
antibody stain, re-examine the positive staining control (Section 15.2.1),
check 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 the
previously-prepared positive staining control to determine whether the
problem is associated with the microscope or the antibody stain.
April 2001 16
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Method 1622 - Cryptosporidium
9.7.4.2 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.
9.7.4.3 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 13.2.2.1, and filter, stain, and
examine the sample concentrate according to Section 11.3.4.
9.7.5 The laboratory should add results that pass the specifications in Section 9.7.3 to initial
and previous ongoing data and update the QC chart to form a graphic representation of
continued laboratory performance. The laboratory should develop a statement of
laboratory accuracy (reagent water, raw surface water) by calculating the average
percent recovery (R) and the standard deviation of percent recovery (sr). Express the
accuracy as a recovery interval from R - 2 ST to R + 2 sr. For example, if R = 95% and sr
= 25%, the accuracy is 45% to 145%.
9.8 The laboratory should periodically analyze an external QC sample, such as a performance
evaluation or standard reference material, when available. The laboratory also should
periodically participate in interlaboratory comparison studies using the method.
9.9 The specifications contained in this method can be met if the analytical system is under control.
The standards used for initial (Section 9.4) and ongoing (Section 9.7) precision and recovery
should be identical, so that the most precise results will be obtained. The microscope in
particular will provide the most reproducible results if dedicated to the settings and conditions
required for the determination of Cryptosporidium by this method.
9.10 Depending on specific program requirements, field replicates may be collected to determine the
precision of the sampling technique, and duplicate spiked samples may be required to determine
the precision of the analysis.
10.0 Microscope Calibration and Analyst Verification
10.1 In a room capable of being darkened to near-complete darkness, assemble the microscope, all
filters, and attachments. The microscope should be placed on a solid surface free from vibration.
Adequate workspace should be provided on either side of the microscope for taking notes and
placement of slides and ancillary materials.
10.2 Using the manuals provided with the microscope, all analysts must familiarize themselves with
operation of the microscope.
10.3 Microscope adjustment and calibration (adapted from Reference 20.6)
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 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.
17 April 2001
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Method 1622 - Cryptosporidium
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.
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.
April 2001 18
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Method 1622 - Cryptosporidium
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.
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.
19 April 2001
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Method 1622 - Cryptosporidium
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 atop 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:
0.6 mm 0.0125 mm
48 ocular micrometer spaces ocular micrometer space
10.3.5.6 Because most measurements of microorganisms are given in um rather
than mm, the value calculated above must be converted to |jm by
multiplying it by 1000 |im /mm. For example:
0.0125 mm 1,000 |i/n 12.5|j,/n
ocular micrometer space mm ocular micrometer space
April 2001 20
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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=
NA =
NA =
NA =
No. of ocular
micrometer
spaces
No. of stage
micrometer
mm1
um /ocular
micrometer
space2
1100 jam/mm
2(Stage micrometer length in mm * (1000 u,m/mm)) •*• no. ocular micrometer
spaces
3N.A. refers to numerical aperature. The numerical aperature value is engraved
on the barrel of the objective.
10.3.6 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 is now adjusted to make it
compatible with the total numerical aperture of the optical system. This
is done by removing an ocular, looking into the tube at the rear focal
plane of the objective, and stopping down the aperture diaphragm iris
leaves until they are visible just inside the rear plane of the objective.
21
April 2001
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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
10.4 Protozoa libraries: Each laboratory is encouraged to develop libraries of photographs and
drawings for identification of protozoa.
10.4.1 Take color photographs of Cryptosporidium oocysts by FA and 4',6-diamidino-2-
phenylindole (DAPI) that the analysts (Section 22.2) determine are accurate (Section
15.2).
10.4.2 Similarly, take color photographs of interfering organisms and materials by FA and
DAPI 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 positive identification of DAPI
positive or negative organisms.
10.5 Verification of performance: Until standard reference materials, such as National Institute of
Standards and Technology standard reference materials, are available that contain a reliable
number of DAPI positive or negative oocysts, this method shall rely upon the ability of the
analyst for identification and enumeration of oocysts.
10.5.1 At least monthly when microscopic examinations are being performed, the laboratory
shall prepare a slide containing 40 to 100 oocysts. More than 50% of the oocysts must
be DAPI positive.
10.5.2 Each analyst shall determine the total number of oocysts and the number that are DAPI
positive or negative using the slide prepared in Section 10.5.1.
10.5.3 The total number and the number of DAPI positive or negative oocysts determined by
each analyst (Section 10.5.2.) must be within ±10% of each other. If the number is not
within this range, the analysts must identify the source of any variability between
analysts' examination criteria, prepare a new slide, and repeat the performance
verification (Sections 10.5.1 to 10.5.2).
10.5.4 Document the date, name(s) of analyst(s), number of total, DAPI positive or negative
oocysts determined by the analyst(s), whether the test was passed/failed and the results
of attempts before the test was passed.
10.5.5 Only after an analyst has passed the criteria in Section 10.5.3, may oocysts in QC
samples and field samples be identified and enumerated.
11.0 Oocyst Suspension Enumeration and Spiking
11.1 This method requires routine analysis of spiked QC samples to demonstrate acceptable initial
and ongoing laboratory and method performance (initial precision and recovery samples
[Section 9.4], matrix spike and matrix spike duplicate samples [Section 9.5], and ongoing
precision and recovery samples [Section 9.7]). The organisms used for these samples must be
enumerated to calculate recoveries and precision. EPA recommends that flow cytometry be used
for this enumeration, rather than manual techniques. Flow cytometer-sorted spikes generally are
characterized by a relative standard deviation of < 2.5%, versus greater variability for manual
enumeration techniques (Reference 20.8). Guidance on preparing spiking suspensions using a
flow cytometer is provided in Section 11.2. Manual enumeration procedures are provided in
Section 11.3. The procedure for spiking bulk samples in the laboratory is provided in Section
11.4.
11.2 Flow cytometry enumeration guidelines. Although it is unlikely that many laboratories
performing Method 1622 will have direct access to a flow cytometer for preparing spiking
suspensions, flow-sorted suspensions are available from commercial vendors and other sources
April 2001 22
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Method 1622 - Cryptosporidium
(Section 7.10.1). The information provided in Sections 11.2.1 through 11.2.4 is simply meant as
a guideline for preparing spiking suspensions using a flow cytometer. Laboratories performing
flow cytometry must develop and implement detailed standardized protocols for calibration and
operation of the flow cytometer.
11.2.1 Spiking suspensions should be prepared using unstained organisms that have not been
heat-fixed or formalin-fixed.
11.2.2 Spiking suspensions should be prepared using Cryptosporidium parvum oocysts <3
months old.
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 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. Laboratories should use flow-cytometer-sorted
spiking suspensions containing live organisms within two weeks of preparation at the
flow cytometry laboratory.
11.3 Manual enumeration procedures. Two sets of manual enumerations are required per
organism before purified Cryptosporidium oocyst stock suspensions (Section 7.9.2.1) received
from suppliers can be used to spike samples in the laboratory. First, the stock suspension must
be diluted and enumerated (Section 11.3.3) to yield a suspension at the appropriate oocyst
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 submitted by 20 different laboratories under the EPA
Protozoa Performance Evaluation Program).
23 April 2001
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Method 1622 - Cryptosporidium
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 ^L of
vortexed suspension per chamber. If this operation has been properly
executed, the liquid should amply fill the entire chamber without
bubbles or overflowing into the surrounding moats. Repeat this step
with a clean, dry hemacytometer and coverslip if loading has been
incorrectly performed. See Section 11.3.3.13, 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 it.
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.
11.3.3.9 Use the following formula to determine the number of organisms per
mL of suspension:
number of organisms counted 10 dilution factor 1000mm3
-, x x x = number of organisms / mL
number of mm counted 1 mm 1 1 mL
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 8000 and 12,000 organisms
per mL (80 to 120 organisms per 10 uL); however, ranges as great as
April 2001 24
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Method 1622 - Cryptosporidium
5000 to 15,000 organisms per mL (50 to 150 organisms per 10 |iL) can
be used.
NOTE: If the diluted stock suspensions (the spiking suspensions) will be enumerated
using hemacytometer chamber counts (Section 11.3.4) or membrane filter counts
(Section 11.3.6), then the stock suspensions should be diluted with 0.01% Tween-20. If
the spiking suspensions will be enumerated using well slide counts (Section 11.3.3),
then the stock suspensions should be diluted in reagent water.
To calculate the volume (in |jL) of stock suspension required per mL of
reagent water (or reagent water/Tween-20, 0.01%), use the following
formula:
required number of organisms x 1000 uL
volume of stock suspension (u.L) required =
number of organisms /mL of stock suspension
If the volume is less than 10 |jL, 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 |jL , use the following formula:
number of organisms required x 10 \iL
predicted number of organisms per 10 ul (80 to 120)
To calculate the volume of reagent water (or reagent water/Tween-20,
0.01%) needed, use the following formula:
reagent water volume (\nL) =
total volume (\nL) - stock suspension volume required (\nL )
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.
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
25 April 2001
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Method 1622 - Cryptosporidium
• 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-uL micropipette without touching the stir bar.
Cover the beaker with a watch glass or petri dish to prevent evaporation
between sample withdrawals.
11.3.4.3 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 |jL (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.
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 Remove well slides from cold storage and lay the slides on a flat surface
for 15 minutes to allow them to warm to room temperature.
11.3.5.2 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.
April 2001 26
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Method 1622 - Cryptosporidium
11.3.5.3 Remove a 10-^L 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 |jL 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 Apply 50-|jL of absolute methanol to each well containing the dried
sample and allow to air-dry for 3 to 5 minutes.
11.3.5.8 Follow the manufacturer's instructions (Section 7.6) in applying the
stain to the slide.
11.3.5.9 Place the slides in a humid chamber in the dark and incubate at room
temperature for approximately 30 minutes. The humid chamber consists
of a tightly sealed plastic container containing damp paper towels on top
of which the slides are placed.
11.3.5.10 Apply one drop of wash buffer (prepared according to the
manufacturer's instructions [Section 7.6]) to each well. Tilt each slide on
a clean paper towel, long edge down. Gently aspirate the excess
detection reagent from below the well using a clean Pasteur pipette or
absorb with a paper towel or other absorbent material. Avoid disturbing
the sample.
NOTE: If using the Merifluor stain (Section 7.6.1), do not allow slides to dry
completely.
11.3.5.11 Add mounting medium (Section 7.8) to each well.
11.3.5.12 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.
11.3.5.13 Record the date and time that staining was completed. If slides will not
be read immediately, store in a humid chamber in the dark at 0 ° C to 8 °C
until ready for examination.
11.3.5.14 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
27 April 2001
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Method 1622 - Cryptosporidium
<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 pH 7.2
(Section 7. 9. 4). Dilute detection reagent (Section 7.7) as per
manufacturer's instructions using sterile PBS. Multiply the anticipated
number of filters to be stained by 100 mL to calculate total volume of
stain required. Divide the total volume required by 5 to obtain the
microliters of antibody necessary. Subtract the volume of antibody from
the total stain volume to obtain the required microliters of sterile PBS to
add to the antibody.
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.
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-|jL aliquots from the
spiking suspension and pipet into the 5 mL of 0.01% Tween 20 standing
April 2001 28
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Method 1622 - Cryptosporidium
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 for the next three samples (if
that the diluted spiking suspension has sat less than 15 minutes, reduce
the suspension vortex time to 60 seconds). Ten, 10-|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-|jL spiking
suspensions have been filtered. The last batch should include a 10-|jL
0.01 Tween 20 blank control and 20 |iL 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 |jL 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
at 0 °C to 8 °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
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
29 April 2001
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Method 1622 - Cryptosporidium
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.
11.4 Procedure for spiking samples in the laboratory with enumerated spiking suspensions.
11.4.1 Arrange a bottom-dispensing container to feed the filter.
11.4.2 For initial precision and recovery (Section 9.4) and ongoing precision and recovery
(Section 9.7) samples, fill the container with 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 Vortex the spiking suspension(s) (Section 11.2 or Section 11.3) for a minimum of 2
minutes.
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 |iL of Antifoam A to 100 mL of reagent water,
and mix well to emulsify.
11.4.3.1.2 Add 5 00 |iL of the diluted antifoam to the tube
containing the spiking suspension and vortex for 2
minutes.
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 Rinse a pipette tip with 0.01% Tween-20 once, then
rinse with the well-mixed spiking suspension a
minimum of five times before pulling an aliquot to be
used to spike the container.
11.4.3.2.2 Add the spiking suspension(s) to the carboy, delivering
the aliquot below the surface of the water.
11.4.3.2.3 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
11.4.4 Allow the spiking suspensions to mix for approximately 1 minute in the container.
11.4.5 Turn on the pump and allow the flow rate to stabilize. Set flow at the rate designated
for the filter being used. As the carboy is depleted, check the flow rate and adjust if
necessary.
April 2001 30
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Method 1622 - Cryptosporidium
11.4.6 When the water level approaches the discharge port of the carboy, tilt the container so
that it is completely emptied. At that time, turn off the pump and add sufficient reagent
water to the container to rinse. Swirl the contents to rinse down the sides.
11.4.7 Turn on the pump. Allow all of the water to flow through the filter and turn off the
pump.
12.0 Sample Filtration and Elution
12.1 A water sample is filtered according to the procedures in Section 12.2. 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).
12.2 Capsule filtration (adapted from Reference 20.9). This procedure was validated using 10-L
sample volumes. Alternate sample volumes may be used, provided the laboratory demonstrates
acceptable performance on initial and ongoing spiked reagent water and source water samples
(Section 9.1.2).
NOTE: The filtration procedures specified in Section 12.2.1-12.2.5.3 are specific to
laboratory filtration of a bulk sample, and reflect the procedures used during the
inter lab oratory validation of this method (Reference 20.10). These procedures may
require modification if samples will be filtered in the field.
12.2.1 Flow rate adjustment
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 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.
12.2.4.2 Place the drain end of the sampling system tubing into an empty
graduated container with a capacity of 10 to 15 L, calibrated at 9.0, 9.5,
10.0, 10.5, and 11.0 L (Section 6.18). 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.
31 April 2001
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Method 1622 - Cryptosporidium
12.2.4.3
12.2.4.4
12.2.5 Disassembly
12.2.5.1
12.2.5.2
12.2.5.3
12.2.6 Elution
Allow the carboy discharge tube and capsule to fill with sample water.
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 upon receipt
in the laboratory, pull the remaining sample volume through the filter
before eluting the filter [Section 12.2.6].)
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 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 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.
12.2.6.1
Setup
12.2.6.1.1
12.2.6.2
12.2.6.1.2
12.2.6.1.3
Elution
12.2.6.2.1
12.2.6.2.2
Assemble the laboratory shaker with the clamps aligned
vertically so that the filters will be aligned horizontally.
Extend the clamp arms to their maximum distance from
the horizontal shaker rods to maximize the shaking
action.
Prepare sufficient elution buffer so that all samples to be
eluted that day can be eluted with the same batch of
buffer. Elution may require up to 275 mL of buffer per
sample.
Designate at least one 250-mL conical centrifuge tube
for each sample and label with the sample number.
Record the 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 and allow the liquid level to stabilize.
Pour elution buffer through the inlet fitting. Sufficient
elution buffer must be added to cover the pleated white
membrane with buffer solution. Replace the inlet cap
and clamp the cap in place.
April 2001
32
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Method 1622 - Cryptosporidium
12.2.6.2.3 Securely clamp the capsule in one of the clamps on the
laboratory shaker with the bleed valve positioned at the
top on a vertical axis (in the 12 o'clock position). Turn
on the shaker and set the speed to maximum
(approximately 900 rpm). 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.
12.2.6.2.4 Remove the filter from the shaker, remove the inlet cap,
and pour the contents of the capsule into the 250-mL
conical centrifuge tube.
12.2.6.2.5 Clamp the capsule vertically with the inlet end up and
add sufficient volume of elution buffer through the inlet
fitting to cover the pleated membrane. Replace the inlet
cap.
12.2.6.2.6 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.
12.2.6.2.7 Remove the filter from the shaker, but leave the elution
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).
13.0 Sample Concentration and Separation (Purification)
13.1 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 may be used if the laboratory first
demonstrates equivalent or superior performance as per Section 9.1.2.
13.2 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.
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).
33 April 2001
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Method 1622 - Cryptosporidium
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 needs to 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
(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. Vortex the tube vigorously for 10 to 15 seconds to completely
resuspend the pellet. Record the resuspended pellet volume on the bench sheet.
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. Proceed 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
April 2001 34
-------
Method 1622 - Cryptosporidium
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, proceed 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 = x 7 00%
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.
13.3 IMS procedure (adapted from Reference 20.11)
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, take 100
|jL of 10X SL-buffer-A and make up to 1 mL with the diluent water. A
volume of 1.5 mL of IX SL-buffer-A will be required per sample or
subsample on which the Dynal IMS procedure is performed.
13.3.1.2 For each 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 10X SL-buffer-A.
13.3.2 Oocyst 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-buffer. 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
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 10 mL. (For
example, if 5 mL was transferred after resuspension of the pellet, 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 10 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 10 mL with reagent water.
Label the flat-sided tube(s) with the sample number (and subsample
letters).
35 April 2001
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Method 1622 - Cryptosporidium
13.3.2.2 Vortex the Dynabeads® anti-Cryptosporidium 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 beads (Section 13.3.2.2) to the sample
tube(s) containing the water sample concentrate and SL-buffer.
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 (MPC-1) 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 MPC-1 is allowed to stand motionless for more than 10
seconds, repeat Section 13.3.2.9 before continuing to Section 13.3.2.11.
13.3.2.9 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.
13.3.2.10 Remove the sample tube from the MPC-1 and resuspend the sample in
1-mL IX SL-buffer-A (prepared from 10X SL-buffer-A
stock—supplied). Mix very gently to resuspend all material in the tube.
Do not vortex.
13.3.2.11 Quantitatively transfer (transfer followed by two rinses) all the liquid
from the sample tube to a labeled, 1.5-mL microcentrifuge tube. Use 1
mL of IX SL-buffer-A to perform the first rinse and 0.5 mL of reagent
water for the second rinse. Liberally rinse down the sides of the
Leighton tube before transferring. 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), with its magnetic strip in place.
13.3.2.13 Without removing the microcentrifuge tube from MPC-M, gently
rock/roll the tube through 180° by hand. Continue for approximately 1
minute with approximately one 180° roll/rock per second. At the end of
this step, the beads should produce a distinct brown dot at the back of
the tube.
April 2001 36
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Method 1622 - Cryptosporidium
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 ^L of 0.1 N HC1, then vortex at the highest setting for
approximately 50 seconds.
NOTE: The laboratory should 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 uL of 1.0 N NaOH to the sample wells of two well slides (add 10
|jL to the sample well of one well slide if the volume from the two
required dissociations will be added to the same slide).
NO TE: The laboratory should 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.
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: If one slide is used, exert extra care when using Dynal Spot-On slides to
ensure that the sample stays within the smaller-diameter wells on these slides.
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 varies from laboratory to laboratory, no minimum time is
specified. However, the laboratory must take care to ensure that the
37 April 2001
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Method 1622 - Cryptosporidium
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.
14.0 Sample Staining
NOTE: The sample must be stained within 72 hours of application of the purified
sample to the slide.
14.1 Prepare positive and negative controls.
14.1.1 For the positive control, pipette 10 |iL of positive antigen or 200 to 400 intact oocysts to
the center of a well.
14.1.2 Forthe negative control, pipette 50 |jL of 150 mM PBS (Section 7.6.4) into the center of
a well and spread it over the well area with a pipette tip.
14.1.3 Air-dry the control slides (see Section 13.3.3.12 for guidance).
14.2 Apply 50-|iL of absolute methanol to each well containing the dried sample and allow to air-dry
for 3 to 5 minutes.
14.3 Follow manufacturer's instructions in applying stain to slide.
14.4 Place the slides in a humid chamber in the dark and incubate at room temperature for
approximately 30 minutes. The humid chamber consists of a tightly sealed plastic container
containing damp paper towels on top of which the slides are placed.
14.5 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.
NOTE: If using the Merifluor stain (Section 7.6.2), do not allow slides to dry
completely.
14.6 Apply 50 uL 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 ug /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.)
14.7 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 stain (Section 7.6.2), do not allow slides to dry
completely.
14.8 Add mounting medium (Section 7.8) to each well.
14.9 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.
14.10 Record the date and time that staining was completed on the bench sheet. If slides will not be
read immediately, store in a humid chamber in the dark at 0 ° C to 8 ° C until ready for
examination.
April 2001 38
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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 and
confirmation should be performed immediately after staining is complete, laboratories
have up to 7 days from completion of sample staining to complete the examination and
confirmation of samples. However, if fading/diffusion ofFITC 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.
15.1 Scanning technique: Scan each well in a systematic fashion. An up-and-down or a side-to-side
scanning pattern may be used (Figure 4).
15.2 Examination 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 shape and measurements must be determined using 1000X magnification
and reported to the nearest 0.5 \im.
Record examination results for Cryptosporidium oocysts on a Cryptosporidium report form. All
oocysts that meet the criteria specified in Section 15.2.2, 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.
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 must
be conducted at a minimum of 1000X. Size, shape, and DIC and DAPI
characteristics of the three Cryptosporidium oocysts must be recorded
by the analyst on a microscope log. The analyst also must indicate on
each sample report 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 report 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 Section 15.2.2.
15.2.2 Sample examination
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 shapes. When brilliant apple-green
fluorescing ovoid or spherical objects 4 to 6 |im in diameter are
observed with brightly highlighted edges, increase magnification to
39 April 2001
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Method 1622 - Cryptosporidium
400X and switch the microscope to the UV filter block for DAPI
(Section 15.2.2.2), then to DIG (Section 15.2.2.3).
15.2.2.2 DAPI 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
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). 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
20.6). 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 |jm), 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.
NOTE: All measurements must be made at 1OOOXmagnification.
15.2.3 Record the date and time that sample examination was completed on the report form.
15.2.4 Report Cryptosporidium concentrations as oocysts/L.
16.0 Analysis of Complex Samples
16.1 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.
16.2 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.
16.3 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.
17.0 Method Performance
17.1 Method acceptance criteria are shown in Table 3 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 20.12). The matrix
spike and matrix spike duplicate criteria are based on spiked source water data generated during
April 2001 40
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Method 1622 - Cryptosporidium
the interlaboratory validation study of Method 1622 involving 12 laboratories and 12 raw surface
water matrices across the U.S. (Reference 20.10).
NOTE: Some sample matrices may prevent the MS acceptance criteria in Table 3 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
18.1 The solutions and reagents used in this method pose little threat to the environment when
recycled and managed properly.
18.2 Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize
the volume of expired materials to be disposed.
19.0 Waste Management
19.1 It is the laboratory's responsibility to comply with all federal, state, and local regulations 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).
19.2 Samples, reference materials, and equipment known or suspected to have viable oocysts attached
or contained must be sterilized prior to disposal.
19.3 For further information on waste management, consult The Waste Management Manual for
Laboratory Personnel and Less is Better: Laboratory Chemical Management for Waste
Reduction, both available from the American Chemical Society's Department of Government
Relations and Science Policy, 1155 16th Street N.W., Washington, D.C. 20036.
41 April 2001
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Method 1622 - Cryptosporidium
20.0 References
20.1 Rodgers, Mark R., Flanigan, Debbie J., and Jakubowski, Walter, \995.Appliedand
Environmental Microbiology £1(10), 3759-3763.
20.2 Fleming, Diane O., et al.(eds.), Laboratory Safety: Principles and Practices, 2nd edition. 1995.
ASM Press, Washington, DC
20.3 "Working with Carcinogens," DREW, PHS, CDC, NIOSH, Publication 77-206, (1977).
20.4 "OSHA Safety and Health Standards, General Industry," OSHA 2206, 29 CFR 1910 (1976).
20.5 "Safety in Academic Chemistry Laboratories," ACS Committee on Chemical Safety (1979).
20.6 ICRMicrobial 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).
20.7 USEPA. EPA Guide to Method Flexibility and Approval of EPA Water Methods, EPA 821-D-96-
004. Office of Water, Engineering and Analysis Division, Washington, DC 20460 (1996).
20.8 Cornell, 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.
20.9 "Envirochek™ Sampling Capsule," PN 32915, Gelman Sciences, 600 South Wagner Road, Ann
Arbor, MI 48103-9019 (1996).
20.10 USEPA. Interlaboratory Validation Study Results for Cryptosporidium Precision and Recovery
for U.S. EPA Method 1622, EPA-821-R-01-027. Office of Water, Office of Science and
Technology, Engineering and Analysis Division, Washington, DC (2001).
20.11 "Dynabeads® GC-Combo," Dynal Microbiology R&D, P.O. Box 8146 Dep., 0212 Oslo, Norway
(September 1998, Revision no. 01).
20.12 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).
20.13 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.
April 2001 42
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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
Collection
Filtration
> 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
> Up to 72 hours are permitted from application of the purified sample to the slide to staining
Staining
> Up to 7 days are permitted between sample staining and examination
Examination
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 modification121
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.
43
April 2001
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Method 1622 - Cryptosporidium
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 20.12). The matrix spike acceptance criteria are
based on data generated through interlaboratory validation of Method 1622
(Reference 20.10).
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
9.4.2
9.4.2
9.7
9.5
9.5.2
9.5.2
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.
Table 4. Distribution of Matrix Spike Recoveries from Multiple Samples Collected from 87
Source Waters During the ICR Supplemental Surveys (Adapted from Reference 20.13)
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%
April 2001
44
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Method 1622- Cryptosporidium
1 mm
1/5 mm
A
D
B
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)
45
April 2001
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Method 1622 - Cryptosporidium
o
0
"
o
o
t
o
*
c
-.
o
O
0
u
Figure 2. Manner of Counting Oocysts in 1 Square mm. Dark
organisms are counted and light organisms are omitted
(after Miale, 1967).
April 2001
46
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Method 1622 - Cryptosporidium
filter
flow
drain
Figure 3. Laboratory Filtration System
47
April 2001
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Method 1622 - Cryptosporidium
\
Figure 4. Methods for Scanning a Well Slide
X\pr/7 200^
48
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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
|jL microliter
< less than
> greater than
% percent
22.1.2 Alphabetical characters
cm centimeter
g gram
G acceleration due to gravity
hr hour
ID inside diameter
in. inch
L liter
m meter
mg milligram
mL milliliter
mm millimeter
mM millimolar
N normal; gram molecular weight of solute divided by hydrogen equivalent of
solute, per liter of solution
RSD relative standard deviation
sr standard deviation of recovery
X average percent recovery
22.2 Definitions, acronyms, and abbreviations (in alphabetical order)
Analyst—The analyst must have at least 2 years of college lecture and laboratory course work in
microbiology or a closely related field. The analyst also must have at least 6 months of
continuous bench experience with environmental protozoa detection techniques and IFA
microscopy, and must have successfully analyzed at least 50 water and/or wastewater samples
for Cryptosporidium. Six months of additional experience in the above areas may be substituted
for two years of college.
Analyte—A protozoan parasite tested for by this method. The analyte in this method is
Cryptosporidium.
Flow cytometer—A particle-sorting instrument capable of counting protozoa.
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.
49 April 2001
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Method 1622 - Cryptosporidium
Initial precision and recovery (IPR)—Four aliquots of spiking suspension analyzed to establish
the ability to generate acceptable precision and accuracy. An IPR is performed prior to the first
time this method is used and any time the method or instrumentation is modified.
Laboratory blank—See Method blank
Laboratory control sample (LCS)—See Ongoing precision and recovery (OPR) standard
Matrix spike (MS)—A sample prepared by adding a known quantity of organisms to a specified
amount of sample matrix for which an independent estimate of target analyte concentration is
available. A matrix spike is used to determine the effect of the matrix on a method's recovery
efficiency.
May—This action, activity, or procedural step is neither required nor prohibited.
May not—This action, activity, or procedural step is prohibited.
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 spiking suspension—See Spiking suspension
Oocyst stock suspension—See Stock suspension
Positive control—See Ongoing precision and recovery standard
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.
April 2001 50
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Method 1622 - Cryptosporidium
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).
51 April 2001
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