&EPA
United States
Environmental Protection
Agency
Method 1623.1: Cryptosporidium and
Giardia in Water by Filtration/I MS/FA
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Office of Water (MS-140)
EPA 816-R-12-001
January 2012
http://www.epa.gov/safewater
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Acknowledgments
This method was originally prepared under the direction of William A. Telliard of the Engineering and
Analysis Division within U.S. Environmental Protection Agency (U.S. EPA) Office of Water. This
original document was prepared by CSC under a U.S. EPA contract, with assistance from its
subcontractor, Interface, Inc. The 2012 version was prepared under the direction of Carrie Miller of the
Technical Support Center within U.S. EPA Office of Water. This version was prepared by CSC and Shaw
Environmental & Infrastructure under U.S. EPA contracts.
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
Phil Berger, Office of Groundwater and Drinking Water, U.S. Environmental Protection Agency
Jennifer Clancy, Clancy Environmental - a Tetra Tech Company
Kevin Connell, CSC
George Di Giovanni, University of Texas School of Public Health El Paso Regional Campus
Ricardo DeLeon, Metropolitan Water District of Southern California
Shirley Dzogan, EnviroTest Laboratories
Mary Ann Feige, Technical Support Center, Office of Ground Water and Drinking Water, U.S.
Environmental Protection Agency
Colin Fricker, CRF Consulting
Vince Hill, Centers for Disease Control and Prevention
Patricia Klonicki, CSC
Alan Lindquist, National Homeland Security Research Center, U.S. Environmental Protection Agency
Carrie Miller, Technical Support Center, Office of Ground Water and Drinking Water, U.S.
Environmental Protection Agency
Stephanie Harris, Manchester Laboratory, U.S. Environmental Protection Agency, Region 10
Dale Rushneck, Interface, Inc.
Frank Schaefer III, National Homeland Security Research Center, U.S. Environmental Protection Agency
Steve Schaub, Health and Ecological Criteria Division (4304), Office of Science and Technology, U.S.
Environmental Protection Agency
Ajaib Singh, City of Milwaukee Health Department
Huw Smith, Department of Bacteriology, Scottish Parasite Diagnostic Laboratory
Timothy Straub, Pacific Northwest National Laboratory
William A. Telliard, Office of Science and Technology, U.S. Environmental Protection Agency
Leah Fohl Villegas, Shaw Environmental & Infrastructure
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Disclaimer
This method has been reviewed by U.S. EPA Office of Water and approved for publication. Mention of
trade names or commercial products does not constitute endorsement or recommendation for use.
Questions regarding this method or its application should be addressed to:
Carrie Miller
Coordinator, Laboratory Quality Assurance Program for the Analysis of Cryptosporidium
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
Technical Support Center, MCI40
26 West Martin Luther King Drive
Cincinnati, OH 45268-1320
(513)569-7919
(513)569-7191 (fax)
miller.carrie@epa.gov
Safe Drinking Water Hotline 1-800-426-4791
http://water.epa.gov/drink/hotline/index.cfm
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Introduction
To support future regulation of protozoa in drinking water, the Safe Drinking Water Act Amendments of
1996 require U.S. Environmental Protection Agency (EPA) to evaluate the risk to public health posed by
drinking water contaminants, including waterborne parasites, such as Cryptosporidium spp. and Giardia
spp. 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.
Note: Throughout the rest of this document, the genera Cryptosporidium spp. and Giardia spp. are
referred to by their generic names (Cryptosporidium and Giardia) without reference to species, and
without providing a scientific authority. This has been done for the sake of clarity and brevity given the
intended audience. The authors acknowledge that this is an atypical example of scientific nomenclature.
Method Development and Validation
EPA initiated an effort in 1996 to identify new and innovative technologies for protozoan monitoring and
analysis. After evaluating potential alternatives to the then-current method through literature searches,
discussions with research and commercial laboratories, and meetings with experts in the field, the
Engineering and Analysis Division within the Office of Science and Technology within EPA's Office of
Water developed draft Method 1622 for Cryptosporidium detection in December 1996. This
Cryptosporidium-on\y method was validated through an interlaboratory study in August 1998, and was
revised as a final, valid method for detecting Cryptosporidium in water in January 1999.
Although development of an acceptable immunomagnetic separation (IMS) system for Giardia lagged
behind development of an acceptable system for Cryptosporidium, an acceptable system was identified in
October 1998, and EPA validated a method for simultaneous detection of Cryptosporidium and Giardia in
February 1999 and developed quality control (QC) acceptance criteria for the method based on this
validation study. To avoid confusion with Method 1622, which already had been validated and was in use
both domestically and internationally as a stand-alone Cryptosporidium-on\y 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.
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
January 2012
Hi
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• Modified concentrate aspiration procedure
• Modified IMS acid dissociation procedure
• Updated QC acceptance criteria for initial precision and recovery (IPR) and ongoing precision and
recovery (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 (LT2 Rule). Changes incorporated 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
QC samples
• Minor clarifications and corrections
• Rejection criteria for sample condition upon receipt
• Guidance on measuring sample temperatures
• Clarification of QC sample requirements and use of QC sample results
• Guidance on minimizing carry-over debris onto microscope slides after IMS
Changes in the December 2005 Versions of the Methods
Both methods were revised again in 2005 to support promulgation of EPA's LT2 Rule. Changes
incorporated include:
• Nationwide approval of the use of portable continuous-flow centrifugation as a modified version
of the method. The product met all method acceptance criteria for Cryptosporidium using 50-L
source water samples (but not Giardia, however, individual laboratories are permitted to
demonstrate acceptable performance for Giardia in their laboratory).
• Addition of BTF EasyStain™ monoclonal antibody stain as an acceptable reagent for staining in
Methods 1622/1623. The product was validated through an interlaboratory validation study using
the Pall Envirochek® HV filter.
• Clarification of the analyst verification procedure
• Clarification of sample condition criteria upon receipt
January 2012
iv
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Changes in Method 1623.1
Method 1623 was revised in January 2012 to become Method 1623.1. Changes incorporated include:
• Nationwide approval of the use of Waterborne AccuSpike™-IR irradiated oocysts and cysts for
use in routine QC samples
• Nationwide approval of the use of Invitrogen's Dynal® MX7 Mixer during IMS procedures
• Clarification of procedural components approved for use; summarized in Table 1
• Deletion of single-laboratory (Tier 1) validated method modifications
• Addition of minimum QC requirements summary (Table 2)
• Clarification of biosafety recommendations (Sections 5.3 and 5.4)
• Updated shipping requirement for samples containing known infectious materials to reflect
changes in U.S. Department of Transportation Hazardous Materials Regulations (HMR; 49 CFR
Parts 171-180) (Section 5.5)
• Updated product and reagent lists with current catalog numbers (Sections 6 and 7)
• Clarification of fluorescein isothiocyanate (FITC) and 4',6-diamidino-2-phenylindole (DAPI)
filter specifications for all microscope vendors (Section 6.7)
• Updated suggestions for measuring sample temperature, to include temperature recorders (Section
8.1.4)
• Clarification of Initial Demonstration of Capability (IDC) (Section 9.2)
• Clarification of control chart procedures (Section 9.12)
• Requirement for flow cytometer-enumerated Cryptosporidium and Giardia spiking suspensions
(Section 11.0)
• Dispersant addition using sodium hexametaphosphate as required for capsule filter elution
procedure (Sections 7.6.1.1 and 12.2.7)
• Requirement for bead pellet wash step during IMS procedure (Section 13.3.2.17)
• Clarified text for adjustment of pellet volume (Section 13.2)
• Clarified requirements for Cryptosporidium and Giardia characterizations (Sections 15.2.2 and
15.2.3)
• Clarified oocyst/cyst reporting requirements (Section 15.2.5)
• Inclusion of updated acceptance criteria for IPR, OPR, and MS/MSD samples (Section 17.0 and
Tables 3 and 4)
• Addition of Figure 4 to clarify Cryptosporidium and Giardia reporting requirements (Section
15.2.2.1, 15.2.3.1, Figure 4)
• Organizational changes to improve ease of use (Sections 9, 10 and Appendices)
To enhance program-wide data quality and consistency, and guard against the use of sample processing
shortcuts that could compromise data quality, the updated method provides laboratories with the
flexibility to select from options for various procedural components that do not require an alternate test
January 2012
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procedure study (ATP) (Table 1). However, each option must be performed according to the procedures
described in the method, which were used during the multi-laboratory validation study or historically
documented SOP. Any additional ATPs should follow a process for conducting side-by-side method
comparisons and for conducting quality control acceptance criteria-based method studies (Reference
20.1).
January 2012
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Table of Contents
1.0 Scope and Application 1
2.0 Summary of Method 2
3.0 Definitions 2
4.0 Contamination, Interferences, and Organism Degradation 3
5.0 Safety 3
6.0 Equipment and Supplies 4
7.0 Reagents and Standards 8
8.0 Sample Collection and Storage 11
9.0 Quality Control 13
10.0 Microscope Calibration 22
11.0 Sample Spiking 25
12.0 Sample Filtration and Elution 27
13.0 Sample Concentration and Separation (Purification) 36
14.0 Sample Staining 46
15.0 Examination 48
16.0 Analysis of Complex Samples 51
17.0 Method Performance 51
18.0 Pollution Prevention 51
19.0 Waste Management 51
20.0 References 52
21.0 Tables and Figures 53
22.0 Glossary of Definitions and Purposes 62
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List of Appendices
Appendix A: Micropipette Calibration A-1
Appendix B: Microscope Protocols B-1
Appendix C: Flow Cytometry-Enumeration Guidelines C-1
January 2012
viii
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Method 1623.1 - Cryptosporidium and Giardia
Method 1623.1: Cryptosporidium and Giardia in Water by
Filtration/IMS/FA
1.0 Scope and Application
1.1 This method is for the detection of Cryptosporidium (CAS Registry number 137259-50-8) and
Giardia (CAS Registry number 137259-49-5) in water by concentration, immunomagnetic
separation (IMS), and immunofluorescence assay (FA) microscopy. Cryptosporidium and
Giardia are further characterized using 4',6-diamidino-2-phenylindole (DAPI) staining and
differential interference contrast (DIC) microscopy.
1.2 This method is designed to meet the survey and monitoring requirements of U.S. Environmental
Protection Agency (EPA). It was originally based on laboratory testing of recommendations by a
panel of experts convened by EPA. The 2012 revisions reflect the improved protocols for
recovery and detection of protozoa evaluated by EPA Office of Ground Water and Drinking
Water's Technical Support Center.
1.3 This method identifies the genera, Cryptosporidium or Giardia, but not the species. The method
cannot determine the host species of origin, nor can it determine the viability or infectivity of
detected oocysts and cysts.
1.4 This method is for use only by persons experienced in the determination of Cryptosporidium and
Giardia by filtration, IMS, and FA. Experienced persons are defined in Section 22.2 as analysts
or principal analysts. Laboratories unfamiliar with analyses of environmental samples by the
techniques in this method must obtain training and experience using water filtration techniques,
IMS, fluorescent antibody staining with monoclonal antibodies, and microscopic examination of
biological particulates using bright-field and DIC microscopy prior to implementing the method.
Training may be obtained through a variety of sources including other laboratories, experienced
analysts, regulatory agencies, on-line modules, etc. Experience may be gained by performing the
method in a variety of matrices under the supervision of an experienced analyst. Document
training and experience according to the individual laboratory's standard operating procedure
(SOP).
1.5 Any modification of the method beyond those expressly permitted is subject to the application
and approval of alternative test procedures (Reference 20.1).
1.6 The laboratory is permitted to select from options for various procedural components in the
method including sample collection, spiking, filtration/elution, concentration/aspiration, IMS, and
staining. The options available for each component are detailed in the method and summarized in
Table 1. To change between options, the laboratory must perform the procedures outlined in the
Initial Demonstration of Capability (IDC, Table 2, Sections 9.5, 9.6, and 9.7) which includes
acceptable performance in at least one matrix of interest and verify that all quality control (QC)
acceptance criteria are met (Sections 9.1 and 21.0, Tables 3 and 4). Although different options
can be used by different laboratories, each option must be performed according to the procedures
specified in this method or the manufacturers' instructions. These procedures were approved by
EPA based on the multi-laboratory validation studies using multiple matrices or historical
demonstration of accuracy and precision at multiple laboratories. To guard against the use of
sample processing shortcuts or cost-cutting that could compromise data quality, no changes to
these validated procedures are permitted without demonstrating acceptability through a multi-
laboratory validation study using multiple matrices, as per Reference 20.1.
January 2012
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Method 1623.1 - Cryptosporidium and Giardia
This description of method flexibility is an abbreviated summation. Specific detail is provided
throughout the method, which supersedes the above general guidance.
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 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 made paramagnetic (magnetic only when exposed to a
magnetic field) by attachment of paramagnetic beads conjugated to anti-
Cryptosporidium and anti-Giardia antibodies. The paramagnetic oocysts and cysts are
separated from the extraneous materials using a magnet, and the extraneous materials
are discarded. The paramagnetic 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 DAPI. The stained sample is examined using fluorescence and 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.
2.3.3 Quantitative analysis is performed by counting the total number of objects on the slide
that meet the FA, DAPI, and DIC criteria for oocysts or cysts.
2.4 Quality is assured through reproducible calibration and testing of the filtration, IMS, staining, and
microscopy systems. Detailed information on these tests is provided in Sections 9.0 and 10.0, and
Appendix B.
3.0 Definitions
3.1 Cryptosporidium is a genus of protozoan parasites potentially found in water and other media.
Cryptosporidium oocysts are defined in this method as objects exhibiting brilliant apple green
fluorescence under UV light [after staining with fluorescein isothiocyanate (FITC)-conjugated
antibodies (FA-positive)], typical size (4 to 6 (im) and shape (round to oval), and no atypical
characteristics by FA, DAPI fluorescence, or DIC microscopy. Examination and characterization
using fluorescence (FITC and DAPI stain) and DIC microscopy are required for exclusion of
atypical organisms (e.g., those possessing spikes, stalks, appendages, pores, one or two large
nuclei filling the cell, red fluorescing chloroplasts, crystals, spores, etc.).
3.2 Giardia is a genus of protozoan parasites potentially found in water and other media. Giardia
cysts are defined in this method as objects exhibiting brilliant apple green fluorescence under UV
light [after staining with FITC-conjugated antibodies (FA-positive)], typical size (8 to 18 (im long
by 5 to 15 (im wide) and shape (oval to round), and no atypical characteristics by FA, DAPI
fluorescence, or DIC microscopy. Examination and characterization by fluorescence (FITC and
DAPI stain) and DIC microscopy are required for exclusion of atypical organisms (e.g., those
January 2012
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Method 1623.1 - Cryptosporidium and Giardia
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 (e.g., clays and algae), chemicals (e.g., iron, alum coagulants) and
polymers added to source waters during the treatment process may result in additional
interference.
4.2 Organisms and debris that autofluoresce or demonstrate non-specific immunofluorescence, such
as algal and yeast cells, when examined by epifluorescent microscopy, may interfere with the
detection of oocysts and cysts and contribute to false positives by FA (Reference 20.2).
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
must be demonstrated to be free from interferences under the conditions of analysis by running a
method blank (negative control sample) initially and a minimum of every week or after changes
in source of reagent water. Specific selection of reagents and purification of solvents and other
materials may be required.
4.4 Freezing samples, filters, eluates, concentrates, or slides may interfere with the detection and/or
identification of oocysts and cysts.
4.5 Clean all equipment according to manufacturers' instructions, or according to SOP used in the
laboratory. Use disposable supplies 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 (Reference 20.3).
5.2 The toxicity or carcinogenicity of each compound or reagent used in this method has not been
precisely determined; however, it is prudent to treat each chemical compound as a potential
health hazard. Reduce exposure to these compounds to the lowest possible level. The laboratory
is responsible for maintaining current knowledge of Occupational Safety and Health
Administration regulations regarding the safe handling of the chemicals specified in this method.
Make a reference file of material safety data sheets available to all personnel involved in these
analyses. Additional information on laboratory safety can be found in References 20.3 through
20.7.
5.3 Samples may contain high concentrations of biohazards and toxic compounds, and must be
handled with gloves. Reference materials and standards containing oocysts 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. Laboratory personnel
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Method 1623.1 - Cryptosporidium and Giardia
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.
5.4 Avoid any potential contamination from concentrated samples of control suspensions that may
results in accidental ingestion. Do not mouth-pipette. Take appropriate precautions to ensure that
no aerosol is generated by any step. Performing the Method in a biological safety cabinet for as
many steps as logistically possible is recommended to prevent exposure (Reference 20.3).
5.5 The U.S. Department of Transportation (DOT) Hazardous Material Regulation (49 CRF Parts
171-178) classifies biological hazards as two categories. Cryptosporidium and Giardia are
Category B: An infectious substance not in a form generally capable of causing permanent
disability, life-threatening, or fatal disease in otherwise healthy humans or animals when
exposure occurs. Any sample known or suspected to contain Cryptosporidium or Giardia should
be shipped as Biological substance, Category B, UN3373 (Reference 20.8) or by regulations
controlling the shipment that are in effect at the time of shipment. Environmental samples that are
not considered to pose a significant health risk are not subject to the requirements of the
Hazardous Material Regulation.
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
when stated as "or equivalent, " but demonstration of equivalent performance that meets the
requirements of this method (Section 9.2 and Table 1) is the responsibility of the laboratory.
6.1 Sample collection equipment for shipment of bulk water samples for laboratory filtration.
Collapsible low-density polyethylene (LDPE) cubitainer for collection of 10-L bulk sample(s)—
Cole Farmer cat. no. U-06100-30 or equivalent. Fill completely to ensure collection of a full 10-L
sample. Discard after one use. Alternatively, use clean, 10-L carboy with bottom delivery port
0/2"), Cole-Parmer cat. no. 06080-42, or equivalent; calibrate to 10.0 L and mark level with
waterproof marker.
6.2 Equipment for sample filtration. Three options have been demonstrated to be acceptable for use
with Method 1623.1: Envirochek® HV Sampling Capsule, Filta-Max® Foam Filter, and Portable
Continuous Flow Centrifuge (PCFC).
6.2.1 Cubitainer spigot to facilitate laboratory filtration of sample—Cole Farmer cat. no. U-
06061-01, or equivalent.
6.2.2 Tubing—Glass, polytetrafluoroethylene (PTFE), high-density polyethylene (HOPE), or
other tubing to which oocysts and cysts will not easily adhere, Tygon formula R-3603,
or equivalent. If rigid tubing (glass, PTFE, HOPE) is used and the sampling system
uses a peristaltic pump, a minimum length of compressible tubing may be used in the
pump. Between samples, the tubing must be autoclaved, thoroughly rinsed with
detergent solution, followed by repeated rinsing with reagent water to minimize sample
contamination. Alternately, decontaminate using hypochlorite solution, sodium
thiosulfate, and multiple reagent water rinses. Dispose of tubing after one use whenever
possible or when wear is evident.
6.2.3 Flow control valve—0.5 gpm (0.03 L/s) Bertram Controls, Plast-O-Matic cat. no.
FC050B!/2-PVor equivalent
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Method 1623.1 - Cryptosporidium and Giardia
6.2.4 Pump—peristaltic, centrifugal, impeller, or diaphragm pump; MasterFlex I/PCS)
EasyLoad® peristaltic pump (Cole-Parmer cat. no. EW-77963-10) with EW-77601-10
pumphead, EW-77410-10 drive unit, and EW-06429-73 Tygon LFL tubing; Dayton,
model number 3YU61 (available through Grainger), Jabsco Flexible Impeller Pump
(Cole-Parmer cat. no.EW-75202-00); Simer, model number M40; or equivalent. It is
recommended that the pump be placed on the effluent side of the filter, to reduce the
risk of contamination and the amount of tubing replaced or cleaned.
6.2.5 Flow meter—Omega flow meter, Stamford, CT, model FTB4105; or equivalent.
Alternatively, use a graduated carboy(s) (See Section 6.16).
6.2.6 Stir bar—Fisher cat. no. 14-513-66, or equivalent
6.2.7 Stir plate—VWR cat. no. 47751-150, or equivalent
6.2.8 Envirochek® HV Sampling Capsule, Pall Corporation, Ann Arbor, MI, part no. 12099
(individual filter) or part no. 12098 (box of 25 filters)
6.2.8.1 Laboratory shaker for agitation of sampling capsules —Pall Corporation
part no. 482lor equivalent
6.2.8.2 Side arms for laboratory shaker
6.2.9 Filta-Max® foam filter, IDEXX, Westbrook, ME. Filter module cat. no. FMC 10603
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.
6.2.9.1 Filta-Max® starter kit, IDEXX, Westbrook, ME, cat. no. FMC 11002
6.2.9.1.1 Manual wash station with clamp set, cat. no. FMC 10101 or
FMC 10106 or Automatic wash station cat. no. FMC 10103
6.2.9.1.2 Quick connect tubing set (includes elution tube, quick connect
kit, concentrator tube, concentrator base (with line tap), steel
tube and all o-rings), cat. no. FMC 10307
6.2.9.1.3 Vacuum set (includes plastic hand pump, waste bottle, tubing
and magnetic stirrer bar), cat. no. FMC 10401
6.2.9.1.4 MKII filter housing with appropriate fittings, cat. no. FMC
10504
6.2.9.1.5 Housing tools cat. no. FMC 10506
6.2.9.2 Filter membranes, 100 pk, IDEXX, Westbrook, ME, cat. no. FMC 10800
6.2.9.3 Forceps—blunt end
6.2.9.4 Rubber stoppers, large and small, IDEXX, Westbrook, ME, cat. no. FMC
10508 and FMC 10302
6.2.10 PCFC requirements (for use with procedures described in Section 12.4). The technique
is based on technology from Haemonetics Corporation, Braintree, MA.
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Method 1623.1 - Cryptosporidium and Giardia
6.2.10.1 Portable continuous-flow centrifuge, Scientific Methods, Inc., Granger, IN,
cat.no. CFC-201
6.2.10.2 Disposable Bowl, 25 pk, Scientific Methods, Inc., Granger, IN, cat. no.
CFC-210
6.2.10.3 Tubing, 25 pk, Scientific Methods, Inc., Granger, IN, cat. no. CFC-220
6.3 IMS apparatus
6.3.1 Sample mixer—Available through IDEXX or Invitrogen, Dynal® Sample Mixer, cat.
no. 94701, or equivalent
6.3.2 Magnetic particle concentrator for 10-mL test tubes—Available through IDEXX or
Invitrogen, Dynal® MPC®-1, cat. no. 12001D or MPC®-6, cat. no. 12002D, or
equivalent
6.3.3 Magnetic particle concentrator for microcentrifuge tubes—Available through IDEXX,
Dynal® MPC®-S, cat. no. A13346 or equivalent
6.3.4 Flat-sided sample tubes—16 x 125 mm Leighton-type tubes with 60 x 10 mm flat-
sided magnetic capture area, available through IDEXX or Invitrogen, Dynal L10, cat.
no. 74003, or equivalent
6.3.5 Microcentrifuge tubes—conical, siliconized (coated with silicone to reduce adhesion to
walls) or low-retention, 1.5-mL, Fisher cat. no. 13-6987-91 or equivalent
6.4 Powder-free latex gloves—Fisher cat. no. 11-394-5B, or equivalent
6.5 Graduated cylinders, autoclavable—10-, 100-, and 1000-mL
6.6 Centrifuges
6.6.1 Centrifuge capable of accepting 50- to 250-mL conical centrifuge tubes and achieving
at least 1500 x G—Allegra® X-15Rbenchtop centrifuge with swinging bucket
SX4750A ARIES™ Smart Balance Rotor with bucket adaptors, Beckman Coulter,
Brea, CA, or equivalent
6.6.2 Centrifuge tubes—Conical, graduated, 50- and 250-mL, Fisher cat. no. 06-443-20 and
05-538-53, or equivalent
6.7 Microscope
6.7.1 Epifluorescence/DIC with stage and ocular micrometers and 20X (N.A.=0.4) to 100X
(N.A.=1.3) objectives.
6.7.2 Excitation/band-pass filters for FA—Filter set to examine FITC (peak excitation 490-
to 495-nm, peak emission 516- to 525-nm), optimized with microscope, DAPI filter
set, and slide preparations; general specifications: 450- to 495-nm excitation filter,
505-nm dichroic mirror, and 515-to 520-nm emission, barrier, or suppression filter; or
equivalent
6.7.3 Excitation/band-pass filters for DAPI—Filter set to examine DAPI (peak excitation
345- to 359-nm, peak emission 455- to 470-nm), optimized with microscope, FITC
filter set, and slide preparations; general specifications: 325- to 380-nm excitation
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Method 1623.1 - Cryptosporidium and Giardia
filter, 390-to 415-nm dichroic mirror, 420-to 485-nm emission, barrier, or suppression
filter; or equivalent
6.8 Ancillary equipment for microscopy
6.8.1 Well slides—Dynal® Spot-On well slides, Invitrogen, cat. no. 740-04; treated, 12-mm
diameter well slides, Meridian Diagnostics Inc., Cincinnati, OH, cat. no. R2206; or
equivalent.
6.8.2 Glass coverslips—22 x 50 mm or appropriate size
6.8.3 Nonfluorescing immersion oil—low fluorescence, Type LDF, HF, or FF Cargille cat.
no. 16241, 16245, 16212, or equivalent
6.8.4 Micropipette, adjustable (See Appendix A):
6.8.4.1 0- to 10-(iL with 0- to 10-^iL tips
6.8.4.2 10- to 100-(iL, with 10- to 200-^L tips
6.8.4.3 100- to 1000-^iL with 100- to 1000-^L tips
6.8.5 Humid chamber—A tightly sealed plastic container containing damp paper towels on
top of which the slides are placed
6.9 Pipettes—Glass or plastic
6.9.1 1-, 5-, 10-, and25-mL
6.9.2 Pasteur, disposable, internal diameter of orifice 0.80 to 1.5 mm
6.10 Balances
6.10.1 Analytical—Capable of weighing 0.1 mg
6.10.2 Top loading—Capable of weighing 10 mg
6.11 pH meter
6.12 Incubator—Fisher Scientific Isotemp™, or equivalent
6.13 Vortex mixer—Scientific Industries Vortex-Genie, or equivalent
6.14 Vacuum source—Capable of maintaining 25 in. Hg, equipped with shutoff valve and vacuum
gauge
6.15 Miscellaneous labware and supplies
6.15.1 Appropriate tube racks
6.15.2 Flasks—Suction, Erlenmeyer, and volumetric, various sizes
6.15.3 Beakers—Glass or plastic, 5-, 10-, 50-, 100-, 500-, 1000-, and 2000-mL
6.15.4 Lint-free tissues
6.16 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
6.17 Filters for filter-sterilizing reagents—Sterile Acrodisc, 0.45 (im, Pall Corporation, cat. no. 4184,
or equivalent
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Method 1623.1 - Cryptosporidium and Giardia
7.0 Reagents and Standards
7.1 Reagents for adjusting pH
7.1.1 Sodium hydroxide (NaOH)—ACS reagent grade, 6.0 N
7.1.2 Hydrochloric acid (HC1) —ACS reagent grade, 6.0 N
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. See Reference 20.9 (Part 9020) for
reagent water requirements.
7.4 Anti-microbial supplies—bleach, 3% hydrogen peroxide, ethanol wipes, and commercial multi-
surface cleaner. Use cleaner appropriate for surface and biohazard (Reference 20.3).
7.5 Phosphate buffered saline (PBS), pH 7.4—Sigma Chemical Co. cat. no. P-3813, or equivalent.
Alternately, prepare 1 X PBS by adding the following to 800 mL of reagent water: 8 g NaCl; 0.2
g KC1; 1.15 g Na2HPO4, anhydrous; and 0.2 g KH2PO4. Adjust pH to 7.4 with HC1 or NaOH.
Dilute to a final volume of 1000 mL using regent water. Store in a plastic or glass container at
room temperature or 4°C. Discard if microbial growth is apparent or after a specific time
determined by the laboratory.
7.6 Reagents for eluting filters
NOTE: Store prepared eluting solution for no more than 1 week or until noticeably
turbid, whichever comes sooner.
7.6.1 Reagents for eluting Envirochek® HV sampling capsules (Section 6.2.8)
7.6.1.1 5% w/v NaHMP solution—Dissolve 50 g of sodium hexametaphosphate
((NaPO3)n- Sigma 71600 or equivalent) in 600 mL of reagent water.
Dilute to final volume of 1000 mL using reagent water. Store in a plastic or
glass container at room temperature for up to 3 months. Discard when
expiration date is reached or sooner if microbial growth is apparent.
7.6.1.2 Laureth-12—Fisher cat. no. NC9253856, 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.
7.6.1.3 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 HC1 or NaOH. Dilute to
a final 1000 mL with reagent water and adjust the final pH. Filter-sterilize
through a 0.2-(im membrane into a sterile plastic container and store at
room temperature. Alternatively, use prepared Tris (Sigma T2194 or
equivalent) or prepared Tris-EDTA (Sigma T9285 or equivalent).
7.6.1.4 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 HC1 or NaOH. Dilute to a final volume of
1000 mL with reagent water and confirm pH. Alternatively, use prepared
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Method 1623.1 - Cryptosporidium and Giardia
EDTA (Sigma E7889 or equivalent) or prepared Tris-EDTA (Sigma T9285
or equivalent).
7.6.1.5 Antifoam A—Sigma Chemical Co. cat. no. 10794, or equivalent
7.6.1.6 Preparation of elution buffer solution—Add the contents of a pre-prepared
Laureth-12 vial (Section 7.6.1.2) 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 7.6.1.3), 2 mL of EDTA solution
(Section 7.6.1.4), (alternatively, if using a manufacturer-combined Tris-
EDTA [100X, Sigma T9285], add 10 mL total), and 150 (iL Antifoam A
(Section 7.6.1.5). Dilute to 1000 mL with reagent water.
7.6.2 Reagents for eluting Filta-Max® foam filters (Section 6.2.9)
7.6.2.1 PBS, pH 7.4 (Section 7.5)
7.6.2.2 TWEEN® 20—Sigma Chemical Co. cat. no. P-7949, or equivalent
7.6.2.3 High-vacuum grease—Fisher cat. no. 14-635-5D, or equivalent
7.6.2.4 Preparation of phosphate buffered saline with TWEEN® (PBST) elution
buffer. Add 100 (iL of TWEEN® 20 to prepared PBS (Section 7.5).
Alternatively, add the contents of one 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.
7.6.3 Reagents for Portable Continuous-Flow Centrifuge (Section 6.2.10)
7.6.3.1 Sodium dodecyl sulfate—Sigma Chemical Co. cat. no. 62862 or equivalent
7.6.3.2 TWEEN® 80—Sigma Chemical Co. cat. no. P1754 or equivalent
7.6.3.3 Antifoam A—Sigma Chemical Co. cat. no. 10794, or equivalent
7.6.3.4 Preparation of concentrated elution buffer. Add above reagents to obtain a
final concentration of 1% sodium dodecyl sulfate, 0.01% TWEEN® 80,
and 0.001% Antifoam A in concentrated sample volume of ~250mL
7.7 Reagents for IMS
7.7.1 Dynabeads® GC-Combo—Available through IDEXX, Westbrook, ME, Dynal cat.
no.730.12, or equivalent
7.7.2 NaOH—ACS reagent grade, 1.0 N, Sigma Chemical Co. cat. no. S2770, or equivalent
7.7.3 HC1—ACS reagent grade, 0.1 N, Sigma Chemical Co. cat. no. 84428, or equivalent
NOTE: Due to the low volumes ofpH-adjusting reagents used during IMS, and the impact that
changes in pH have on the FA, the laboratory must purchase standards at the required normality
directly from a vendor. Normality must not be adjusted by the laboratory.
7.8 Direct antibody labeling reagents for detection of oocysts and cysts. Store reagents between 1°C
and 10°C and return promptly to this temperature after each use. Do not allow any of the reagents
to freeze. Protect the reagents from exposure to light. Discard diluted, unused working reagents
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Method 1623.1 - Cryptosporidium and Giardia
after 48 hours or following manufacturer's instructions. Discard reagents after the expiration date
is reached. The labeling reagents in Sections 7.8.1-7.8.4 have been approved for use with this
method.
7.8.1 MeriFluor® Cryptosporidium/Giardia, Meridian Diagnostics cat. no. 250050,
Cincinnati, OH, or equivalent
7.8.2 Aqua-Glo™ G/C Direct FL, Waterborne, Inc. cat. no. A100FLK, New Orleans, LA, or
equivalent
7.8.3 Crypt-a-Glo™ and Giardi-a-Glo™, Waterborne, Inc. cat. nos. A400FLK and
A300FLK, respectively, New Orleans, LA, or equivalent
7.8.4 EasyStain™C&G, BTF Pty Limited, Sydney, Australia or equivalent
NOTE: If a laboratory will use multiple types of labeling reagents, the laboratory must
demonstrate equivalent or superior performance through an IDC per Section 9.2 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. Monitor
the performance of each labeling reagent used in each source water type through MS
samples (Section 9.6.1).
7.9 DAPI stain (4',6-diamidino-2-phenylindole )—Sigma Chemical Co. cat. no. D9542, or equivalent
7.9.1 Stock solution—Purchase smallest amount possible (typically 1 mg vials) to eliminate
weighing a portion of the powder. Dissolve 2 mg/mL DAPI in absolute methanol by
adding 0.5 mL of methanol to 1 mg vial of DAPI. Store between 1°C and 10°C in the
dark. Do not allow to freeze. Discard unused solution when positive staining control
fails or after specified time determined by laboratory.
7.9.2 Staining solution—Follow antibody kit manufacturer's instructions. Add 10 \\L of
2 mg/mL DAPI stock solution to 50 mL of PBS for use with Aqua-Glo™ G/C Direct
FL or MeriFluor® Cryptosporidium/Giardia. Add 50 \\L of 2 mg/mL DAPI stock
solution to 50 mL of PBS for use with EasyStain™. Prepare working solution daily and
store between 1°C and 10°C (do not allow to freeze). DAPI is light sensitive; therefore,
store in the dark except when staining. The DAPI concentration may be increased if
fading/diffusion of DAPI staining is encountered, but the staining solution must be
tested first on expendable environmental samples to confirm that staining intensity is
appropriate.
7.10 Mounting medium
7.10.1 DABCO/glycerol mounting medium (2%)—Dissolve 2 g of DABCO (Sigma Chemical
Co. cat. no. D27802, or equivalent) in 95 mL of warm glycerol/PBS (60% glycerol,
40% PBS). After the DABCO has dissolved completely, adjust the solution volume to
100 mL by adding an appropriate volume of glycerol/PBS solution. Alternately,
dissolve the DABCO in 40 mL of PBS, then add azide (1 mL of 100X, or 10%
solution), then 60 mL of glycerol.
7.10.2 Mounting medium supplied with MeriFluor® Cryptosporidium/Giardia, Meridian
Diagnostics cat. no. 250050, or equivalent (Section 7.8.1)
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Method 1623.1 - Cryptosporidium and Giardia
7.10.3 Mounting medium supplied with Aqua-Glo™ G/C Direct FL kit, Waterborne, Inc. cat.
no. A100FLK, cat. no. M101, or equivalent (Section 7.8.2)
7.10.4 Mounting medium supplied with EasyStain™C&G, BTF Pty Limited or equivalent
(Section 7.8.4)
7.10.5 Elvanol® or equivalent permanent, non-fade archiving mounting medium
7.11 Clear fingernail polish or clear fixative, Fisher, cat. no. NC0154994, or equivalent
7.12 Oocyst and cyst suspensions for spiking
7.12.1 Enumerated spiking suspensions prepared by flow cytometer
7.12.1.1 Live, flow cytometer-sorted oocysts and cysts—Wisconsin State
Laboratory of Hygiene Flow Cytometry Unit, or equivalent
7.12.1.2 Irradiated, flow cytometer-sorted oocysts and cysts -EasySeed™ BTF Pty
Limited, or AccuSpike™-IR Waterborne, Inc., or equivalent.
7.12.2 Storage procedure—Store oocyst and cyst suspensions between 1°C and 10°C, until
ready to use or follow manufacturer's instructions; do not allow to freeze
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 on ice for processing through the entire method, or are filtered in the field
and shipped to the laboratory on ice for processing from elution (Section 12.2.6 or
12.3.2) 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.
Chill all samples to reduce biological activity, and preserve the state of source water
samples between collection and analysis. Samples analyzed by an off-site laboratory
should be shipped on ice via overnight service on the day they are collected.
NOTE: See transportation precautions in Section 5.5.
8.1.2.1 If samples are collected early in the day, chill samples by storing in a
refrigerator between 1°C and 10°C or pre-icing the sample in a cooler. If
the sample is pre-iced before shipping, replace with fresh ice immediately
before shipment.
8.1.2.2 If samples are collected later in the day, these samples may be chilled
overnight in a refrigerator between 1°C and 10°C. Consider overnight
refrigeration for bulk water samples that will be shipped off-site, as this
minimizes the potential for water samples collected during the summer to
melt the ice in which they are packed and arrive at the laboratory at >20°C.
8.1.2.3 If samples are shipped after collection at >20°C with no chilling, the
sample will not maintain the temperature during shipment at <20°C.
8.1.2.4 Public water systems shipping samples to off-site laboratories for analysis
should include in the shipping container a means for monitoring the
temperature of the sample during shipping to verify that the sample did not
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Method 1623.1 - Cryptosporidium and Giardia
freeze or exceed 20°C. Suggested approaches for monitoring sample
temperature during shipping are discussed in Section 8.1.4.
8.1.3 Sample receipt—Upon receipt, the laboratory must record the sample temperature.
Samples that were not collected the same day they were received, and that are received
at >20°C or frozen, or samples that the laboratory has determined exceeded >20°C or
froze during shipment, must be rejected. After receipt, samples must be stored at the
laboratory between 1°C and 10°C, and not frozen, until processed.
8.1.4 Suggestions on measuring sample temperature—Given the importance of
maintaining sample temperatures for Cryptosporidium and Giardia determination,
laboratories performing analyses using this method must establish acceptance criteria
for receipt of samples transported to their laboratory. As with other laboratory
equipment, all temperature measurement devices must be calibrated routinely to ensure
accurate measurements. See EPA Manual for the Certification of Laboratories
Analyzing Drinking Water (Reference 20.10) for more information. Several options are
available to measure sample temperature upon receipt at the laboratory and, in some
cases, during shipment:
8.1.4.1 Temperature sample—One option, for filtered samples only (not for 10-L
bulk samples), is for the sampler to fill a small, inexpensive sample bottle
with water and pack this "temperature sample" next to the filtered sample.
The temperature of this extra sample volume is measured upon receipt to
estimate the temperature of the filter. Temperature sample bottles are not
appropriate for use with bulk samples because of the potential effect that
the difference in sample volume may have in temperature equilibration in
the sample cooler. Example product: Cole Farmer cat. no. U-06252-20.
8.1.4.2 Thermometer vial—A similar option is to use a thermometer that is
securely housed in a liquid-filled vial. Unlike temperature samples, the
laboratory does not need to perform an additional step to monitor the
temperature of the vial upon receipt, but instead just needs to read the
thermometer. The thermometer vial is appropriate for use with filtered
samples not bulk samples. Example product: Eagle-Picher Sentry
Temperature Vial 3TR-40CS-F or 3TR-40CS.
8.1.4.3 Temperature Recorders—Option allows the measurement and recording
of sample temperature during shipment and upon receipt. These small,
waterproof devices contain a computer chip that can be programmed to
record temperature at different time intervals. The information is then
downloaded from the temperature recorder onto a computer. Place the
temperature recorder in a temperature sample, rather than loose in the
cooler, or attached to the sample container. This option is appropriate for
use with both filtered and bulk samples. Example products: Fisherbrand
Exact-Temp Temperature Datalogger 15-059-201, Thermocron® iButtons,
distributors at http://www.maxim-ic.com/products/ibutton/ 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
(cubitainer or filter) upon receipt at the laboratory. This option does not
measure temperature as precisely as the other options, but provides an
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Method 1623.1 - Cryptosporidium and Giardia
indication of sample temperature to verify that the sample temperature is
acceptable. This option is appropriate for use with both filtered and bulk
samples. Example product: Cole Farmer cat. no. U-90316-00.
8.1.4.5 Infrared thermometers—The thermometer is pointed at the sample, and
measures the temperature without coming in contact with the sample
volume. This option is appropriate for use with both filtered and bulk
samples. Example product: Cole Farmer cat. no. EW-39641-00.
8.2 Sample holding times—Samples must be processed or examined within each of the holding
times specified in Sections 8.2.1 through 8.2.4. Complete sample processing as soon as possible.
The laboratory should complete sample filtration, elution, concentration, purification, and
staining the day the sample is received whenever 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 5, 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
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 it is preferable to perform FA and DAPI and DIC
microscopy examination and characterization immediately after staining is complete,
laboratories have up to 168 hours (7 days) from the completion of sample staining to
perform the examination and verification of samples. However, if fading/diffusion of
FITC or DAPI staining is noticed, the laboratory must reduce this holding time. In
addition the laboratory may adjust the concentration of the DAPI staining solution
(Sections 7.9.2) so that fading/diffusion does not occur.
8.3 Spiking suspension enumeration holding times—Flow cytometer-sorted spiking suspensions
(Sections 7.12.1 and Appendix C) used for spiked QC samples (Section 9) must be used within
the expiration date noted on the suspension. Oocyst and cyst suspensions must be stored between
1°C and 10°C or following manufacturer's instructions, 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 that addresses and documents data quality, instrument and equipment maintenance and
performance, reagent quality and performance, analyst training and certification, and records
storage and retrieval. General requirements and recommendations for QA and QC procedures for
microbiology laboratories are provided in References 20.9 and 20.10. The minimum analytical
requirements of this program consist of an IDC through performance of the initial precision and
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Method 1623.1 - Cryptosporidium and Giardia
recovery (IPR) test (Section 9.5), matrix spike/matrix spike duplicate (MS/MSD) test along with
unspiked field sample (Section 9.6) and method blank (MB, Section 9.7), and ongoing
demonstration of laboratory capability and method performance through the MS test along with
unspiked field sample (Section 9.6.1), the MB test (Section 9.7), the ongoing precision and
recovery (OPR) test (Section 9.8), staining controls (Sections 9.9, 14.1, and 15.2.1), and analyst
verification tests (Section 9.10). A principal analyst (Section 22.2) verifies the quality and
accuracy of all sample results. Laboratory performance is compared to established performance
criteria to determine if the results of analyses meet the performance characteristics of the method.
Table 2 summarizes the minimum QC requirements.
9.2 Prior to first use of the method, or if a laboratory changes to another option for a method
procedural component, the laboratory must demonstrate acceptable performance through an IDC
(Table 2) which consists of acceptable performance in four IPR samples (Section 9.5), a
MS/MSD (Section 9.6), an unspiked field sample, and one MB (Section 9.7). The laboratory is
permitted to choose options for each procedural component as listed in Table 1.
NOTE: Only consider method procedural component changes 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
must not be used.
9.3 The laboratory is required to maintain records of modifications or procedural component changes
made to this method. These records include the following, at a minimum:
9.3.1 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.
9.3.2 A listing of the analyte(s) measured (Cryptosporidium and Giardia).
9.3.3 A narrative stating reason(s) for the modification.
9.3.4 Results from all QC tests comparing the modified method to this method, including:
IPR (Section 9.5), MS/MSD (Section 9.6), and analysis of method blanks (Section 9.7).
9.3.5 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
7.12)
(c) Spike enumeration date and time
(d) Spiking suspension values (Section 11.0 and Appendix C)
(e) Sample spiking dates and times
(f) Volume filtered (Section 12.2.4.5 and 12.3.1.5.5)
(g) Filtration and elution dates and times (Section 12.2.3, 12.2.7.2, 12.3.1.4, 12.3.2.1)
(h) Pellet volume, resuspended concentrate volume, resuspended concentrate volume
transferred to IMS, and all calculations required to verify the percent of concentrate
examined (Section 13.2)
(i) Purification completion dates and times (Section 13.3.3.11)
(j) Staining completion dates and times (Section 14.10)
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Method 1623.1 - Cryptosporidium and Giardia
(k) Staining control results (Section 15.2.1)
(1) All required examination information (Sections 15.2.2 and 15.2.3)
(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
9.4 Microscope adjustment and calibration—Adjust the microscope as specified in Section 10.0
and Appendix B. All of the requirements in Section 10.0 and Appendix B must be met prior to
analysis of IPRs, method blanks, OPRs, field samples, and MS/MSDs.
9.5 Initial precision and recovery (IPR)—To establish the ability to demonstrate control over the
analytical system and to generate acceptable precision and recovery through an IDC or if
equipment/supplies are changed, the laboratory must perform the following operations:
9.5.1 Using the spiking procedure in Section 11.2 and flow cytometer-enumerated spiking
suspensions (Section 7.12.1), spike, filter, elute, concentrate, separate (purify), stain,
and examine the four reagent water samples spiked with -100-500 oocysts and -100-
500 cysts.
9.5.1.1 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). Note that the variability of four
measurements performed on multiple days or using multiple analysts or
reagent lots may be greater than the variability of measurements performed
on the same day with the same analysts and reagent lots. As a result, the
laboratory is at a greater risk of generating unacceptable IPR results if the
test is performed across multiple days, analysts, and /or reagent lots.
9.5.1.2 If more than one option will be used for a procedural component, a
separate set of IPR samples must be prepared for each option as part of an
IDC.
9.5.1.3 The set of four IPR samples must be accompanied by analysis of an
acceptable method blank (Section 9.7).
9.5.2 For each organism, calculate the percent recovery (R) using the following equation:
R= 100 x
T
where:
R = the percent recovery
N = the number of oocysts or cysts counted
T = the number of oocysts or cysts spiked
This calculation assumes that the total volume spiked was processed and examined.
9.5.3 Using percent recovery (R) of the four analyses, calculate the mean percent recovery
and the relative standard deviation (RSD) of the recoveries for Cryptosporidium and for
Giardia. The RSD is the standard deviation divided by the mean, times 100.
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Method 1623.1 - Cryptosporidium and Giardia
9.5.4 Compare the mean and RSD to the corresponding method performance acceptance
criteria for initial precision and recovery in Tables 3 and 4 in Section 21.0. If the mean
and RSD for recovery meet the acceptance criteria, system performance is acceptable
and analysis of blanks and samples may begin. If the mean or RSD falls outside the
range for recovery, system performance is unacceptable. In this event, troubleshoot the
problem by starting at the end of the method (see guidance in Section 9.8.7), correct the
problem and repeat the IPRtest (Section 9.5.1).
9.5.5 Examine and document the IPR slides following the procedure in Section 15.0. The
first three Cryptosporidium oocysts and first three Giardia cysts identified in each IPR
sample must be characterized (size, shape, DAPI category, and DIC category) and
documented on the examination form, as well as any additional comments on
organisms appearance, if notable.
9.5.6 Using 200X to 400X magnification, more than 50% of the oocysts or cysts must appear
undamaged and morphologically intact; otherwise, the organisms in the spiking
suspension may be of unacceptable quality or the analytical process may be damaging
the organisms. If the quality of the organisms on the IPRtest slides is unacceptable,
examine the spiking suspension organisms directly (by centrifuging, if possible, to
concentrate the organisms in a volume that can be applied directly to a slide). If the
unprocessed organisms appear undamaged and morphologically intact under DIC,
determine the step or reagent that is causing damage to the organisms. Correct the
problem (see Section 9.8.7) and repeat the IPRtest.
9.6 Matrix spike (MS) and matrix spike duplicate (MSD)
9.6.1 MS — The laboratory must spike a separate sample aliquot from the same source to
determine the effect of the matrix on the method's oocyst and cyst recovery. The
laboratory must analyze a 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 (Tables 3 and 4). If an MS sample cannot be
analyzed on the first sampling event, the first MS sample must be analyzed as soon as
possible to identify potential method performance issues with the matrix. The
laboratory must analyze MS samples at a minimum frequency of 1 MS sample per 20
field samples, or portions thereafter, for each individual source analyzed.
9.6.2 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 analyzed must be within 10% of the field sample volume. Every MS
must be associated with an acceptable OPR and MB.
9.6.2.1 Analyze an unspiked field sample according to the procedures in Sections
12.0 to 15.0. Using the spiking procedure in Section 1 1.2 and flow
cytometry-enumerated spiking suspensions (Section 7.12.1), spike, filter,
elute, concentrate, separate (purify), stain, and examine a second field
sample aliquot with a similar number of organisms as that used in the IPR
or OPR tests (Sections 9.5 and 9.8).
9.6.2.2 For each organism, calculate the percent recovery (R) using the following
equation.
N - N
T
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Method 1623.1 - Cryptosporidium and Giardia
where:
R is the percent recovery
N is the number of oocysts or cysts counted in the spiked sample
Ns is the number of oocysts or cysts counted in the unspiked sample
T is the true value of the oocysts or cysts spiked
9.6.2.3 Compare the recovery for each organism with the acceptance criteria in
Tables 3 and 4 in Section 21.0. Add results to the MS control chart
(Section 9.12.2).
NOTE: Some sample matrices may prevent the MS acceptance criteria in Tables 3 and 4
from being met. Close evaluation of mean MS/MSD recovery (Section 9.6.3) and RSD
should be performed if the MS recovery is below acceptance criteria. Repeated failure to
meet the MS quality control acceptance criteria, when all other QCpasses, warrants
corrective action from quality conscience laboratories to systematically review control
charts and modify analytical protocols to improve matrix spike recoveries. An
assessment of the distribution of MS recoveries across 4 30 MS samples from 87 sites
during the ICR Supplemental Surveys is provided in Table 6.
9.6.3 MSB analysis is required for: 1) an IDC; and 2) as part of multi-laboratory validation
study using multiple matrices (Section 1.6) to demonstrate that the modified version of
this method produces results equal or superior to results produced by the method as
written. As noted above, an MSB is also performed as a corrective action when quality
control criteria are not met. The laboratory spikes and analyzes another field sample
aliquot (MSB) at the same time the laboratory spikes and analyzes the MS field sample
aliquot (Section 9.6.2.1). The MS, MSB 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 and MSB sample volumes analyzed must be
within 10% of the field sample volume.
9.6.3.1 For each organism, calculate the percent recovery (R) in the MSB using
the equation in Section 9.6.2.2.
9.6.3.2 Calculate the mean of the number of oocysts or cysts in the MS and MSB
as follows: (Xmean) = ([MS+MSB]/2).
9.6.3.3 Calculate the relative percent difference (RPB) of the recoveries using the
following equation:
RPD = 100 x
where
MSD
v
AMEAN
RPB is the relative percent difference
NMS is the number of oocysts or cysts
NMSD is the number of oocysts or cysts counted in the MSB
NMS is the number of oocysts or cysts counted in the MS
tne mean number of oocysts or cysts counted in the MS and
MSB
9.6.3.4 Compare the mean MS/MSB recovery and RPB with the acceptance
criteria in Tables 3 and 4 in Section 21.0 for each organism. Add results to
the MS control chart (Section 9.12.2).
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Method 1623.1 - Cryptosporidium and Giardia
9.7 Method blank (MB)—Method blanks serve as the negative control sample, as well as the
laboratory blank. Reagent water blanks are routinely analyzed to demonstrate the absence of
contamination throughout the analytical process. Analyze the blank immediately after analysis of
the IPRtest (Section 9.5) and OPRtest (Section 9.8) and prior to analysis of samples for the week
to demonstrate freedom from contamination. A method blank must be analyzed each week (7 day
or 168 hours time period that begins with processing the OPR) in which samples are analyzed if
20 or fewer field samples are analyzed during this period. If more than 20 samples are analyzed in
a week (7 days or 168 hours), process and analyze one reagent water method blank for every 20
samples or portions thereafter.
9.7.1 Filter, elute, concentrate, separate (purify), stain, and examine at least one reagent
water method blank per week according to the procedures in Sections 12.0 to 15.0.
9.7.2 If Cryptosporidium oocysts, Giardia cysts, or potentially interfering organisms or
materials that may be misidentified as oocysts or cysts are not found in the method
blank, the method blank test is acceptable and analysis of samples may proceed.
9.7.3 If Cryptosporidium oocysts, 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. 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.
9.7.4 Every field sample and MS must be associated with an acceptable method blank.
9.8 Ongoing precision and recovery (OPR)—The OPR serves as the positive control sample, as
well as the laboratory control sample. Using the spiking procedure in Section 11.2 and flow
cytometry-enumerated spiking suspensions (Section 7.12.1), 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 samples are analyzed to verify all performance criteria. The
laboratory must analyze one OPR sample for every 20 samples, or portions thereafter, 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 must be performed until all performance criteria are met.
9.8.1 Examine the slide from the OPR prior to analysis of samples from the same batch.
9.8.1.1 Using 200X to 400X magnification, more than 50% of the oocysts or cysts
must appear undamaged and morphologically intact; otherwise, the
organisms in the spiking suspension may be of unacceptable quality or the
analytical process may be damaging the organisms. Examine the spiking
suspension organisms directly (by centrifuging, if possible, to concentrate
the organisms in a volume that can be applied directly to a slide). If the
organisms appear undamaged and morphologically intact under DIG,
determine the step or reagent that is causing damage to the organisms.
Correct the problem and repeat the OPR test.
9.8.1.2 Identify and enumerate each organism using epifluorescence microscopy.
The first three Cryptosporidium oocysts and three Giardia cysts identified
in the OPR sample must be examined using FITC, DAPI, and DIC, as per
Section 15.2, and the detailed characteristics (size, shape, DAPI category,
and DIC category) reported on the Cryptosporidium and Giardia
examination form, as well as any additional comments on organism
appearance, if notable.
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Method 1623.1 - Cryptosporidium and Giardia
9.8.2 For each organism, calculate the percent recovery (R) using the following equation:
R = 100x-
T
where:
R = the percent recovery
N = the number of oocysts or cysts detected
T = the number of oocysts or cysts spiked
9.8.3 Compare the recovery with the acceptance criteria for ongoing precision and recovery
in Tables 3 and 4 in Section 21.0 for each organism. Add results to the OPR control
chart (Section 9.12.1).
9.8.4 If the recoveries for Cryptosporidium and Giardia meet the acceptance criteria, system
performance is acceptable and analysis of samples may proceed.
9.8.5 If the recovery for Cryptosporidium or Giardia falls outside of the criteria, system
performance is unacceptable. Analysis of additional samples must be halted until the
analytical system is brought under control. Troubleshoot the problem using the
procedures at Section 9.8.7 as a guide. After assessing the issue, perform another OPR
test and verify that Cryptosporidium and Giardia recoveries meet the acceptance
criteria.
9.8.6 Every field sample must be associated with a MS, an acceptable OPR and MB.
9.8.7 Troubleshooting—If an OPR sample has failed, and the cause of the failure is not
known, then identify the problem by working backward in the analytical process from
the microscopic examination to filtration.
9.8.7.1 Quality of spiked organisms—Examine the flow cytometry-enumerated
spiking suspension organisms (Section 7.12.1) 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, determine whether the problem is associated with
the microscope system or antibody stain (Section 9.8.7.2).
9.8.7.2 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 and DAPI. If results are unacceptable, re-
examine a previously-prepared positive staining control to determine
whether the problem is associated with the microscope or the antibody
stain.
9.8.7.3 Separation (purification) system—To determine if the failure of the OPR
test is attributable to the separation system, check system performance by
adding a flow cytometry-enumerated spiking suspension of-100 oocysts
and cysts (Section 7.12.1) along with the appropriate rinses (reagent water
or elution buffer) to a flat-sided tube and processing the sample through the
IMS, staining, and examination procedures in Sections 13.3 through 15.0.
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Method 1623.1 - Cryptosporidium and Giardia
If recoveries are less than 70%, further troubleshooting of the IMS system
may be necessary.
9.8.7.4 Filtration/elution/concentration system—If the failure of the OPRtest 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 13.2.3, and stain the oocysts in
suspension, filter through a 0.8 (im porosity, 13 mm diameter
polycarbonate filter, and enumerate by microscopy.
9.9 Staining controls—These controls are performed to confirm appropriate performance of the
immunofluorescent and DAPI staining reagents and the microscope. The controls also
demonstrate proper staining technique and the absence of contamination through the staining
process. The laboratory must prepare a positive and negative staining control (Section 14.1) each
time samples are stained. Positive and negative staining controls must be acceptable before
proceeding with sample examination.
9.9.1 Examine the negative staining control to confirm that it does not contain any
fluorescent oocysts or cysts (Section 15.0). Indicate on each sample examination form
whether the negative staining control was acceptable. If the negative staining control is
acceptable, examination of samples may proceed.
9.9.2 Examine the positive staining control(s) to confirm that it contains oocysts and cysts
with the appropriate fluorescence for FA and DAPI (Section 15.0). Indicate on each
sample examination form whether the positive staining control(s) was acceptable. If
the positive staining control is acceptable, examination of samples may proceed.
9.9.3 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 at the beginning of
each microscope examination session. FA examination must be conducted at a
minimum of 200X total magnification, DAPI examination must be conducted at a
minimum of 400X, and DIG examination and size measurements must be conducted at
a minimum of 1000X. Size, shape, and DIC and DAPI characteristics of three
Cryptosporidium oocysts and three Giardia cysts must be recorded by the analyst on a
microscope log.
9.10 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 relies upon the
ability of the analyst for identification and enumeration of oocysts and cysts. The goal of analyst
verification is to encourage comparison and discussion among analysts to continually refine the
consistency of characterizations between analysts.
9.10.1 At least monthly when microscopic examinations are being performed, the laboratory
must prepare or purchase a slide containing 40 to 200 oocysts and 40 to 200 cysts.
More than 50% of the oocysts and cysts must be DAPI positive and undamaged under
DIC.
9.10.2 Each analyst must determine the total number of oocysts and cysts detected by FITC on
the entire slide meeting the criteria in Section 9.10.1. For the same 10 oocysts and 10
cysts, each analyst must determine the DAPI category (DAPI negative, DAPI positive
internal intense blue and DAPI positive number of nuclei) and the DIC category
(empty, containing amorphous structures, or containing identifiable internal structures)
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Method 1623.1 - Cryptosporidium and Giardia
of each. The DAPI/DIC comparisons may be performed on the slide prepared or
purchased in Section 9.10.1, OPR slide, MS slide, or a positive staining control slide.
9.10.3 Requirements for laboratories with multiple analysts
9.10.3.1 The total number of oocysts and cysts determined by each analyst (Section
9.10.2) 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 ana-
lysts' examination criteria, prepare a new slide, and repeat the performance
verification (Sections 9.10.1 to 9.10.2). It is recommended that the DAPI
and DIC categorization of the same 10 oocysts and 10 cysts occur with all
analysts at the same time, i.e. each analyst determines the categorizations
independently, then the differences in the DAPI and DIC categorizations
among analysts are discussed and resolved, and these resolutions
documented. Alternatively, organism coordinates may be recorded for each
analyst to locate and categorize the organisms at different times.
Differences among analysts must be discussed and resolved.
9.10.3.2 Document the date, name(s) of analyst(s), number of total oocysts and
cysts, and DAPI and DIC categories determined by the analyst(s), whether
the test was passed / failed and the results of attempts before the test was
passed.
9.10.3.3 Only after an analyst has passed the criteria in Section 9.10.3 may oocysts
and cysts in QC samples and field samples be identified and enumerated.
9.10.4 Laboratories with only one analyst should maintain a protozoa library (Section 9.11)
and compare the results of the examinations performed in Sections 9.10.1 and 9.10.2 to
photographs of oocysts and cysts and interfering organisms to verify that examination
results are consistent with these references. This analyst must perform repetitive counts
of a single verification slide for FITC demonstrating counts within ±10% of each other.
Alternatively, laboratories may coordinate with other laboratories to share slides and
compare counts or purchase blind spiked slides from vendor.
9.11 Protozoa libraries—Each laboratory is encouraged to develop libraries of photographs and
drawings for identification of protozoa.
9.11.1 Take color photographs of Cryptosporidium oocysts and Giardia cysts by FA, DAPI,
and DIC that the analysts (Section 22.2) determine are accurate (Section 15.2).
9.11.2 Similarly, take color photographs of interfering organisms and materials by FA, DAPI,
and DIC that the analysts believe are not Cryptosporidium oocysts or Giardia cysts.
Quantify the size, shape, microscope settings, and other characteristics that can be used
to differentiate oocysts and cysts from interfering debris and that will result in accurate
identification of positive or negative organisms.
9.12 Control charts— The laboratory must maintain graphic records to define quality of OPR and MS
data generated over time. Control Charts confirm high and improving performance but also alert
laboratories of trends toward failing quality control acceptance criteria limits and the potential for
sample analysis failures when performance degrades. Failure to meet OPR quality control
acceptance criteria (Tables 3 and 4) indicates systemic problems the laboratory must address prior
to processing any samples. Repeated failure to meet the MS/MSD quality control acceptance
criteria (Tables 3 and 4), when all other QC passes, warrants corrective action from quality
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Method 1623.1 - Cryptosporidium and Giardia
conscience laboratories to systematically review control charts and modify analytical protocols to
improve matrix spike recoveries.
9.12.1 As part of the QA program for the laboratory, laboratory precision must be assessed,
records maintained, and typical control charting procedures followed. The laboratory
must add OPR results to initial and/or previous ongoing data and update the QC chart
to form a graphic representation of continued laboratory performance. The laboratory
should develop a statement of accuracy by calculating the mean percent recovery (R)
and the standard deviation of percent recovery (sr). Express the accuracy as a recovery
interval from R - 2 sr to R + 2 sr using the most recent 20 - 30 data points. For example,
if R = 95% and sr = 25%, the accuracy is 45% to 145%.
9.12.2 As part of the QA program for the laboratory, method accuracy and precision for MS
samples must be assessed and records maintained.
9.12.2.1 After the analysis of five MS samples, the laboratory must calculate the
mean percent recovery (P) and the standard deviation of the percent
recovery (sr). The precision assessment should be updated regularly (e.g.,
monthly, after each sample, etc.) across all MS samples and stratified by
MS samples for each source.
9.12.2.2 In addition the laboratory must develop a frequency distribution of MS
recoveries as a table or graph (histogram) using intervals of 10%. The
laboratory must chart their distribution of MS recoveries compared with
Table 6. Overall MS recoveries are anticipated to follow a similar pattern
as the frequency distribution of recoveries shown in Table 6.
9.13 The laboratory may routinely process an IMS control (e.g. each lot of beads, each proficiency
testing round) to monitor the recovery of the separation (purification) system (Section 9.8.7.3).
IMS control results may be added to the QC chart (Section 9.12.1) to monitor laboratory
performance.
9.14 External QC samples, such as proficiency testing or standard reference material are to be
analyzed when available. Laboratory participation in interlaboratory comparison studies using
the method is encouraged to permit laboratories to gain, maintain and demonstrate proficiency
with the method.
9.15 The specifications contained in this method can be met if the analytical system is under control.
Use of identical standards for initial (Section 9.5) and ongoing (Section 9.8) precision and
recovery samples will permit the most precise results to be obtained. The microscope in particular
will provide the most reproducible results if dedicated to the settings and conditions required for
the determination of Cryptosporidium and Giardia by this method.
9.16 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
10.1 In a room capable of being darkened to near-complete darkness, assemble the microscope, all
filters, and attachments. Place the microscope on a solid surface free from vibration. Provide
adequate workspace on either side of the microscope for taking notes and placement of slides and
ancillary materials.
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Method 1623.1 - Cryptosporidium and Giardia
10.2 Using the manuals provided with the microscope, all analysts must familiarize themselves with
operation of the microscope. Write SOPs for the specific microscopes in use and make these
available to analysts.
10.3 The microscopy portion of this procedure depends upon proper alignment and adjustment of very
sophisticated optics. Without proper alignment and adjustment, the microscope will not function
at maximal efficiency, and reliable identification and enumeration of oocysts and cysts will not be
possible. Consequently, it is imperative that all portions of the microscope from the light sources
to the oculars must be properly adjusted.
10.4 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 and Appendix B work for a particular instrument.
10.5 The sections below and Appendix B 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.
10.6 When using the microscope, corrective lenses must be worn by people with astigmatism.
WARNING: Never look at the ultraviolet (UV) light from the mercury lamp, lamp house,
or the UV image without a barrier filter in place. UV radiation can cause serious eye
damage.
10.7 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 10.7.2 assumes use of a microscope with both oculars adjustable; Section
10.7.3 assumes use of a microscope with a single adjustable ocular. The procedure must be
followed each time an analyst uses the microscope.
10.7.1 Interpupillary distance
10.7.1.1 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.
10.7.1.2 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.
10.7.2 Ocular adjustment for microscopes capable of viewing a photographic frame through
the viewing binoculars: This procedure assumes both oculars are adjustable.
10.7.2.1 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.
10.7.2.2 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 1623.1 - Cryptosporidium and Giardia
10.7.3 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.
10.7.3.1 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.
10.7.3.2 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.
10.8 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 Giardia cysts. If more than one objective is to be used for DIC,
then each time the objective is changed, Kohler illumination should be reestablished for the new
objective lens. Previous sections addressed ocular adjustment and Appendix B addressed
adjusting 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.
10.8.1 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.
10.8.2 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.
10.8.3 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.
10.8.4 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.
10.8.5 The aperture diaphragm of the condenser should now be adjusted to make it compatible
with the total numerical aperture of the optical system. This is done by removing an
ocular, looking into the tube at the rear focal plane of the objective, and stopping down
the aperture diaphragm iris leaves until they are visible just inside the rear plane of the
objective.
10.8.6 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.
10.9 Microscope cleaning procedure
10.9.1 Use canned air to remove dust from the lenses, filters, and microscope body.
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Method 1623.1 - Cryptosporidium and Giardia
10.9.2 Use a lint-free tissue 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 lint-free tissue.
10.9.3 Protocol for cleaning oculars and condenser
10.9.3.1 Use a new, clean, dust-free cotton-tipped stick dampened with MCS to
clean each lens. Start at the center of the lens and spiral the cotton tip
outward using little to no pressure. Rotate the cotton tip while spiraling to
ensure a clean surface is always contacting the lens.
10.9.3.2 Repeat the procedure using a new, dry cotton-tipped stick.
10.9.3.3 Repeat Sections 10.9.3.1 and 10.9.3.2.
10.9.3.4 Remove the ocular and repeat the cleaning procedure on the bottom lens of
the ocular.
10.9.4 Protocol for cleaning objective lenses
10.9.4.1 Wipe 100X oil objective with lens paper to remove the bulk of the oil from
the objective.
10.9.4.2 Hold a new clean, dust-free cotton-tipped stick dampened with MCS at a
45° angle on the objective and twirl.
10.9.4.3 Repeat Section 10.9.4.2 with a new, dry cotton-tipped stick.
10.9.4.4 Repeat Sections 10.9.4.2 and 10.9.4.3.
10.9.4.5 Clean all objectives whether they are used or not.
10.9.5 Protocol for cleaning light source lens and filters
10.9.5.1 Using lens paper dampened with MCS, wipe off the surface of each lens
and filter.
10.9.5.2 Repeat the procedure using dry lens paper.
10.9.5.3 Repeat Sections 10.9.5.1 and 10.9.5.2.
10.9.6 Protocol for cleaning microscope stage
10.9.6.1 Using a lint-free tissue dampened with microscope cleaning solution, wipe
off the stage and stage clip.
10.9.6.2 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.
10.9.7 Use commercial multi-surface cleaner and a paper towel to clean the bench top
surrounding the microscope.
10.9.8 Frequency: Clean microscope after every session
11.0 Sample Spiking
11.1 This method requires routine analysis of spiked QC samples to demonstrate acceptable initial and
ongoing laboratory and method performance (IPR samples [Section 9.5], MS/MSD [Section 9.6],
OPR samples [Section 9.8], and IMS controls [Section 9.13]). The organisms used for these
samples must be enumerated to calculate recoveries and precision, and monitor method
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Method 1623.1 - Cryptosporidium and Giardia
performance. Laboratories must use flow cytometry-enumerated spiking suspensions, rather than
manually enumerated suspensions. Guidance on preparing spiking suspensions using a flow
cytometer is provided in Appendix C. The procedure for spiking bulk samples in the laboratory is
provided in Section 11.2.
11.2 Procedure for spiking samples in the laboratory with flow cytometry-enumerated spiking
suspensions—Three optional spiking suspensions have been demonstrated to be acceptable for
use with Method 1623.1: 1) Wisconsin State Laboratory of Hygiene, 2) BTF EasySeed™, and 3)
Waterborne AccuSpike™-IR.
11.2.1 Arrange a disposable cubitainer or bottom-dispensing container with a spigot to feed
the filter or insert a pipette connected to the influent end of the tube attached to the
filter through the top of a carboy to allow siphoning of the sample.
11.2.2 For initial precision and recovery (Section 9.5) and ongoing precision and recovery
(Section 9.8) 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 MS samples (Section 9.6), 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).
11.2.3 Follow the procedures in Section 11.2.3.1 or manufacturer's instructions for flow
cytometer-enumerated suspensions. Adjust volume of antifoam and rinses in
proportion to volume of spiking suspension; instructions below assume ~45 mL of
spiking suspension in 50-mL tube.
11.2.3.1 Add 400 (iL of Antifoam A to 100 mL of reagent water, and mix well to
emulsify.
11.2.3.2 Add 500 (iL of the diluted antifoam to the tube containing the spiking
suspension and vortex for 30 seconds.
11.2.3.3 Pour the suspension into the sample container.
11.2.3.4 Add 20 mL of reagent water to the empty tube, cap, vortex 10 seconds to
rinse, and add the rinsate to the carboy.
11.2.3.5 Repeat this rinse using another 20 mL of reagent water.
11.2.3.6 Record the estimated number of organisms spiked, the date and time the
sample was spiked, and the sample volume spiked on a bench sheet.
11.2.4 Allow the spiked sample to mix for approximately 1 minute in the container.
11.2.5 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.
11.2.6 When the water level approaches the discharge port of the carboy, tilt the container so
that it is completely emptied. At that time, turn off the pump and add 1 L of reagent
water to the 10-L carboy to rinse (5 L of reagent water rinse to 50-L carboy). Swirl the
contents to rinse down the sides. Additional rinses may be performed.
11.2.7 Turn on the pump. Allow all of the water to flow through the filter and turn off the
pump.
11.2.8 Proceed to filter disassembly.
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Method 1623.1 - Cryptosporidium and Giardia
12.0 Sample Filtration and Elution
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).
12.1 Three optional filtration/elution procedures have been demonstrated to be acceptable for
use with Method 1623.1—1) Envirochek® HV Sampling Capsule (Section 12.2), 2) Filta-Max®
Foam Filter (Section 12.3), and 3) Portable Continuous Flow Centrifuge (PCFC, Section 12.4)
(Table 1). Laboratories may use a different optional procedure if the laboratory first demonstrates
that the optional procedure provides equivalent or superior performance per Section 9.2. Alternate
procedures and products may be added to the optional procedures in Table 1 only after
demonstrating equivalent or superior performance through a multi-laboratory validation study
using multiple matrices, as per Reference 20.1.
12.2 Filtration/Elution Option 1—Capsule filtration using the Envirochek® F£V (adapted from
Reference 20.11). This procedure was validated using 50-L sample volumes and historically with
10-L sample volumes. Alternate sample volumes may be used, provided the laboratory
demonstrates equivalent or superior performance through an IDC per Section 9.2.
12.2.1 Flow rate adj ustment
12.2.1.1 Connect the sampling system, minus the capsule, to a carboy filled with
reagent water (Figure 1).
12.2.1.2 Turn on the pump and adjust the flow rate to 2.0 L/min.
12.2.1.3 Allow 2 to 10 L of reagent water to flush the system. Adjust the pump
speed as required during this period. Turn off the pump when the flow rate
has been adjusted.
12.2.2 Install the capsule filter in the line, securing the inlet and outlet ends with the
appropriate clamps/fittings.
12.2.3 Record the sample number, sample turbidity (if not provided with the field sample),
sample type, sample filtration start date and time, and name of analyst filtering the
sample on a bench sheet.
12.2.4 Filtration
12.2.4.1 Mix the sample well by shaking, add stir bar and place on stir plate. Turn
on stir plate to lowest setting needed to keep sample thoroughly mixed.
Connect the sampling system to the field carboy of sample water, or
transfer the sample water to the laboratory carboy used in Section 12.2.1.1.
If the sample will be filtered from a field carboy, a spigot (Section 6.2.1)
can be used with the carboy to facilitate sample filtration.
NOTE: If the bulk field sample is transferred to a laboratory carboy, the laboratory
carboy must be cleaned and disinfected before it is used with another field sample.
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 6.16). This container will be used to determine the
January 2012
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Method 1623.1 - Cryptosporidium and Giardia
sample volume filtered. Alternately, determine the volume by weight or
connect a flow meter (Section 6.2.5) 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 the entire sample has passed through the filter, turn off the pump and
stir plate. Allow the pressure to decrease until flow stops. (If the sample
was filtered in the field, and excess sample remains in the filter capsule
upon receipt in the laboratory, pull the remaining sample volume through
the filter before eluting the filter [Section 12.2.6].)
12.2.4.5 Based on the water level in the graduated container and !/2-L hash marks,
the volume determined by weight, or meter reading, record the volume
filtered on the bench sheet to the nearest quarter liter. Discard the contents
of the graduated container.
12.2.4.6 Add 1 L reagent water rinse (to 10-L carboy) or 5 L reagent water rinse (to
50-L carboy). Swirl or shake the carboy to rinse down the side walls.
12.2.4.7 Reconnect to pump, turn on pump and allow pump to pull all water
through filter; turn off pump.
12.2.5 Di sassembly
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. If needed,
restart the pump and allow as much water to drain as possible. Turn off the
pump.
12.2.5.2 Loosen the outlet fitting, then cap the inlet and outlet fittings.
12.2.6 Elution Setup
NOTE: The laboratory must complete the elution, concentration, and purification
(Sections 12.2.7 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 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.
12.2.6.2 Prepare sufficient quantity of elution buffer to elute all samples that are
associated with the OPR/MB which used that batch of elution buffer.
Elution may require up to 275 mL of buffer per sample.
12.2.6.3 Designate at least one 250-mL conical centrifuge tube for each sample and
label with the sample number.
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Method 1623.1 - Cryptosporidium and Giardia
12.2.6.4 Prepare sufficient quantity of 5% sodium hexametaphosphate (NaHMP)
solution to pre-treat all of the designated filters associated with the
OPR/MB used for that batch of elution buffer. Pre-treatment may require
up to 150 mL of NaHMP per sample.
12.2.7 Di spersant Addition
12.2.7.1 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.
NOTE: Dispersant Addition cannot be performed on a sampling capsule through which water
can no longer be filtered (i.e. clogged). Record on the bench sheet that the sampling capsule is
clogged, and proceed to Section 12.2.8.2.
12.2.7.2 Remove the inlet cap, pour NaHMP solution through the inlet opening, and
allow the liquid level to stabilize. Sufficient NaHMP solution must be
added to cover the pleated white membrane with NaHMP solution or
NaHMP solution may be measured to 125 mL. Replace the inlet cap.
12.2.7.3 Securely clamp the capsule in one of the clamps on the laboratory shaker
with the bleed valve positioned at the top on a vertical axis (in the 12
o'clock position). Turn on the shaker and set the speed to maximum (700 -
900 rpm or per manufacturer's instructions). Agitate the capsule for
approximately 5 minutes. Time the agitation using a lab timer, rather than
the timer on the shaker to ensure accurate time measurement.
12.2.7'.4 Remove the filter from the shaker, remove the outlet cap, and attach the
capsule filter outlet to tubing, upstream of a pump. Holding the filter
upright, remove the inlet cap, being careful not to pour any liquid from the
inlet, turn on the pump and allow pump to pull all the NaHMP through the
filter, turn off pump. Do not allow the filter pleats to collapse during the
pumping process.
12.2.7.5 Fill the capsule with reagent water, pinching the outlet hose if necessary, to
cover the white pleated membrane and the plastic above the membrane;
allow the liquid level to stabilize. Sufficient reagent water must be added to
cover the pleated white membrane. Turn on the pump and allow pump to
pull all the water through the filter. Turn off the pump.
12.2.7.6 Replace the inlet cap. Disconnect the outlet tubing from the filter, and
replace the outlet cap. Proceed directly to elution within the same working
day.
12.2.8 Elution
12.2.8.1 Using a ring stand or other means, clamp each capsule in a vertical position
with the inlet end up.
12.2.8.2 Remove the inlet cap, pour elution buffer through the inlet fitting, and
allow the liquid level to stabilize. Sufficient elution buffer must be added
to cover the pleated white membrane with buffer solution or elution buffer
may be measured to ensure the use of one 250-mL centrifuge tube. Replace
the inlet cap.
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Method 1623.1 - Cryptosporidium and Giardia
12.2.8.3 Securely clamp the capsule in one of the clamps on the laboratory shaker
with the bleed valve positioned at the top on a vertical axis (in the 12
o'clock position). Turn on the shaker and set the speed to maximum
(approximately 700 - 900 rpm or per manufacturer's instructions). Agitate
the capsule for approximately 5 minutes. Time the agitation using a lab
timer, rather than the timer on the shaker to ensure accurate time
measurement.
12.2.8.4 Remove the filter from the shaker, remove the inlet cap, and pour the
contents of the capsule into the 250-mL conical centrifuge tube.
12.2.8.5 Clamp the capsule vertically with the inlet end up and add sufficient
volume of elution buffer through the inlet fitting to cover the pleated
membrane. Replace the inlet cap.
12.2.8.6 Return the capsule to the shaker with the bleed valve positioned at the 4
o'clock position. Turn on the shaker and agitate the capsule for
approximately 5 minutes.
12.2.8.7 Leaving the elution buffer in the capsule, re-align the capsule in the shaker
with the bleed valve now at the 8 o'clock position. Turn on the shaker and
agitate the capsule for a final 5 minutes.
12.2.8.8 Remove the filter from the shaker, remove the inlet cap 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
with a disposable tip or a serological pipette 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. Replace the inlet cap.
Manually swing the filter capsule through an arc of-180° to retrieve more
of the eluate from the filter.
12.2.9 Proceed to Section 13.0 for concentration and separation (purification).
12.3 Filtration/Elution Option 2—Sample filtration using the Filta-Max® foam filter. This procedure
was validated using 50-L sample volumes and historically with 10-L sample volumes. Alternate
sample volumes may be used, provided the laboratory demonstrates equivalent or superior
performance through an IDC per Section 9.2.
NOTE: The filtration procedures specified in Sections 12.3.1.2 - 12.3.1.6.2 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 Preparation
12.3.1.1 Flow rate adjustment
12.3.1.1.1 Connect the sampling system, minus the filter housing, to a
carboy filled with reagent water (Figure 2).
12.3.1.1.2 Place the peristaltic pump upstream of the filter housing.
12.3.1.1.3 Turn on the pump and adjust the flow rate to 1 to 4 L per
minute.
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Method 1623.1 - Cryptosporidium and Giardia
NOTE: A head pressure of 0.5 bar (7.5 psi) is required to create flow through the filter,
and the recommended pressure of 5 bar (75 psi) should produce the flow rate of 3 to 4 L
per minute. The maximum operating pressure of 8 bar (120 psi) should not be exceeded.
12.3.1.1.4 Allow 2 to 10 L of reagent water to flush the system. Adjust
the pump speed as necessary during this period. Turn off the
pump when the flow rate has been adjusted.
12.3.1.2 Place filter module into the filter housing bolt head down and secure lid,
hand tighten housings, apply gentle pressure to create the seal between the
module and the 'O' rings in the base and the lid of the housing. Excessive
tightening is not necessary, and may shorten the life of the 'O' rings. Tools
may be used to tighten housing to the alignment marks (refer to
manufacturer's instructions). Lightly grease the 'O' rings 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), sample type, sample filtration start date and time, and the name of
the analyst filtering the sample on a bench sheet.
12.3.1.5 Filtration
NOTE: If the bulk fleld sample is transferred to a laboratory carboy, the laboratory
carboy must be cleaned and disinfected before it is used with another field sample.
12.3.1.5.1 Mix the sample well by shaking, add stir bar and place on stir
plate. Turn on stir plate to lowest setting needed to keep
sample thoroughly mixed. Connect the sampling system to the
field carboy of sample water, or transfer the sample water to
the laboratory carboy used in Section 12.3.1.1.1. If the sample
will be filtered from a field carboy, a spigot can be used with
the carboy to facilitate sample filtration.
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.
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Method 1623.1 - Cryptosporidium and Giardia
12.3.1.5.4 After the entire sample has passed through the filter, turn off
the pump and stir plate. Allow the pressure to decrease until
flow stops.
12.3.1.5.5 Based on the water level in the graduated container and 1A -L
marks, the volume determined by weight, or the meter reading,
record the volume filtered on a bench sheet to the nearest
quarter liter. Discard the contents of the graduated container.
12.3.1.5.6 Add 1 L reagent water rinse (to 10-L carboy) or 5 L reagent
water to rinse (to 50-L carboy). Swirl or shake the carboy to
rinse down the side walls.
12.3.1.5.7 Reconnect to pump, turn on pump and allow pump to pull all
water through filter; turn off pump.
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. If needed, restart the pump and allow
as much water to drain as possible. Turn off the pump.
12.3.1.6.2 Loosen the outlet fitting and seal the filter housing with rubber
plugs.
NOTE: Prevent filters 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 using the Filta-Max®
wash station (manual or automatic), which moves a plunger up and down a
tube containing the filter and eluting solution (Section 12.3.2.2). If the
Filta-Max® automatic wash station is used please see the manufacturer's
operator's guide for instructions on its use. If Filta-Max® Quick Connect
kit is used please follow manufacturer's instructions. Record the elution
date and time on the bench sheet.
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 concentra-
tor 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.
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Method 1623.1 - Cryptosporidium and Giardia
(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 trans-
portation 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) will "click" when the plunger is
correctly locked in position.
(f) Remove the filter module bolt by turning the adapted
alien key (provided) in a clockwise direction (as seen
from above). Attach the steel tube to the elution tube
base.
(g) Add 600 mL of PBST to the assembled concentrator. If
more than 50 mL of liquid has been recovered from the
shipped filter module, reduce the volume of PBST
accordingly. Screw the concentrator tube onto the base
beneath the elution tube. Release the locking pin.
NOTE: When functioning correctly, the pin is easily released by gentle pressure on the
lever, coupled with a pulling action on the locking pin.
(h) Wash the foam disks by moving the plunger up and down
20 times. Gentle movements of the plunger are recom-
mended 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, concentrate the eluate from the
first wash using the Filta-Max® apparatus according to
Section 12.3.3.2.1.
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Method 1623.1 - Cryptosporidium and Giardia
12.3.2.2.2 Second wash
(a) Add an additional 600 mL of PBST to the concentrator
module, remove the plug from the end of the steel tube
and screw the concentrator tube back onto the elution
module base. Release the locking pin.
(b) Wash the foam disks by moving the plunger up and down
10 times. Gentle movements of the plunger are
recommended to avoid generating excess foam.
(c) Concentrate the eluate using the Filta-Max® apparatus
according to Section 12.3.3.2.2.
12.3.3 Concentration
12.3.3.1 The eluate is 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.
12.3.3.2 The Filta-Max® concentrator procedure
12.3.3.2.1 Concentration of first wash
(a) 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 cm Hg (11.8 in Hg).
(b) 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
reagent 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) Add the concentrate, in the 50-mL tube, retained from the
first concentration (Section 12.3.3.2.1 (b)) to the 600 mL
of eluate from the second wash, then repeat concentration
steps in Section 12.3.3.2.1. 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.
(b) Remove the magnetic stirrer. Insert the empty
concentrator module into the jaws of the wash station and
twist off the concentrator tube.
(c) Transfer the membrane from the concentrator base to the
bag provided using membrane forceps.
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Method 1623.1 - Cryptosporidium and Giardia
12.3.3.2.3 Membrane elution
(a) 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.
(b) Using a pipette, transfer the eluate to a 50-mL tube
(c) Repeat the membrane wash with another 5 mL of PBST
and transfer the eluate to the 50-mL tube. (Optional:
Perform a third wash using another 5 mL of PBST, by
hand-kneading an additional minute or placing the bag on
a flat-headed vortexer and vortexing for one minute.
Transfer the eluate to the 50-mL tube.)
NOTE: Mark the bag with an "X" to note which side of the membrane has the oocysts to
encourage the hand-kneading to focus on the appropriate side of the membrane.
12.3.3.2.4 If the membrane filter clogs before concentration is complete,
replace the membrane as often as necessary. Filter membranes
may be placed smooth side up during the second concentration
step. Separate 50-mL tubes may be used for the eluate of each
membrane or the eluate from multiple membranes may be
combined into one 50-mL tube.
(a) Disassemble the concentrator tube and pour any
remaining eluate back into a pooling beaker. Remove the
membrane using membrane forceps, placing it in the bag
provided. Place a new membrane in the concentrator tube
smooth side up, reassemble, return the eluate to the
concentrator tube, rinse the pooling beaker and add rinse
to the eluate, and continue the concentration. Replace the
membrane as often as necessary.
12.3.3.2.5 If multiple 50-mL tubes have been used, the sample can be
further concentrated by centrifugation or each 50-mL tube may
be processed separately following Section 13.0.
(a) Make sure that the centrifuge tubes are balanced.
Centrifuge the tubes containing the eluate at 1500 x G for
15 minutes. Allow the centrifuge to coast to a stop.
(b) 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.
(c) Vortex each 50-mL tube vigorously until pellet is
completely re suspended. Swirl the centrifuge tube gently
to reduce any foaming after vortexing. Combine the
contents of each 50-mL centrifuge tube into one 50-mL
January 2012
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Method 1623.1 - Cryptosporidium and Giardia
centrifuge tube. Rinse each of the 50-mL centrifuge tubes
with PBST and add the rinse to the final 50-mL
centrifuge tube.
12.3.3.3 Proceed to Section 13.0 for centrifugation and separation (purification) of
the eluate.
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. Re-lubricate all O-rings. 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 reassemble the plunger head.
12.4 Filtration/Elution Option 3—Sample collection (filtration and concentration) using portable
continuous-flow centrifugation. Please follow manufacturer's instructions. This procedure was
validated for the detection of Cryptosporidium using 50-L sample volumes and historically for
the detection of Cryptosporidium and Giardia using 10-L sample volumes. Alternate sample
volumes may be used, provided the laboratory demonstrates equivalent or superior performance
through IDC per Section 9.2.
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 IMS. Optional
procedures listed in Table 1 may be used if the laboratory first demonstrates that the optional
procedure provides equivalent or superior performance per Section 9.2. Alternate procedures and
products may be added to the optional procedures in Table 1 only after demonstrating equivalent
or superior performance through a multi-laboratory validation study using multiple matrices, as
per Reference 20.1.
13.2 Adjustment of pellet volume
13.2.1 Balance the tubes to within 0.5 g of each other prior to centrifugation and/or use a self-
balancing rotor. Centrifuge the 250-mL (or 50-mL) centrifuge tube containing the
January 2012
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Method 1623.1 - Cryptosporidium and Giardia
eluate at a minimum of 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: Historical demonstration at multiple laboratories indicates that recoveries may be
improved if centrifugation force is increased to 1800-2000 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 If the packed pellet volume is < 0.5 mL, using a pipette, carefully aspirate the
supernatant to 5 mL above the pellet. Aspirate at the air/water interface from the center
of the tube using gentle and steady low vacuum pressure (e.g., <5 in. Hg vacuum).
Vacuum pressure may be reduced when 30 mL of supernatant remains. 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).
NOTE: Recoveries may be improved if aspiration is performed using a Pasteur pipette or
serological pipette with an internal diameter of orifice ranging from 1.2 to 1.5 mm.
13.2.2.1 Vortex the tube vigorously for 10-15 seconds and/or pipette mix until
pellet is completely resuspended. Swirl the centrifuge tube gently to reduce
any foaming after vortexing. Record the resuspended pellet volume,
volume transferred to IMS, and the number of any subsamples on the
bench sheet. Proceed to Section 13.3. Be sure pellet is completely
homogenized immediately before transfer. Visually inspect to ensure
complete homogenization and lack of debris aggregates. This is
particularly important for samples with high clay content.
NOTE: 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.
13.2.3 If the packed pellet volume is > 0.5 mL, the concentrate must be separated into
multiple subsamples: a subsample is equivalent to no greater than 0.5 mL of packed
pellet material. No more than 0.5 mL of pellet must be processed at a time. Aspirate the
supernatant from the centrifuge tube leaving 5 mL of fluid for every 0.5 mL of pellet or
portion of 0.5 mL of pellet. For example, if the packed pellet volume is 1.2 mL, the
total volume required is 15 mL. Aspirate at the air/water interface from the center of
the tube using gentle and steady low vacuum pressure (e.g., <5 in. Hg vacuum).
Vacuum pressure may be reduced when 30 mL of supernatant remains. Use the
following formula to determine the total volume required in the centrifuge tube before
separating the concentrate into two or more subsamples, rounding the result up to the
nearest multiple of 5:
pellet volume
total volume (ml) required = x 5 ml
0.5 ml
Record total volume in centrifuge tube, which includes the pellet and supernatant, on
the bench sheet.
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Method 1623.1 - Cryptosporidium and Giardia
NOTE: 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.
13.2.3.1 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:
13.2.3.1.1 Calculate number of subsamples: Divide the total volume in
the centrifuge tube by 5 mL. Record the number of subsamples
on the bench sheet.
13.2.3.1.2 Process subsamples through IMS. Vortex the tube
vigorously for 10 to 15 seconds and/or pipette mix to
completely resuspend the pellet. 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 all flat-sided tubes on
the bench sheet. Be sure pellet is completely homogenized
immediately before transfer. Visually inspect to ensure
complete homogenization and lack of debris aggregates. This
is particularly important for samples with high clay content.
13.2.3.2 Analysis of partial sample. If not all of the concentrate will be examined,
vortex the tube vigorously for 10 to 15 seconds and/or pipette mix to
completely resuspend the pellet. 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 all flat-sided tubes and the number
of subsamples on the bench sheet. Be sure pellet is completely
homogenized immediately before transfer. Visually inspect to ensure
complete homogenization and lack of debris aggregates. This is
particularly important for samples with high clay content. 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 13.2.3
Then multiply the volume filtered (Section 12.2.4.5 or 12.3.1.5.5) by this
percentage to determine the volume analyzed.
January 2012
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Method 1623.1 - Cryptosporidium and Giardia
13.3 IMS procedure (adapted from Reference 20.12)
NOTE: The IMS procedure should be performed with samples and IMS buffers at room
temperature, ranging from 15°C to 25 °C.
13.3.1 Preparation and addition of reagents
13.3.1.1 Prepare a IX dilution of SL-buffer-A from the 10X SL-buffer-A (clear,
colorless solution) supplied. Use reagent water (demineralized;
Section 7.3) as the diluent. For every 1 mL of IX SL-buffer-A required,
mix 100 (iL of 10X SL-buffer-A and 0.9 mL diluent water. A volume of
1.5 mL of IX SL-buffer-A will be required per sample or subsample on
which the Dynal IMS procedure is performed.
13.3.1.2 For each 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 6.3.4).
13.3.1.3 For each sample or subsample, add 1 mL of the 10X SL-buffer-B
(supplied— magenta solution) to the flat-sided tube containing the 10X
SL-buffer-A.
NOTE: The volumes of IMS reagents listed above are a requirement for this method.
Changes in reagent concentrations and volumes are not allowed.
13.3.2 Oocyst and cyst capture
13.3.2.1 Use a graduated, 5- or 10-mL pipette that has been pre-rinsed with elution
buffer to measure and 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). Rinse twice with half the volume needed to bring the total
volume in the flat-sided sample tube to 12 mL (including the buffers added
in Sections 13.3.1.2 and 13.3.1.3). (For example, if the tube contained 1
mL of SL-buffer-A and 1 mL of SL-buffer-B, and 5 mL of sample was
transferred after resuspension of the pellet, for a total of 7 mL, the
centrifuge tube would be rinsed twice with 2.5 mL of reagent water to
bring the total volume in the flat-sided tube to 12 mL.) Visually inspect the
centrifuge tube after completing the transfer to ensure that no concentrate
remains. If multiple subsamples will be processed, bring the volume in the
remaining flat-sided tubes to 12 mL with reagent water. Label the flat-
sided tube(s) with the sample number (and subsample letters).
13.3.2.2 Vortex the Dynabeads®Crypto-Combo vial from the IMS kit for
approximately 10 seconds to suspend the beads. Ensure that the beads are
fully resuspended by tilting the sample tube and making sure that there is
no residual pellet at the bottom.
13.3.2.3 Add 100 (iL of the resuspended DynabeadsDCrypto-Combo (Section
13.3.2.2) to the sample tube(s) containing the water sample concentrate and
SL-buffers.
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Method 1623.1 - Cryptosporidium and Giardia
13.3.2.4 Vortex the Dynabeads®G/'ara?/'a-Combo vial from the IMS kit for
approximately 10 seconds to suspend the beads. Ensure that the beads are
fully resuspended by tilting the tube and making sure that there is no
residual pellet at the bottom.
13.3.2.5 Add 100 (iL of the resuspended Dynabeads®G/ara?/a-Combo (Section
13.3.2.4) to the sample tube(s) containing the water sample concentrate,
Dynabeads®Crypto-Combo, and SL-buffers.
13.3.2.6 Affix the sample tube(s) to a rotating mixer and rotate at approximately 18
rpm for 1 hour at room temperature.
13.3.2.7 After rotating for 1 hour, remove each sample tube from the mixer and
place the tube in the magnetic particle concentrator (MPC®-1 or MPC®-6)
with flat side of the tube toward the magnet.
13.3.2.8 Make sure that the tube is snug and flat against the magnet; push the tube
flat and hold there if necessary.
13.3.2.9 Gently rock the sample tube by hand end-to-end through approximately
90° (180° for MPC®-6), tilting the cap-end and base-end of the tube up
and down in turn. Continue the tilting action for 2 minutes with
approximately one tilt per second.
13.3.2.10 Ensure that the tilting action is continued throughout this period to prevent
binding of low-mass, magnetic or magnetizable material. If the sample in
the MPC®-1 or MPC®-6 is allowed to stand motionless for more than 10
seconds, remove the flat-sided tube from the MPC®-1 or MPC®-6, gently
resuspend all material, replace the sample tube in the MPC®-1 or MPC®-6
and repeat Section 13.3.2.9 before continuing to Section 13.3.2.11.
13.3.2.11 Return the MPC®-1 or MPC®-6 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 or MPC®-6 into a suitable container. Allow more supernatant
to settle; aspirate additional supernatant with Pasteur pipette. Do not shake
the tube and do not remove the tube from MPC®-1 or MPC®-6 during this
step. Use a clean, lint-free tissue to blot the end of flat-sided tube after
decanting to remove more matrix debris. With the MPC®-6, the
supernatant may be decanted from 1 to 3 flat-sided tubes at one time;
maximum 3 tubes at once. Rock each side through a 90° angle 3 times
before decanting the remaining tubes.
13.3.2.12 Remove the sample tube from the MPC®-1 or MPC®-6 and resuspend the
sample in 0.5 mL IX SL-buffer-A (prepared from 10X SL-buffer-A
stock—supplied). Add IX SL Buffer A directly to the flat side of the tube;
avoid any debris present on the round side of the tube. Mix very gently
(using a 1.0 mL pipette) to resuspend bead pellet in the tube; release the
liquid down the flat side of the tube to further rinse the tube. Do not vortex.
13.3.2.13 Place a labeled, 1.5-mL microcentrifuge tube into the second magnetic
particle concentrator, MPC®-S, with its magnetic strip in the vertical
(back) position.
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Method 1623.1 - Cryptosporidium and Giardia
13.3.2.14 Quantitatively transfer (transfer followed by two rinses) all the liquid from
the sample tube to the labeled, 1.5-mL microcentrifuge tube in the MPC®-
S. Use 0.5 mL of IX SL-buffer-A to perform the first rinse and 0.5 mL of
IX SL-buffer-A for the second rinse. Mix each rinse gently (using a 1.0
mL pipette) and release the liquid down the flat side of the tube to further
rinse the flat side of the tube. Avoid transferring debris present on the
round side of the 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.
13.3.2.15 Without removing the microcentrifuge tube from MPC®-S, gently
rock/roll the tube through 180° by hand. Continue for approximately 1
minute with approximately one 180° roll/rock per second. The magnet is
rocked 180° in one second in one direction and then rocked back the
following second. At the end of this step, the beads will produce a distinct
brown dot at the back of the tube. If this brown dot is not visible, check
the magnet and microcentrifuge tube placement in the MPC®-S to assure
that it has been assembled correctly and repeat this step.
13.3.2.16 Immediately aspirate the supernatant from the tube and cap held in the
MPC®-S. 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®-S
while conducting these steps.
13.3.2.17 Rinse the beads inside the microcentrifuge tube held in the MPC®-S.
13.3.2.17.1 Gently add 1.0 mL of IX PBS to the tube. Take care not to
disturb the bead pellet attached to the wall of the tube
adjacent to the magnet.
13.3.2.17.2 Remove the magnet from the MPC®-S and gently rock the
sample 8-10 times 180° until the beads are resuspended.
13.3.2.17.3 Replace the magnetic strip in the vertical (back) position in
the MPC®-S.
13.3.2.17.4 Repeat Sections 13.3.2.15 and 13.3.2.16.
13.3.2.18 Let the tube stand undisturbed for 1 minute, allowing any residual liquid to
flow to the bottom of the tube. Using a Pasteur pipette, gently mix
residual liquid/debris and aspirate. Do not shake the tube. Do not remove
the tube from MPC®-S 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®-S.
13.3.3.2 Add 50 (iL of 0.1 N HC1, then vortex at the highest setting for
approximately 50 seconds.
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Method 1623.1 - Cryptosporidium and Giardia
NOTE: The laboratory must use 0.1-N standards purchased directly from a vendor,
rather than adjusting the normality in-house.
13.3.3.3 Keep the tube in the MPC®-S without the magnetic strip in place and
allow it 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 the entire sample is at the base of the tube. Place the
microcentrifuge tube in the MPC®-S.
13.3.3.6 Replace magnetic strip in the slanted (front) position of the MPC®-S 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. See Section
13.4.5 for suggestions for dirty samples.
13.3.3.8 Add 5 (iL of 1.0 N NaOH to two sample wells on same or separate well
slides (add 10 \\L to one sample well if the volume from the two required
dissociations will be added to the same well).
NOTE: The laboratory must use 1.0-N standards purchased directly from a vendor
rather than adjusting the normality in-house.
13.3.3.9 Without removing the microcentrifuge tube from the MPC®-S, transfer the
entire sample from the microcentrifuge tube in the MPC®-S to the sample
well with the NaOH. Do not disturb the beads at the back wall of the tube.
Ensure that all of the fluid is transferred.
13.3.3.10 Do not discard the beads or microcentrifuge tube after transferring the
volume from the first acid dissociation to the well slide. Perform the steps
in Sections 13.3.3.1 through 13.3.3.9 a second time. The volume from the
second dissociation can be added to the well containing the volume from
the first dissociation, or can be applied to a second well on the same slide
or a separate slide.
13.3.3.11 Record the date and time the purified sample was applied to the well
slide(s).
13.3.3.12 Air-dry the sample on the well slide(s). Because temperature and humidity
vary from laboratory to laboratory, no minimum time is specified.
However, the laboratory must take care to ensure that the sample has dried
completely and to stain manufacturers' instructions before staining to
prevent losses during the rinse steps. A slide warmer set at 35°C to 42°C
also can be used.
13.4 Additional IMS techniques for use with complex samples.
13.4.1 Removing Magnetic Materials - Some source water samples can contain high
concentrations of iron and other magnetic material that may interfere with IMS. Some
laboratories have determined the removal of extraneous magnetic material from the
sample, prior to the addition of beads, can improve recoveries. The sample is processed
through filtration, elution, and concentration then extraneous magnetic material is
January 2012
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Method 1623.1 - Cryptosporidium and Giardia
removed. The remaining sample is carried through the IMS process. Process sample(s)
according to Method 1623.1 through Section 13.2.3.2; then proceed at 13.3.1 with the
following substitutions and additions:
13.3.1 Preparation and addition of reagents
13.3.1.1 Same as method
13.3.1.2 Same as method
13.3.1.3 Same as method with following steps added:
13.3.1.3.1 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 another
flat-sided tube(s) not containing IMS 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). Rinse with two
rinses each of which is comprised of half the volume
needed to bring the total volume in the flat-sided
sample tube to 8 mL. (For example, if 5 mL of
sample was transferred after resuspension of the
pellet, 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 8 mL.) Visually
inspect the centrifuge tube after completing the
transfer to ensure that no concentrate remains. If
multiple subsamples will be processed, bring the
volume in the remaining flat-sided tubes to 8 mL
with reagent water. Label the flat-sided tube(s) with
the sample number (and subsample letters).
13.3.1.3.2 Place the flat-sided tube in the MPC®-1 or MPC®-
6; check the tube is tight against the magnet.
13.3.1.3.3 Rock the magnet and tube gently and smoothly
through a 90° angle for 2 minutes with
approximately one 90° rock per second. Ensure the
tilting action is continued throughout the 2 minute
period.
13.3.1.3.4 If the sample is allowed to stand motionless for more
than 10 seconds, remove the tube from the magnet,
shake to resuspend all materials, replace the sample
tube in the magnet, and repeat the 2 minute rocking.
13.3.1.3.5 Return tube to upright position and immediately
remove the cap.
13.3.1.3.6 Keeping the flat side of tube on top, pour off
supernatant into another flat-sided tube containing
Buffers A and B (Section 13.3.1.3). Without
January 2012
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Method 1623.1 - Cryptosporidium and Giardia
removing the tube from the MFC, rinse the round
side of tube twice with 1 mL of reagent water. This
tube now contains 12 mL of the sample and buffers
ready to continue through the IMS process. Label
this flat-sided tube(s) with the sample number (and
subsample letters).
13.3.2 Oocyst and cyst capture
13.3.2.1 Omit this step
13.3.2.2 Same as method, to completion of the method.
NOTE: The flat-sided tube remaining in the magnet may contain extraneous iron and
other magnetic material removed by the magnet. This extraneous material may be
discarded as waste and the tube either discarded or cleaned for reuse per laboratory
SOP.
13.4.2 Adjusting pH - Some source water samples may produce a pellet with non-neutral pH
characteristics. Low recoveries could result if acidity or alkalinity of the pellet is not
adequately buffered during the IMS process. Some laboratories have determined the
addition of HC1 or NaOH to neutralize the sample, after buffers have been added, can
improve recoveries. Process sample(s) according to Method 1623.1 through Section
13.3.2.1, transferring all sample(s) to flat-sided tube(s) with buffers; then proceed with
the following additions:
13.3.2.1 Same as method
13.3.2.1.1 Gently mix the buffers with the transferred sample
by inverting the flat-sided tube 3 times.
13.3.2.1.2 Read and record the pH of the suspension.
13.3.2.1.3 Adjust the pH of the suspension with IN HC1 or
IN NaOH as needed to establish pH = 7.
13.3.2.2 Same as method, to completion of the method.
NOTE: The pH of the sample could also be checked and adjusted before buffers are
added (Sections 13.2.2.1, 13.2.3.1.2, or 13.2.3.2), and after the 1-hour rotation (Section
13.3.2.7) to ensure the pH is stable. Some laboratories have used IQ Scientific
Instruments-handheld pH meter with Micro Probe (PHI 7-SS) to check the pH in the flat-
sided tube. Alternative pH measurement techniques may be used. Ensure technique used
prevents cross-contamination of samples.
13.4.3 Additional Rinse of Flat-sided Tube - When source water pellets contain visible
excess debris after aspiration of the supernatant (Section 13.3.2.11), debris carryover
may interfere with recoveries and an additional rinse of the flat-sided tube may be
performed. Process sample(s) according to Method 1623.1 through Section 13.3.2.11,
remove supernatant from flat-sided tube; then proceed with the following additions:
13.3.2.11 Same as method with following steps added:
13.3.2.11.1 Orient the tube to almost horizontal with bead
pellet and magnet on top.
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Method 1623.1 - Cryptosporidium and Giardia
13.3.2.11.2 Gently add between 2.5 and 10 mL of reagent
water, PBS to the rounded side of the tube
opposite the beads; do not disturb the bead pellet.
13.3.2.11.3 Gently tip the rinse solution over the side of the
tube opposite of the bead pellet, three times.
13.3.2.11.4 Decant while continuing to keep magnet and flat-
side of tube up.
13.3.2.12 Same as method, to completion of the method.
13.4.4 Heat Dissociation - The addition of acid to some source water matrices for
dissociation may drive chemical reactions that interfere with the method, e.g., bead
clumping. Heat can be used instead of acid to inhibit reformation of the bead oo/cyst
complexes and potentially improve recoveries. Process sample(s) according to Method
1623.1 through Section 13.3.3.1; then revise 13.3.3.2 through 13.3.3.10 as follows:
13.3.3.2 Add 50 \\L of reagent water [instead of HC1], then vortex at the
highest setting for approximately 50 seconds.
13.3.3.3 Place tube(s) in heat block stabilized at 80°C for 10 minutes.
13.3.3.4 Remove tube(s) from heat block, and vortex at the highest
setting for approximately 30 seconds.
13.3.3.5 Same as method
13.3.3.6 Same as method
13.3.3.7 Same as method
13.3.3.8 DELETE this step in the method.
13.3.3.9 Same as method except omit "with the NaOH".
13.3.3.10 Replace "acid" with "heat"
13.3.3.11 Same as method, to completion of the method.
NOTE: Some laboratories have used a Multi Block Heater, Model 2050 or Grant UBD1
heat block. The Grant UBD1 heat block has options of various block sizes to
accommodate different plasticware including a 1.5 mL microtube interchangeable block
BB-E1 that can hold 24 tubes at once.
13.4.5 Increasing Surface Area for Sample Application to Slide - Matrix debris may result
in loss of organisms during the staining process and visual obstruction of oo/cysts on
the slide. Increasing the surface area for sample application to slides can reduce
interference from debris.
• Use slides with larger diameter wells to spread the debris and organisms over a
larger surface area. Commercially available microscope slides have well diameters
ranging from 9 mm to 15 mm.
• Split the dissociation volumes from each sample evenly between two wells.
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Method 1623.1 - Cryptosporidium and Giardia
To split each dissociation volume, process sample(s) according to Method 1623.1
through Section 13.3.3.6 (sample is in the microcentrifuge tube in the MPC®-S); then
proceed at Section 13.3.3.7 with the following substitutions and additions:
13.3.3.7 Prepare two separate wells (on same or separate slides) for each
sample.
13.3.3.8 OMIT this step
13.3.3.9 Same as method, except apply half of the dissociation volume,
25 \\L, to one well and the second half to a second well (See
Figure A; apply 25 (iL to Well A and 25 (iL to Well B)
13.3.3.10 Same as method, except apply splits of the dissociation volume
to the two wells as before (modified 13.3.3.9 above, See Figure
A).
13.3.3.11 Add 5 (iL of 1.0 N NaOH to each of the two wells after applying
the samples. Record the date and time the purified sample was
applied to the well slide(s).
13.3.3.12 Same as method, to completion of the method.
25
25
1st Dissociation
Well A
WellB
2nd Dissociation
25 [
25 (
Figure A.
14.0 Sample Staining
NOTE: The sample must be stained within 72 hours of application of the purified sample
to the slide.
NOTE: If a laboratory will use multiple types of labeling reagents, the laboratory must
demonstrate equivalent or superior performance through an IDC per Section 9.2 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 is monitored in each source water type
through MS samples (Section 9.6.1).
14.1 Prepare positive and negative controls
14.1.1 For the positive control, pipette 10 (iL of positive antigen or >200 intact oocysts and
>200 cysts to the center of a well.
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Method 1623.1 - Cryptosporidium and Giardia
14.1.2 For the negative control, pipette 50 (iL of PBS (Section 7.5) 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 at the same time and following the same procedure as used
for field samples (see Section 13.3.3.12 for guidance).
NOTE: Multiple control slides may be prepared if a 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. These control slides are prepared along
with the field slides and examined, alternating between the positive controls when
performing the positive control check.
14.2 Four optional stains have been demonstrated to be acceptable for use with Method 1623.1—
1) MeriFluor® Cryptosporidium/Giardia, 2) Aqua-Glo™G/C Direct FL, 3) Crypt-a-Glo™ and
Giardi-a-Glo™, and 4) EasyStain™C&G. 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 7.8]) 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.
14.6 Apply 50 (iL of DAPI staining solution (Section 7.9.2) 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
7.8]) to each well. Tilt each slide on a clean paper towel, long edge down. Gently aspirate the
excess DAPI staining solution from below the well using a clean Pasteur pipette or absorb with
paper towel or other absorbent material placed at edge of slide. Avoid disturbing the sample.
NOTE: If using the MeriFluor® Cryptosporidium/Giardia (Section 7.8.1), do not allow
slides to dry completely.
14.8 Add mounting medium (Section 7.10) 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 between 1°C and 10°C until ready for
examination.
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Method 1623.1 - Cryptosporidium and Giardia
15.0 Examination
NOTE: Although it is preferable to perform FA and DAPI and DIC microscopy
examination and characterization immediately after staining is complete, laboratories
have up to 168 hours (7 days) to complete the examination and verification of samples.
However, if fading/diffusion of FITC or DAPI fluorescence is noticed, the laboratory
must reduce this holding time. In addition, the laboratory may adjust the concentration of
the DAPI staining solution (Sections 7.9.2) 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 3).
15.2 Examination using FA, DAPI staining characteristics, and DIC microscopy. The minimum
magnification requirements for each type of examination are noted below.
NOTE: All characterization (DAPI and DIC) and size measurements must be determined
using 1000X magnification and reported to the nearest 0.5 pm.
Record examination results for Cryptosporidium oocysts on a Cryptosporidium examination
form; record examination results for Giardia cysts on a Giardia examination results form. All
organisms that meet the criteria specified in Sections 15.2.2 and 15.2.3, less atypical organisms
specifically identified as non-target organisms by DIC or DAPI (e.g., possessing spikes, stalks,
appendages, pores, one or two large nuclei filling the cell, red fluorescing chloroplasts, crystals,
spores, etc), must be reported.
15.2.1 Positive and negative staining control. Positive and negative staining controls must be
acceptable before proceeding with examination of field sample slides.
15.2.1.1 Each analyst must characterize a minimum of three Cryptosporidium
oocysts 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 three
Cryptosporidium oocysts and three Giardia cysts must be recorded by the
analyst on a microscope log. The analyst also must indicate on each sample
examination form whether the positive staining control was acceptable.
15.2.1.2 Examine the negative staining control to confirm that it does not contain
any oocysts or cysts (Section 14.1). Indicate on each sample examination
form whether the negative staining control was acceptable.
15.2.1.3 If the positive staining control contains oocysts 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 A positive result is a Cryptosporidium oocyst which exhibits all of the
following: 1) typical FA fluorescence, 2) typical size and shape, 3) nothing
atypical on DAPI fluorescence, and 4) nothing atypical on DIC microscopy
January 2012
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Method 1623.1 - Cryptosporidium and Giardia
(Figure 4). Each positive result must be characterized and assigned to
one of the DAPI and DIC categories in Sections 15.2.2.3 and 15.2.2.4
and recorded on an examination form.
15.2.2.2 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 15.2.2.3), then to
DIC (Section 15.2.2.4) at 1000X.
15.2.2.3 DAPI fluorescence examination (the analyst must use a minimum of
400X total magnification). Using the UV filter block for DAPI, the object
will exhibit one of the following characteristics:
(a) Light blue internal staining (no distinct nuclei) with a green rim
(b) Intense blue internal staining
(c) Up to four distinct, sky-blue nuclei
Look for atypical DAPI fluorescence, e.g., more than four stained nuclei,
size of stained nuclei, and wall structure and color. If atypical structures are
not observed, then categorize each object meeting the criteria defined as a
positive result in Section 15.2.2.1 and record oocysts in category (a) as
DAPI-negative; record oocysts in categories (b) and (c) as DAPI-positive.
15.2.2.4 DIC examination (the analyst must use a minimum of 1000X total
magnification [oil immersion lens]). Using DIC microscopy, 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, crystals, spores, etc.). If atypical structures
are not observed, then categorize each object meeting the criteria defined
as a positive result in Section 15.2.2.1 and specified in Sections 15.2.2.2
and 15.2.2.3 as one of the following, based on DIC examination:
(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 (im), and number of sporozoites (if applicable) for each apple-
green fluorescing object meeting the size and shape characteristics.
Although not a defining characteristic, surface oocyst folds may be
observed in some specimens.
15.2.3 Sample examination—Giardia
15.2.3.1 A positive result is a Giardia cyst which exhibits all of the following: 1)
typical FA fluorescence, 2) typical size and shape, 3) nothing atypical on
DAPI fluorescence, and 4) nothing atypical on DIC microscopy (Figure 4).
Each positive result must be characterized and assigned to one of the
DAPI and DIC categories in Sections 15.2.3.3 and 15.2.3.4 and
recorded on an examination form.
January 2012
49
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Method 1623.1 - Cryptosporidium and Giardia
15.2.3.2 FITC examination (the analyst must use a minimum of 200X total
magnification). When brilliant apple-green fluorescing round to ovoid
objects (8-18 (im long by 5 - 15 (im wide) are observed with brightly
highlighted edges, increase magnification to 400X and switch the
microscope to the UV filter block for DAPI (Section 15.2.3.3) then to DIG
(Section 15.2.3.4) at 1000X.
15.2.3.3 DAPI fluorescence examination (the analyst must use a minimum of
400X total magnification). Using the UV filter block for DAPI, the object
will exhibit one or more of the following characteristics:
(a) Light blue internal staining (no distinct nuclei) and a green rim
(b) Intense blue internal staining
(c) Up to four distinct, sky-blue nuclei
Look for atypical DAPI fluorescence, e.g., more than four stained nuclei,
size of stained nuclei, and wall structure and color. If atypical structures
are not observed, then categorize each object meeting the criteria defined
as a positive result in Section 15.2.3.1 and record cysts in category (a) as
DAPI negative; record cysts in categories (b) and (c) as DAPI positive.
15.2.3.4 DIC examination (the analyst must use a minimum of 1000X total
magnification [oil immersion lens]). Using DIC microscopy, 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, crystals, spores, etc.). If atypical structures are not observed, then
categorize each object meeting the criteria defined as a positive result in
Section 15.2.3.1 and specified in Sections 15.2.3.2 and 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 (im), 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.
15.2.4 Record the date and time that sample examination was completed on the examination
form.
15.2.5 Report Cryptosporidium and Giardia concentrations as oocysts/L and cysts/L,
respectively. If no oocysts or cysts, as defined in Section 15.2.2.1 and 15.2.3.1, are
detected, report zero organisms.
15.2.6 Record analyst name
January 2012
50
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Method 1623.1 - Cryptosporidium and Giardia
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 See the Training modules available for downloading at
http://water.epa.gov/lawsregs/rulesregs/sdwa/mdbp/training.cfm for further suggestions.
17.0 Method Performance
Method acceptance criteria are shown in Tables 3 and 4 in Section 21.0. The criteria listed for
Cryptosporidium in Table 3 and Giardia in Table 4 were based on results generated from 56
spiked reagent and 53 spiked raw surface water samples from 14 sites across the U.S. analyzed
during the inter-laboratory validation of Method 1623.1 involving 14 laboratories (Reference
20.13).
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 6.
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 Prepare solutions and reagents in volumes consistent with laboratory use to minimize the volume
of expired materials that need to be discarded.
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.
January 2012
51
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Method 1623.1 - Cryptosporidium and Giardia
20.0 References
20.1 USEPA. EPA Microbiological Alternate Test Procedure (ATP) Protocol for Drinking Water,
Ambient Water, Wastewater, and Sewage Sludge Monitoring Methods. EPA-821-B-10-001.
Office of Science and Technology, U.S. Environmental Protection Agency, 1200 Pennsylvania
Avenue, NW, Washington, DC (2010).
20.2 Rodgers, Mark R., Flanigan, Debbie J., and Jakubowski, Walter, 1995. Applied and
Environmental Microbiology 61 (10), 3759-3763.
20.3 "Biosafety in Microbiological and Biomedical Laboratories (5th ed.)," DHHS, CDC, NIH,
Publication 21-1112 (2007).
20.4 Fleming, Diane O., et al.(eds.), Laboratory Safety: Principles and Practices, 2nd edition. 1995.
ASM Press, Washington, DC
20.5 "Working with Carcinogens," DREW, PHS, CDC, NIOSH, Publication 77-206, (1977).
20.6 "OSHA Safety and Health Standards, General Industry," OSHA 2206, 29 CFR 1910 (1976).
20.7 "Safety in Academic Chemistry Laboratories, Volume 1: Accident Prevention for College and
University Students, 7th Edition" American Chemical Society Committee on Chemical Safety.
Washington, DC (2003).
20.8 "Hazardous Materials: Infectious Substances; Harmonization with the United Nations
Recommendations. " 49 Federal Register Parts 171-178 (2 June, 2006).
20.9 APHA, AWWA, and WEF. 2005. Standard Methods for the Examination of Water and
th
Wastewater; 21 Edition. American Public Health Association, American Water Works
Association, Washington, D.C.
20.10 USEPA. Manual for the Certification of Laboratories Analyzing Drinking Water; Criteria and
Procedures; Quality Assurance. Fifth Edition. EPA 815-R-05-004. Office of Ground Water and
Drinking Water, U.S. Environmental Protection Agency, 26 West Martin Luther King Drive,
Cincinnati, OH (2005).
20.11 "Envirochek® HV Sampling Capsule Protocol," PN 33210, Pall Corporation, 25 Harbor Park
Drive, Port Washington, NY, 11050 (Revision Date 08/25/10).
20.12 "Dynabeads® GC-Combo," 730.02, Invitrogen Dynal, Oslo, Norway (2007, Revision no. 014).
20.13 USEPA. Results of the Inter-laboratory Method Validation Study Using U.S. Environmental
Protection Agency Method 1623.1: Cryptosporidium and Giardia in Water by
Filtration/IMS/FA. EPA-816-R-12-002. Office of Water, Office of Ground Water and Drinking
Water, Technical Support Center, Cincinnati, OH (2012).
20.14 USEPA. Implementation and Results of the Information Collection Rule Supplemental Surveys.
EPA-815-R-01-003. Office of Water, Office of Ground Water and Drinking Water, Standards and
Risk Management Division, Washington, DC (2001).
20.15 Connell, K., J. Scheller, K. Miller, and C.C. Rodgers, 2000. Performance of Methods 1622 and
1623 in the ICR Supplemental Surveys. Proceedings, American Water Works Association Water
Quality Technology Conference, November 5-9, 2000, Salt Lake City, UT.
January 2012
52
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Method 1623.1 - Cryptosporidium and Giardia
21.0 Tables and Figures
Table 1. Method 1623.1 Procedural Component Options
Method
1623.1
Procedural
Component
Sample
Collection
Spiking
Suspension
Filtration/
Elution
Concentration
/Aspiration
Optional Component or
Processing Step
Field filter- Envirochek® HV;
10Lor50L
Field filter - Filta-Max®; 10 L
or SOL
Bulk; 10 L
Wisconsin State Laboratory
of Hygiene
BTF EasySeed™
Waterborne AccuSpike™-IR
Envirochek® HV/shaker
(700-900 opm)/NaHMP/LA-12
Filta-Max®/manual wash
station/PBST/concentrator
tube
Filta-Max®/automatic wash
station/PBST/concentrator
tube
PCFC
Centrifugation (minimum
1500 xQ; 1800-2000 x G)
Concentrator
Membrane/Centrifugation
Aspiration
Protocol
(Section references to Method 1623.1)
Section 12.2 - 12.2.5; LT2 Rule Cryptosporidium
and £. co// Sample Collection Recommendations
Training Module and Pocket Guide
Section 12.3-12.3.1.6; LT2 Rule
Cryptosporidium and £. co// Sample Collection
Recommendations Training Module and Pocket
Guide
Section 8.1.1; LT2 Rule Cryptosporidium and £.
co// Sample Collection Recommendations
Training Module and Pocket Guide
Section 11.0; Manufacturer's instructions
Section 11.0; Manufacturer's instructions
Section 11.0; Manufacturer's instructions
Section 12.2
Section 12.3-12.3.1.6.2; 12.3.2.1 -12.3.2.2,
12.3.4
Manufacturer's instructions
Manufacturer's instructions
Section 13.2.1
Section12.3.3; 13.2.1
Sections 13.2.2-13.2.3
Reference*
NA
NA
Validation study
Validation study
Historically
documented SOP;
Validation study
Validation study
Historically
documented SOP;
Validation study
Validation study
refer to manual wash
validation study
Validation study
Historically
documented SOP;
Validation study
Validation study
Validation Study
January 2012
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Method 1623.1 - Cryptosporidium and Giardia
Method
1623.1
Procedural
Component
IMS
Staining
Equipment or
supply
changes
Optional Component or
Processing Step
Dynabeads® GC Combo with
MPC®-1 or MPC®-6 and
MPC®-S
Transfer to microcentrifuge
tube -0.7, 0.3, 0.3-mL
Multiple wells; on same or
separate slides
MeriFluor®
Cryptosporidium/Giardia
Aqua-Glo™G/C Direct FL
Crypt-a-Glo™ and Giardi-a-
Glo™
EasyStain™C&G
Using different
equipment/supplies within
procedural component, such
as
• pumps
• lab shaker
• IMS magnets
• flat-sided tubes
• microcentrifuge tubes
• slides
• mounting medium
• microscope
Protocol
(Section references to Method 1623.1)
Sections 13.3- 13.4
Sections 13.3.2.12 and 13.3.2.14
Section 13.3.3.10
Section 14.0, Manufacturer's instructions
Section 14.0, Manufacturer's instructions
Section 14.0, Manufacturer's instructions
Section 14.0, Manufacturer's instructions
Follow same procedure in Method 1623.1,
manufacturer's instructions or historically
documented SOP
Reference*
Historically
documented SOP;
Validation study
Historically
documented SOP;
Validation study
Validation study
Validation study
Validation study
Validation study
Historically
documented SOP;
Validation study
NA
*Multi-laboratory validation studies or historical demonstration of accuracy and precision at multiple laboratories.
January 2012
54
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Method 1623.1 - Cryptosporidium and Giardia
Table 2. Summary of Routine QC Requirements
Method
1623.1
Reference
Section 9.5, 9.6,
9.7
Section 9.5
Section 9.7
9121
Section 9.6.1
Section 9.6.3
Sections 9.9,
14.1 and 15.2.1
Sections 9.9,
14.1 and 15.2.1
Section 9. 10
QC sample
or
procedure
IDC
IPR
Method
Blank
OPR
MS
MS/MSD
staining
control
Negative
staining
control
Analyst
Performance
Matrix
Reagent water
and water
matrix of
interest
Reagent water
Reagent water
Reagent water
Water matrix
of interest
Water matrix
of interest
none
none
Reagent
Water
Number
of
samples
8
4
1
1
1, plus 1
unspiked
field
sample
2, plus 1
unspiked
field
sample
1
1
N/A
Frequency
Initial use of method
and each procedural
component change
Each equipment/supply
change
Each IPR and OPR set
At least each week
samples are processed
or every 20 samples,
whichever is more
frequent
For each water matrix -
initial sampling and
every 20 samples
Each IDC, and multi-
laboratory validation of
Process each time
samples are stained;
examine each
microscope session
Each time samples are
stained
Monthly
Purpose
To demonstrate control over the
analytical system with new or
different procedural component;
consists of IPR set, Method
blank, field sample and
MS/MSD
To establish control over the
analytical system and
demonstrate acceptable method
performance (recovery and
precision) as part of IDC and
with new equipment or supplies
To demonstrate the absence of
contamination throughout the
analytical process
To demonstrate ongoing control
of the analytical system and
verify continuing method
performance (recovery and
precision)
To determine the effect of the
matrix on (oo)cyst recoveries;
must be accompanied by an
unspiked field sample collected
at the same time as the MS
sample
To estimate the (oo)cyst
recovery precision with the
effect of the matrix; must be
accompanied by an unspiked
field sample collected at the
same time as MS/MSD samples
To demonstrate ongoing control
of the staining process and
performance of reagents and
microscope
To demonstrate the absence of
contamination through staining
process
Refine consistency of organism
enumeration and
characterizations between
analysts
Charts
No
No
No
Required
Required
Required
No
No
No
January 2012
55
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Method 1623.1 - Cryptosporidium and Giardia
Table 3. Quality Control Acceptance Criteria for Cryptosporidium
Performance test
Initial precision and recovery1 (IPR)
Mean recovery (percent)
Precision (as maximum relative standard deviation)
Ongoing precision and recovery1 (OPR as percent)
MS/MSD (for method modifications)
Mean recovery2-3 (as percent)
Precision (as maximum relative percent difference)
Section
9.5
9.5.2
9.5.3
9.8
9.6
9.6.3.2
9.6.3.3
Acceptance criteria
38-100
37
33-100
32-100
46
(1) Failure to meet IPR or OPR quality control acceptance criteria indicates systemic problems the laboratory must
address prior to processing any samples.
(2) The acceptance criteria for mean MS/MSD recovery serve as the acceptance criteria for MS recovery during
routine use of the method (Section 9.6.1).
(3) Some sample matrices may prevent the acceptance criteria from being met. Repeated failure to meet the
MS/MSD quality control acceptance criteria, when all other QC passes, warrants corrective action from quality
conscience laboratories to systematically review control charts and modify analytical protocols to improve matrix
spike recoveries. An assessment of the distribution of MS recoveries from multiple MS samples from 87 sites
during the ICR Supplemental Surveys is provided in Table 6. The laboratory must chart their distribution of MS
and MSD recoveries compared with Table 6 (Section 9.12.2.2).
Table 4. Quality Control Acceptance Criteria for Giardia
Performance test
Initial precision and recovery1 (IPR)
Mean recovery (percent)
Precision (as maximum relative standard deviation)
Ongoing precision and recovery1 (OPR as percent)
MS/MSD (for method modifications)
Mean recovery 2' 3 (as percent)
Precision (as maximum relative percent difference)
Section
9.5
9.5.2
9.5.3
9.8
9.6
9.6.3.2
9.6.3.3
Acceptance criteria
27-100
39
22-100
8-100
97
Footnotes 1, 2, and 3 are the same as Table 3.
NOTE: The criteria listed for Cryptosporidium and Giardia were based on data
generated from 56 spiked reagent and 53 spiked raw surface water samples from 14 sites
across the U.S. analyzed during the interlaboratory validation of Method 1623.1
involving 14 laboratories (Reference 20.13.).
January 2012
56
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Method 1623.1 - Cryptosporidium and Giardia
Table 5. Method Holding Times (See Section 8.2 for details)
Maximum Allowable Time for Sample Processing Steps
4 days (96 hours) between collection/filtration and elution
1 working day between elution and application of sample to slide
3 days (72 hours) between application to slide and staining
7 days (168 hours) between staining and examination
Table 6. Distribution of
During the ICR
MS Recoveries from Multiple Samples Collected from 87 Source Waters
Supplemental Surveys (Adapted from References 20.14 and 20.15)
MS Recovery Range
<10%
>10%-20%
>20% - 30%
>30% - 40%
>40% - 50%
>50% - 60%
>60% - 70%
>70% - 80%
>80% - 90%
>90%
Percent of 430 Cryptosporidium MS
Samples in Recovery Range
6.7%
6.3%
14.9%
14.2%
18.4%
17.4%
11.2%
8.4%
2.3%
0.2%
Percent of 270 Giardia MS Samples in
Recovery Range
5.2%
4.8%
7.0%
8.5%
17.4%
16.3%
16.7%
14.1%
6.3%
3.7%
January 2012
57
-------
Method 1623.1 - Cryptosporidium and Giardia
Sample
Flew rate meter '
with vali/e \
/
Flaw totalizer
]_ (Valve)
Influent
tubing
SS Cerrtrifcgal
tubing
pump
BRECr OKI CF FLOW
Out el tubing
Flow rate meter'—
with valve
Effluent tubing
Effluent tubing
Pressure
regulator
Envinxhek™
capsule
Flow totalizer
mtvm 01 FLOW
Fbw control
value may be
used h ptee* of
fta* rale meter
Figure 1.
Filtration Systems for Envirochek® HV Capsule
(unpressurized source - top, pressurized source -
bottom)
January 2012
58
-------
Method 1623.1 - Cryptosporidium and Giardia
Flow rate
meter
Flo iv:ota Liar Effluent tubir»9
(V^lve)
Sample
Infl uent tu bing Inlet tubing
Qutlel tubing
DIRECTION OF FLOW
Effluent tubing
Pressure In let tubing I
regutalor Filta-UaxT"
fitter housing
Flow tolalizer
DRECT1CW OF FLOW
Ftow conW
value may he
flow rale meter
Figure 2. Filtration Systems for Filta-Max® filters (unpressurized
source - top, pressurized source - bottom)
January 2012
59
-------
Method 1623.1 - Cryptosporidium and Giardia
'• X
Figure 3. Methods for Scanning a Well Slide
January 2012
60
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Method 1623.1 - Cryptosporidium and Giardia
Method 1623.1 Microscopy Visual Guide
Cryptosporidium oocyst criteria:
• Brilliant apple-green fluorescence • Brightly highlighted edges
• 4 - 6 |jm size • Spherical to ovoid shape
• No atypical characteristics by FA, DAPI fluorescence or DIG microscopy
0 10
Giardia cyst criteria:
• Brilliant apple-green fluorescence • Brightly highlighted edges
• 8 - 18 urn long by 5 - 15 urn wide • Spherical to ovoid shape
• No atypical characteristics by FA, DAPI fluorescence or DIG microscopy
DIG
image
Image
not in DIG
Figure 4. Method 1623.1 Microscopy Visual Guide - Photographs courtesy of
The City of San Diego Water Quality Laboratory; Texas AgriLife Research Center at El Paso,
Texas A&M University System; University of Iowa Hygienic Laboratory
January 2012
61
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Method 1623.1 - 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
jiL microliter
< less than
> greater than
% percent
22.1.2 Alphabetical characters
cm centimeter
g gram
G acceleration due to gravity
hr hour
ID inside diameter
in. inch
L liter
m meter
mg milligram
mL milliliter
mm millimeter
mM millimolar
N normal; gram molecular weight of solute divided by hydrogen equivalent of
solute, per liter of solution
RSD relative standard deviation
sr standard deviation of recovery
X mean percent recovery
22.2 Definitions, acronyms, and abbreviations (in alphabetical order)
4',6-diamidino-2-phenylindole (DAPI) - A nucleic acid stain which fluoresces blue and can show
the position and number of nuclei present in the cyst/oocyst.
Analyst—The analyst participates in a monthly analyst verification (Section 9.10), establishes
Kohler illumination for the microscope, may perform the same duties as a technician, and is able
to examine samples using the microscope. An analyst has at least 2 years of college education in
microbiology, or an equivalent field and a minimum of 6 months of continuous bench experience
with Cryptosporidium and FA microscopy. In addition, an analyst must have a minimum of 3
months experience using EPA Method 1623 or 1623.1 analyzing aminimum of 50 samples using
either method. "Grandfathering" analysts with >10 years experience of continuous protozoan
identification duties may be substituted for college education.
Analyte—A protozoan parasite tested for by this method. The analytes in this method are
Cryptosporidium and Giardia.
January 2012
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Method 1623.1 - Cryptosporidium and Giardia
Axoneme—An internal flagellar structure that occurs in some protozoa, such as Giardia,
Spironucleous, and Trichomonas.
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.
Fluorescein isothiocyanate (FITC) - The fluorochrome used to label the monoclonal antibodies
targeted against the cell wall antigens of both Giardia and Cryptosporidium which fluoresces a
brilliant apple green color.
Immunofluorescence assay (FA) - A microscopic assay technique in which a fluorochrome is
conjugated to an antibody molecule that selectively binds to the organism of interest which is
detected using fluorescent microscopy.
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 demonstration of capability (IDC)—Four IPR samples, one MB, and MS/MSD/unspiked
field sample analyzed to establish the ability to generate acceptable precision and accuracy. An
IDC is performed prior to the first time this method is used and any time the method is modified.
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 as part of the IDC
or when equipment/supply changes are made to this method.
Laboratory blank—See Method blank
Laboratory control sample —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.
Matrix spike duplicate (MSD)—A second 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 duplicate is used to determine the precision of a
method's recovery efficiency in a particular matrix type.
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. These structures can be a crescent-shaped or round.
January 2012
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Method 1623.1 - Cryptosporidium and Giardia
Method blank (MB)—An aliquot of reagent water that is treated exactly as a sample, including
exposure to all glassware, equipment, solvents, and procedures that are used with samples. The
method blank is used to determine if analytes or interferences are present in the laboratory
environment, the reagents, or the apparatus.
Must—This action, activity, or procedural step is required.
Negative control—See Method blank
Nucleus—A membrane-bound organelle containing genetic material. Nuclei are a prominent
internal structure seen both in Cryptosporidium oocysts and Giardia cysts. In Cryptosporidium
oocysts, there is one nucleus per sporozoite. One to four nuclei can be seen in Giardia cysts.
Oocyst—The encysted zygote of some sporozoa; e.g., Cryptosporidium. The oocyst is a phase or
form of the organism produced as a normal part of the life cycle of the organism. It is
characterized by a thick and environmentally resistant outer wall.
Ongoing precision and recovery (OPR) standard—A method blank spiked with known quantities
of analytes. The OPR is analyzed exactly like a sample. Its purpose is to assure that the results
produced by the laboratory remain within the limits specified in this method for precision and
recovery.
Oocyst and cyst spiking suspension—See Spiking suspension
Positive control—See Ongoing precision and recovery (OPR) standard
Principal analyst—The principal analyst participates in a monthly analyst verification (Section
9.10), supervises and verifies the processing and microscopy in the laboratory, and may perform
the same duties as an analyst. A principal analyst has a BS/BA in microbiology or closely related
field and a minimum of 1 year of continuous bench experience with Cryptosporidium and FA
microscopy. In addition to formal training in microbiology, the principal analyst must also have a
minimum of 6 months experience using EPA Method 1623 or 1623.1 and have analyzed a
minimum of 100 samples using EPA Method 1623 or 1623.1. "Grandfathering" principal analysts
with >10 years experience of continuous protozoan identification duties may be substituted for
college education.
PTFE—Polytetrafluoroethylene
Quality control (QC) - Operational techniques and activities used for maintaining standards.
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
January 2012
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Method 1623.1 - Cryptosporidium and Giardia
Relative standard deviation (RSD)—The standard deviation divided by the mean times 100.
Should—This action, activity, or procedural step is suggested but not required.
Spiking suspension—Flow cytometry-enumerated 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.
Technician—The technician filters samples, performs centrifugation, elution, concentration, and
purification using IMS, and prepares purified samples on slides for microscopic examination, but
does not perform microscopic protozoan detection and identification. No minimum education or
experience requirements with Cryptosporidium and IFA microscopy apply to the technician. At
least 3 months of experience in filter extraction and processing of protozoa samples by EPA
Method 1623 or 1623.1 and successful processing of aminimum of 50 samples using EPA
Method 1623 or 1623.1 are required to become a technician
January 2012
65
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Appendix A
Micropipette Calibration
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Method 1623.1 - Cryptosporidium and Giardia
A.O Micropipette Calibration
A. 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.
A.2 Internal and external calibration records must be kept on file in the laboratory's QA logbook.
A. 3 If a micropipette calibration problem is suspected, the laboratory should tare an empty weighing
boat on the analytical balance and pipette the following volumes of reagent water into the weigh
boat using the pipette in question: 100% of the maximum dispensing capacity of the micropipette,
50% of the capacity, and 10% of the capacity. Ten replicates should be performed at each weight.
Record the weight of the water (assume that 1.00 mL of reagent water weighs 1.00 g) and
calculate the relative standard deviation (RSD) for each. If the weight of the reagent water is
within 1% of the desired weight (mL) and the RSD of the replicates at each weight is within 1%,
then the pipette remains acceptable for use.
A. 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 A. 3. If problems
with the pipette persist, the laboratory must send the pipette to the manufacturer for recalibration.
A-1
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Appendix B
Microscope Protocols
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Method 1623.1 - Cryptosporidium and Giardia
B.O Microscope Protocols
CAUTION: In the procedures below, do not touch the quartz portion of the mercury bulb
with your bare fingers. Finger oils can cause rapid degradation of the quartz and
premature failure of the bulb.
WARNING: Never look at the ultraviolet (UV) light from the mercury lamp, lamp house,
or the UV image without a barrier filter in place. UV radiation can cause serious eye
damage.
Section 10 and 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.
B.1 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.
B.1.1 Remove the diffuser lens between the lamp and microscope or swing it out of the
transmitted light path.
B.1.2 Using a prepared microscope slide, adjust the focus so the image in the oculars is
sharply defined.
B.1.3 Replace the slide with a business card or a piece of lens paper.
B.1.4 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.
B. 1.5 Mount the mercury lamp house on the microscope without the UV diffuser lens in
place and turn on the mercury bulb.
B.1.6 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.
B.1.7 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.
B. 1.8 Reattach the objective to the nosepiece.
B. 1.9 Insert the diffuser lens into the light path between the mercury lamp house and the
microscope.
B. 1.10 Turn off the transmitted light and replace the card with a slide of fluorescent material.
Check the field for even fluorescent illumination. Adjustment of the diffuser lens
probably will be required. Additional slight adjustments as in Section B.I.7 may be
required.
B.1.11 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 by the manufacturer.
B-1
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Method 1623.1 - Cryptosporidium and Giardia
B.2 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.
B.2.1 Remove the diffuser lens between the lamp and microscope or swing it out of the
transmitted light path.
B.2.2 Using a prepared microscope slide and a 40X (or similar) objective, adjust the focus so
the image in the oculars is sharply defined.
B.2.3 Without the ocular or Bertrand optics in place, view the pupil and filament image at the
bottom of the tube.
B.2.4 Focus the lamp filament image with the appropriate adjustment on the lamp house.
B.2.5 Similarly, center the lamp filament image within the pupil with the appropriate
adjustment(s) on the lamp house.
B.2.6 Insert the diffuser lens into the light path between the transmitted lamp house and the
microscope.
B.3 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, leave the ocular reticle in place. The
more an ocular is manipulated the greater the probability is for it to become contaminated with
dust particles. Calibrate each objective in use on the microscope following this procedure. 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.
B.3.1 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.
B.3.2 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.
B.3.3 Without changing the stage adjustment, find a point as distant as possible from the two
"0" lines where two other lines are exactly superimposed.
B.3.4 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.
B.3.5 Calculate the number of mm/ocular micrometer space. For example:
0.6mm 0.0125mm
48 ocular micrometer spaces ocular micrometer space
B-2
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Method 1623.1 - Cryptosporidium and Giardia
B.3.6 Because most measurements of microorganisms are given in um rather than mm, the
value calculated above must be converted to um by multiplying it by 1000 um/mm. For
example:
0.0125 mm
1,000
12.5
ocular micrometer space
mm
ocular micrometer space
B.3.7 Follow the procedure for each objective. Record the information as shown in the
example below. Make this information available at the microscope. One way of
making the information available is to print the chart below on plain paper, fill it in,
and affix the chart to the microscope or table with tape.
Item
no.
1
2
3
4
Objective
power
10X
20X
40X
100X
Description
N.A3 =
N.A.=
N.A.=
N.A.=
No. of ocular
micrometer
spaces
No. of stage
micrometer
mm1
(xm/ocular
micrometer
space2
1000 |jm/mm
(Stage micrometer length in mm * (1000 pm/mm)) •*• no. ocular micrometer spaces
N.A. refers to numerical aperture. The numerical aperture value is engraved on the barrel of the
objective.
B-3
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Appendix C
Flow Cytometry-Enumeration Guidelines
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Method 1623.1 - Cryptosporidium and Giardia
C.O Flow Cytometry-Enumeration Guidelines
Flow cytometry-sorted suspensions are available from commercial vendors and other sources (Section
7.12.1). The information provided in Sections C.I through C.5 is simply meant as a guideline for the
preparation of spiking suspensions using a flow cytometer. Laboratories or vendors performing flow
cytometry must develop and implement detailed standardized protocols for calibration and operation of
the flow cytometer.
C.1 Spiking suspensions should be prepared using Cryptosporidium oocysts <3 months old, and
Giardia cysts <2 weeks old.
C.2 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 must be documented and should
be < 2.5%. If the RSD is > 2.5%, the laboratory or vendor should perform the initial calibration
again, until the RSD of the 10 counts is < 2.5%. In addition to counting the organisms, the
laboratory or vendor should evaluate the quality of the organisms using DAPI fluorescence and
DIG to confirm that the organisms are in good condition. Oocyst or cyst preparations with many
(>10%) atypical organisms must be discarded.
C.3 Ongoing calibration. When sorting the spiking suspensions for use in QC samples, the
laboratory or vendor should perform ongoing calibration samples at a 10% frequency, at a
minimum. The laboratory or vendor 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 or vendor should discard the batch.
C.4 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 C.3).
C.5 Holding time criteria. Flow cytometer-sorted spiking suspensions (Section 7.12.1) used for
spiked QC samples (Section 9) must be used within the expiration date noted on the suspension.
The holding time specified by the flow cytometry laboratory or vendor should be determined
based on a holding time study.
C-7
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