oEPA
United States
Environmental Protection
Agency
Method 1623: Cryptosporidium and
Giardia in Water by Filtration/IMS/FA
June 2003
Draft for Comment
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Office of Water (4607)
EPA 815-R-03-XXX
htto: //www .epa. gov/microbe s/
June 2003
Printed on Recycled Paper
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Acknowledgments
This method was prepared under the direction of William A. Telliard of the Engineering and Analysis
Division within the U.S. Environmental Protection Agency (U.S. EPA) Office of Water. This document
was prepared by DynCorp under a U.S. EPA contract, with assistance from its subcontractor, Interface,
Inc.
The U.S. EPA Office of Water gratefully acknowledges the contributions of the following persons and
organizations to the development of this method:
Mike Arrowood, Centers for Disease Control and Prevention, Division of Parasitic Diseases (MS-F13),
4770 Buford Highway, N.E., Atlanta, GA 30341-3724, USA
Phil Berger, Office of Groundwater and Drinking Water, U.S. Environmental Protection Agency, 401 M
Street, S.W., Washington, DC 20460, USA
Jennifer Clancy, Clancy Environmental Consultants, Inc., P.O. Box 314, St. Albans, VT 05478, USA
Kevin Connell, DynCorp, 6101 Stevenson Avenue, Alexandria, VA 22314, USA
Ricardo DeLeon, Metropolitan Water District of Southern California, 700 Moreno Avenue, LaVerne, CA
91760, USA
Shirley Dzogan, EnviroTest Laboratories, 745 Logan Avenue, Winnipeg, Manitoba R3E 3L5, Canada
Mary Ann Feige, Technical Support Center, Office of Ground Water and Drinking Water, U.S.
Environmental Protection Agency, 26 W. Martin Luther King Drive, Cincinnati, OH 45268-1320,
USA
Colin Fricker, Thames Water Utilities, Manor Farm Road, Reading, Berkshire, RG2 0JN, England
Carrie Hancock, CH Diagnostic & Consulting Service, Inc., 214 S.E. Nineteenth Street, Loveland, CO
80537, USA
Stephanie Harris,Manchester Laboratory, U.S. Environmental Protection Agency, Region 10, 7411
Beach Drive East, Port Orchard, WA 98366, USA
Dale Rushneck, Interface, Inc., 3194 Worthington Avenue, Fort Collins, CO 80526, USA
Frank Schaefer III, National Exposure Research Laboratory, U.S. Environmental Protection Agency, 26
W. Martin Luther King Drive, Cincinnati, OH 45268-1320, USA
Steve Schaub, Health and Ecological Criteria Division (4304), Office of Science and Technology, U.S.
Environmental Protection Agency, 401 M Street, S.W., Washington, DC 20460, USA
Ajaib Singh, City of Milwaukee Health Department, 841 North Broadway, Milwaukee, WI 53202, USA
Huw Smith, Department of Bacteriology, Scottish Parasite Diagnostic Laboratory, Stobhill NHS Trust,
Springburn, Glasgow, G21 3UW, Scotland
Timothy Straub, Lockheed Martin, 7411 Beach Drive East, Port Orchard, WA 98366, USA
William A. Telliard, Office of Science and Technology, U.S. Environmental Protection Agency, 401 M
Street, S.W., Washington, DC 20460, USA
Cryptosporidium cover photo courtesy of the U.S. Centers for Disease Control
Giardia cover photo courtesy of CH Diagnostic & Consulting Service, Inc.
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Disclaimer
This method has been reviewed by the U.S. EPA Office of Water and approved for publication. Mention of
trade names or commercial products does not constitute endorsement or recommendation for use.
Questions regarding this method or its application should be addressed to:
Laboratory Quality Assurance Program for the Analysis of Cryptosporidium in Water Coordinator
Office of Ground Water and Drinking Water Technical Support Center
U.S. EPA Office of Water
26 West Martin Luther King Drive
Cincinnati, OH 45268-1320
William A. Tclliard
U.S. EPA Office of Water
Analytical Methods Staff
MailCodc 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 and Giardia.
To implement these requirements, EPA must assess Cryptosporidium and Giardia occurrence in raw
surface waters used as source waters for drinking water treatment plants. EPA Method 1623 was
developed to support this assessment.
Method Development and Validation
EPA initiated an effort in 1996 to identify new and innovative technologies for protozoan monitoring and
analysis. After evaluating potential alternatives to the then-current method through literature searches,
discussions with research and commercial laboratories, and meetings with experts in the field, the
Engineering and Analysis Division within the Office of Science and Technology within EPA's Office of
Water developed draft Method 1622 for ('rypiosporidiiim detection in December 1996. This
Cryptosporidium-only method was validated through an interlaboratory study in August 1998, and was
revised as a final, valid method for detecting Cryptosporidium in water in January 1999.
Although development of an acceptable immunomagnetic separation system for Giardia lagged behind
development of an acceptable system for Cryptosporidium, an acceptable system was identified in
October 1998, and EPA validated a method for simultaneous detection of ('rypiosporidiiim and Giardia
in February 1999 and developed quality control (QC) acceptance criteria for the method based on this
validation study. To avoid confusion with Method 1622, which already had been validated and was in use
both domestically and internationally as a stand-alone Cryptosporidium-only detection method, EPA
designated the new combined procedure EPA Method 1623.
The interlaboratory validated versions of Method 1622 (January 1999; EPA-821-R-99-001) and Method
1623 (April 1999; EPA-821-R-99-006) were used to analyze approximately 3,000 field and QC samples
during the Information Collection Rule Supplemental Surveys (ICRSS) between March 1999 and
February 2000. Method 1622 was used to analyze samples from March 1999 to mid-July 1999; Method
1623 was used from mid-July 1999 to February 2000. The April 2001 revision of both methods include
updated QC acceptance criteria based on analysis of the QC samples analyzed during the ICRSS.
Changes in the April 2001 Versions of the Methods
Both methods were revised in April 2001, after completion of the ICRSS and multiple meetings with
researchers and experienced laboratory staff to discuss potential method updates. Changes incorporated in
the April 2001 revisions of the methods (EPA-821-R-01-025 and EPA-821-R-01-026) included the
following:
• Nationwide approval of modified versions of the methods using the following components:
(a) Whatman Nuclepore CrypTest™ filter
(b) IDEXX Filta-Max™ filter
(c) Waterborne Aqua-Glo™ G/C Direct FL antibody stain
(d) Waterborne Crypt-a-Glo™ and Giardi-a-Glo™ antibody stains
• Clarified sample acceptance criteria
• Modified capsule filter elution procedure
• Modified concentrate aspiration procedure
/'/'/
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• Modified IMS acid dissociation procedure
• Updated QC acceptance criteria for IPR and OPR tests
• Addition of a troubleshooting section for QC failures
• Modified holding times
• Inclusion of flow cytometry-sorted spiking suspensions
Changes in the June 2003 Versions of the Methods
Both methods were revised again in June 2003 to support proposal of EPA's Long Term 2 Enhanced
Surface Water Treatment Rule. Changes incorporated into the December 2002 versions include:
Nationwide approval of a modified version of the methods using the Pall Gelman Envirochek™ HV
filter
Removal of Whatman Nuclepore CrypTest™ filter from the methods as a result of discontinuation of
the product by the manufacturer
Nationwide approval of the use of BTF EasySeed™ irradiated oocysts and cysts for use in routine
quality control (QC) samples
Minor clarifications and corrections
Rejection criteria for sample condition upon receipt
Guidance on measuring sample temperatures
Clarification of QC sample requirements and use of QC sample results
Guidance on minimizing carry-over debris onto microscope slides after IMS
Performance-Based Method Concept and Modifications Approved for Nationwide Use
EPA Method 1623 is a performance-based method applicable to the determination of Cryptosporidium
and Giardia in aqueous matrices. EPA Method 1623 requires filtration, immunomagnetic separation of
the oocysts and cysts from the material captured, and enumeration of the target organisms based on the
results of immunofluorescence assay, 4',6-diamidino-2-phenylindole (DAPI) staining results, and
differential interference contrast microscopy.
The interlaboratory validation of EPA Method 1623 conducted by EPA used the Pall Gelman capsule
filtration procedure, Dynal immunomagnetic separation (IMS) procedure, and Meridian sample staining
procedure described in this document. Alternate procedures are allowed, provided that required quality
control tests are performed and all quality control acceptance criteria in this method are met.
Since the interlaboratory validation of EPA Method 1623, interlaboratory validation studies have been
performed to demonstrate the equivalency of modified versions of the method using the following
components:
Whatman Nuclepore CryptTest™ filter (no longer available)
• IDEXX Filta-Max™ filter
• Pall Gelman Envirochek™ HV filter
Waterborne Aqua-Glo™ G/C Direct FL antibody stain
Waterborne Crypt-a-Glo™ and Giardi-a-Glo™ antibody stains
BTF EasySeed™ irradiated oocysts and cysts for use in routine QC samples
iv
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The validation studies for these modified versions of the method met EPA's performance-based
measurement system Tier 2 validation for nationwide use (see Section 9.1.2 for details), and have been
accepted by EPA as equivalent in performance to the original version of the method validated by EPA.
The equipment and reagents used in these modified versions of the method are noted in Sections 6 and 7
of the method; the procedures for using these equipment and reagent options are available from the
manufacturers.
Because this is a performance-based method, other alternative components not listed in the method may
be available for evaluation and use by the laboratory. Confirming the acceptable performance of a
modified version of the method using alternate components in a single laboratory does not require that an
interlaboratory validation study be conducted. However, method modifications validated only in a single
laboratory have not undergone sufficient testing to merit inclusion in the method. Only those modified
versions of the method that have been demonstrated as equivalent at multiple laboratories on multiple
water sources through a Tier 2 interlaboratory study will be cited in the method.
v
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Table of Contents
I.0 Scope and Application 1
2.0 Summary of Method 1
3.0 Definitions 2
4.0 Contamination, Interferences, and Organism Degradation 2
5.0 Safety 3
6.0 Equipment and Supplies 3
7.0 Reagents and Standards 8
8.0 Sample Collection and Storage 10
9.0 Quality Control 13
10.0 Microscope Calibration and Analyst Verification 20
II.0 Oocyst and Cyst Suspension Enumeration and Spiking 26
12.0 Sample Filtration and Elution 35
13.0 Sample Concentration and Separation (Purification) 44
14.0 Sample Staining 49
15.0 Examination 50
16.0 Analysis of Complex Samples 53
17.0 Method Performance 53
18.0 Pollution Prevention 53
19.0 Waste Management 53
20.0 References 54
21.0 Tables and Figures 55
22.0 Glossary of Definitions and Purposes 63
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Method 1623: Cryptosporidium and Giardia in Water
by Filtration/IMS/FA
1.0 Scope and Application
1.1 This method is for determination of the identity and concentration of Cryptosporidium (CAS
Registry number 137259-50-8) and Giardia (CAS Registry number 137259-49-5) in water by
filtration concentration, immunomagnetic separation (IMS), and immunofluorescence assay (FA)
microscopy. Cryptosporidium and Giardia may be confirmed using 4',6-diamidino-2-
phenylindole (DAPI) staining and differential interference contrast (DIC) microscopy. The
method has been validated in surface water, but may be used in other waters, provided the
laboratory demonstrates that the method's performance acceptance criteria are met.
1.2 This method is designed to meet the survey and monitoring requirements of the U.S.
Environmental Protection Agency (EPA). It is based on laboratory testing of recommendations by
a panel of experts convened by EPA. The panel was charged with recommending an improved
protocol for recovery and detection of protozoa that could be tested and implemented with
minimal additional research.
1.3 This method will not identify the species of ('rypiosporidiiim or Giardia or the host species of
origin, nor can it determine the viability or infectivity of detected oocysts and cysts.
1.4 This method is for use only by persons experienced in the determination of ('rypiosporidiiim and
Giardia by filtration, IMS, and FA. Experienced persons are defined in Section 22.2 as analysts
(and principal analysts, in applicable programs). Laboratories unfamiliar with analyses of
environmental samples by the techniques in this method should gain experience using water
filtration techniques, IMS, fluorescent antibody staining with monoclonal antibodies, and
microscopic examination of biological particulates using bright-field and DIC microscopy.
1.5 Any modification of the method beyond those expressly permitted is subject to the application
and approval of alternative test procedures under 40 CFR Part 141.27.
2.0 Summary of Method
2.1 A water sample is filtered and the oocysts, cysts, and extraneous materials are retained on the
filter. Although EPA has only validated the method using laboratory filtration of bulk water
samples shipped from the field, field-filtration also can-may be used.
2.2 Elution and separation
2.2.1 Materials on the filter are eluted and the eluate is centrifuged to pellet the oocysts and
cysts, and the supernatant fluid is aspirated.
2.2.2 The oocysts and cysts are magnetized by attachment of magnetic beads conjugated to
anti-Cryptosporidium and anti-Giardia antibodies. The magnetized oocysts and cysts are
separated from the extraneous materials using a magnet, and the extraneous materials are
discarded. The magnetic bead complex is then detached from the oocysts and cysts.
2.3 Enumeration
2.3.1 The oocysts and cysts are stained on well slides with fluorescently labeled monoclonal
antibodies and 4',6-diamidino-2-phenylindole (DAPI). The stained sample is examined
using fluorescence and differential interference contrast (DIC) microscopy.
2.3.2 Qualitative analysis is performed by scanning each slide well for objects that meet the
size, shape, and fluorescence characteristics of Cryptosporidium oocysts or Giardia
cysts. Potential oocysts or cysts are confirmed through DAPI staining characteristics and
1
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Method 1623 - Cryptosporidium and Giardia
DIC microscopy. Oocysts and cysts are identified when the size, shape, color, and
morphology agree with specified criteria and examples in a photographic library.
2.3.3 Quantitative analysis is performed by counting the total number of objects on the slide
confirmed as oocysts or cysts.
2.4 Quality is assured through reproducible calibration and testing of the filtration, immunomagnetic
separation (IMS), staining, and microscopy systems. Detailed information on these tests is
provided in Section 9.0.
3.0 Definitions
3.1 Cryptosporidium is defined as a protozoan parasite potentially found in water and other media.
The six species of Cryptosporidium and their potential hosts are C. pctrvum (mammals, including
humans); C. bctileyi and C. melectgridis (birds); C. muris (rodents); C. serpentis (reptiles); and C.
nasorum (fish). Cryptosporidium is defined in this method as those determined by brilliant apple
green fluorescence under UV light, size (4 to 6 and shape (round to oval), excluding
atypical organisms specifically identified as other microbial organisms by FITC and DIC (for
example, those possessing spikes, stalks, appendages, pores, one or two large nuclei filling the
cell, red fluorescing chloroplasts, crystals, spores, etc.).
3.2 Giardia is a protozoan parasite potentially found in water and other media. The two species of
Giardia and their potential hosts are G. intestinalis (humans) and G. muris (mice). Giardia is
defined in this method as those determined by brilliant apple green fluorescence under UV light,
size (8 to 18 (.un long by 5 to 15 |_im wide), and shape (oval), excluding atypical organisms
specifically identified as other microbial organisms by FITC and DIC (for example, those
possessing spikes, stalks, appendages, pores, one or two large nuclei filling the cell, red
fluorescing chloroplasts, crystals, spores, etc.).
3.3 Definitions for other terms used in this method are given in the glossary (Section 22.0).
4.0 Contamination, Interferences, and Organism Degradation
4.1 Turbidity caused by inorganic and organic debris can interfere with the concentration, separation,
and examination of the sample for Cryptosporidium oocysts and Giardia cysts. In addition to
naturally-occurring debris, such as clays and algae, chemicals, such as iron and alum coagulants
and polymers, may be added to finished waters during the treatment process, which may result in
additional interference.
4.2 Organisms and debris that autofluoresce or demonstrate non-specific fluorescence, such as algal
and yeast cells, when examined by epifluorescent microscopy, may interfere with the detection of
oocysts and cysts and contribute to false positives by immunofluorescence assay (FA) (Reference
20.1).
4.3 Solvents, reagents, labware, and other sample-processing hardware may yield artifacts that may
cause misinterpretation of microscopic examinations for oocysts and cysts. All materials used
shall be demonstrated to be free from interferences under the conditions of analysis by running a
method blank (negative control sample) initially and a minimum of every week or after changes
in source of reagent water. Specific selection of reagents and purification of solvents and other
materials may be required.
4A Interferences co-extracted from samples will vary considerably from source to source, depending
on the water being sampled. Experience suggests that high levels of algae, bacteria, and other
protozoa can interfere in the identification of oocysts and cysts (Reference 20.1).
June 2003
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Method 1623 - Cryptosporidium and Giardia
4.4 Freezing samples, filters, eluates, concentrates, or slides may interfere with the detection and/or
identification of oocysts and cysts.
4.5 All equipment should be cleaned according to manufacturers" instructions. Disposable supplies
should be used wherever possible.
5.0 Safety
5.1 The biohazard associated with, and the risk of infection from, oocysts and cysts is high in this
method because live organisms are handled. This method does not purport to address all of the
safety problems associated with its use. It is the responsibility of the laboratory to establish
appropriate safety and health practices prior to use of this method. In particular, laboratory staff
must know and observe the safety procedures required in a microbiology laboratory that handles
pathogenic organisms while preparing, using, and disposing of sample concentrates, reagents and
materials, and while operating sterilization equipment.
5.2 The toxicity or carcinogenicity of each compound or reagent used in this method has not been
precisely determined; however, each chemical compound should be treated as a potential health
hazard. Exposure to these compounds should be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file of Occupational Safety and
Health Administration regulations regarding the safe handling of the chemicals specified in this
method. A reference file of material safety data sheets should be made available to all personnel
involved in these analyses. Additional information on laboratory safety can be found in
References 20.2 through 20.5.
5.3 Samples may contain high concentrations of biohazards and toxic compounds, and must be
handled with gloves and opened in a biological safety cabinet to prevent exposure. Reference
materials and standards containing oocysts and cysts must also be handled with gloves and
laboratory staff must never place gloves in or near the face after exposure to solutions known or
suspected to contain oocysts and cysts. Do not mouth-pipette.
5.4 Laboratory personnel must change gloves after handling filters and other contaminant-prone
equipment and reagents. Gloves must be removed or changed before touching any other
laboratory surfaces or equipment.
5.5 Centers for Disease Control (CDC) regulations (42 CFR 72) prohibit interstate shipment of more
than 4 L of solution known to contain infectious materials (see
htto://www.cdc.gov/od/ohs/biosftv/shipregs.htm for details). State regulations may contain
similar regulations for intrastate commerce. Unless the sample is known or suspected to contain
Cryptosporidium, Giardia, or other infectious agents (e.g., during an outbreak), samples should
be shipped as noninfectious and should not be marked as infectious. If a sample is known or
suspected to be infectious, and the sample must be shipped to a laboratory by a transportation
means affected by CDC or state regulations, the sample should be shipped in accordance with
these regulations.
6.0 Equipment and Supplies
NOTE: Brand names, suppliers, and part numbers are for illustrative purposes only. No
endorsement is implied. Equivalent performance may be achieved using apparatus and
materials other than those specified here, but demonstration of equivalent performance that
meets the requirements of this method is the responsibility of the laboratory.
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June 2003
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Method 1623 - Cryptosporidium and Giardia
6.1 Sample collection equipment for shipment of bulk water samples for laboratory filtration.
Collapsible LDPE cubitainer for collection of 10-L bulk sample(s)—Cole Parmer cat. no. U-
06100-30 or equivalent. Fill completely to ensure collection of a full 10-L sample. Discard after
one use.
6.2 Equipment for sample filtration. Three options have been demonstrated to be acceptable for use
with Method 1623. Other options may be used if their acceptability is demonstrated according to
the procedures outlined in Section 9.1.2.
6.2.1 Cubitainer spigot to facilitate laboratory filtration of sample (for use with any filtration
option)—Cole Parmer cat. no. U-06061-01, or equivalent.
6.2.2 Original Envirochek™ sampling capsule or Envirochek™ HV sampling capsule
equipment requirements (for use with the procedure described in Section 12.2). The
versions of the method using this filter was these filters were validated using 10-L and
50-L sample volumes, respectively. Alternate sample volumes may be used, provided
the laboratory demonstrates acceptable performance on initial and ongoing spiked
reagent water and source water samples (Section 9.1.2).
6.2.2.1 Sampling capsule
6.2.2.1.1 Envirochek™, Pall Gelman Laboratory, Ann Arbor, MI,
product no. 12110 (individual filter) and or product
no.12107 (box of 25 filters) (www.pall.com/gelman or
(800) 521-1520 ext. 2)
6.2.2.1.2 Envirochek™ HV, Pall Gelman Laboratory, Ann Arbor,
MI, product no. 12099 (individual filter) or product
no. 12098 (box of 25 filters) (www.pall.com/gelman or
(800) 521-1520 ext. 2)
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
6.2.3 Filta-Max™ foam filter equipment requirements (for use with the procedure described in
Section 12.3). The version of the method using this filter was validated using 50-L
June 2003
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Method 1623 - Cryptosporidium and Giardia
sample volumes; alternate sample volumes may be used, provided the laboratory
demonstrates acceptable performance on initial and ongoing spiked reagent water and
matrix samples (Section 9.1.2).
6.2.3.1 Foam filter—Filta-Max™, IDEXX, Westbrook, ME. Filter module 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.
6.2.3.2 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).
6.3 Ancillary sampling equipment
6.3.1 Tubing—Glass, polytetrafluoroethylene (PTFE), high-density polyethylene (HDPE), or
other tubing to which oocysts and cysts will not easily adhere—Tygon formula R-3603,
or equivalent. If rigid tubing (glass, PTFE, HDPE) is used and the sampling system uses
a peristaltic pump, a minimum length of compressible tubing may be used in the pump.
Before use, the tubing must be autoclaved, thoroughly rinsed with detergent solution,
followed by repeated rinsing with reagent water to minimize sample contamination.
Alternately, decontaminate using hypochlorite solution, sodium thiosulfate, and multiple
reagent water rinses; dispose of tubing when wear is evident. Dispose of tubing after one
use whenever possible.
6.3.2 Flow control valve—0.5 gpm (0.03 L/s), Bertram Controls, Plast-O-Matic cat. no.
FC050B!/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, YA, cat. no. 2PG13 Simer, model number,
M40, or equivalent.
6.3.4 Flow meter—Sameco cold water totalizer, E. Clark and Associates, Northboro, MA,
product no. WFU 10.110, or equivalent
6.4 Equipment for spiking samples in the laboratory
6.4.1 10-L carboy with bottom delivery port (Yi")—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
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Method 1623 - Cryptosporidium and Giardia
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-(im-pore-size, 25-mm-
diameter, Fisher cat. no. A12SP02500, or equivalent
6.4.8.4 Polycarbonate track-etch hydrophilic membrane filter—l-(im-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. * 3 in or 2 in. * 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
6.5.2 Magnetic particle concentrator for 10-mL test tubes—Dynal MPC-1® , cat. no. 120.01,
or equivalent
6.5.3 Magnetic particle concentrator for microcentrifuge tubes—Dynal MPC-M®, cat. no.
120.09, or equivalent
6.5.4 Flat-sided sample tubes—16 * 125 mm Leighton-type tubes with 60 x 10 mm flat-sided
magnetic capture area, Dynal L10, cat. no. 740.03, or equivalent
6.6 Powder-free latex gloves—Fisher cat no. 113945B, or equivalent
6.7 Graduated cylinders, autoclavable—10-, 100-, and 1000-mL
6.8 Centrifuges
6.8.1 Centrifuge capable of accepting 15- to 250-mL conical centrifuge tubes and achieving
1500 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
6.9 Microscope
6.9.1 Epifluorescence/differential interference contrast (DIC) with stage and ocular
micrometers and 20X (N.A.=0.4) to 100X (N.A.=1.3) objectives—Zeiss™ Axioskop,
Olympus™ BH, or equivalent
6.9.2 Excitation/band-pass filters for immunofluorescence assay (FA)—Zeiss™ 487909 or
equivalent, including, 450- to 490-nm exciter filter, 510-nm dicroic beam-splitting
mirror, and 515- to 520-nm barrier or suppression filter
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Method 1623 - Cryptosporidium and Giardia
6.9.3 Excitation/band-pass filters for DAPI—Filters cited below (Chroma Technology,
Brattleboro, V"
"), or equivalent
Microscope
model
Fluoro-chrome
Excitation
filter (nm)
Dichroic beam-
splitting mirror
(nm)
Barrier or
suppression
filter (nm)
Chroma
catalog
number
Zeiss™ -
Axioskop
DAPI (UV)
340-380
400
420
CZ902
Zeiss™ -IM35
DAPI (UV)
340-380
400
420
CZ702
Olympus™ BH
DAPI (UV)
340-380
400
420
11000
Filter holder
91002
Olympus™ BX
DAPI (UV)
340-380
400
420
11000
Filter holder
91008
Olympus™
DAPI (UV)
340-380
400
420
11000
IMT2
Filter holder
91003
6.10 Ancillary equipment for microscopy
6.10.1 Well slides— Spot-On well slides, Dynal cat. no. 740.04; treated, 12-mm diameter well
slides, Meridian Diagnostics Inc., Cincinnati, OH, cat. no. R2206; or equivalent
6.10.2 Glass coverslips—22 * 50 mm
6.10.3 Nonfluorescing immersion oil—Type FF, Cargille cat. no. 16212, or equivalent
6.10.4 Micropipette, adjustable: 0- to 10-|uL with 0- to 10-|uL tips
10- to 100-(iL, with 10- to 200-(iL tips
100-to 1000-fiLwith 100-to 1000-fiLtips
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
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Method 1623 - Cryptosporidium and Giardia
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 |_im. 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 immuno fluorescence assay, the laboratory should
purchase standards at the required normality directly from a vendor. Normality should not
be adjusted by the laboratory.
7.2 Solvents—Acetone, glycerol, ethanol, and methanol, ACS reagent grade
7.3 Reagent water—Water in which oocysts and cysts and interfering materials and substances,
including magnetic minerals, are not detected by this method
7.4 Reagents for eluting filters
NOTE: Laboratories should store prepared eluting solution for no more than 1 week or
when noticeably turbid, whichever comes sooner.
7.4.1 Reagents for eluting Envirochek™ sampling capsules (Section 6.2.2)
7.4.1.1 Laureth-12—PPG Industries, Gurnee, IL, cat. no. 06194, or equivalent.
Store Laureth-12 as a 10% solution in reagent water. Weigh 10 g of
Laureth-12 and dissolve using a microwave or hot plate in 90 mL of
reagent water. Dispense 10-mL aliquots into sterile vials and store at
room temperature for up to 2 months, or in the freezer for up to a year.
7.4.1.2 1 M Tris, pH 7.4—Dissolve 121.1 g Tris (Fisher cat. no. BP152) in 700
mL of reagent water and adjust pH to 7.4 with 1 N HC1 or NaOH. Dilute
to a final 1000 mL with reagent water and adjust the final pH. Filter-
sterilize through a 0.2-f.im 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
of reagent water and adjust pH to 8.0 with 6.0 N HC1 or NaOH. Dilute to
a final volume of 1000 mL with reagent water and adjust to pH 8.0 with
1.0NHC1 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 |aL Antifoam A
(Section 7.4.1.4). Dilute to 1000 mL with reagent water.
7.4.2 Reagents for eluting CiypTest™ capsule filters (Section 6.2.3). To 900 mL of reagent
water add 8.0 g NaCl, 0.2 g iaLPO4r^-g^teJffO4-^m0) 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.
June 2003
8
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Method 1623 - Cryptosporidium and Giardia
cat. no. A5758, or equivalent). Adjust volume to 1 L with reagent water and adjust pll to
7.4 willi 1 N NaOII ui IIC1.
7.4.2 Reagents for eluting Filta-Max™ foam filters (Section 6.2.3)
7.4.2.1 Phosphate buffered saline (PBS), pH 7.4—Sigma Chemical Co. cat. no.
P-3813, or equivalent. Alternately, prepare PBS by adding the following
to 1 L of reagent water: 8 g NaCl; 0.2 g KC1; 1.15 g NaoHP04,
anhydrous; and 0.2 g KH2P04.
7.4.2.2 Tween 20—Sigma Chemical Co. cat. no. P-7949, or equivalent
7.4.2.3 High-vacuum grease—BDH/Merck. cat. no. 636082B, or equivalent
7.4.2.4 Preparation of PBST elution buffer. Add 100 (.iL of Tween 20 to
prepared PBS (Section 7.4.3.1 7.4.2.1). Alternatively, add the contents of
one sachet packet of PBS to 1.0 L of reagent water. Dissolve by stirring
for 30 minutes. Add 100 (.iL of Tween 20. Mix by stirring for 5 minutes.
7.5 Reagents for immunomagnetic separation (IMS)—Dynabeads® GC-Combo, Dynal cat. nos.
730.02, 730.12, or equivalent
7.6 Direct antibody labeling reagents for detection of oocysts and cysts. Store reagents at 0°C to 8°C
and return promptly to this temperature after each use. Do not allow any of the reagents to freeze.
The reagents should be protected from exposure to light. Diluted, unused working reagents
should be discarded after 48 hours. Discard reagents after the expiration date is reached. The
labeling reagents in Sections 7.6.1-7.6.3 have been approved for use with this method.
7.6.1 Merifluor Cryptosporidium/Giardia, Meridian Diagnostics cat. no. 250050, Cincinnati,
OH, or equivalent
7.6.2 Aqua-Glo™ G/C Direct FL, Waterborne cat. no. A100FLR, New Orleans, LA, or
equivalent
7.6.3 Crypt-a-Glo™ and Giardi-a-Glo™, Waterborne cat. nos. A400FLR and A300FLR,
respectively, New Orleans, LA, or equivalent
NOTE: If a laboratory> will use multiple types of labeling reagents, the laboratory must
demonstrate acceptable performance through an initial precision and recovery test (Section
9.4) for each type, and must perform positive and negative staining controls for each batch
of slides stained using each product. However, the laboratory is not required to analyze
additional ongoing precision and recovery samples or method blank samples for each type.
The performance of each labeling reagent used also should be monitored in each source
water type.
7.6.4 Diluent for labeling reagents—Phosphate buffered saline (PBS) (Section 7.4.1). 7-pH
9-4—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 ICCl; 1.15 g NaJiP04-
anhydrous; and 0.2 g KILPQ4. Filter sterilize (Section 6.19) or autoclave. Discard if
growth is detected or after 6 months, whichever comes first.
7.7 4',6-diamidino-2-phenylindole (DAPI) stain—Sigma Chemical Co. cat. no. D9542, or equivalent
7.7.1 Stock solution—Dissolve 2 mg/mL DAPI in absolute methanol. Prepare volume
consistent with minimum use. Store at 0°C to 8°C in the dark. Do not allow to freeze.
Discard unused solution when positive staining control fails.
7.7.2 Staining solution (1/5000 dilution in PBS [Section 7.6.4])—Add 10 (.iL 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 [ag/in L 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.
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Method 1623 - Cryptosporidium and Giardia
7.8 Mounting medium
7.8.1 DABCO/glycerol mounting medium (2%)—Dissolve 2 g of DABCO (Sigma Chemical
Co. cat no. D-2522, or equivalent) in 95 mL of warm glycerol/PBS (60% glycerol, 40%
PBS [Section 7.6.4]). After the DABCO has dissolved completely, adjust the solution
volume to 100 mL by adding an appropriate volume of glycerol/PBS solution.
Alternately, dissolve the DABCO in 40 mL of PBS, then add azide (1 mL of 100X, or
10% solution), then 60 mL of glycerol.
7.8.2 Mounting medium supplied with Merifluor direct labeling kit (Section 7.6.1)
7.9 Clear fingernail polish or clear fixative, PGC Scientifics, Gaithersburg, MD, cat. no. 60-4890, or
equivalent
7.10 Oocyst and cyst suspensions for spiking
7.10.1 Enumerated spiking suspensions prepared by flow cytometer—not heat-fixed or
formalin fixed: Wisconsin State Laboratory of Hygiene Flow Cytometry Unit or
equivalent.
7.10.1.1 Live, flow cytometer-sorted oocysts and cysts—Wisconsin State
Laboratory of Hygiene Flow Cytometry Unit ([608] 224-6260), or
equivalent
7.10.1.2 Irradiated, flow cytometer-sorted oocysts and cysts—flow
cytometer-sorted oocysts and cysts—BTF EasySeed™
(www.biotechnologyfrontiers.com). or equivalent
7.10.2 Materials for manual enumeration of spiking suspensions
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 Purified Giardia cyst stock suspension for manual enumeration—not
heat-fixed or formalin-fixed: Waterborne, Inc., New Orleans, LA;
Hyperion Research, Medicine Hat, Alberta, Canada; or equivalent
7.10.2.3 Tween-20, 0.01%—Dissolve 1.0 mL of a 10% solution of Tween-20 in 1
L of reagent water
7.10.3 Storage procedure—Store oocyst and cyst suspensions at 0°C to &10°C, until ready to
use; do not allow to freeze
7.11 Additional reagents for enumeration of spiking suspensions using membrane filtration (Section
11.3.6)—Sigmacote® Sigma Company Product No. SL-2, or equivalent
8.0 Sample Collection and Storage
8.1 Sample collection, shipment, and receipt
8.1.1 Sample collection. Samples are collected as bulk samples and shipped to the laboratory
for processing through the entire method, or are filtered in the field and shipped to the
laboratory for processing from elution (Section 12.2.6) onward.
8.1.2 Sample shipment. Ambient water samples are dynamic environments and, depending
on sample constituents and environmental conditions, Cryptosporidium oocysts or
Giardia cysts present in a sample can degrade, potentially biasing analytical results.
Samples that are not analyzed the same day they are collected should be chilled to
reduce biological activity, and preserve the state of source water samples between
collection and analysis. Samples must analyzed by an off-site laboratory should be
shipped via overnight service on the day they are collected. Overnight service may not
be necessary if the samples are maintained below 10°C (but not frozen) and holding
times are met.
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Method 1623 - Cryptosporidium and Giardia
NOTE: See transportation precautions in Section 5.5.
8.1.2.1 If samples are collected early in the day, chill samples as much as
possible between collection and shipment by storing in a refrigerator or
pre-icing the sample in a cooler. If the sample is pre-iced before
shipping, replace with fresh ice immediately before shipment.
8.1.2.2 If samples are collected later in the day, these samples may be held
overnight in a refrigerator. This should be considered for bulk water
samples that will be shipped off-site, as this minimizes the potential for
water samples collected during the summer to melt the ice in which they
are packed and arrive at the laboratory at >10°C.
8.1.2.3 Samples should be shipped at O^C to < 10°C $^6 and not frozen, unless
the time required to chill the sample to 8^6 10°C would prevent the
sample from being shipped overnight for receipt at the laboratory the day
after collection. Samples must not be allowed to freeze.
8.1.2.4 Public water systems shipping samples to off-site laboratories for
analysis should include in the shipping container a means for monitoring
the temperature of the sample during shipping to verify that the sample
did not freeze or exceed 10°C. Suggested approaches for monitoring
sample temperature during shipping are discussed in Section 8.1.4.
8.1.3 Sample receipt. Upon receipt, the laboratory must record the temperature. Samples that
were not collected the same day they were received, and that are received at >10°C or
frozen, or samples that the laboratory has determined exceeded >10°C or froze during
shipment, must be rejected. After receipt, samples must be stored at the laboratory at <
10°C, and not frozen, until processed. Results from samples shipped overnight to the
laboratory and received at > 8gC should be qualified by the laboratory..
8.1.4 Suggestions on measuring sample temperature. Given the importance of maintaining
sample temperatures for ('rypiosporicliiim and Giardia determination, laboratories
performing analyses using this method must establish acceptance criteria for receipt of
samples transported to their laboratory. Several options are available to measure sample
temperature upon receipt at the laboratory and, in some cases, during shipment:
8.1.4.1 Temperature sample. One option, for filtered samples only (not for 10-
L bulk samples), is for the sampler to fill a small, inexpensive sample
bottle with water and pack this "temperature sample" next to the filtered
sample. The temperature of this extra sample volume is measured upon
receipt to estimate the temperature of the filter. Temperature sample
bottles are not appropriate for use with bulk samples because of the
potential effect that the difference in sample volume may have in
temperature equilibration in the sample cooler. Example product: Cole
Parmer cat. no. U-06252-20.
8.1.4.2 Thermometer vial. A similar option is to use a thermometer that is
securely housed in a liquid-filled vial. Unlike temperature samples, the
laboratory does not need to perform an additional step to monitor the
temperature of the vial upon receipt, but instead just needs to read the
thermometer. Example product: Eagle-Picher Sentry Temperature Vial
3TR-40CS-F or 3TR-40CS.
8.1.4.3 iButton. Another option for measuring the sample temperature during
shipment and upon receipt is a Thermocron® iButton. An iButton is a
small, waterproof device that contains a computer chip that can be
programmed to record temperature at different time intervals. The
information is then downloaded from the iButton onto a computer. The
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June 2003
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Method 1623 - Cryptosporidium and Giardia
iButton should be placed in a temperature sample (Section 8.1.4.1),
rather than placed loose in the cooler. Information on Thermocron®
iButtons is available from http://www.ibutton.com/. Distributors include
http://www.pointsix.com/. http://www.rdsdistributing.com. and
http: //www. scigiene. com/.
8.1.4.4 Stick-on temperature strips. Another option is for the laboratory to
apply a stick-on temperature strip to the outside of the sample container
upon receipt at the laboratory. This option does not measure temperature
as precisely as the other options, but still mitigates the risk of sample
contamination while providing an indication of sample temperature to
verify that the sample temperature is acceptable. Example product: Cole
Parmer cat. no. U-90316-00.
8.1.4.5 Infrared thermometers. A final option is to measure the temperature of
the surface of the sample container or filter using an infrared
thermometer. The thermometer is pointed at the sample, and measures
the temperature without coming in contact with the sample volume.
Example product: Cole Parmer cat. no. EW-39641-00.
As with other laboratory equipment, all temperature measurement devices must be
calibrated routinely to ensure accurate measurements. See the EPA Manual, for the
Certification of Laboratories Analyzing Drinking Water (Reference 20.6) for more
information.
8.2 Sample holding times. Samples must be processed or examined within each of the holding times
specified in Sections 8.2.1 - 8.2.4. Sample processing should be completed as soon as possible by
the laboratory. The laboratory should complete sample filtration, elution, concentration,
purification, and staining the day the sample is received wherever possible. However, the
laboratory is permitted to split up the sample processing steps if processing a sample completely
in one day is not possible. If this is necessary, sample processing can be halted after filtration,
application of the purified sample onto the slide, or staining. Table 1, in Section 21.0 provides a
breakdown of the holding times for each set of steps. Sections 8.2.1 through 8.2.4 provide
descriptions of these holding times.
8.2.1 Sample collection and filtration. Sample elution must be initiated within 96 hours of
sample collection (if shipped to the laboratory as a bulk sample) or filtration (if filtered
in the field).
8.2.2 Sample elution, concentration, and purification. The laboratory must complete the
elution, concentration, and purification (Sections 12.2.6 through 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 IIovv-ca toinctcr-
June 2003
12
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Method 1623 - Cryptosporidium and Giardia
sorted spiking suspensions containing live organisms within two weeks of preparation at the flow
cytometry laboratory. Manually enumerated spiking suspensions must be used within 24 hours of
enumeration of the spiking suspension if the hemacytometer chamber technique is used (Section
11.3.4); or within 24 hours of application of the spiking suspension to the slides if the well slide
or membrane filter enumeration technique is used (Sections 11.3.5 and 11.3.6). Oocyst and cyst
suspensions must be stored at 0°C to 10°C, until ready to use; do not allow to freeze
9.0 Quality Control
9.1 Each laboratory that uses this method is required to operate a formal quality assurance (QA)
program (Reference 20.7). The minimum requirements of this program consist of an initial
demonstration of laboratory capability through performance of the initial precision and recovery
(IPR) test (Section 9.4), analysis of spiked samples to evaluate and document data quality, and
analysis of standards and blanks as tests of continued performance. Laboratory performance is
compared to established performance criteria to determine if the results of analyses meet the
performance characteristics of the method.
9.1.1 A test of the microscope used for detection of oocysts and cysts is performed prior to
examination of slides. This test is described in Section 10.0.
9.1.2 In recognition of advances that are occurring in analytical technology, the laboratory is
permitted to modify certain method procedures to improve recovery or lower the costs
of measurements, provided that all required quality control (QC) tests are performed and
all QC acceptance criteria are met. Method procedures that can be modified include
front-end techniques, such as filtration or immunomagnetic separation (IMS). The
laboratory is not permitted to use an alternate determinative technique to replace
immunofluorescence assay in this method (the use of different determinative techniques
are considered to be different methods, rather than modified version of this method).
However, the laboratory is permitted to modify the immunofluorescence assay
procedure, provided that all required QC tests are performed (Section 9.1.2.1) and all
QC acceptance criteria are met (see guidance on the use of multiple labeling reagents in
Section 7.6).
NOTE: Method modifications should be considered only to improve method performance,
reduce cost, or reduce sample processing time. Method modifications that reduce cost or
sample processing time, but that result in poorer method performance should not be used.
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
single laboratory, the laboratory must, at a minimum,
validate the modification according to Tier 1 of EPA's
performance-based measurement system (PBMS) (Table
2 and Reference 20.8) to demonstrate that the
modification produces results equivalent or superior to
results produced by this method as written. Briefly, each
time a modification is made to this method, the
laboratory is required to demonstrate acceptable
modified method performance through the IPR test
(Section 9.4). IPR results must meet the QC acceptance
criteria in Tables 3 and 4 in Section 21.0, and should be
comparable to previous results using the unmodified
procedure. Although not required, the laboratory also
should perform a matrix spike/matrix spike duplicate
(MS/MSD) test to demonstrate the performance of the
modified method in at least one real-world matrix before
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Method 1623 - Cryptosporidium and Giardia
analyzing field samples using the modified method. The
laboratory is required to perform MS samples using the
modified method at the frequency noted in Section 9.1.8.
If the modified method involves changes that cannot be
adequately evaluated through these tests, additional tests
may be required to demonstrate acceptability.
9.1.2.1.2 Method modifications for nationwide approval. If the
laboratory or a manufacturer seeks EPA approval of a
method modification for nationwide use, the laboratory
or manufacturer must, at a minimum, validate the
modification according to Tier 2 of EPA's PBMS (Table
2 and Reference 20.8). Briefly, at least three laboratories
must perform IPR tests (Section 9.4) and MS/MSD
(Section 9.5) tests using the modified method, and all
tests must meet the QC acceptance criteria specified in
Tables 3 and 4 in Section 21.0. Upon nationwide
approval, laboratories electing to use the modified
method still must demonstrate acceptable performance in
their own laboratory according to the requirements in
Section 9.1.2.1.1. If the modified method involves
changes that cannot be adequately evaluated through
these tests, additional tests may be required to
demonstrate acceptability.
9.1.2.2 The laboratory is required to maintain records of modifications made to
this method. These records include the following, at a minimum:
9.1.2.2.1 The names, titles, addresses, and telephone numbers of
the analyst(s) who performed the analyses and
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
and Gicirdici).
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
June 2003
14
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Method 1623 - Cryptosporidium and Giardia
IMS, and all calculations required to verify the
percent of concentrate examined (Section 13.2)
(i) Purification completion dates and times (Section
13.3.3.11)
(j) Staining completion dates and times (Section
14.10)
(k) Staining control results (Section 15.2.1)
(1) All required examination information (Section
15.2.2)
(m) Examination completion dates and times
(Section 15.2.4)
(n) Analysis sequence/run chronology
(o) Lot numbers of elution, IMS, and staining
reagents
(p) Copies of bench sheets, logbooks, and other
recordings of raw data
(q) Data system outputs, and other data to link the
raw data to the results reported
9.1.3 The laboratory shall spike a separate sample aliquot from the same source to monitor
method performance. 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 MS samples (Section 9.5.1) at a minimum frequency of 1
MS sample per 20 field samples and at least 12 months must elapse between the first and
last MS sample. The laboratory should analyze an MS sample when samples are first
received from a PWS for which the laboratory has never before analyzed samples to
identify potential method performance issues with the matrix (Section 9.5.1; Tables 3
and 4). If an MS sample cannot be analyzed on the first sampling event, the first MS
sample should be analyzed as soon as possible to identify potential method performance
issues with the matrix.
9.2 Micropipette calibration
9.2.1 Micropipettes must be sent to the manufacturer for calibration annually. Alternately, a
qualified independent technician specializing in micropipette calibration can be used, or
the calibration can be performed by the laboratory, provided the laboratory maintains a
detailed procedure that can be evaluated by an independent auditor. Documentation on
the precision of the recalibrated micropipette must be obtained from the manufacturer or
technician.
9.2.2 Internal and external calibration records must be kept on file in the laboratory's QA
logbook.
9.2.3 If a micropipette calibration problem is suspected, the laboratory shall tare an empty
weighing boat on the analytical balance and pipette the following volumes of reagent
water into the weigh boat using the pipette in question: 100% of the maximum
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June 2003
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Method 1623 - Cryptosporidium and Giardia
dispensing capacity of the micropipette, 50% of the capacity, and 10% of the capacity.
Ten replicates should be performed at each weight. Record the weight of the water
(assume that 1.00 mL of reagent water weighs 1.00 g) and calculate the relative standard
deviation (RSD) for each. If the weight of the reagent water is within 1% of the desired
weight (mL) and the RSD of the replicates at each weight is within 1%, then the pipette
remains acceptable for use.
9.2.4 If the weight of the reagent water is outside the acceptable limits, consult the
manufacturer's instruction manual troubleshooting section and repeat steps described in
Section 9.2.3. If problems with the pipette persist, the laboratory must send the pipette to
the manufacturer for recalibration.
9.3 Microscope adjustment and certification: Adjust the microscope as specified in Section 10.0. All
of the requirements in Section 10.0 must be met prior to analysis of IPRs, blanks, OPRs, field
samples, and MS/MSDs.
9.4 Initial precision and recovery (IPR)—To establish the ability to demonstrate control over the
analytical system and to generate acceptable precision and recovery, the laboratory shall perform
the following operations:
9.4.1 Using the spiking procedure in Section 11.4 and enumerated spiking suspensions
(Section 7.10.1 or Section 11.3), spike, filter, elute, concentrate, separate (purify), stain,
and examine four reagent water samples spiked with 100 to 500 oocysts and 100 to 500
cysts.
9.4.1.1 The laboratory is permitted to analyze the four spiked reagent samples on
the same day or on as many as four different days (provided that the
spiked reagent samples are analyzed consecutively), and also may use
different analysts and/or reagent lots for each sample (however, the
procedures used for all analyses must be identical). Laboratories should
note that the variability of four measurements performed on multiple
days or using multiple analysts or reagent lots may be greater than the
variability of measurements performed on the same day with the same
analysts and reagent lots. As a result, the laboratory is at a greater risk of
generating unacceptably variable IPR results if the test is performed
across multiple days, analysts, and /or reagent lots.
9.4.1.2 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.
9.4.1.3 The set of four IPR tests samples must be accompanied by analysis of a
an acceptable method blank (Section 9.6).
9.4.2 Using results of the four analyses, calculate the average percent recovery and the
relative standard deviation (RSD) of the recoveries for Cryptosporidium and for
Giardia. The RSD is the standard deviation divided by the mean, times 100.
9.4.3 Compare the RSD and the mean with the corresponding limits for initial precision and
recovery in Tables 3 and 4 in Section 21.0. If the RSD and the mean meet the
acceptance criteria, system performance is acceptable and analysis of blanks and
samples may begin. If the RSD or the mean falls outside the range for recovery, system
performance is unacceptable. In this event, trouble-shoot the problem by starting at the
end of the method (see guidance in Section 7.9.5 9.7.5), correct the problem and repeat
the test (Section 9.4.1).
9.5 Matrix spike (MS) and matrix spike duplicate (MSD):
9.5.1 Matrix spike—The laboratory shall spike and analyze a separate field sample aliquot to
determine the effect of the matrix on the method's oocyst and cyst recovery. The MS
and field sample must be collected from the same sampling location as split samples or
as samples sequentially collected immediately after one another. The MS sample volume
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Method 1623 - Cryptosporidium and Giardia
analyzed must be within 10% of the field sample volume. The MS shall be analyzed
according to the frequency in Section 9.1.8.
9.5.1.1 Analyze an unspiked field sample according to the procedures in
Sections 12.0 to 15.0. Using the spiking procedure in Section 11.4 and
enumerated spiking suspensions (Section 7.10.1 or Section 11.3), spike,
filter, elute, concentrate, separate (purify), stain, and examine a second
field sample aliquot with a similar the number of organisms as that used
in the IPR or OPR tests (Sections 9.4 and 9.7).
9.5.1.2 For each organism, calculate the percent recovery (R) using the
following equation.
NSP - Ns
R = 100 x
where
R is the percent recovery
Nsp is the number of oocysts or cysts detected in the spiked
sample
Ns is the number of oocysts or cysts detected in the unspiked
sample
T is the true value of the oocysts or cysts spiked
9.5.1.3 Compare the recovery for each organism with the corresponding limits in
Tables 3 and 4 in Section 21.0.
NOTE: Some sample matrices may prevent the acceptance criteria in Tables 3 and 4 from
being met. An assessment of the distribution of MS recoveries across 430 MS samples from
87 sites during the ICR Supplemental Surveys is provided in Table 5.
9.5.1.4 As part of the QA program for the laboratory, method precision for
samples should be assessed and records maintained. After the analysis of
five samples for which the spike recovery for each organism 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—MSD analysis is required as part of nationwide approval of a
modified version of this method to demonstrate that the modified version of this method
produces results equal or superior to results produced by the method as written (Section
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 For each organism, 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 or cysts in the MS and
MSD (Xmean) (= [MS+MSD]/2).
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Method 1623 - Cryptosporidium and Giardia
9.5.2.3 Calculate the relative percent difference (RPD) of the recoveries using
the following equation:
I Nms " Nmsd I
RPD = 100 x
Xmean
where
RPD is the relative percent difference
Nms is the number of oocysts or cysts detected in the MS
Nmsd is the number of oocysts or cysts detected in the MSD
Xmean is the mean number of oocysts or cysts detected in the MS
and MSD
9.5.2.4 Compare the mean MS/MSD recovery and RPD with the corresponding
limits in Tables 3 and 4 in Section 21.0 for each organism.
9.6 Method blank (negative control sample, laboratory blank): Reagent water blanks are analyzed to
demonstrate freedom from contamination. Analyze the blank immediately prior to after analysis
of the IPR test (Section 9.4) and OPR test (Section 9.7) and prior to analysis of samples for the
week to demonstrate freedom from contamination.
9.6.1 Filter, elute, concentrate, separate (purify), stain, and examine at least one reagent water
blank per week (Section 9.1.7) according to the procedures in Sections 12.0 to 15.0. If
more than 20 samples are analyzed in a week, process and analyze one reagent water
blank for every 20 samples.
9.6.2 Actions
9.6.2.1 If Cryptosporidium oocysts, Gicirdici cysts, or potentially interfering
organisms or materials that may be misidentified as oocysts or cysts are
not found in the method blank, the method blank test is acceptable and
analysis of samples may proceed Any method blank in which oocysts or
cysts are not detected is assumed to be uncontaminated and may be
reported.
9.6.2.2 If Cryptosporidium oocysts; or Giardia cysts (as defined in Section 3) or
any potentially interfering organism or materials that may be
misidentified as oocysts or cysts are found in the method blank, the
method blank test is unacceptable. Any field sample in a batch associated
with an unacceptable method blank a contaminated blank that shows the
presence of one or more oocysts or cysts is assumed to be contaminated
and should be recollected, if possible. Analysis of additional samples is
halted until the source of contamination is eliminated, the method blank
test is performed again, and no evidence of contamination is detected.
9.7 Ongoing precision and recovery (OPR; positive control sample; laboratory control sample):
Using the spiking procedure in Section 11.4 and enumerated spiking suspensions (Section 7.10.1
or Section 11.3), filter, elute, concentrate, separate (purify), stain, and examine at least one
reagent water sample spiked with 100 to 500 oocysts and 100 to 500 cysts each week to verify all
performance criteria. The laboratory must analyze one OPR sample for every 20 samples if more
than 20 samples are analyzed in a week. If multiple method variations are used, separate OPR
samples must be prepared for each method variation. Adjustment and/or recalibration of the
analytical system shall be performed until all performance criteria are met. Only after all
performance criteria are met may should samples be analyzed.
9.7.1 Examine the slide from the OPR prior to analysis of samples from the same batch.
9.7.1.1 Using 200X to 400X magnification, more than 50% of the oocysts or
cysts must appear undamaged and morphologically intact; otherwise, the
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Method 1623 - Cryptosporidium and Giardia
organisms in the spiking suspension may be of unacceptable quality or
the analytical process is may be damaging the organisms. Examine the
spiking suspension organisms directly (by centrifuging, if possible, to
concentrate the organisms in a volume that can be applied directly to a
slide). If the organisms appear undamaged and morphologically intact
under DIC, determine the step or reagent that is causing damage to the
organisms. Correct the problem and repeat the OPRtest.
9.7.1.2 Identify and enumerate each organism using epifluorescence microscopy.
The first three presumptive Cryptosporidium oocysts and three Gicirdici
cysts identified in the OPR sample must be examined using FITC, DAPI,
and DIC, as per Section 15.2, and the detailed characteristics (size,
shape, DAPI category, and DIC category) reported on the
('rypiosporidiiim and Giardia report form, as well as any additional
comments on organism appearance, if notable.
9.7.2 For each organism, calculate the percent recovery (R) using the following equation:
N
R = 100 x
T
where:
R = the percent recovery
N = the number of oocysts or cysts detected
T = the number of oocysts or cysts spiked
9.7.3 Compare the recovery with the limits for ongoing precision and recovery in Tables 3
and 4 in Section 21.0.
9.7.4 Actions
9.7.4.1 If the recoveries for ('rypiosporidiiim and Giardia meet the acceptance
criteria, system performance is acceptable and analysis of blanks and
samples may proceed.
9.7.4.2 If, however, the recovery for Cryptosporidium or Giardia falls outside of
the criteria range given, system performance is unacceptable. Any
sample in a batch associated with an unacceptable OPR sample is
unacceptable. Analysis of additional samples is halted until the analytical
system is brought under control. In this event, there may be a problem
with the microscope or with the filtration or separation systems.
Troubleshoot the problem using the procedures at Section 9.7.5 as a
guide. After assessing the issue, perform another OPRtest and verify that
('rypiosporidiiim and Giardia recoveries meet the acceptance criteria. AH
samples must be associated with an OPR that passes the criteria in
Section 21.0. Samples that are not associated with an acceptable OPR
must be flagged accordingly.
9.7.5 Troubleshooting. If an OPR sample has failed, and the cause of the failure is not
known, the laboratory generally should identify the problem working backward in the
analytical process from the microscopic examination to filtration.
9.7.5.1 Quality of spiked organisms. Examine the spiking suspension
organisms directly (by centrifuging, if possible, to concentrate the
organisms in a volume that can be applied directly to a slide). If the
organisms appear damaged under DIC, obtain fresh spiking materials. If
the organisms appear undamaged and morphologically intact, determined
whether the problem is associated with the microscope system or
antibody stain (Section 9.7.5.2).
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Method 1623 - Cryptosporidium and Giardia
9.7.5.2 Microscope system and antibody stain: To determine if the failure of
the OPR test is due to changes in the microscope or problems with the
antibody stain, re-examine the positive staining control (Section 15.2.1),
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.
9.7.5.3 Separation (purification) system: To determine if the failure of the
OPR test is attributable to the separation system, check system
performance by spiking a 10-mL volume of reagent water with 100-500
oocysts and cysts and processing the sample through the IMS, staining,
and examination procedures in Sections 13.3 through 15.0. Recoveries
should be greater than 70%.
9.7.5.4 Filtration/elution/concentration system: If the failure of the OPR test
is attributable to the filtration/elution/concentration system, check system
performance by processing spiked reagent water according to the
procedures in Section 12.2 through 13.2.2.1 13.2.2, and filter, stain, and
examine the sample concentrate according to Section 11.3.6.
9.7.6 The laboratory should add results that pass the specifications in Section 9.7.3 to initial
and previous ongoing data and update the QC chart to form a graphic representation of
continued laboratory performance. The laboratory should develop a statement of
laboratory accuracy (reagent water, raw surface water) by calculating the average
percent recovery (R) and the standard deviation of percent recovery (sr). Express the
accuracy as a recovery interval from R 2 sr to R + 2 sr. For example, if R = 95% and sr
= 25%, the accuracy is 45% to 145%.
9.8 The laboratory should periodically analyze an external QC sample, such as a performance
evaluation or standard reference material, when available. The laboratory also should periodically
participate in interlaboratory comparison studies using the method.
9.9 The specifications contained in this method can be met if the analytical system is under control.
The standards used for initial (Section 9.4) and ongoing (Section 9.7) precision and recovery
should be identical, so that the most precise results will be obtained. The microscope in particular
will provide the most reproducible results if dedicated to the settings and conditions required for
the determination of Cryptosporidium and Gicirdici by this method.
9.10 Depending on specific program requirements, field replicates may be collected to determine the
precision of the sampling technique, and duplicate spiked samples may be required to determine
the precision of the analysis.
10.0 Microscope Calibration and Analyst Verification
10.1 In a room capable of being darkened to near-complete darkness, assemble the microscope, all
filters, and attachments. The microscope should be placed on a solid surface free from vibration.
Adequate workspace should be provided on either side of the microscope for taking notes and
placement of slides and ancillary materials.
10.2 Using the manuals provided with the microscope, all analysts must familiarize themselves with
operation of the microscope.
10.3 Microscope adjustment and calibration (adapted from Reference 20.7)
10.3.1 Preparations for adjustment
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
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Method 1623 - Cryptosporidium and Giardia
efficiency, and reliable identification and enumeration of oocysts and
cysts will not be possible. Consequently, it is imperative that all portions
of the microscope from the light sources to the oculars are properly
adjusted.
10.3.1.2 While microscopes from various vendors are configured somewhat
differently, they all operate on the same general physical principles.
Therefore, slight deviations or adjustments may be required to make the
procedures below work for a particular instrument.
10.3.1.3 The sections below assume that the mercury bulb has not exceeded time
limits of operation, that the lamp socket is connected to the lamp house,
and that the condenser is adjusted to produce Kohler illumination.
10.3.1.4 Persons with astigmatism should always wear contact lenses or glasses
when using the microscope.
CAUTION: In the procedures below, do not touch the quartz portion of the mercury
bulb with your bare fingers. Finger oils can cause rapid degradation of the quartz and
premature failure of the bulb.
WARNING: Never look at the ultraviolet (UV) light from the mercury lamp, lamp house,
or the UV image without a barrier filter in place. UV radiation can cause serious eye
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.
21 June 2003
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Method 1623 - Cryptosporidium and Giardia
Adjustment of the diffuser lens probably will be required. Additional
slight adjustments as in Section 10.3.2.7 above may be required.
10.3.2.11 Maintain a log of the number of hours the UV bulb has been used. Never
use the bulb for longer than it has been rated. Fifty-watt bulbs should not
be used longer than 100 hours; 100-watt bulbs should not be used longer
than 200 hours.
10.3.3 Transmitted bulb adjustment: The purpose of this procedure is to center the filament and
ensure even field illumination. This procedure must be followed when the bulb is
changed.
10.3.3.1 Remove the diffuser lens between the lamp and microscope or swing it
out of the transmitted light path.
10.3.3.2 Using a prepared microscope slide and a 40X (or similar) objective,
adjust the focus so the image in the oculars is sharply defined.
10.3.3.3 Without the ocular or Bertrand optics in place, view the pupil and
filament image at the bottom of the tube.
10.3.3.4 Focus the lamp filament image with the appropriate adjustment on the
lamp house.
10.3.3.5 Similarly, center the lamp filament image within the pupil with the
appropriate adjustment(s) on the lamp house.
10.3.3.6 Insert the diffuser lens into the light path between the transmitted lamp
house and the microscope.
10.3.4 Adjustment of the interpupillary distance and oculars for each eye: These adjustments
are necessary so that eye strain is reduced to a minimum, and must be made for each
individual using the microscope. Section 10.3.4.2 assumes use of a microscope with
both oculars adjustable; Section 10.3.4.3 assumes use of a microscope with a single
adjustable ocular. The procedure must be followed each time an analyst uses the
microscope.
10.3.4.1 Interpupillary distance
10.3.4.1.1 Place a prepared slide on the microscope stage, turn on
the transmitted light, and focus the specimen image
using the coarse and fine adjustment knobs.
10.3.4.1.2 Using both hands, move the oculars closer together or
farther apart until a single circle of light is observed
while looking through the oculars with both eyes. Note
interpupillary distance.
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.
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Method 1623 - Cryptosporidium and Giardia
10.3.4.3 Ocular adjustment for microscopes without binocular capability: This
procedure assumes a single focusing ocular. The following procedure
assumes that only the right ocular is capable of adjustment.
10.3.4.3.1 Place a card between the right ocular and eye keeping
both eyes open. Using the fine adjustment, focus the
image for the left eye to its sharpest point.
10.3.4.3.2 Transfer the card to between the left eye and ocular.
Keeping both eyes open, bring the image for the right
eye into sharp focus by adjusting the ocular collar at the
top of the ocular without touching the coarse or fine
adjustment.
10.3.5 Calibration of an ocular micrometer: This section assumes that a reticle has been
installed in one of the oculars by a microscopy specialist and that a stage micrometer is
available for calibrating the ocular micrometer (reticle). Once installed, the ocular reticle
should be left in place. The more an ocular is manipulated the greater the probability is
for it to become contaminated with dust particles. This calibration should be done for
each objective in use on the microscope. If there is a top lens on the microscope, the
calibration procedure must be done for the respective objective at each top lens setting.
The procedure must be followed when the microscope is first used and each time the
objective is changed.
10.3.5.1 Place the stage micrometer on the microscope stage, turn on the
transmitted light, and focus the micrometer image using the coarse and
fine adjustment knobs for the objective to be calibrated. Continue
adjusting the focus on the stage micrometer so you can distinguish
between the large (0.1 mm) and the small (0.01 mm) divisions.
10.3.5.2 Adjust the stage and ocular with the micrometer so the "0" line on the
ocular micrometer is exactly superimposed on the "0" line on the stage
micrometer.
10.3.5.3 Without changing the stage adjustment, find a point as distant as possible
from the two 0 lines where two other lines are exactly superimposed.
10.3.5.4 Determine the number of ocular micrometer spaces as well as the number
of millimeters on the stage micrometer between the two points of
superimposition. For example: Suppose 48 ocular micrometer spaces
equal 0.6 mm.
10.3.5.5 Calculate the number of mm/ocular micrometer space. For example:
0.6 mm 0.0125 mm
48 ocular micrometer spaces ocular micrometer space
10.3.5.6 Because most measurements of microorganisms are given in |_im rather
than mm, the value calculated above must be converted to |_im by
multiplying it by 1000 (im/mm. For example:
0.0125 mm 1,000 |jm 12.5 |jm
x =
ocular micrometer space mm ocular micrometer space
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Method 1623 - Cryptosporidium and Giardia
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
Item
no.
Objective
power
Description
No. of ocular
micrometer
spaces
No. of stage
micrometer
mm1
jjm/ocular
micrometer
space2
1
10X
n.a.3=
2
20X
N.A.=
3
40X
N.A.=
4
100X
N.A.=
11000 pm/mm
2(Stage micrometer length in mm * (1000 pm/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 DIC objective that will be used to identify internal morphological
characteristics in Cryptosporidium oocysts and Gicirdici cysts. If more than one objective
is to be used for DIC, 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 DIC will not work to its maximal potential. These steps need
to become second nature and must be practiced regularly until they are a matter of reflex
rather than a chore. The procedure must be followed each time an analyst uses the
microscope and each time the objective is changed.
10.3.6.1 Place a prepared slide on the microscope stage, place oil on the slide,
move the 100X oil objective into place, turn on the transmitted light, and
focus the specimen image using the coarse and fine adjustment knobs.
10.3.6.2 At this point both the radiant field diaphragm in the microscope base and
the aperture diaphragm in the condenser should be wide open. Now close
down the radiant field diaphragm in the microscope base until the lighted
field is reduced to a small opening.
10.3.6.3 Using the condenser centering screws on the front right and left of the
condenser, move the small lighted portion of the field to the center of the
visual field.
10.3.6.4 Now look to see whether the leaves of the iris field diaphragm are
sharply defined (focused) or not. If they are not sharply defined, then
they can be focused distinctly by changing the height of the condenser up
and down with the condenser focusing knob while you are looking
through the binoculars. Once you have accomplished the precise
focusing of the radiant field diaphragm leaves, open the radiant field
diaphragm until the leaves just disappear from view.
10.3.6.5 The aperture diaphragm of the condenser should is now be adjusted to
make it compatible with the total numerical aperture of the optical
system. This is done by removing an ocular, looking into the tube at the
rear focal plane of the objective, and stopping down the aperture
diaphragm iris leaves until they are visible just inside the rear plane of
the objective.
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Method 1623 - Cryptosporidium and Giardia
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 DIC.
10.4 Microscope cleaning procedure
10.4.1 Use canned air to remove dust from the lenses, filters, and microscope body.
10.4.2 Use a Kimwipe-dampened with a microscope cleaning solution (MCS) (consisting of 2
parts 90% isoproponal and 1 part acetone) to wipe down all surfaces of the microscope
body. Dry off with a clean, dry Kimwipe.
10.4.3 Protocol for cleaning oculars and condenser
10.4.3.1 Use a new, clean Q-tip dampened with MCS to clean each lense. Start at
the center of the lens and spiral the Q-tip outward using little to no
pressure. Rotate the Q-tip head while spiraling to ensure a clean surface
is always contacting the lens.
10.4.3.2 Repeat the procedure using a new, dry Q-tip.
10.4.3.3 Repeat Sections 10.4.3.1 and 10.4.3.2.
10.4.3.4 Remove the ocular and repeat the cleaning procedure on the bottom lens
of the ocular.
10.4.4 Protocol for cleaning objective lenses
10.4.4.1 Wipe 100X oil objective with lense paper to remove the bulk of the oil
from the objective.
10.4.4.2 Hold a new Q-tip dampened with MCS at a 45° angle on the objective
and twirl.
10.4.4.3 Repeat Sections 10.4.4.2 with a new, dry Q-tip.
10.4.4.4 Repeat Sections 10.4.4.2 and 10.4.4.3.
10.4.4.5 Clean all objectives whether they are used or not.
10.4.5 Protocol for cleaning light source lens and filters
10.4.5.1 Using a Kimwipe dampened with microscope cleaning solution, wipe off
the surface of each lens and filter.
10.4.5.2 Repeat the procedure using a dry Kimwipe.
10.4.5.3 Repeat Sections 10.4.5.1 and 10.4.5.2.
10.4.6 Protocol for cleaning microscope stage
10.4.6.1 Using a Kimwipe dampened with microscope cleaning solution, wipe off
the stage and stage clip. Be sure to clean off any residual immersion oil
or fingernail polish. Remove the stage clip if necessary to ensure that it is
thoroughly cleaned.
10.4.7 Use 409 and a paper towel to clean the bench top surrounding the microscope.
10.4.8 Frequency
10.4.8.1 Perform Sections 10.4.2, 10.4.3, 10.4.4, 10.4.5 and 10.4.7 after each
microscope session.
10.4.8.2 Perform complete cleaning each week.
10.5 Protozoa libraries: Each laboratory is encouraged to develop libraries of photographs and
drawings for identification of protozoa.
10.5.1 Take color photographs of Cryptosporidium oocysts and Gicirdici cysts by FA, and 4',6-
diamidino-2-phenylindole (DAPI), and DIC that the analysts (Section 22.2) determine
are accurate (Section 15.2).
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Method 1623 - Cryptosporidium and Giardia
10.5.2 Similarly, take color photographs of interfering organisms and materials by FA, and
DAPI, and DIC that the analysts believe are not Cryptosporidium oocysts or Gicirdici
cysts. Quantify the size, shape, microscope settings, and other characteristics that can be
used to differentiate oocysts and cysts from interfering debris and that will result in
accurate positive identification of DAPI positive or negative organisms.
10.6 Verification of analyst performance: Until standard reference materials, such as National Institute
of Standards and Technology standard reference materials, are available that contain a reliable
number of DAPI positive or negative oocysts and cysts, this method shall rely upon the ability of
the analyst for identification and enumeration of oocysts and cysts.
10.6.1 At least monthly when microscopic examinations are being performed, the laboratory
shall prepare a slide containing 40 to 100 oocysts and 40 to 100 cysts. More than 50% of
the oocysts and cysts must be DAPI positive and undamaged under DIC.
10.6.2 Each analyst shall determine the total number of oocysts and cysts and the number that
are DAPI positive and DAPI negative for the entire slide. For 10 oocysts and 10 cysts,
each analyst shall determine using the slide prepared in Section 10.5.1. the number of
nuclei by DAPI and the DIC category (empty, containing amorphous structures, or
containing identifiable internal structures) of each.
10.6.3 Requirements for laboratories with multiple analysts
10.6.3.1 The total number and the number of DAPI positive errand DAPI negative
oocysts and cysts determined by each analyst (Section 10.5.2.) must be
within ±10% of each other. If the number is not within this range, the
analysts must identify the source of any variability between analysts"
examination criteria, prepare a new slide, and repeat the performance
verification (Sections 10.5.1 to 10.5.2). Differences in the number of
nuclei by DAPI and in DIC categorizations among analysts must be
discussed and resolved, and these resolutions must be documented.
10.6.3.2 Document the date, name(s) of analyst(s), number of total, DAPI positive
or negative oocysts and cysts determined by the analyst(s), whether the
test was passed/failed and the results of attempts before the test was
passed.
10.6.3.3 Only after an analyst has passed the criteria in Section 10.6.3, may
oocysts and cysts in QC samples and field samples be identified and
enumerated.
10.6.4 Laboratories with only one analyst should maintain a protozoa library (Section 10.5) and
compare the results of the examinations performed in Sections 10.6.1 and 10.6.2 to
photographs of oocysts and cysts and interfering organisms to verify that examination
results are consistent with these references. These laboratories also should perform
repetitive counts of a single verification slide for FITC and DAPI. These laboratories
should also coordinate with other laboratories to share slides and compare counts.
11.0 Oocyst and Cyst Suspension Enumeration and Spiking
11.1 This method requires routine analysis of spiked QC samples to demonstrate acceptable initial and
ongoing laboratory and method performance (initial precision and recovery samples [Section
9.4], matrix spike and matrix spike duplicate samples [Section 9.5], and ongoing precision and
recovery samples [Section 9.7]). The organisms used for these samples must be enumerated to
calculate recoveries and precision. EPA recommends that flow cytometry be used for this
enumeration, rather than manual techniques. Flow cytometer-sorted spikes generally are
characterized by a relative standard deviation of <2.5%, versus greater variability for manual
enumeration techniques (Reference 20.8). Guidance on preparing spiking suspensions using a
flow cytometer is provided in Section 11.2. Manual enumeration procedures are provided in
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Method 1623 - Cryptosporidium and Giardia
Section 11.3. The procedure for spiking bulk samples in the laboratory is provided in Section
11.4.
11.2 Flow cytometry enumeration guidelines. Although it is unlikely that many laboratories
performing Method 1623 will have direct access to a flow cytometer for preparing spiking
suspensions, flow-sorted suspensions are available from commercial vendors and other sources
(Section 7.10.1). The information provided in Sections 11.2.1 through 11.2.4 is simply meant as a
guideline for preparing spiking suspensions using a flow cytometer. Laboratories performing
flow cytometry must develop and implement detailed standardized protocols for calibration and
operation of the flow cytometer.
11.2.1 Spiking suspensions should be prepared using unstained organisms that have not been
heat-fixed or formalin-fixed.
11.2.2 Spiking suspensions should be prepared using Cryptosporidium pctrvum oocysts <3
months old, and Gicirdici intestinalis cysts <2 weeks old.
11.2.3 Initial calibration. Immediately before sorting spiking suspensions, an initial
calibration of the flow cytometer should be performed by conducting 10 sequential sorts
directly onto membranes or well slides. The oocyst and cyst levels used for the initial
calibration should be the same as the levels used for the spiking suspensions. Each initial
calibration sample should be stained and manually counted microscopically and the
manual counts used to verify the accuracy of the system. The relative standard deviation
(RSD) of the 10 counts should be < 2.5%. If the RSD is > 2.5%, the laboratory should
perform the initial calibration again, until the RSD of the 10 counts is < 2.5%. In
addition to counting the organisms, the laboratory also should evaluate the quality of the
organisms using DAPI and DIC to confirm that the organisms are in good condition.
11.2.4 Ongoing calibration. When sorting the spiking suspensions for use in QC samples, the
laboratory should perform ongoing calibration samples at a 10% frequency, at a
minimum. The laboratory should sort the first run and every eleventh sample directly
onto a membrane or well slide. Each ongoing calibration sample should be stained and
manually counted microscopically and the manual counts used to verify the accuracy of
the system. The mean of the ongoing calibration counts also should be used as the
estimated spike dose, if the relative standard deviation (RSD) of the ongoing calibration
counts is < 2.5%. If the RSD is > 2.5%, the laboratory should discard the batch.
11.2.5 Method blanks. Depending on the operation of the flow cytometer, method blanks
should be prepared and examined at the same frequency as the ongoing calibration
samples (Section 11.2.4).
11.2.6 Holding time criteria. Flow-cytometer-sorted spiking suspensions (Sections 7.10.1 and
11.2) used for spiked quality control (QC) samples (Section 9) must be used within the
expiration date noted on the suspension. The holding time specified by the flow
cytometry laboratory should be determined based on a holding time study. Laboratories
should use flow-cytometer-sorted spiking suspensions containing live organisms within
two weeks of preparation at the flow cytometry laboratory.
11.3 Manual enumeration procedures. Two sets of manual enumerations are required per organism
before purified Cryptosporidium oocyst and Giardia cyst stock suspensions (Sections 7.9.2.1 and
7.9.2.2 Section 7.10.2) received from suppliers can be used to spike samples in the laboratory.
First, the stock suspension must be diluted and enumerated (Section 11.3.3) to yield a suspension
at the appropriate oocyst or cyst concentration for spiking (spiking suspension). Then, 10 aliquots
of spiking suspension must be enumerated to calculate a mean spike dose. Spiking suspensions
can be enumerated using hemacytometer chamber counting (Section 11.3.4), well slide counting
(Section 11.3.5), or membrane filter counting (Section 11.3.6).
11.3.1 Precision criteria. The relative standard deviation (RSD) of the calculated mean spike
dose for manually enumerated spiking suspensions must be < 16% for Cryptosporidium
and < 19% for Giardia before proceeding (these criteria are based on the pooled RSDs of
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Method 1623 - Cryptosporidium and Giardia
105 manual Cryptosporidium enumerations and 104 manual Giardia enumerations
submitted by 20 different laboratories under the EPA Protozoa Performance Evaluation
Program).
11.3.2 Holding time criteria. Manually enumerated spiking suspensions must be used within
24 hours of enumeration of the spiking suspension if the hemacytometer chamber
technique is used (Section 11.3.4); or within 24 hours of application of the spiking
suspension or membrane filter to the slides if the well slide or membrane filter
enumeration technique is used (Sections 11.3.5 and 11.3.6).
11.3.3 Enumerating and diluting stock suspensions
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 (iL 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
or cysts to settle for 2 minutes. Do not attempt to adjust the coverslip,
apply clips, or in any way disturb the chamber after it has been filled.
11.3.3.4 Use 200X magnification.
11.3.3.5 Move the chamber so the ruled area is centered underneath it the
objective.
11.3.3.6 Move the objective close to the coverslip while watching it from the side
of the microscope, rather than through the microscope.
11.3.3.7 Focus up from the coverslip until the hemacytometer ruling appears.
11.3.3.8 At each of the four corners of the chamber is a 1-square-mm area divided
into 16 squares in which organisms are to be counted (Figure 1).
Beginning with the top row of four squares, count with a hand-tally
counter in the directions indicated in Figure 2. Avoid counting organisms
twice by counting only those touching the top and left boundary lines.
Count each square millimeter in this fashion.
11.3.3.9 Use the following formula to determine the number of organisms per mL
of suspension:
number of 1Q dilution ieeemm2 number of
organisms counted factor organisms
number of mm2
counted
1 mm 1 1 pLmt pLmt
11.3.3.10 Record the result on a hemacytometer data sheet.
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Method 1623 - Cryptosporidium and Giardia
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 +0-|_iL): however, ranges as great as
5000 to 15,000 organisms per mL (50 to 150 organisms per +0-|_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 11.3.5), then the stock suspensions
should be diluted in reagent water.
To calculate the volume (in (.iL) 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 |jL
volume of stock suspension (ML) required = number Qf organisms/ MfflL of stock
suspension
If the volume is less than 10 (.iL. 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 (.iL. use the following formula:
number of organisms required x 1 0|jL
total volume (|jL) =
predicted number of organisms per 10|jL (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 (|jL) = total volume (|jL) - stock suspension volume required (|jL)
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.
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Method 1623 - Cryptosporidium and Giardia
11.3.3.14 Several factors are known to introduce errors into hemacytometer counts,
including:
• Inadequate mixing of suspension before flooding the chamber
• Irregular filling of the chamber, trapped air bubbles, dust, or oil
on the chamber or coverslip
• Total number of organisms counted is too low to provide
statistical confidence in the result
• Error in recording tally
• Calculation error; failure to consider dilution factor, or area
counted
• Inadequate cleaning and removal of organisms from the previous
count
• Allowing filled chamber to sit too long, so that the chamber
suspension dries and concentrates.
11.3.4 Enumerating spiking suspensions using a hemacytometer chamber
NOTE: Spiking suspensions enumerated using a hemacytometer chamber must be used
within 24 hours of enumeration.
11.3.4.1 Vortex the tube containing the spiking suspension (diluted stock
suspension; Section 11.3.3) for a minimum of 2 minutes. Gently invert
the tube three times.
11.3.4.2 To an appropriate-size beaker containing a stir bar, add enough spiking
suspension to perform all spike testing and the enumeration as described.
The liquid volume and beaker relationship should be such that a spinning
stir bar does not splash the sides of the beaker, the stir bar has unimpeded
rotation, and there is enough room to draw sample from the beaker with a
10-jj.L 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-jj.L aliquot and carefully
load one side of the hemacytometer. Count all organisms on the platform,
at 200X magnification using phase-contrast or darkfield microscopy. The
count must include the entire area under the hemacytometer, not just the
four outer 1-mm2 squares. Repeat this procedure nine times. This step
allows confirmation of the number of organisms per 10 jj.L (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 and < 19% for Giardia before proceeding. If the RSD is
unacceptable, or the mean number is outside the expected range, add
additional oocysts from stock suspension or dilute the contents of the
beaker appropriately with reagent water. Repeat the process to confirm
counts. Refer to Section 11.3.3.14 for factors that may introduce errors.
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Method 1623 - Cryptosporidium and Giardia
11.3.5 Enumerating spiking suspensions using well slides
NOTE: Spiking suspensions enumerated using well slides must be used within 24 hours of
application of the spiking suspension to the slides.
11.3.5.1 Prepare well slides for sample screening and label the slides. 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.
11.3.5.3 Remove a 10-j_iL aliquot from the spiking suspension and apply it to the
center of a well.
11.3.5.4 Before removing subsequent aliquots, cap the tube and gently invert it
three times to ensure that the oocysts or cysts 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 or cysts to the center
of a well and distribute evenly over the well area.
11.3.5.6.2 For the negative control, pipette 50 (iL of PBS onto the
center of a well and spread it over the well area with a
pipette tip.
11.3.5.6.3 Air-dry the control slides.
11.3.5. 7 Apply 50-f.iL of absolute methanol to each well containing the dried
sample and allow to air-dry for 3 to 5 minutes.
11.3.5.7 Follow the manufacturer's instructions (Section 7.6) in applying the stain
to the slide.
11.3.5.8 Place the slides in a humid chamber in the dark and incubate according
to manufacturer's directions, 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.
11.3.5.9 Apply one drop of wash buffer (prepared according to the manufacturer's
instructions [Section 7.6]) to each well. Tilt each slide on a clean paper
towel, long edge down. Gently aspirate the excess detection reagent from
below the well using a clean Pasteur pipette or absorb with a paper towel
or other absorbent material. Avoid disturbing the sample.
NOTE: If using the Merifluor stain (Section 7.6.1), do not allow slides to dry completely.
11.3.5.10 Add mounting medium (Section 7.8) to each well.
11.3.5.11 Apply a cover slip. Use a tissue to remove excess mounting fluid from
the edges of the coverslip. Seal the edges of the coverslip onto the slide
using clear nail polish.
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Method 1623 - Cryptosporidium and Giardia
11.3.5.12 Record the date and time that staining was completed. If slides will not
be read immediately, store in a humid chamber in the dark at 0°C to 8°C
< 10°C and not frozen until ready for examination.
11.3.5.13 After examination of the 10 wells, calculate the mean, standard
deviation, and RSD of the 10 replicates. Record the counts and the
calculations on a spiking suspension enumeration form. The relative
standard deviation (RSD) of the calculated mean spike dose must be
< 16% for Cryptosporidium and < 19% for Gicirdici 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 * 15 mm petri dishes.
11.3.6.3 Filter-sterilize (Section 6.19) approximately 10 mL of PBS pll 7.2
(Section 7. 9. 4 7.6.4). Dilute detection reagent (Section 7.7) as per
manufacturer's instructions using sterile PBS. Multiply the anticipated
number of filters to be stained by 100 mL to calculate total volume of
stain required. Divide the total volume required by 5 to obtain the
microliters of antibody necessary. Subtract the volume of antibody from
the total stain volume to obtain the required microliters of sterile PBS to
add to the antibody.
11.3.6.4 Label the tops of foil-covered, 60 * 15 mm petri dishes for 10 spiking
suspensions plus positive and negative staining controls and multiple
filter blanks controls (one negative control, plus a blank after every five
sample filters to control for carry-over). Create a humid chamber by
laying damp paper towels on the bottom of a stain tray (the inverted 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 12-\xm 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.
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Method 1623 - Cryptosporidium and Giardia
11.3.6.9
11.3.6.10
11.3.6.11
11.3.6.12
11.3.6.13
11.3.6.14
11.3.6.15
11.3.6.16
11.3.6.17
11.3.6.18
11.3.6.19
11.3.6.20
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.
Using a micropipettor, sequentially remove two, 10-|iL aliquots from the
spiking suspension and pipet into the 5 mL of 0.01% Tween 20 standing
in the unit. Rinse the pipet tip twice after each addition. Apply 10 (iL of
0.01% Tween 20 to the third unit to serve as the negative control. Apply
vacuum at 2" Hg and allow liquid to drain to miniscus, then close off
vacuum. Pipet 10 mL of reagent water into each funnel and drain to
miniscus, closing off the vacuum. Repeat the rinse and drain all fluid,
close off the vacuum.
Pipet 100 mL of diluted antibody to the center of the bottom of a 60 * 15
mm petri dish for each sample.
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.
Reclamp the top funnels, apply vacuum and rinse each three times, each
time with 20 mL of reagent water.
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-f_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.
Repeat Sections 11.3.6.4 through 11.3.6.10 until the 10-f_iL spiking
suspensions have been filtered. The last batch should include a 10-j_iL
0.01 Tween 20 blank control and 20 |_iL of positive control antigen as a
positive staining control.
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.
To each slide, add 20 |_iL of mounting medium (Section 7.8).
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.)
Record the date and time that staining was completed. If slides will not
be read immediately, store sealed slides in a closed container in the dark
at 0"C to < 8°C (but not frozen) until ready for examination.
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
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Method 1623 - Cryptosporidium and Giardia
< 16% for Cryptosporidium and < 19% for Giardia before proceeding. If
the RSD is unacceptable, or the mean number is outside the expected
range, add additional oocysts from stock suspension or dilute the
contents of the beaker appropriately with reagent water. Repeat the
process to confirm counts.
11.3.6.21 If oocysts or cysts are detected on the filter blanks, modify the rinse
procedure to ensure that no carryover occurs and repeat enumeration.
11.4 Procedure for spiking samples in the laboratory with enumerated spiking suspensions.
11.4.1 Arrange a bottom-dispensing container to feed the filter or insert the influent end of the
tube connected to the filter through the top of a carboy to allow siphoning of the sample.
11.4.2 For initial precision and recovery (Section 9.4) and ongoing precision and recovery
(Section 9.7) samples, fill the container with 10 L of reagent water or a volume of
reagent water equal to the volume of the field samples analyzed in the analytical batch.
For matrix spike samples (Section 9.5), fill the container with the field sample to be
spiked. Continuously mix the sample (using a stir bar and stir plate for smaller-volume
samples and alternate means for larger-volume samples).
11.4.3 Follow the procedures in Section 11.4.3.1 for flow cytometer-enumerated suspensions
and the procedures in Section 11.4.3.2 for manually enumerated suspensions. 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 500 (.iL of the diluted antifoam to the tube
containing the spiking suspension and vortex for 30
seconds 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 Vortex the spiking suspension(s) (Section 11.2 or
Section 11.3) for a minimum of 30 seconds.
11.4.3.2.2 Rinse a pipette tip with 0.01% Tween-20 once, then
rinse with the well-mixed spiking suspension a minimum
of five times before pulling an aliquot to be used to spike
the container.
11.4.3.2.3 Add the spiking suspension(s) to the carboy, delivering
the aliquot below the surface of the water.
11.4.3.2.4 Record the estimated number of organisms spiked, the
date and time the sample was spiked, and the sample
volume spiked on a bench sheet. Proceed to Section
11.4.4
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Method 1623 - Cryptosporidium and Giardia
11.4.4 Allow the spiking suspensions to mix for approximately 1 minute in the container.
11.4.5 Turn on the pump and allow the flow rate to stabilize. Set flow at the rate designated for
the filter being used. As the carboy is depleted, check the flow rate and adjust if
necessary.
11.4.6 When the water level approaches the discharge port of the carboy, tilt the container so
that it is completely emptied. At that time, turn off the pump and add sufficient reagent
water to the container to rinse. Swirl the contents to rinse down the sides. Additional
rinses may be performed.
11.4.7 Turn on the pump. Allow all of the water to flow through the filter and turn off the
pump.
12.0 Sample Filtration and Elution
12.1 A water sample is filtered according to the procedures in Section 12.2 or Section 12.3. Alternate
procedures may be used if the laboratory first demonstrates that the alternate procedure provides
equivalent or superior performance per Section 9.1.2.
NOTE: Sample elution must be initiated within 96 hours of sample collection (if shipped to
the laboratory as a bulk sample) or filtration (iffiltered in the field).
12.2 Capsule filtration (adapted from Reference 20.9). This procedure was validated using 10-L
sample volumes (for the original Envirochek™ filter) and 50-L sample volumes (for the
Envirochek™ HV filter). Alternate sample volumes may be used, provided the laboratory
demonstrates acceptable performance on initial and ongoing spiked reagent water and source
water samples (Section 9.1.2).
NOTE: The filtration procedures specified in Section 12.2.1 -12.2.5.3 are specific to
laboratory filtration of a bulk sample, and reflect the procedures used during the
interlaboratoiy 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 Allow2to 10 L of reagent water to flush the system. Adjust the pump
speed as required during this period. Turn off the pump when the flow
rate has been adjusted.
12.2.2 Install the capsule filter in the line, securing the inlet and outlet ends with the
appropriate clamps/fittings.
12.2.3 Record the sample number, sample turbidity (if not provided with the field sample),
sample type, and sample filtration start date and time on a bench sheet.
12.2.4 Filtration
12.2.4.1 Mix the sample well by shaking and 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.
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Method 1623 - Cryptosporidium and Giardia
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.
12.2.4.3 Allow the carboy discharge tube and capsule to fill with sample water by
gravity. Vent residual air using the bleed valve/vent port, gently shaking
or tapping the capsule, if necessary. Turn on the pump to start water
flowing through the filter. Verify that the flow rate is 2 L/min.
12.2.4.4 After all of the sample has passed through the filter, turn off the pump.
Allow the pressure to decrease until flow stops. (If the sample was
filtered in the field, and excess sample remains in the filter capsule upon
receipt in the laboratory, pull the remaining sample volume through the
filter before elutingthe filter [Section 12.2.6].)
12.2.5 Disassembly
12.2.5.1 Disconnect the inlet end of the capsule filter assembly while maintaining
the level of the inlet fitting above the level of the outlet fitting to prevent
backwashing and the loss of oocysts and cysts from the filter. Restart the
pump and allow as much water to drain as possible. Turn off the pump.
12.2.5.2 Based on the water level in the graduated container and 'A-L hash marks
or meter reading, record the volume filtered on the bench sheet to the
nearest quarter liter. Discard the contents of the graduated container.
12.2.5.3 Loosen the outlet fitting, then cap the inlet and outlet fittings.
12.2.6 Elution
NOTE: The laboratory must complete the elution, concentration, and purification (Sections
12.2.6 through 13.3.3.11) in one workday. It is critical that these steps be completed in one
work day to minimize the time that any target organisms present in the sample sit in eluate or
concentrated matrix. This process ends with the application of the purified sample on the
slide for drying.
12.2.6.1 Setup
12.2.6.1.1
12.2.6.1.2
12.2.6.1.3
12.2.6.2 Elution
12.2.6.2.1
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.
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Method 1623 - Cryptosporidium and Giardia
Pour elution buffer through the inlet fitting. Remove the
inlet cap and allow the liquid level to stabilize. 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.
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.
Remove the filter from the shaker, remove the inlet cap,
and pour the contents of the capsule into the 250-mL
conical centrifuge tube.
Clamp the capsule vertically with the inlet end up and
add sufficient volume of elution buffer through the inlet
fitting to cover the pleated membrane. Replace the inlet
cap.
Return the capsule to the shaker with the bleed valve
positioned at the 4 o'clock position. Turn on the shaker
and agitate the capsule for approximately 5 minutes.
Remove the filter from the shaker, but leave the elution
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.
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).
12.3 Sample filtration using the Filta-Max™ foam filter. This procedure was validated using 50-L
sample volumes. Alternate sample volumes may be used, provided the laboratory demonstrates
acceptable performance on initial and ongoing spiked reagent water and source water samples
(Section 9.1.2).
NOTE: The filtration procedures specified in Sections 12.3.1.2-12.3.1.6.3 are specific to
laboratory filtration of a bulk sample. These procedures may require modification if samples
will be filtered in the field.
12.3.1 Filtration
12.3.1.1 Flow rate adj ustment
12.3.1.1.1 Connect the sampling system, minus the filter housing,
to a carboy filled with reagent water.
12.3.1.1.2 Place the peristaltic pump upstream of the filter housing.
12.2.6.2.2
12.2.6.2.3
12.2.6.2.4
12.2.6.2.5
12.2.6.2.6
12.2.6.2.7
12.2.6.2.8
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Method 1623 - Cryptosporidium and Giardia
12.3.1.1.3 Turn on the pump and adjust the flow rate to 1 to 4 L per
minute.
NOTE: A head pressure of 0.5 bar (7.5 psi) is required to create flow through the filter,
and the recommended pressure of 5 bar (75 psi) should produce the flow rate of 3 to 4 L per
minute. The maximum operating pressure of 8 bar (120 psi) should not be exceeded.
12.3.1.1.4 Allow 2 to 10 L of reagent water to flush the system.
Adjust the pump speed as necessary during this period.
Turn off the pump when the flow rate has been adjusted.
12.3.1.2 Place filter module into the filter housing and secure lid, hand tighten
housings, apply gentle pressure to create the seal between the module
and the 'O' rings in the base and the lid of the housing. Excessive
tightening is not necessary, and may shorten the life of the 'O' rings.
Tools may be used to tighten housing to the alignment marks (refer to
manufacturer's instructions). O' rings should be lightly greased before
use (refer to manufacturer's instructions).
12.3.1.3 Install the filter housing in the line, securing the inlet and outlet ends
with the appropriate clamps/fittings. Verify that the filter housing is
installed so that the end closest to the screw top cap is the inlet and the
opposite end is the outlet.
12.3.1.4 Record the sample number, sample turbidity (if not provided with the
field sample), and the name of the analyst filtering the sample on a bench
sheet.
12.3.1.5 Filtration
12.3.1.5.1 Connect the sampling system to the field carboy of
sample water, or transfer the sample water to the
laboratory carboy used in Section 12.3.1.1.1. If the
sample will be filtered from a field carboy, a spigot can
be used with the carboy to facilitate sample filtration.
NOTE: If the bulk field sample is transferred to a laboratory carboy, the laboratory carboy
must be cleaned and disinfected before it is used with another field sample.
12.3.1.5.2 Place the drain end of the sampling system tubing into
an empty graduated container with a capacity greater
than or equal to the volume to be filtered. This container
will be used to determine the sample volume filtered.
Alternately, connect a flow meter downstream of the
filter, and record the initial meter reading.
12.3.1.5.3 Allow the carboy discharge tube and filter housing to fill
with sample water. Turn on the pump to start water
flowing through the filter. Verify that the flow rate is
between 1 and 4 L per min.
12.3.1.5.4 After all of the sample has passed through the filter, turn
off the pump. Allow the pressure to decrease until flow
stops.
12.3.1.6 Disassembly
12.3.1.6.1 Disconnect the inlet end of the filter housing assembly
while maintaining the level of the inlet fitting above the
level of the outlet fitting to prevent backwashing and the
loss of oocysts and cysts from the filter. Restart the
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Method 1623 - Cryptosporidium and Giardia
pump and allow as much water to drain as possible. Turn
off the pump.
12.3.1.6.2 Based on the water level in the graduated container or
the meter reading, record the volume filtered on a bench
sheet to the nearest quarter liter.
12.3.1.6.3 Loosen the outlet fitting, the filter housing should be
sealed with rubber plugs.
NOTE: Filters should be prevented from drying out, as this can impair their ability to
expand when decompressed.
12.3.2 Elution
12.3.2.1 The filter is eluted to wash the oocysts from the filter. This can be
accomplished using the Filta-Max™ wash station, which moves a
plunger up and down a tube containing the filter and eluting solution
(Section 12.3.2.2), or a stomacher, which uses paddles to agitate the
stomacher bag containing the foam filter in the eluting solution (Section
12.3.2.3). If the Filta-Max™ automatic wash station is used please see
the manufacturer's operator's guide for instructions on its use.
12.3.2.2 Filta-Max™ wash station elution procedure
12.3.2.2.1 First wash
(a) Detach the removable plunger head using the tool
provided, and remove the splash guard.
(b) Place the filter membrane flat in the concentrator
base with the rough side up. Locate the concentrator
base in the jaws of the wash station and screw on the
concentrator tube (the longer of the two tubes),
creating a tight seal at the membrane. Take the
assembled concentrator out of the jaws and place on
the bench.
(c) Replace the splash guard and temporarily secure it at
least 15 cm above the end of the rack. Secure the
plunger head with the tool provided ensuring that the
lever is fully locked down.
(d) Remove the filter module from the filter housing or
transportation container. Pour excess liquid into the
assembled concentrator, then rinse the housing or
container with PBST and add the rinse to the
concentrator tube. Screw the filter module onto the
base of the plunger. Locate the elution tube base in
the jaws of the wash station and screw the elution
tube (the shorter of the two tubes) firmly in place.
(e) Pull the plunger down until the filter module sits at
the bottom of the elution tube; the locking pin (at the
top left of the wash station) should "click" to lock
the plunger in position.
(f) Remove the filter module bolt by turning the
adapted alien key (provided) in a clockwise direction
(as seen from above). Attach the steel tube to the
elution tube base.
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Method 1623 - Cryptosporidium and Giardia
(g) Add 600 mL of PBST to the assembled concentrator.
If more than 50 mL of liquid has been recovered
from the shipped filter module, reduce the volume of
PBST accordingly. Screw the concentrator tube onto
the base beneath the elution tube. Release the
locking pin.
NOTE: Gentle pressure on the lever, coupled with a pulling action on the locking pin
should enable the pin to be easily released.
(h) Wash the foam disks by moving the plunger up and
down 20 times. Gentle movements of the plunger are
recommended to avoid generating excess foam.
NOTE: The plunger has an upper movement limit during the wash process to prevent it
popping out of the top of the chamber.
(i) Detach the concentrator and hold it such that the
stainless steel tube is just above the level of the
liquid. Purge the remaining liquid from the elution
tube by moving the plunger up and down 5 times,
then lock the plunger in place. To prevent drips,
place the plug provided in the end of the steel tube.
(j) Prior to the second wash the eluate from the first
wash can be concentrated using the Filta-Max™
apparatus according to Section 12.3.3.2.1 or the
eluate can be decanted into a 2-L pooling beaker and
set aside.
13.3.2.2.2 Second wash
(a) Add an additional 600 mL of PBST to the
concentrator module, remove the plug from the end
of the steel tube and screw the concentrator tube
back onto the elution module base. Release the
locking pin.
(b) Wash the foam disks by moving the plunger up and
down 10 times. Gentle movements of the plunger are
recommended to avoid generating excess foam.
(c) The eluate can be concentrated using the Filta-
Max™ apparatus according to Section 12.3.3.2.2 or
the eluate can be decanted into the 2-L pooling
beaker containing the eluate from the first wash and
concentrated using centrifugation, as described in
Section 12.3.3.3.
12.3.2.3 Stomacher elution procedure
12.3.2.3.1 First wash
(a) Place the filter module in the stomacher bag then use
the alien key to remove the bolt from the filter
module, allowing the rings to expand. Remove the
end caps from the stomacher bag and rinse with
PBST into the stomacher bag.
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Method 1623 - Cryptosporidium and Giardia
(b) Add 600 mL of PBST to stomacher bag containing
the filter pads. Place bag in stomacher and wash for
5 minutes on a normal setting.
(c) Remove the bag from the stomacher and decant the
eluate into a 2-L pooling beaker.
12.3.2.3.2 Second wash
(a) Add a second 600-mL aliquot of PBST to the
stomacher bag. Place bag in stomacher and wash for
5 minutes on a normal setting. Remove the bag from
the stomacher and decant the eluate from the
stomacher bag into the 2-L pooling beaker. Wring
the stomacher bag by hand to remove eluate from
the foam filter and add to the pooling beaker.
Remove the foam filter from the bag and using a
squirt bottle, rinse the stomacher bag with reagent
water and add the rinse to the pooling beaker.
(b) Proceed to concentration (Section 12.3.3).
12.3.3 Concentration
12.3.3.1 The eluate can be concentrated using the Filta-Max™ concentrator
apparatus, which pulls most of the eluate through a membrane filter
leaving the oocysts concentrated in a small volume of the remaining
eluting solution (Section 12.3..2), or by directly centrifuging all of the
eluting solution used to wash the filter (Section 12.3.2.3).
12.3.3.2 The Filta-Max™ concentrator procedure
12.3.3.2.1 Concentration of first wash
(a) If the stomacher was used to elute the sample
(Section 12.3.2.3), transfer 600 mL of eluate from
the pooling beaker to the concentrator tube.
Otherwise proceed to Step (b).
(b) Stand the concentrator tube on a magnetic stirring
plate and attach the lid (with magnetic stirrer bar).
Connect the waste bottle trap and hand or electric
vacuum pump to the valve on the concentrator base.
Begin stirring and open the tap. Increase the vacuum
using the hand pump.
NOTE: The force of the vacuum should not exceed 30 cmHg.
(c) Allow the liquid to drain until it is approximately
level with the middle of the stirrer bar then close the
valve. Remove the magnetic stirrer, and rinse it with
PBST or distilled water to recover all oocysts.
Decant the concentrate into a 50-mL tube, then rinse
the sides of the concentration tube and add the
rinsate to the 50-mL tube.
12.3.3.2.2 Concentration of second wash
(a) If the stomacher was used to elute the sample
(Section 12.3.2.3), transfer the remaining 600 mL of
eluate from the pooling beaker to the concentrator
tube. Otherwise proceed to Step (b).
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June 2003
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Method 1623 - Cryptosporidium and Giardia
(b) Add the concentrate, in the 50-mL tube, retained
from the first concentration (Section 12.3.3.2.1 (c))
to the 600 mL of eluate from the second wash, then
repeat concentration steps from Sections 12.3.3.2.1
(b) and 12.3.3.2.1 (c). The final sample can be
poured into the same 50-mL tube used to retain the
first concentrate. Rinse the sides of the concentrator
tube with PBST and add the rinse to the 50-mL tube.
(c) Remove the magnetic stirrer. Insert the empty
concentrator module into the jaws of the wash
station and twist off the concentrator tube.
(d) Transfer the membrane from the concentrator base to
the bag provided using membrane forceps.
12.3.3.2.3 Membrane elution. The membrane can be washed
manually or using a stomacher:
Manual wash. Add 5 mL of PBST to the bag
containing the membrane. Rub the surface of the
membrane through the bag until the membrane
appears clean. Using a pipette, transfer the eluate to
a 50-mL tube. Repeat the membrane wash with
another 5 mL of PBST and transfer the eluate to the
50-mL tube. (Optional: Perform a third wash using
another 5 mL of PBST, by hand-kneading an
additional minute or placing the bag on a fiat-headed
vortexer and vortexing for one minute. Transfer the
eluate to the 50-mL tube.)
NOTE: Mark the bag with an "X" to note which side of the membrane has the oocysts to
encourage the hand-kneading to focus on the appropriate side of the membrane.
Stomacher wash. Add 5 mL of PBST to the bag
containing the membrane. Place the bag containing
the membrane into a small stomacher and stomach
for 3 minutes. Using a pipette transfer the eluate to a
50-mL tube. Repeat the wash two times using the
stomacher and 5-mL aliquots of PBST. (Optional:
Perform a fourth wash using another 5 mL of PBST,
by hand-kneading an additional minute or placing
the bag on a flat-headed vortexer and vortexing for
one minute. Transfer the eluate to the 50-mL tube.)
12.3.3.2.4 If the membrane filter clogs before concentration is
complete, there are two possible options for completion
of concentration. One option is replacing the membrane
as often as necessary. Filter membranes may be placed
smooth side up during the second concentration step.
Another option is concentrating the remaining eluate
using centrifugation. Both options are provided below.
Using multiple membranes. Disassemble the
concentrator tube and pour any remaining eluate
back into the pooling beaker. Remove the membrane
using membrane forceps, placing it in the bag
provided. Place a new membrane in the concentrator
June 2003
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Method 1623 - Cryptosporidium and Giardia
tube smooth side up, reassemble, return the eluate to
the concentrator tube, rinse the pooling beaker and
add rinse to the eluate, and continue the
concentration. Replace the membrane as often as
necessary.
• Centrifuging remaining volume. Decant the
remaining eluate into a 2-L pooling beaker. Rinse
the sides of the concentrator tube and add to the
pooling beaker. Remove the filter membrane and
place it in the bag provided. Wash the membrane as
described in Section 12.3.3.2.3, then concentrate the
sample as described in Section 12.3.3.3.1.
12.3.3.3 If the Filta-Max™ concentrator is not used for sample concentration, or
if the membrane filter clogs before sample concentration is complete,
then the procedures described in Section 12.3.3.3.1 should be used to
concentrate the sample. If less than 50 mL of concentrate has been
generated, the sample can be further concentrated, as described in
Section 12.3.3.3.2, to reduce the volume of sample to be processed
through IMS.
NOTE: The volume must not be reduced to less than 5 mL above the packed pellet. The
maximum amount ofpellet that should be processed through IMS is 0.5 mL. If the packed
pellet is greater than 0.5 mL then the pellet may be subsampled as described in Section
13.2.4.
12.3.3.3.1 Centrifugation of greater than 50 mL of eluate
(a) Decant the eluate from the 2-L pooling beaker into
250-mL conical centrifuge tubes. Make sure that the
centrifuge tubes are balanced.
(b) Centrifuge the 250-mL centrifuge tubes containing
the eluate at 1500 * G for 15 minutes. Allow the
centrifuge to coast to a stop.
(c) Using a Pasteur pipette, carefully aspirate off the
supernatant to 5 mL above the pellet. If the sample is
reagent water (e.g. initial or ongoing precision and
recovery sample) extra care must be taken to avoid
aspirating oocysts and cysts during this step.
(d) Vortex each 250-mL tube vigorously until pellet is
completely resuspended. Swirl the centrifuge tube
gently to reduce any foaming after vortexing.
Combine the contents of each 250-mL centrifuge
tube into a 50-mL centrifuge tube. Rinse each of the
250-mL centrifuge tubes with PBST and add the
rinse to the 50-mL tube.
(e) Proceed to Section 12.3.3.3.2.
12.3.3.3.2 Centrifugation of less than 50 mL of eluate
(a) Centrifuge the 50-mL centrifuge tube containing the
combined concentrate at 1500 x G for 15 minutes.
Allow the centrifuge to coast to a stop. Record the
initial pellet volume (volume of solids) and the date
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Method 1623 - Cryptosporidium and Giardia
and time that concentration was completed on a
bench sheet.
(b) Proceed to Section 13.0 for concentration and
separation (purification).
12.3.4 Maintenance and cleaning
12.3.4.1 Maintenance of O-rings
12.3.4.1.1 Check all rubber O-rings for wear or deterioration prior
to each use and replace as necessary.
12.3.4.1.2 Lubricate the plunger head O-ring inside and out with
silicon before each use.
12.3.4.1.3 Lubricate all other O-rings (concentrator tube set, filter
housing) regularly in order to preserve their condition.
12.3.4.2 Cleaning
12.3.4.2.1 All components of the Filta-Max™ system can be
cleaned using warm water and laboratory detergent.
After washing, rinse all components with oocyst and
cyst free reagent water and dry them. All O-rings should
be re-lubricated. Alternatively a mild (40°C) dishwasher
cycle without bleach or rinse aid can be used.
12.3.4.2.2 To wash the detachable plunger head slide the locking
pin out and wash the plunger head and locking pin in
warm water and laboratory detergent. Rinse the plunger
head and locking pin with oocyst and cyst free reagent
water and dry. Lightly lubricate the locking pin and re-
assemble the plunger head.
13.0 Sample Concentration and Separation (Purification)
13.1 During concentration and separation, the filter eluate is concentrated through centrifugation, and
the oocysts and cysts in the sample are separated from other particulates through
immunomagnetic separation (IMS). Alternate procedures and products may be used if the
laboratory first demonstrates 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 and/or cysts 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 and cysts during this step,
particularly if the sample is reagent water (e.g. initial or ongoing precision and recovery
sample).
13.2.3 If the packed pellet volume is < 0.5 mL, vortex the tube vigorously until pellet is
completely resuspended. Swirl the centrifuge tube gently to reduce any foaming after
vortexing. Record the resuspended pellet volume on the bench sheet. Proceed to Section
13.3.
June 2003
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Method 1623 - Cryptosporidium and Giardia
NOTE: Extra care must be taken with samples containing sand or other gritty material
when vortexing to ensure that the condition of any oocysts and/or cysts in the sample is not
compromised.
13.2.4 If the packed pellet volume is > 0.5 mL, the concentrate needs to must be separated
into multiple subsamples (a subsample is equivalent to no greater than 0.5 mL of packed
pellet material, the recommended maximum amount of particulate material to process
through the subsequent purification and examination steps in the method). Use the
following formula to determine the total volume required in the centrifuge tube before
separating the concentrate into two or more subsamples:
pellet volume
total volume (mL) required = x 5 mL
0.5 mL
(For example, if the packed pellet volume is 1.2 mL, the total volume required is 12
mL.) Add reagent water to the centrifuge tube to bring the total volume to the level
calculated above. 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. Vortex the tube
vigorously for 10 to 15 seconds to completely resuspend
the pellet. Record the resuspended pellet volume on the
bench sheet. Proceed immediately to Section 13.3, and
transfer aliquots of the resuspended concentrate
equivalent to the volume in the previous step to multiple,
flat-sided sample tubes in Section 13.3.2.1. Process the
sample as multiple, independent subsamples from
Section 13.3 onward, including the preparation and
examination of separate slides for each aliquot. Record
the volume of resuspended concentrate transferred to
IMS on the bench sheet (this will be equal to the volume
recorded in Section 13.2.4). Also record the number of
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June 2003
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Method 1623 - Cryptosporidium and Giardia
subsamples processed independently through the method
on the bench sheet.
13.2.4.2 Analysis of partial sample. If not all of the concentrate will be
examined, vortex the tube vigorously for 10 to 15 seconds to completely
resuspend the pellet. Record the resuspended pellet volume on the bench
sheet. Proceed immediately to Section 13.3, and transfer one or more 5-
mL aliquots of the resuspended concentrate to one or more flat-sided
sample tubes in Section 13.3.2.1. Record the volume of resuspended
concentrate transferred to IMS on the bench sheet. To determine the
volume analyzed, calculate the percent of the concentrate examined
using the following formula:
total volume of resuspended concentrate transferred to IMS
percent examined = x 100%
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 1 OX 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
(.iL 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 and cyst capture
13.3.2.1 Use a graduated, 10-mL pipette that has been pre-rinsed with elution
buffer to transfer the water sample concentrate from Section 13.2 to the
flat-sided tube(s) containing the SL-buffers. If all of the concentrate is
used, rinse the centrifuge tube twice with reagent water and add the
rinsate to the flat-sided tube containing the concentrate (or to the tube
containing the first subsample, if multiple subsamples will be processed).
Each of the two rinses should be half the volume needed to bring the
volume in the flat-sided sample tube to 10 mL (including the buffers
added in Sections 13.3.1.2 and 13.3.1.3). (For example, if the tube
contained 1 mL of SL-buffer-A and 1 mL of SL-buffer-B, and 5 mL of
sample was transferred after resuspension of the pellet, for a total of 7
mL, the centrifuge tube would be rinsed twice with 1.5 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
June 2003
46
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Method 1623 - Cryptosporidium and Giardia
13.3.2.2
13.3.2.3
13.3.2.4
13.3.2.5
13.3.2.6
13.3.2.7
13.3.2.8
13.3.2.9
13.3.2.10
13.3.2.11
13.3.2.12
13.3.2.13
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).
Vortex the Dynabeads®Crypto-Combo vial from the IMS kit for
approximately 10 seconds to suspend the beads. Ensure that the beads are
fully resuspended by inverting the sample tube and making sure that
there is no residual pellet at the bottom.
Add 100 |_iL of the resuspended Dynabeads®Crypto-Combo (Section
13.3.2.2) to the sample tube(s) containing the water sample concentrate
and SL-buffer.
Vortex the Dynabeads®Giardia-Combo vial from the IMS kit for
approximately 10 seconds to suspend the beads. Ensure that the beads are
fully resuspended by inverting the tube and making sure that there is no
residual pellet at the bottom.
Add 100 |_iL of the resuspended Dynabeads®Giardia-Combo (Section
13.3.2.4) to the sample tube(s) containing the water sample concentrate,
Dynabeads®Crypto-Combo, and SL-buffer.
Affix the sample tube(s) to a rotating mixer and rotate at approximately
18 rpm for 1 hour at room temperature.
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.
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.
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.
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, remove the flat-sided tube from the MPC-1, shake the tube to
resuspend all material, replace the sample tube in the MPC-1 and repeat
Section 13.3.2.9 before continuing to Section 13.3.2.11.
Return the MPC-1 to the upright position, sample tube vertical, with cap
at top. Immediately remove the cap and, keeping the flat side of the tube
on top, pour off all of the supernatant from the tube held in the MPC-1
into a suitable container. Do not shake the tube and do not remove the
tube from MPC-1 during this step. Allow more supernatant to settle;
aspirate additional supernatant with pipette.
Remove the sample tube from the MPC-1 and resuspend the sample in -b
mfe-0.5 mL IX SL-buffer-A (prepared from 10X SL-buffer-A
stock—supplied). Mix very gently to resuspend all material in the tube.
Do not vortex.
Quantitatively transfer (transfer followed by two rinses) all the liquid
from the sample tube to a labeled, 1.5-mL microcentrifuge tube. Use +
rrrfc-0.5 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
Lcighton tube before transferring and transfer the rinsate to the
47
June 2003
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Method 1623 - Cryptosporidium and Giardia
microcentrifuge tube. 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.
13.3.2.14 Place the microcentrifuge tube into the second magnetic particle
concentrator (MPC-M), with its magnetic strip in place.
13.3.2.15 Without removing the microcentrifuge tube from MPC-M, gently
rock/roll the tube through 180° by hand. Continue for approximately 1
minute with approximately one 180° roll/rock per second. At the end of
this step, the beads should produce a distinct brown dot at the back of the
tube.
13.3.2.16 Immediately aspirate the supernatant from the tube and cap held in the
MPC-M. If more than one sample is being processed, conduct three 90°
rock/roll actions before removing the supernatant from each tube. Take
care not to disturb the material attached to the wall of the tube adjacent to
the magnet. Do not shake the tube. Do not remove the tube from MPC-M
while conducting these steps.
13.3.3 Dissociation of beads/oocyst/cyst complex
NOTE: Two acid dissociations are required.
13.3.3.1
Remove the magnetic strip from the MPC-M.
13.3.3.2
Add 50 (.iL of 0.1 N HC1, then vortex at the highest setting for
approximately 50 seconds.
NOTE: The laboratory should must use 0.1-Nstandards purchased directly from a vendor,
rather than adjusting t
he normality in-house.
13.3.3.3
Place the tube in the MPC-M without the magnetic strip in place and
allow to stand in a vertical position for at least 10 minutes at room
temperature.
13.3.3.4
Vortex vigorously for approximately 30 seconds.
13.3.3.5
Ensure that all of the sample is at the base of the tube. Place the
microcentrifuge tube in the MPC-M.
13.3.3.6
Replace magnetic strip in MPC-M and allow the tube to stand
undisturbed for a minimum of 10 seconds.
13.3.3.7
Prepare a well slide for sample screening and label the slide.
13.3.3.8
Add 5 (iL of 1.0 N NaOH to the sample wells of two well slides (add 10
(.iL to the sample well of one well slide if the volume from the two
required dissociations will be added to the same slide).
NOTE: The laboratory should must use 1.0-Nstandards 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
June 2003
48
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Method 1623 - Cryptosporidium and Giardia
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 The wells on Dvnal Spot-On slides
are likely to be too small to accommodate the volumes from both dissociations.
13.3.3.11 Record the date and time the purified sample was applied to the slide(s).
13.3.3.12 Air-dry the sample on the well slide(s). Because temperature and
humidity varyies from laboratory to laboratory, no minimum time is
specified. However, the laboratory must take care to ensure that the
sample has dried completely before staining to prevent losses during the
rinse steps. A slide warmer set at 35°C to 42°C also can be used.
13.3.4 Tips for minimizing carry-over of debris onto microscope slides after IMS
• Make sure the resuspended pellet is fully homogenized before placing the tube in the
MPC-1 or MPC-M to avoid trapping "clumps" or a dirty layer between the beads and
the side of the tube.
• When using the MPC-1 magnet, make sure that the tube is snugged flat against the
magnet. Push the tube flat if necessary. Sometimes the magnet is not flush with the
outside of the holder and, therefore, the attraction between the beads and the magnet
is not as strong as it should be. However, it can be difficult to determine this if you
do not have more than one MPC-1 to make comparisons.
• After the supernatant has been poured off at Section 13.3.2.11, leave the tube in the
MPC-1 and allow time for any supernatant remaining in the tube to settle down to the
bottom. Then aspirate the settled supernatant and associated particles from the bottom
of the tube. The same can be done at Section 13.3.2.16 with the microcentrifuge tube.
• An additional rinse can also be performed at Section 13.3.2.11. After the supernatant
has been poured off and any settled material is aspirated off the bottom, leave the
tube in the MPC-1 and add an additional 10 mL of reagent water or PBS to the tube
and repeat Sections 13.3.2.9 and 13.3.2.11. Although labs have reported successfully
using this technique to reduce carryover, because the attraction between the MPC-1
and the beads is not as great as the attraction between the MPC-M and the beads, the
chances would be greater for loss of cysts and oocysts doing the rinse at this step
instead of at Section 13.3.2.16.
• After the supernatant has been aspirated from the tube at Section 13.3.2.16, add 0.1
mL of PBS, remove the tube from the MPC-M, and resuspend. Repeat Sections
13.3.2.15 and 13.3.2.16.
• Use a slide with the largest diameter well available to spread out the sample as much
as possible.
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
and 200 to 400 cysts to the center of a well.
14.1.2 For the negative control, pipette 50 |aL 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).
49
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Method 1623 - Cryptosporidium and Giardia
NOTE: If the laboratory has a large batch of slides that will be examined over several-
days, and is concerned that a single positive control may fade, due to multiple examinations,
the laboratory should prepare multiple control slides with the batch of field slides and
alternate between the positive controls when performing the positive control check.
14.2—Apply 50-^L of absolute methanol to each well containing the dried sample and allow to air-dry
for 3 to 5 minutes.
14.2 Follow manufacturer's instructions in applying stain to slides.
14.3 Place the slides in a humid chamber in the dark and incubate at room temperature for
approximately 30 minutes. The humid chamber consists of a tightly sealed plastic container
containing damp paper towels on top of which the slides are placed.
14.4 Remove slides from humid chamber and allow condensation to evaporate, if present.
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 7.6.1), do not allow slides to dry completely.
14.6 Apply 50 |_iL of 4',6-diamidino-2-phenylindole (DAPI) staining solution (Section 7.7.2) to each
well. Allow to stand at room temperature for a minimum of 1 minute. (The solution concentration
may be increased up to 1 (ig/mL if fading/diffusion of DAPI staining is encountered, but the
staining solution must be tested first on expendable environmental samples to confirm that
staining intensity is appropriate.)
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 7.6.1), 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 (but not frozen) until ready for
examination.
15.0 Examination
NOTE: Although immunofluorescence assay (FA) and 4',6-diamidino-2-phenylindole
(DAPI) and differential interference contrast (DIC) microscopy examination ttrtd
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).
June 2003
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Method 1623 - Cryptosporidium and Giardia
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 characterization and size measurements must be determined using 1000X
magnification and reported to the nearest 0.5 /xm.
Record examination results for Cryptosporidium oocysts on a Cryptosporidium report
examination results form; record examination results for Giardia cysts on a Giardia report
examination results form. All oocysts and cysts that meet the criteria specified in Sections 15.2.2
and 15.2.3, less atypical organisms specifically identified as non-target organisms by DIC or
DAPI (e.g. possessing spikes, stalks, appendages, pores, one or two large nuclei filling the cell,
red fluorescing chloroplasts, crystals, spores, etc), must be reported.
15.2.1 Positive and negative staining control. Positive and negative staining controls must be
acceptable.
15.2.1.1 Each analyst must characterize a minimum of three Cryptosporidium
oocysts and three Giardia cysts on the positive staining control slide
before examining field sample slides. This characterization must be
performed by each analyst during each microscope examination session.
FITC examination must be conducted at a minimum of 200X total
magnification, DAPI examination must be conducted at a minimum of
400X, and DIC examination and size measurements must be conducted
at a minimum of 1000X. Size, shape, and DIC and DAPI characteristics
of the three Cryptosporidium oocysts and Giardia cysts must be recorded
by the analyst on a microscope log. The analyst also must indicate on
each sample report examination results 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 or cysts (Section 14.1). Indicate on each sample report
examination results form whether the negative staining control was
acceptable.
15.2.1.3 If the positive staining control contains oocysts and cysts within the
expected range and at the appropriate fluorescence for both FA and
DAPI, and the negative staining control does not contain any oocysts or
cysts (Section 14.1), proceed to Sections 15.2.2 and 15.2.3.
15.2.2 Sample examination—Cryptosporidium
15.2.2.1 FITC examination (the analyst must use a minimum of 200X total
magnification). Use epifluorescence to scan the entire well for apple-
green fluorescence of oocyst and cyst shapes. When brilliant apple-green
fluorescing ovoid or spherical objects 4 to 6 |_im in diameter are observed
with brightly highlighted edges, increase magnification to 400X and
switch the microscope to the UV filter block for DAPI (Section
15.2.2.2), then to DIC (Section 15.2.2.3) at 1000X.
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.
51
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Method 1623 - Cryptosporidium and Giardia
15.2.2.3 DIC examination (the analyst must use a minimum of 1000X total
magnification [oil immersion lens]). Using DIC, look for external or
internal morphological characteristics atypical of Cryptosporidium
oocysts (e.g., spikes, stalks, appendages, pores, one or two large nuclei
filling the cell, red fluorescing chloroplasts, crystals, spores, etc.)
(adapted from Reference 20.7). 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 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 1000Xmagnification.
15.2.3 Sample examination—Giardia
15.2.3.1 FITC examination (the analyst must use a minimum of 200X total
magnification). When brilliant apple-green fluorescing round to oral-
ovoid objects (8 - 18 |_im long by 5 - 15 |_im wide) are observed, increase
magnification to 400X and switch the microscope to the UV filter block
for DAPI (Section 15.2.3.2) then to DIC (Section 15.2.3.3) at 1000X.
15.2.3.2 DAPI examination (the analyst must use a minimum of 400X total
magnification). Using the UV filter block for DAPI, the object will
exhibit one or more of the following characteristics:
(a) Light blue internal staining (no distinct nuclei) and a green rim
(b) Intense blue internal staining
(c) Two to four sky-blue nuclei
Record cysts in category (a) as DAPI negative; record cysts in categories
(b) and (c) as DAPI positive.
15.2.3.3 DIC examination (the analyst must use a minimum of 1000X total
magnification [oil immersion lens]). Using DIC, look for external or
internal morphological characteristics atypical of Giardia cysts (e.g.,
spikes, stalks, appendages, pores, one or two large nuclei filling the cell,
red fluorescing chloroplasts, crystals, spores, etc.) (adapted from
Reference 20.7). If atypical structures are not observed, then categorize
each object meeting the criteria specified in Sections 15.2.3.1 - 15.2.3.3
as one of the following, based on DIC examination:
(a) An empty Giardia cyst
(b) A Giardia cyst with amorphous structure
(c) A Giardia cyst with one type of internal structure (nuclei,
median body, or axonemes), or
(d) A Giardia cyst with more than one type of internal structure
Using 1000X total magnification, record the shape, measurements (to the
nearest 0.5 and number of nuclei and presence of median body or
axonemes (if applicable) for each apple-green fluorescing object meeting
the size and shape characteristics.
NOTE: All measurements must be made at 1000Xmagnification.
June 2003
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Method 1623 - Cryptosporidium and Giardia
15.2.4 Record the date and time that sample examination was completed on the report
examination results form.
15.2.5 Report Cryptosporidium and Gicirdici concentrations as oocysts/L and cysts/L.
15.2.6 Record analyst name
16.0 Analysis of Complex Samples
16.1 Some samples may contain high levels (>1000/L) of oocysts and cysts and/or interfering
organisms, substances, or materials. Some samples may clog the filter (Section 12.0); others will
not allow separation of the oocysts and cysts from the retentate or eluate; and others may contain
materials that preclude or confuse microscopic examination.
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 Tables 3 and 4 in Section 21.0. The initial and ongoing
precision and recovery criteria are based on the results of spiked reagent water samples analyzed
during the Information Collection Rule Supplemental Surveys (Reference 20.8). The matrix spike
and matrix spike duplicate criteria are based on spiked source water data generated during the
interlaboratory validation study of Method 1623 involving 11 laboratories and 11 raw surface
water matrices across the U.S. (Reference 20.10).
NOTE: Some sample matrices may prevent the MS acceptance criteria in Tables 3 and
4 to be met. An assessment of the distribution of MS recoveries across 430 MS samples
from 87 sites during the ICR Supplemental Surveys is provided in Table 5.
18.0 Pollution Prevention
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 that need to be discarded 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 or cysts
attached or contained must be sterilized prior to disposal.
19.3 For further information on waste management, consult The Waste Management Manual for
Laboratory^ Personnel and Less is Better: Laboratory Chemical Management for Waste
Reduction, both available from the American Chemical Society's Department of Government
Relations and Science Policy, 1155 16th Street N.W., Washington, D.C. 20036.
53
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Method 1623 - Cryptosporidium and Giardia
20.0 References
20.1 Rodgers, Mark R., Flanigan, Debbie J., and Jakubowski, Walter, 1995. Applied and
Environmental Microbiology 6_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," DHEW, 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 USEPA. Manual, for the Certification of Laboratories Analyzing Drinking Water: Criteria and
Procedures; Quality Assurance: Fourth Edition, EPA 815-B-97-001, Office of Ground Water
and Drinking Water, U.S. Environmental Protection Agency, 26 West Martin Luther King Drive,
Cincinnati, OH 45268 (1997).
20.7 ICR Microbial Laboratory Manual, EPA/600/R-95/178, National Exposure Research Laboratory,
Office of Research and Development, U.S. Environmental Protection Agency, 26 Martin Luther
King Drive, Cincinnati, OH 45268 (1996).
20. 8 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 Connell, K, C.C. Rodgers, H.L. Shank-Givens, J Scheller, M.L Pope, and K. Miller, 2000.
Building a Better Protozoa Data Set. Journal AWWA, 92:10:30.
20.9 "Envirochek™ Sampling Capsule," PN 32915, Gelman Sciences, 600 South Wagner Road, Ann
Arbor, MI 48103-9019 (1996).
20.10 USEPA. Results of the Interlaboratory Method Validation Study for Determination of
Cryptosporidium and Giardia Using USEPA Method 1623, EPA-821-R-01-028. Office of Water,
Office of Science and Technology, Engineering and Analysis Division, Washington, DC (2001).
20.11 "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.
June 2003
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Method 1623 - Cryptosporidium and Giardia
21.0 Tables and Figures
Table 1. Method Holding Times (See Section 8.2 for details)
Sample Processing Step
Maximum Allowable Time between Breaks
(Samples should be processed as soon as possible)
Collection
Filtration
^ 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
55
June 2003
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Method 1623 - Cryptosporidium and Giardia
Table 2. Tier 1 and Tier 2 Validation/Equivalency Demonstration Requirements
Test
Description
Tier 1 modification'1'
Tier 2 modification'2'
IPR
(Section 9.4)
4 replicates of spiked
reagent water
Required. Must be accompanied by a method
blank.
Required per laboratory
Method blank
(Section 9.6)
Unspiked reagent
water
Required
Required per laboratory
MS
(Section 9.5.1)
Spiked matrix water
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
Not required
MS/MSD
(Section 9.5)
2 replicates of spiked
matrix water
Recommended, but not required. Must be
accompanied by an unspiked field sample
collected at the same time as the MS sample
Required per laboratory.
Each laboratory must
analyze a different water.
(1) If a modification will be used only in one laboratory, these tests must be performed and the results must meet all
of the QC acceptance criteria in the method (these tests also are required the first time a laboratory uses the
validated version of the method)
(2) If nationwide approval of a modification is sought for one type of water matrix (such as surface water), a
minimum of 3 laboratories must perform the tests and the results from each lab individually must meet all QC
acceptance criteria in the method. If more than 3 laboratories are used in a study, a minimum of 75% of the
laboratories must meet all QC acceptance criteria.
NOTE: The initial precision and recovery and ongoing precision and recovery (OPR)
acceptance criteria listed in Tables 3 and 4 are based on results from 293
Cryptosporidium OPR samples and 186 Giardia OPR samples analyzed by six
laboratories during the Information Collection Rule Supplemental Surveys (Reference
20.13). The matrix spike acceptance criteria are based on data generated through
interlaboratory validation of Method 1623 (Reference 20.11).
June 2003
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Method 1623 - Cryptosporidium and Giardia
Table 3. Quality Control Acceptance Criteria for Cryptosporidium
Performance test
Section
Acceptance criteria
Initial precision and recovery
9.4
Mean recovery (percent)
9.4.2
24 -100
Precision (as maximum relative standard deviation)
9.4.2
55
Ongoing precision and recovery (percent)
9.7
11-100
Matrix spike/matrix spike duplicate (for method modifications)
9.5
Mean recovery1,2 (as percent)
9.5.2
13-111
Precision (as maximum relative percent difference)
9.5.2
61
(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 5.
Table 4. Quality Control Acceptance Criteria for Giardia
Performance test
Section
Acceptance criteria
Initial precision and recovery
9.4
Mean recovery (percent)
9.4.2
24 -100
Precision (as maximum relative standard deviation)
9.4.2
49
Ongoing precision and recovery (percent)
9.7
14 -100
Matrix spike/matrix spike duplicate (for method modifications)
9.5
Mean recovery* (as percent)
9.5.2
15-118
Precision (as maximum relative percent difference)
9.5.2
30
(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 across multiple MS samples from 87 sites during the ICR Supplemental Surveys is provided in
Table 5.
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Method 1623 - Cryptosporidium and Giardia
Table 5. Distribution of Matrix Spike Recoveries from Multiple Samples Collected from 87
Source Waters During the ICR Supplemental Surveys (Adapted from Reference 20.14)
MS Recovery Range
Percent of 430 Cryptosporidium MS
Samples in Recovery Range
Percent of 270 Giardia MS
Samples in Recovery Range
<10%
6.7%
5.2%
>10%-20%
6.3%
4.8%
>20% - 30%
14.9%
7.0%
>30% - 40%
14.2%
8.5%
>40% - 50%
18.4%
17.4%
>50% - 60%
17.4%
16.3%
>60% - 70%
11.2%
16.7%
>70% - 80%
8.4%
14.1%
>80% - 90%
2.3%
6.3%
>90%
0.2%
3.7%
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Method 1623 - Cryptosporidium and Giardia
1 mm
1
i
?
1/5 mm
A
B
E
D
C
i
1
i
T .
J .
Figure 1. Hemacytometer Platform Ruling. Squares 1, 2, 3, and 4 are
used to count stock suspensions of Cryptosporidium
oocysts and Giardia cysts (after Miale, 1967)
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Method 1623 - Cryptosporidium and Giardia
Figure 2. Manner of Counting Oocysts and Cysts in 1 Square mm.
Dark organisms are counted and light organisms are
omitted (after Miale, 1967).
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Method 1623 - Cryptosporidium and Giardia
capsule
filter
sample
^==0.13
flow
controller pump
drain
Figure 3. Laboratory Filtration System
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Method 1623 - Cryptosporidium and Giardia
\ ~/ v ~/ \ ~/
\* / /
~ ~ ~
I w
Figure 4. Methods for Scanning a Well Slide
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Method 1623 - Cryptosporidium and Giardia
22.0 Glossary of Definitions and Purposes
These definitions and purposes are specific to this method but have been conformed to common
usage as much as possible.
22.1 Units of weight and measure and their abbreviations
22.1.1 Symbols
°C degrees Celsius
l_iL microliter
< less than
> greater than
% percent
22.1.2 Alphabetical characters
cm centimeter
g gram
G acceleration due to gravity
hr hour
ID inside diameter
in. inch
L liter
m meter
MCS microscope cleaning solution
mg milligram
mL milliliter
mm millimeter
mM millimolar
N normal; gram molecular weight of solute divided by hydrogen equivalent of
solute, per liter of solution
RSD relative standard deviation
sr standard deviation of recovery
X average percent recovery
22.2 Definitions, acronyms, and abbreviations (in alphabetical order)
Analyst—The analyst should have at least 2 years of college in microbiology or equivalent or
closely related field. The analyst also should have a minimum of 6 months of continuous bench
experience with Cryptosporidium and IFA microscopy. The analyst shouldhave a minimum of 3
months experience using EPA Method 1622 and/or EPA Method 1623 and should have
successfully analyzed a minimum of 50 samples using EPA Method 1622 and/or EPA Method
1623. 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 and Giardia. Six months of additional experience in the above areas may be
substituted for two years of college.
Analyte—A protozoan parasite tested for by this method. The analytes in this method are
Cryptosporidium and Giardia.
Axoneme—An internal flagellar structure that occurs in some protozoa, such as Giardia,
Spironucleous, and Trichonmonas.
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Method 1623 - Cryptosporidium and Giardia
Cyst—A phase or a form of an organism produced either in response to environmental conditions
or as a normal part of the life cycle of the organism. It is characterized by a thick and
environmentally resistant cell wall.
Flow cytometer—A particle-sorting instrument capable of counting protozoa.
Immunomagnetic separation (IMS)—A purification procedure that uses microscopic,
magnetically responsive particles coated with an antibodies targeted to react with a specific
pathogen in a fluid stream. Pathogens are selectively removed from other debris using a magnetic
field.
Initial precision and recovery (IPR)—Four aliquots of spiking suspension analyzed to establish
the ability to generate acceptable precision and accuracy. An IPR is performed prior to the first
time this method is used and any time the method or instrumentation is modified.
Laboratory blank—See Method blank
Laboratory control sample (LCS)—See Ongoing precision and recovery (OPR) standard
Matrix spike (MS)—A sample prepared by adding a known quantity of organisms to a specified
amount of sample matrix for which an independent estimate of target analyte concentration is
available. A matrix spike is used to determine the effect of the matrix on a method's recovery
efficiency.
May—This action, activity, or procedural step is neither required nor prohibited.
May not—This action, activity, or procedural step is prohibited.
Median bodies—Prominent, dark-staining, paired organelles consisting of microtubules and
found in the posterior half of Giardia. In G. intestinalis (from humans), these structures often
have a claw-hammer shape, while in G. muris (from mice), the median bodies are round.
Method blank—An aliquot of reagent water that is treated exactly as a sample, including
exposure to all glassware, equipment, solvents, and procedures that are used with samples. The
method blank is used to determine if analytes or interferences are present in the laboratory
environment, the reagents, or the apparatus.
Must—This action, activity, or procedural step is required.
Negative control—See Method blank
Nucleus—A membrane-bound organelle containing genetic material. Nuclei are a prominent
internal structure seen both in Cryptosporidium oocysts and Giardia cysts. In Cryptosporidium
oocysts, there is one nucleus per sporozoite. One to four nuclei can be seen in Giardia cysts.
Oocyst—The encysted zygote of some sporozoa; e.g., Cryptosporidium. The oocyst is a phase or
form of the organism produced as a normal part of the life cycle of the organism. It is
characterized by a thick and environmentally resistant outer wall.
Ongoing precision and recovery (OPR) standard—A method blank spiked with known quantities
of analytes. The OPR is analyzed exactly like a sample. Its purpose is to assure that the results
produced by the laboratory remain within the limits specified in this method for precision and
recovery.
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Method 1623 - Cryptosporidium and Giardia
Oocyst and cyst spiking suspension—See Spiking suspension
Oocyst and cyst stock suspension—See Stock suspension
Positive control—See Ongoing precision and recovery standard
Principle analyst—The principle analyst (may not be applicable to all monitoring programs)
should have a BS/BA in microbiology or closely related field and a minimum of 1 year of
continuous bench experience with Cryptosporidium and IFA microscopy. The principle analyst
also should have a minimum of 6 months experience using EPA Method 1622 and/or EPA
Method 1623 and should have analyzed a minimum of 100 samples using EPA Method 1622
and/or EPA Method 1623.
PTFE—Polytetrafluoroethylene
Quantitative transfer—The process of transferring a solution from one container to another using
a pipette in which as much solution as possible is transferred, followed by rinsing of the walls of
the source container with a small volume of rinsing solution (e.g., reagent water, buffer, etc.),
followed by transfer of the rinsing solution, followed by a second rinse and transfer.
Reagent water—Water demonstrated to be free from the analytes of interest and potentially
interfering substances at the method detection limit for the analyte.
Reagent water blank—see Method blank
Relative standard deviation (RSD)—The standard deviation divided by the mean times 100.
RSD—See Relative standard deviation
Should—This action, activity, or procedural step is suggested but not required.
Spiking suspension—Diluted stock suspension containing the organism(s) of interest at a
concentration appropriate for spiking samples.
Sporozoite—A motile, infective stage of certain protozoans; e.g., Cryptosporidium. There are
four sporozoites in each Cryptosporidium oocyst, and they are generally banana-shaped.
Stock suspension—A concentrated suspension containing the organism(s) of interest that is
obtained from a source that will attest to the host source, purity, authenticity, and viability of the
organism(s).
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