United States
Environmental Protection
Agency
Office of Water
Washington, DC 20460
EPA-821-R-97-023
December 1997
DRAFT
SEPA Method 1622: Cryptosporidium in
Water by Filtration/I MS/FA npAPT
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United States
Environmental Protection
Agency
Office of Water
Washington, DC 20460
EPA821-R-97-023
December 1997
DRAFT
SEPA Method 1622: Cryptosporidium in
Water by Filtration/I MS/FA
December 1997 Draft
-------
Acknowledgments
'\ ' • ' ,,i " • ',,.'' i , S1 T,, ' f '*'. ' '"' ','- ,, •(. ''
This method was prepared under the direction of William A. felliard of the Engineering and Analysis
Division within EPA's Office of Water. This document was prepared under EPA Contract No. 68-C3-0337
by DynCorp, Inc., with assistance from its subcontractor, Interface, Inc.
The contributions of the following persons and organizations to the development of this method are
gratefully acknowledged:
Mike Arrowood, Centers for Disease Control, Division of Parasitic Diseases (MS-F13), 4770 Buford
Highway, N.E., Atlanta, GA 30341-3724, USA
Phil Berger, Office of Groundwater and Drinking Water, U.S. Environmental Protection Agency, 401 M
Street, S.W., Washington, DC 20460, USA
Jennifer Clancy, Clancy Environmental Consultants, Inc., P.O. Box 314, St. Albans, VT 05478, USA
Ricardo DeLeon, Metropolitan Water District of Southern California, 700 Moreno Avesnue, LaVerne, CA
91760, USA
Colin Pricker, Thames Water Utilities, Manor Farm Road, Reading, Berkshire, RG2 OJN, England
Frank Schaefer m, National Exposure Research Laboratory, U.S. Environmental Protection Agency, 26
W. Martin Luther King Drive, Cincinnati, OH 45268-1320, USA
Steve Schaub, Health and Ecological Criteria Division (4304), Office of Science and Technology, U.S.
Environmental Protection Agency, 401 M Street, S.W., Washington, DC 20460, USA
Huw Smith, Department of Bacteriology, Scottish Parasite Diagnostic Laboratory, Stobhill NHS Trust,
Springburn, Glasgow, G21 3UW, Scotland
Disclaimer
This method has been reviewed by the Office of Water, U.S. Environmental Protection Agency, 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:
William A. Telliard
U.S. EPA Office of Water
Analytical Methods Staff
Mail Code 4303
Washington, DC 20460
Phone: 202/260-7120
Fax: 202/260-7185
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Introduction
The occurrence of Cryptosporidium outbreaks in drinking water have brought about an increased need for
detection at levels necessary to protect human health. Method 1622 was developed to determine
Cryptosporidium reliably at low concentrations.
EPA initiated an effort in 1996 to identify new and innovative technologies for protozoan monitoring and
analysis. After evaluating potential alternatives to current methods through literature searches, discussions
with research and commercial laboratories, and meetings with experts, the Office of Water developed an
initial draft of Method 1622 in December 1996. The draft method was revised in January, May, and
November 1997, based on comments from experts, multiple in-laboratory peer reviews, and two single-
laboratory validation studies.
Method 1622 is a performance-based method applicable to the determination of Cryptosporidium in
aqueous matrices. Method 1622 requires filtration, immunomagnetic separation of the oocysts from the
material captured, and immunofluorescence assay for determination of oocyst concentrations, with
confirmation through vital dye staining and differential interference contrast microscopy.
This revision of Method 1622 includes several procedures for filtration and IMS, however, only the
capsule filtration procedure (Section 12.2) and the Dynal IMS procedure (Section 13.3) have been
validated. Alternate techniques are allowed, provided that required quality control tests are performed and
all quality control acceptance criteria in this method are met.
These alternate techniques currently include the vortex-flow filtration procedure listed in Section 12.4, the
membrane disk filtration procedure listed at Section 12.5, and the ImmuCell IMS procedure listed at
Section 13.5. These techniques have been tested on a preliminary basis, but have not been validated.
NOTE: The quality control acceptance criteria listed in Table 1 are
based on validation studies using the capsule filtration procedure (Section
12.2) and the Dynal IMS procedure (Section 13.3). No validation studies have
been conducted using the vortex flow filtration procedure (Section 12.4),
membrane disk filtration procedure (Section 12.5), or ImmuCell IMS procedure
(Section 13.5). As a result, these procedures must be demonstrated to meet
Method 1622 's performance-based requirements before use.
in
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1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
17.0
18.0
19.0
20.0
21.0
Table of Contents
Scope and Application
Summary of Method
Definitions
Contamination, Interferences, and Oocyst Degradation ...
Safety,
Equipment and Supplies
Reagents and Standards
Sample Collection and Storage
Quality Control
Microscope Calibration and Analyst Verification ..,
Oocyst Suspension Enumeration and Spiking
Sample Filtration and Elution . .• ,
Sample Concentration and Separation (Purification)
Sample Staining
Examination
Analysis of Complex Samples
Method Performance
Pollution Prevention
Waste Management
References
Tables and Figures
Table 1. Quality control acceptance criteria
Figure 1. Hemacytometer platform ruling
Figure 2. Manner of counting Cryptosporidium oocysts in 1 square mm
Figure 3. Laboratory filtration system for capsule filter or membrane disk filter
Figure 4. Vortex-flow filter concentrator drive assembly
Figure 5. Vortex-flow filter system
Figure 6. Membrane disk filter assembly
Figure 7. Methods for scanning a well slide
1
1
, 2
, 2
. 3
. 3
. 7
. 9
10
15
21
26
33
37
38
39
39
39
39
40
40
40
41
42
43
44
45
46
47
22.0 Glossary of Definitions and Purposes 48
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VI
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Method 1622: Cryptosporidium in Water by Filtration/IMS/FA
December 1997 Draft
1.0 Scope and Application
1.1 This method is for determination of the identity and concentration of Cryptosporidium (CAS
Registry number 137259-50-8) in untreated and treated drinking water and in other waters by
filtration, immunomagnetic separation (IMS), and immunofluorescence assay microscopy (FA).
Cryptosporidium may be confirmed using 4',6-diamidino-2-phenylindole (DAPI) vital dye staining
and differential interference contrast (D.I.C.) microscopy.
1.2 This method is designed to meet the survey and monitoring requirements of the U.S.
Environmental Protection Agency (EPA). It is based on laboratory testing of recommendations by
a panel of experts convened by EPA. The panel was charged with recommending an improved
protocol for recovery and detection of Cryptosporidium that could be tested and implemented with
minimal additional research.
1.3 This method will not identify the species of Cryptosporidium or the host species of origin, nor can
it determine the viability or infectivity of detected oocysts.
1.4 A method detection limit (MDL) of 4 oocysts/L typifies the minimum concentration of oocysts that
can be detected in a 10-L sample with no interferences present.
1.5 This method is for use only by persons experienced in the determination of Cryptosporidium by
filtration, IMS, and FA. Experienced persons are defined in the glossary at the end of this method
as the principal analyst/supervisor, analyst, and technician. Laboratories unfamiliar with analyses
of environmental samples by the techniques in this method should gain experience using water
filtration techniques, IMS, fluorescent antibody staining with monoclonal antibodies, and
microscopic examination of biological particulates using bright-field and D.I.C. microscopy.
1.6 Any modification of the method beyond those expressly permitted is subject to the application and
approval of alternative test procedures under 40 CFR Part 141.27.
2.0 Summary of Method
2.1 A 10-L volume of water is collected in a carboy in the field and shipped to the laboratory. The
sample is filtered in the laboratory and the oocysts and extraneous materials are captured on the
filter.
2.2 Elution and separation
2.2.1 Materials on the filter are removed by extraction with an aqueous buffered salt and
detergent solution. The salt/detergent solution from the filter is centrifuged to settle the
oocysts, and the supernatant fluid is decanted.
2.2.2 The oocysts are magnetized by attachment of magnetic beads conjugated to an antibody.
The magnetized oocysts are separated from the extraneous materials using a magnet, and
the extraneous materials are discarded. The magnetic bead complex is then detached
from the oocysts.
2.3 Enumeration
2.3.1 The oocysts are stained on well slides with fluorescently labeled monoclonal antibodies
and vital dye. The stained sample is examined using fluorescence and differential
interference contrast (D.I.C.) microscopy.
December 1997 Draft
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Method 1622 - Draft
2.3.2 Qualitative analysis is performed by scanning each slide well for objects that meet the
size, shape, and fluorescence characteristics of Cryptosporidium oocysts. Potential
oocysts are confirmed through 4',6-diamidino-2-phenylindole (DAPI) vital dye staining
characteristics and D.I.C. microscopy. An oocyst is identified when size, shape color, and
morphology agree with specified criteria and examples in a photographic library.
2.3.3 Quantitative analysis is performed by counting the total number of objects on the slide
confirmed as oocysts.
2.4 Quality is assured through reproducible calibration and testing of the filtration, immunomagnetic
separation (IMS), staining, and microscopy systems. Detailed information on these tests is
provided in Section 9.
3.0 Definitions
3.1 Cryptosporidium is defined as a protozoan parasite potentially found in water iand other media.
The six species of Cryptosporidium and their potential hosts are C. parvum (mammals, including
humans); C. baileyi and C. meleagridis (birds); C. muris (rodents); C. serpentis (reptiles); and C.
nasorum (fish).
3.2 Definitions for other terms used in this method are given in the glossary at the end of the method.
4.0 Contamination, Interferences, and Oocyst Degradation
4.1 Turbidity caused by inorganic and organic debris can interfere with the concentration, separation,
and examination of the sample for Cryptosporidium oocysts. In addition to naturally-occurring
debris, such as clays and algae, chemicals, such as iron and alum coagulants and polymers, may be
added to water during the treatment process, which may result in additional interference.
4.2 Organisms and debris that autofluoresce or demonstrate non-specific fluorescence, such as algal
and yeast cells, when examined by epifluorescent microscopy, may interfere with the detection of
oocysts and contribute to false positives by immunofluorescent assay (FA).
4.3 Solvents, reagents, labware, and other sample-processing hardware may yield artifacts that may
cause misinterpretation of microscopic examinations for oocysts. All materials used shall be
demonstrated to be free from interferences under the conditions of analysis by running a method
blank (negative control sample) initially and a minimum of every week or after changes in source
of reagent water. Specific selection of reagents and purification of solvents and other materials
may be required.
4.4 Interferences co-extracted from samples will vary considerably from source to source, depending
on the diversity of the water being sampled. Experience suggests that high levels of algae, bacteria,
and other protozoa can interfere in the identification of oocysts (Reference 20.1).
4.5 Freezing 10-L samples, filters, eluates, concentrates, or slides may interfere with the detection
and/or identification of oocysts.
4.6 All equipment should be autoclaved after use and before washing. Clean equipment by scrubbing
with warm detergent solution and exposing to hypochlorite solution (minimum of 5%) for at least
30 minutes at room temperature. Rinse the equipment with reagent water and place in an oocyst-
free environment until dry. Disposable supplies should be used wherever possible.
December 1997 Draft
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Method 1622 - Draft
5.0 Safety
5.1 The biohazard associated with, and the risk of infection from, oocysts is high in this method
because live organisms are handled. This method does not purport to address all of the safety
problems associated with its use. It is the responsibility of the laboratory to establish appropriate
safety and health practices prior to use of this method. In particular, the analyst/technician must
know and observe the safety procedures required in a microbiology laboratory that handles
pathogenic organisms while preparing, using, and disposing of sample concentrates, reagents and
materials, and while operating sterilization equipment.
5.2 The toxicity or carcinogenicity of each compound or reagent used in this method has not been
precisely determined; however, each chemical compound should be treated as a potential health
hazard. Exposure to these compounds should be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file of Occupational Safety and
Health Administration regulations regarding the safe handling of the chemicals specified in this
method. A reference file of material safety data sheets should be made available to all personnel
involved in these analyses. Additional information on laboratory safety can be found in References
20.2 through 20.5.
5.3 Samples may contain high concentrations of biohazards and toxic compounds, and must be
handled with gloves and opened in a biological safety cabinet to prevent exposure. Reference
materials and standards containing oocysts must also be handled with gloves and the
analyst/technician must never place gloves in or near the face after exposure to solutions known or
suspected to contain oocysts. Do not mouth-pipette.
5.4 Laboratory personnel must change gloves after handling filters and other contaminant-prone
equipment and reagents. Gloves must be removed or changed before touching any other laboratory
surfaces or equipment.
6.0 Equipment and Supplies
NOTE: Brand names, suppliers, and part numbers are for illustrative
purposes only. No endorsement is implied. Equivalent performance may be
achieved using apparatus and materials other than those specified here, but
demonstration of equivalent performance that meets the requirements of this
method is the responsibility of the laboratory.
6.1 Equipment for spiking samples in the laboratory
6.1.1 10-L carboy with bottom delivery port C/z")—Cole-Palmer cat. no. 06080-42, or
equivalent; 'calibrate to 10.0 L and mark level with waterproof marker
6.1.2 Stir bar—Fisher cat. no. 14-511-93, or equivalent
6.1.3 Stir plate—Fisher cat. no. 14-493-120S, or equivalent
6.1.4 Hemacytometer—Hausser Scientific, Horsham, PA, cat. no. 3200 or 1475, or equivalent
6.1.5 Hemacytometer coverslip—Hauser Scientific, cat. no. 5000 (for hemacytometer cat. no.
3200) or 1461 (for hemacytometer cat. no 1475), or equivalent
6.1.6 Lens paper without silicone—Fisher cat. no. 11-995, or equivalent
6.1.7 Polystyrene or polypropylene conical tubes with screw caps—15- and 50-mL
December 1997 Draft
-------
Method 1622 - Draft
6.1.8 Sonicator—Fisher Scientific Ultrasonic Cleaner cat. no. 1533532, 44- to 48-kHz peak
output/frequency, 3-qt tank capacity, or equivalent
6.2 Equipment for laboratory filtration of samples
6.2.1 Capsule filter system
6.2.1.1 Sampling capsule, approximately 6-cm in diameter x 21-cm long with
approximately 1300 cm2 polyethersulfone filter media, and '/2-in. inlet and
outlet fittings—Gelman Sciences, Inc., Ann Arbor, MI, Envirochek™
Sampling Capsule, product 12110, or equivalent
6.2.1.2 Wrist-action shaker with arms for agitation of sampling capsules
6.2.1.2.1 Wrist-action shaker—Lab-Line model 3589, VWR
Scientific cat. no. 57039-055, Fisher cat. no. 14260-11,
or equivalent
6.2.1.2.2 Side arms for wrist action shaker—Lab-Line Model
3587-4, VWR Scientific cat. no. 57039-045, Fisher cat.
no. 14260-13, or equivalent
Vortex-flow filter (VFF) system—alternate procedure requiring demonstration of
performance prior to use
6.2.2.1
6.2.2
VFF concentrator—ClearWater, Portland, ME, cat. no. VC-1, or
equivalent
6.2.2.2 Membrane cartridge—ClearWater Mb 10, or equivalent
6.2.2.3 Peristaltic pump—Masterflex Pump System Complete Pump A, Cole
Parmercat. no. E-77910-10, or equivalent
6.2.2 A Pump tubing—Masterflex E 96400-16, or equivalent
6.2.2.5 Pressurized gas (nitrogen or air)
6.2.2.6 Pressure reservoir 10- to 20-L—Millipore no. XX6700P10 or no.
X6700P20, or equivalent
6.2.3 Membrane disk filter system—alternate procedure requiring demonstration of
performance prior to use
NOTE: The membrane disk filter systems listed below may be acceptable for
waters with low turbidity but may clog with higher turbidity waters.
6.2.3.1 Filter housing assembly, membrane disk, 142- or 293-mm, including
stand, inlet and outlet plates, support screen, O-rings, fittings,
etc.—Micro-filtration Systems, Pleasanton CA, KS142/302000 (142-mm
housing), KS293/302600 (293-mm housing), or equivalent.
6.2.3.2 Sonicator—-Section 6.1.7
6.2.3.3 Membrane disk filters
6.2.3.3.1 142-mm diameter: 1-, 2-, or 3-^m pore size: Corning
Separations Division•#! 12110, #112111, #112112,
respectively, or equivalent
December 1997 Draft
4
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Method 1622 - Draft
6.2.3.3.2 293-mm diameter: 1-, 2-, or 3-ywm pore size: Coming
Separations Division #112810, #112811, or#112812,
respectively, or equivalent
6.2.3.4 Membrane filter drain disks
6.2.3.4.1 Drain disks for 142-mm filters—Corning Separations
Division #231700, or equivalent
6.2.3.4.2 Drain disks for 293-mm filters—Corning Separations
Division #232500, or equivalent
6.2.3.5 Washing machine hose—-standard household type
6.2.3.6 Specimen cups for elution
6.2.3.6.1 125-mL (for elution of 142-mm membrane disk
filters)—Cole-Parmer cat. no. E-06101-31, or equivalent
6.2.3.6.2 250-mL (for elution of 293-mm membrane disk
filters)—Cole-Parmer cat. no. E-06101-40, or equivalent
6.3 Ancillary sampling equipment using capsule or membrane disk filter
6.3.1 Tubing—Glass, polytetrafluoroethylene (PTFE), high-density polyethylene (HOPE), or
other tubing to which oocysts 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.
Before use, the tubing must be thoroughly rinsed with detergent solution, followed by
repeated rinsing with reagent water to minimize sample contamination.
6.3.2 Flow control valve—0.5 gpm (0.03 L/s), Bertram Controls, Plast-O-Matic cat. no.
FC050BV4-PV, or equivalent; or 0.4- to 4-Lpm flow meter with valve—Alamo Water
Treatment, San Antonio, TX, cat. no. R5310, or equivalent
6.3.3 Centrifugal pump—Grainger, Springfield, VA, cat. no. 2P613, or equivalent
6.4 Immunomagnetic separation (IMS) apparatus
6.4.1 Dynal IMS system
6.4.1.1 Sample mixer—Dynal Inc., Lake Success, NY, no. 947.01, or end-over-
end rotator, Barnstead-Thermolyne 400110, or equivalent
6.4.1.2 Magnetic particle concentrator—MPC-1, for 10-mL test tubes, Dynal no.
120.01, or equivalent
6.4.1.3 Magnetic particle concentrator—MPC-M, for microcentrifuge tubes,
Dynal no. 120.09, or equivalent
6.4.1.4 16 x 125 mm Leighton type tubes—Dynal® L10, no. 740.03, or
equivalent
6.4.2 ImmuCell IMS system—alternate procedure requiring demonstration of performance
prior to use
6.4.2.1 Petri dish, polymethylpentane—100 x 15 mm, Nalgene no. 5500-0010,
Fisher 0875715C, or equivalent
December 1997 Draft
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Method 1622 - Draft
6.5
6.6
6.7
6.8
6.4.2.2 Orbital shaker—Bellco #7744-01010, orbital shaker adaptor VWR
20903-879, or mini-orbital shaker adaptor VWR 20903-875, or
equivalent
6.4.2.3 Magnetic panning device (magnet)—ClearWater 1400, or equivalent
6.4.2.4 End-over-end rotator—Barnstead-Thermolyne 4002110, or equivalent
6.4.2.5 Polypropylene tubes—50-mL
Powder-free latex gloves—Fisher cat no. 113945B, or equivalent
Graduated cylinders, autoclavable—10-, 100-, and 1000-mL
Centrifuges
6.7.1 Centrifuge capable of accepting 15- to 250-mL conical centrifuge tubes and achieving
1000 x G —International Equipment Company, Needham Heights, MA, Centrifuge Size
2, Model K with swinging bucket, or equivalent
6.7.2 Centrifuge tubes—conical, graduated, 1.5-, 2-, 5-, 15-, 25- 50-, and 250-mL
Microscope
6.8.1 Epifluorescence/differential interference contrast (D.I.C.) with stage and ocular
micrometers and 20X (N.A.=0.4) to 100X (N.A.=1.3) objectives—Zeiss™ Axioskop,
Olympus™ BH, or equivalent
6.8.2 Excitation/band-pass filters for immunofluorescent assay (FA)—Zeiss™ 487909 or
equivalent, including, 450- to 490-nm exciter filter, 510 nm dicroic beam-splitting
mirror, and 515- to 520-nm barrier or suppression filter
6.8.3 Excitation/band-pass filters for DAPI—filters cited below (Chroma Technology,
Brattleboro, VT), or equivalent
Microscope
model
Zeiss™ -Axioskop
Zeiss™ -IM35
Olympus™ BH
Olympus™ BX
Olympus™ IMT2
Fluoro-
chrome
DAPI (UV)
DAPI (UV)
DAPI (UV)
Excitation
filter (nm)
340-380
340-380
340-380
Dichroic beam-
splitting mirror (nm)
400
400
400
Barrier or suppression
filter (nm)
420
420
420
Filter holder
DAPI (UV)
DAPI(UV)
340-380
400
Filter holder
340-380
400
420
420
Filter holder
Chroma catalog
number
CZ902
CZ702
11000
91002
11000
91008
11000
91003
6.9
Ancillary equipment for microscopy
6.9.1 Well slides—treated, 12-mm diameter, Meridian Diagnostics Inc., Cincinnati, OH, cat.
no. R2206, or equivalent
6.9.2 Glass coverslips—22 x 50 mm
6.9.3 Fingernail polish—clear or clear fixative, PGC Scientifics, Gaithersburg, MD, cat. no.
60-4890, or equivalent
6.9.4 Nonfluorescing immersion oil
December 1997 Draft
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Method 1622-Draft
6.9.5 Micropipette, adjustable: 0- to IQ-/J.L with 0- to 10-//L tips
10- to 100-//L, with 10- to 200-//L tips
100- to 1000-/^L with 100- to 1000-^L tips
6.9.6 Forceps—splinter, fine tip
6.9.7 Forceps—blunt-end
6.9.8 Desiccant—Drierite™ Absorbent, Fisher cat. no. 07-577-1A, or equivalent
6.10 Pipettes—glass or plastic
6.10.1 5-, 10-, and 25-mL
6.10.2 Pasteur, disposable
6.11 Balances
6.11.1 Analytical—capable of weighing 0.1 mg
6.11.2 Top loading—capable of weighing 10 mg
6.12 pH meter
6.13 Incubator—Fisher Scientific Isotemp™, or equivalent
6.14 Vortex mixer—Fisons Whirlmixer, or equivalent
6.15 Vacuum source—capable of maintaining 25 in. Hg, equipped with shutoff valve and vacuum
gauge
6.16 Miscellaneous labware and supplies
6.16.1 Test tubes and rack
6.16.2 Flasks—suction, Erlenmeyer, and volumetric, various sizes
6.16.3 Beakers—glass or plastic, 5-, 10-, 50-, 100-, 500-, 1000-, and 2000-mL
6.16.4 Lint-free tissues
6.17 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
6.18 Equipment for field sampling and shipping
6.18.1 -10-L carboy—Cole Farmer cat. no. 06100-33, or equivalent
6.18.2 Shipping container—Cole Farmer cat. no. 06100-03, or equivalent
7.0 Reagents and Standards
7.1 Reagents for adjusting pH
7.1.1 Sodium hydroxide (NaOH)—ACS reagent grade, 6.0 N and 1.0 N in reagent water
7.1.2 Hydrochloric acid (HC1)—ACS reagent grade, 6.0 N and 0.1 N in reagent water
7.2 Solvents—acetone, glycerol, ethanol, and methanol, ACS reagent grade
7.3 Reagent water—water in which oocysts and interfering materials and substances, including
magnetic minerals, are not detected by this method
7.4 Reagents for eluting capsule filters
7.4.1 Laureth-12—PPG Industries, Gurnee, EL, cat. no. 06194, or equivalent
December 1997 Draft
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Method 1622 - Draft
7.4.2 1 M Tris, pH 7.4—Dissolve 121.1 g Tris (Fisher cat. no. BP152) in 700 mL of reagent
water and adjust pH to 7.4 with 1 N HC1 or NaOH. Filter-sterilize through a 2-/u.m
membrane into a sterile plastic container and store at room temperature.
7.4.3 0.5 M EDTA, 2 Na, pH 8.0—Dissolve 186.1 g ethylenediamine tetraacetic acid,
disodium salt dihydrate (Fisher cat. no. S311) in 800 mL and adjust pH to 8.0 with 6.0 N
HC1 or NaOH for initial adjustment and 1.0 N HC1 or NaOH for final adjustment.
7.4.4 Antifoam A—Sigma Chemical Co. cat. no. A5758, or equivalent
7.4.5 Preparation of buffer solution—Weigh 1 g of Laureth-12 in a glass beaker and add 100
mL of reagent water. Heat the beaker to melt the Laureth-12 using a hot plate or
microwave and transfer the solution to a 1000-mL graduated cylinder. Rinse the beaker
several times to ensure the transfer of the detergent to the cylinder. Add 10 mL of Tris
solution, pH 7.4; 2 mL of EDTA solution, pH 8.0; and 150 (A. Antifoam A. Dilute to
1000 mL with reagent water.
7.5 Reagents for eluting membrane disk filters—alternate procedure requiring demonstration of
performance prior to use
7.5.1 Sodium chloride (NaCl), ACS
7.5.2 Potassium dihydrogen phosphate (KH2PO4), ACS
7.5.3 Hydrated disodium hydrogen phosphate (Na2HPO4( 12H2O)), ACS
7.5.4 Potassium chloride (KC1), ACS
7.5.5 Tween 80—Sigma Chemical Co. cat. no. P1754, or equivalent
7.5.6 Antifoam A—Sigma Chemical Co. cat. no. A5758, or equivalent
7.5.7 Sodium Lauryl Sulfate (SDS)—Sigma Chemical Co. cat. no. L4509, or equivalent
7.5.8 Preparation of buffer solution—Dissolve 8 g NaCl, 0.2 g KH2PO4, 2.9 g
Na2HPO4(12H2O), 0.2 g KC1, 1 g SDS, 1 mL Tween 80, and 100 //L Antifoam A in 750
mL of reagent water. After dissolution, adjust the volume to 1 L, mix thoroughly, and
adjust the pH to 7.4 with 1 N NaOH or HC1. Prepare weekly.
7.6 Reagents for immunomagnetic separation (IMS)
7.6.1 Dynal IMS system—Dynabeads® anti-Cryptosporidium kit, cat no. 7:30.01, or equivalent
7.6.2 ImmuCell IMS system—Crypto-Scan™ water diagnostic test kit #R10, or
equivalent—alternate procedure requiring demonstration of performance prior to use
7.7 Sample examination solutions
7.7.1 DABCO/glycerol mounting medium (2%)—Dissolve 2 g of DABCO (Sigma Chemical
Co. cat no. D-2522, or equivalent) in 95 mL of warm glycerol/PBS (60% glycerol, 40%
150 mM PBS). After the DABCO has dissolved completely, adjust the solution volume
to 100 mL by adding an appropriate volume of glycerol/PBS solution,,
7.7.2 Bovine serum albumin (BSA) (1 %)—Add 1 g of BSA crystals (Sigma Chemical Co. cat.
no. A7030) to 95 mL of 150 mM PBS (pH 7.2 [Section 7.9.3]). After the crystals have
dissolved completely, adjust the solution volume to 100 mL by adding an appropriate
volume of 150 mM PBS (pH 7.2). Filter-sterilize the solution with a 0.2-yum membrane
filter into a sterile container. Store at 0°C to 8°C and discard after 6 months or when
contamination is evident. Do not allow to freeze.
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Method 1622 - Draft
7.8
7.7.3 Hanks Balanced Salt Solution (HBSS)—Sigma Chemical Co. cat. no. H9269, or
equivalent
Detection kit—Store the kit at 0°C to 8°C and return it promptly to this temperature after each
use. Do not allow any of the reagents in this kit to freeze. The labeling reagents should be
protected from exposure to light. Diluted, unused working reagents should be discarded after 48
hours. Discard the kit after the expiration date is reached.
7.8.1 Direct labeling kit for detection of oocysts—Crypt-a-Glo™, Waterborne, Inc., New
Orleans, LA, cat. no. A400FL, or equivalent
4',6-diamidino-2-phenylindole (DAPI) vital-dye stain
7.9
7.8.2
7.8.2.1
7.8.2.2
Stock solution—Add 2 mg/mL in absolute methanol. Prepare volume
consistent with minimum use. Store at 0°C to 8°C in the dark. Discard
unused solution after 2 weeks. Do not allow to freeze.
Staining solution (1/5000 dilution in PBS)—Add 10 pL of 2 mg/mL
DAPI stock solution to 50 mL of 150 mM PBS. Prepare daily. Store at
0°C to 8°C in the dark except when staining. Do not allow to freeze.
Oocyst suspension for spiking
7.9.1 Purified, live Cryptosporidium oocyst stock suspension—not formalin-fixed
Tween 20, 0.01%—Dissolve 1.0 mL of a 10% solution of Tween 20 in reagent water.
Phosphate buffered saline (PBS), 150 mM—Add 1.07 g Na2HPO4, 0.39 g
NaH2PO4.2H2O, and 8.5 g NaCl to 800 mL reagent water. Dissolve and adjust to 1 L
volume with reagent water. Adjust pH to 7.2 with NaOH or HC1. Prepare weekly.
Storage procedure—Store oocyst suspensions at 0°C to 8°C, until ready to use. Do not
allow to freeze. Enumerated oocyst spiking suspension (Section 11.3 or 11.4) must be
"used within 24 hours of counting.
7.9.2
7.9.3
7.9.4
8.0 Sample Collection and Storage
8.1 Samples are collected in plastic 10-L carboys and shipped to the laboratory for filtration, elution,
concentration, immunomagnetic separation (IMS), staining, and examination. Samples must be
shipped to the laboratory the day that they are collected and must arrive at the laboratory within 24
hours of sample collection. Store 10-L carboys at 0°C to 8°C between collection and shipment to
the laboratory and upon receipt at the laboratory until ready for filtration. Do not allow to freeze.
NOTE: U.S. Department of Transportation (DOT) regulations (49 CFR 172)
prohibit interstate shipment of more than 4 L of solution known to contain
infectious materials. State regulations may contain similar regulations for
intrastate commerce. This method requires a minimum of 10 L to achieve the
method detection limit listed in Section 1.4. Unless the sample is known or
suspected to contain Cryptosporidium or other infectious agents (e.g.,
during an outbreak), samples should be shipped as noninfectious and should
not be marked as infectious. If a sample is known or suspected to be
infectious, and the sample must be shipped to a laboratory by a
December 1997 Draft
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Method 1622 - Draft
transportation means affected by DOT or state regulations, it is recommended
that the sample be collected in the field using a capsule or membrane disk
filter, and that the filter be shipped to the laboratory to avoid violating
transport regulations.
8.2 Sample holding times: Laboratory filtration, elution, and concentration of a sample received in a
carboy must be completed within 72 hours of sample collection. At this point, a break may be
inserted if the laboratory will not progress immediately to the immunomagnetic separation (IMS)
procedure. If a break is inserted at this point, the concentrate must be stored at 0°C to 8 °C. Do not
allow to freeze.
8.3 Concentrate holding times: IMS and sample staining must be completed within 24 hours of
completion of sample concentration. Stained slides must be stored at 0°C to 8°C in the dark. Do
not allow to freeze.
8.4 Stained sample holding times: Immunofluorescence assay (FA) and 4',6-diamidino-2-phenylindole
(DAPI) and differential interference contrast (D.I.C.) microscopy examination and confirmation
must be completed within 72 hours of completion of sample staining.
9.0 Quality Control
9.1 Each laboratory that uses this method is required to operate a formal quality assurance (QA)
program (Reference 20.6). The minimum requirements of this program consist of an initial
demonstration of laboratory capability, analysis of spiked samples to evaluate and document data
quality, and analysis of standards and blanks as tests of continued performance. Laboratory
performance is compared to established performance criteria to determine if the results of analyses
meet the performance characteristics of the method.
9.1.1 A test of the microscope used for detection of oocysts is performed prior to examination
of slides. This test is described in Section 10.
9.1.2 In recognition of advances that are occurring in analytical technology, the laboratory is ._
permitted certain options to improve detection or lower the costs of measurements,
provided that all quality control acceptance criteria are met. If an anab/tical technique
other than the techniques specified in this method is used, that technique must have a
specificity equal to or better than the specificity of the techniques in this method for
Cryptosporidium in the sample of interest. Specificity is defined as producing results
equivalent to the results produced by this method for Cryptosporidium in reagent water
and environmental samples, and that meet all of the quality control (QC) acceptance
criteria stated in this method.
9.1.2.1 Each time a modification is made to this method, the analyst is required to
repeat the initial precision and recovery (IPR) test in Section 9.4.2 to
demonstrate that the modification produces results equivalent to or
superior to results produced by this method. If the detection limit of the
method will be affected by the modification, the analyst must demonstrate
that the method detection limit (MDL) (40 CFR 136, Appendix B) is less
than or equal to the MDL in this method or one-third the regulatory
compliance level, whichever is higher. The tests required for this
equivalency demonstration are given in Section 9.4.
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Method 1622 - Draft
9.1.3
9.1.4
9.1.5
9.1.6
9.1.2.2 The laboratory is required to maintain records of modifications made to
this method. These records include the following, at a minimum:
9.1.2.2.1 The names, titles, addresses, and telephone numbers of
the analyst(s) who performed the analyses and
modification, and of the quality control officer who
witnessed and will verify the analyses and modification.
9.1.2.2.2 A listing of the analyte measured (Cryptosporidium).
9.1.2.2.3 A narrative stating reason(s) for the modification.
9.1.2.2.4 Results from all QC tests comparing the modified method
to this method, including:
(a) Microscope calibration (Section 10)
(b) Calibration verification (Section 10)
(c) Initial precision and recovery (Section 9.4.2)
(d) Analysis of blanks (Section 9.6)
(e) Accuracy assessment (Section 9.4)
(f) Ongoing precision and recovery (Section 9.7)
9.1.2.2.5 Data that will allow an independent reviewer to validate
each determination by tracing the following processing
and analysis steps leading to the final result:
(a) Sample numbers and other identifiers
(b) All spiking suspension enumeration calculations
(Section 11)
(c) Volume filtered (Section 12)
(d) Filtration and concentration dates and times
(e) Initial and final pellet volumes (Section 13)
(f) Staining dates and times (Section 13 and 14)
(g) Examination and confirmation dates and times
(h) Analysis sequence/run chronology
(i) Make and model of microscope
(j) Copies of bench sheets, logbooks, and other
recordings of raw data
(k) Data system outputs, and other data to link the
raw data to the results reported
The laboratory shall spike a separate aliquot of samples from the same source to monitor
method performance. This test is described in Section 9.7. When results of these spikes
indicate atypical method performance, the sample is diluted before spiking to bring
method performance within acceptable limits (Section 16).
Analyses of blanks are required to demonstrate freedom from contamination. The
procedures and criteria for analysis of a blank are described in Section 9.6.
The laboratory shall, on an ongoing basis, demonstrate through analysis of the ongoing
precision and recovery (OPR) sample that the analysis system is in control. These
procedures are described in Section 9.7.
The laboratory shall maintain records to define the quality of data that is generated.
Development of accuracy statements is described in Section 9.5.4 and 9.7.3.
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Method 1622 - Draft
9.1.7 The laboratory shall analyze 1 laboratory blank (Section 9.6) and 1 OPR sample (Section
9.7) each week during which samples are analyzed if 20 or fewer field samples are
analyzed during this period. The laboratory shall analyze 1 laboratory blank and 1 OPR
sample for every 20 samples if more than 20 samples are analyzed in a week.
9.1.8 The laboratory shall analyze one matrix spike (MS) sample (Section 9.5) when samples
are first received from a utility for which the laboratory has never before analyzed
samples. The MS analysis should be performed on an extra sample sent from the utility.
If the laboratory routinely analyzes samples from 1 or more utilities, 1 MS analysis must
be performed per 20 field samples. For example, when a laboratory receives the first
sample from a given site, the laboratory must obtain a second aliquot of this sample to be
used for the MS. When the laboratory receives the 21st sample from this site, a separate
aliquot of this 21st sample must be collected and spiked.
9.2 Micropipette calibration
9.2.1 Micropipettes must be sent to manufacturer for calibration annually. Alternately, a
qualified independent technician specializing in micropipette calibration can be used.
Documentation on the precision of the recalibrated micropipette must be obtained from
the manufacturer or technician.
9.2.2 Internal and external calibration records must be kept on file in the laboratory's QA
logbook.
9.2.3 If a micropipette calibration problem is suspected, the laboratory shall tare an empty
weighing boat on the analytical balance and pipette the following volumes of reagent
water into the weigh boat using the pipette in question: 100% of the maximum
dispensing capacity of the micropipette, 50% of the capacity, and 109& of the capacity.
Record the weight of the water (assume that 1.00 mL of reagent water weighs 1.00 g). If
the weight of the reagent water is within 1 % of the desired weight (mL) then the pipette
remains acceptable for use.
9.2.4 If the weight of the reagent water is outside the acceptable limits, consult the
manufacturer's instruction manual troubleshooting section and repeat steps described in
Section 9.2.3. If problems with the pipette persist, the laboratory must send the pipette to
the manufacturer for recalibration.
9.3 Microscope adjustment and certification: Adjust the microscope as specified in Section 10. All of
the requirements in Section 10 must be met prior to analysis of samples, blanks, OPRs, and MSs.
9.4 Initial demonstration of laboratory capability
9.4.1 Method detection limit (MDL)—To establish the ability to detect Cryptosporidium
oocysts, the laboratory shall determine the MDL in reagent water per the procedure in 40
CFR 136, Appendix B using the apparatus, reagents, and standards that will be used in
the practice of this method. An MDL less than or equal to the MDL in Section 1.4 must
be achieved prior to the practice of this method.
9.4.2 Initial precision and recovery (IPR)—To establish the ability to demonstrate control over
the analysis system and to generate acceptable precision and accuracy, the laboratory
shall perform the following operations:
9.4.2.1 Using the spiking procedure in Section 11.5 and enumerated oocyst
spiking suspension containing 500 to 1000 oocysts (Section 11.3 or 11.4),
the laboratory must filter, elute, concentrate, separate (purify), stain, and
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Method 1622 - Draft
examine four 10-L aliquots. If more than one filtration and/or separation
process will be used for filtration/separation of samples, a separate set of
IPR aliquots must be prepared for each process.
9.4.2.2 Using results of the four analyses, compute the average percent recovery
(X) and the relative standard deviation of the recovery (sr) for
Cryptosporidium.
9.4.2.3 Compare sr and X with the corresponding limits for initial precision and
recovery in Table 1. If Sr and X meet the acceptance criteria, system
performance is acceptable and analysis of blanks and samples may begin.
If, however, any individual sr exceeds the precision limit or any individual
X falls outside the range for recovery, system performance is
unacceptable for Cryptosporidium. In this event, correct the problem and
repeat the test (Section 9.4.2).
9.5 Matrix spike (MS): The laboratory shall spike and analyze a separate field sample aliquot to
determine the effect of the matrix on the method's recovery efficiency. MSs shall be analyzed
according to the frequency in Section 9.1.8.
9.5.1 Analyze an unspiked field sample according to the method beginning in Section 12.
Using the spiking procedure in Section 11.5 and an appropriate volume of the
enumerated oocyst spiking suspension (Section 11.3 or 11.4), spike the second field
sample aliquot to produce five times the number of oocysts detected in the unspiked
sample or the number used in the IPR/OPR, whichever is greater.
9.5.2 Compute the percent recovery (R) of the oocysts using the following equation.
Nm -AT,
fl=100x-
"sp
9.5.3
9.5.4
where
R is the percent recovery
Nsp is the number of oocysts detected in the spiked sample
Ns is the number of oocysts detected in the unspiked sample
T is the true value of the oocysts spiked
Compare the oocyst recovery with the corresponding limits in Table 1. If the recovery for
Cryptosporidium falls outside its limit, method performance is unacceptable for that
sample. If the results for the blank (Section 9.6) and for the OPR sample (Section 9.7)
associated with this batch of samples are within their respective control limits, a matrix
interference may be causing the poor recovery. See Section 16 for instructions for dealing
with matrix interferences. If the results for the blank and OPR are not within their control
limits, the laboratory is not in control. The problem must be identified and corrected and
a fresh sample should be collected and reanalyzed.
As part of the QA program for the laboratory, method precision for samples should be
assessed and records maintained. After the analysis of five samples for which the spike
recovery for Cryptosporidium passes the tests in Section 9.5.3, the laboratory should
compute the average percent recovery (P) and the standard deviation of l&e percent
recovery (sr) for Cryptosporidium. Express the precision assessment as a percent recovery
interval from P - 2 sr to P + 2 Sr for each matrix. For example, if P = 80% and sr = 30%,
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Method 1622 - Draft
the accuracy interval is expressed as 20% to 140%. The precision assessment should be
updated on a regular basis (e.g., after each 5 to 10 new accuracy measurements).
9.6 Blank (negative control sample): Reagent water blanks are analyzed to demonstrate freedom from
contamination. Analyze the blank immediately prior to analysis of the OPR (Section 9.7) and prior
to analysis of samples for the week to demonstrate freedom from contamination,.
9.6.1 Filter, elute, concentrate, separate (purify), stain, and examine one reagent water blank
per week (Section 9.1.7) using the procedures in Sections 12 to 15. If more than 20
samples are analyzed in a week period, process and analyze 1 reagent water blank for
every 20 samples.
9.6.2 If a single Cryptosporidium oocyst or any potentially interfering organism or material is
found in the blank, analysis of additional samples is halted until the source of
contamination is eliminated and a blank shows no evidence of contamination. Any
sample in a batch associated with a contaminated blank that shows the presence of one or
more oocysts is assumed to be contaminated and must be recollected. Any sample in
which oocysts are not detected is assumed to be uncontaminated and may be reported.
9.7 Ongoing precision and recovery ([OPR]; positive control sample; laboratory control sample):
Using the spiking procedure in Section 11.5 and enumerated oocyst spiking suspension containing
500 to 1000 oocysts (Section 11.3 or 11.4), filter, elute, concentrate, separate (purify), stain, and
examine one reagent water sample at least once per week to verify all performance criteria. The
laboratory must analyze one OPR sample for every 20 samples if more than 20 samples are
analyzed in a week. Adjustment and/or recalibration of the analytical system shiill be performed
until all performance criteria are met. Only after all performance criteria are melt may samples be
analyzed.
9.7.1 Examine the slide from the OPR prior to analysis of samples from the same batch.
9.7.1.1 More than 50% of the oocysts must be appear undamaged and
morphologically intact; otherwise, the analytical process is damaging the
oocysts. Determine the step or reagent that is causing damage to the
oocysts. Correct the problem and repeat the OPR test.
9.7.1.2 Identify and enumerate each oocyst using epifluorescence microscopy.
Each oocyst must meet the identification criteria in Section 15.
9.7.2 Compute the percent recovery of the total number of oocysts using the following
equation:
= 100 x
N
where
N = the number of oocysts detected
T = the number of oocysts spiked
9.7.2.1 Compare the recovery with the limits for ongoing precision and recovery
in Table 1. If the recovery meets the acceptance criteria, system
performance is acceptable and analysis of blanks and samples may
proceed. If, however, the recovery falls outside of the range given, system
performance is unacceptable. In this event, there may be a problem with
the microscope or with the filtration or separation systems. Reanalyze the
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Method 1622 - Draft
OPR sample and recollect and reanalyze samples. All samples must be
associated with an OPR that passes the criteria in Table 1.
9.7.2.2 Microscope system: To determine if the failure of the OPR test (Section
9.7.2.1) is due to changes in the microscope, examine a slide containing a
known number of freshly prepared oocysts, check Kohler illumination,
and check the fluorescence of the fluorescein-labeled monoclonal
antibody (Mab) and 4',6-diamidino-2-phenylindole (DAPI).
9.7.2.3 Filtration/ elution/ concentration system: If the failure of the OPR test
(Section 9.7.2.1) is attributable to the filtration/ elution/ concentration
system, these systems may not be in control. Check filtration/ elution/
concentration system performance using spiked reagent water, eluting the
filter, and, analyzing the concentrated sample without separation
(purification) using FA.
9.7.2.4 Separation (purification) system: If the failure of the OPR test (Section
9.7.2.1) is attributable to the separation system, this system may not be in
control. Check separation system performance using spiked reagent water
with IMS and analyzing the purified sample using FA.
9.7.3 The laboratory should add results that pass the specifications in Section 9.7.2.1 to initial
and previous ongoing data and update the QC chart to form a graphic representation of
continued laboratory performance. The laboratory should develop a statement of
laboratory accuracy (reagent water, raw water) by calculating the average percent
recovery (R) and the standard deviation of percent recovery (sr). Express the accuracy as
a recovery interval from R - 2 sr to R + 2 sr. For example, if R = 95% and sr = 25%, the
accuracy is 45% to 145%.
9.8 The laboratory should periodically analyze an external QC sample, such as a performance
evaluation or standard reference material, when available. The laboratory also should periodically
participate in interlaboratory comparison studies using the method.
9.9 The specifications contained in this method can be met if the analytical system is maintained under
control. The standards used for initial (Section 9.4) and ongoing (Section 9.7) precision and
recovery should be identical, so that the most precise results will be obtained. The microscope in
particular will provide the most reproducible results if dedicated to the settings and conditions
required for the determination of Cryptosporidium by this method.
9.10 Depending on specific program requirements, field replicates may be collected to determine the
precision of the sampling technique, and duplicate spiked samples may be required to determine
the precision of the analysis.
10.0 Microscope Calibration and Analyst Verification
10.1 In a room capable of being darkened to near-complete darkness, assemble the microscope and all
filters and attachments. The microscope should be placed on a solid surface free from vibration.
Adequate workspace should be provided on either side of the microscope for taking notes and
placement of slides and ancillary materials.
10.2 Using the manuals provided with the microscope, the principal analyst/supervisor and all analysts
must familiarize themselves with operation of the microscope.
10.3 Microscope adjustment and calibration (adapted from Reference 20.6)
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Method 1622 - Draft
10.3.1 Preparations for adjustment
10.3.1.1 The microscopy portion of this procedure depends upon proper alignment
and adjustment of very sophisticated optics. Without proper alignment
and adjustment, the microscope will not function at maximal efficiency,
and reliable identification and enumeration of oocysts will not be
possible. Consequently, it is imperative that all portions of the microscope
from the light sources to the oculars are properly adjusted.
10.3.1.2 While microscopes from various vendors are configured somewhat
differently, they all operate on the same general physical principles.
Therefore, slight deviations or adjustments may be required to make the
procedures below work for a particular instrument.
10.3.1.3 The sections below assume that the mercury bulb has not exceeded time
limits of operation, that the lamp socket is connected to the lamp house,
and that the condenser is adjusted to produce Kohler illumination.
10.3.1.4 Persons with astigmatism should always wear contact lenses or glasses
when using the microscope.
CAUTION: In the procedures below, do not touch the quartz portion of the
mercury bulb with your bare fingers. Finger oils can cause rapid degradation
of the quartz and premature failure of the bulb.
WARNING: Never look at the ultraviolet (UV) light from the mercury lamp,
lamp house, or the UV image without a barrier filter in place. UV radiation
can cause serious eye damage.
10.3.2 Epifluorescent mercury bulb adjustment: The purpose of this procedure is to insure even
field illumination. This procedure must be followed when the microscope is first used,
when replacing bulbs, and if problems such as diminished fluorescence or uneven field
illumination are experienced.
10.3.2.1 Remove the diffuser lens between the lamp and microscope or swing it
out of the transmitted light path.
10.3.2.2 Using a prepared microscope slide, adjust the focus so the image in the
oculars is sharply defined.
10.3.2.3 Replace the slide with a business card or a piece of lens paper.
10.3.2.4 Close the field diaphragm (iris diaphragm in the microscope base) so only
a small point of light is visible on the card. This dot of light tells you
where the center of the field of view is.
10.3.2.5 Mount the mercury lamp house on the microscope without the UV
diffuser lens in place and turn on the mercury bulb.
10.3.2.6 Remove the objective in the light path from the nosepiece. A primary
(brighter) and secondary image (dimmer) of the mercury bulb arc should
appear on the card after focusing the image with the appropriate
adjustment.
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Method 1622 - Draft
10.3.2.7 Using the lamp house adjustments, adjust the primary and secondary
mercury bulb images so they are side by side (parallel to each other) with
the transmitted light dot in between them.
10.3.2.8 Reattach the objective to the nosepiece.
10.3.2.9 Insert the diffuser lens into the light path between the mercury lamp house
and the microscope.
10.3.2.10 Turn off the transmitted light and replace the card with a slide of
fluorescent material. Check the field for even fluorescent illumination.
Adjustment of the diffuser lens will most likely be required. Additional
slight adjustments as in Section 10.3.2.7 above may be required.
10.3.2.11 Maintain a log of the number of hours the UV bulb has been used. Never
use the bulb for longer than it has been rated. Fifty-watt bulbs should not
be used longer than 100 hours; 100-watt bulbs should not be used longer
than 200 hours.
10.3.3 Transmitted bulb adjustment: The purpose of this procedure is to center the filament and
insure even field illumination. This procedure must be followed when the bulb is
changed.
10.3.3.1 Remove the diffuser lens between the lamp and microscope or swing it
out of the transmitted light path.
10.3.3.2 Using a prepared microscope slide and a 40X (or similar) objective,
adjust the focus so the image in the oculars is sharply defined.
10.3.3.3 Without the ocular or Bertrand optics in place, view the pupil and
filament image at the bottom of the tube.
10.3.3.4 Focus the lamp filament image with the appropriate adjustment on the
lamp house.
10.3.3.5 Similarly, center the lamp filament image within the pupil with the
appropriate adjustment(s) on the lamp house.
10.3.3.6 Insert the diffuser lens into the light path between the transmitted lamp
house and the microscope.
10.3.4 Adjustment of the interpupillary distance and oculars for each eye: These adjustments are
necessary so that eye strain is reduced to a minimum, and must be made for each
individual using the microscope. Section 10.3.4.2 assumes use of a microscope with both
oculars adjustable; Section 10.3.4.3 assumes use of a microscope with a single adjustable
ocular. The procedure must be followed each time an analyst uses the microscope.
10.3.4.1 Interpupillary distance
10.3.4.1.1 Place a prepared slide on the microscope stage, turn on
the transmitted light, and focus the specimen image using
the coarse and fine adjustment knobs.
10.3.4.1.2 Using both hands, move the oculars closer together or
farther apart until a single circle of light is observed
while looking through the oculars with both eyes. Note
interpupil distance.
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Method 1622 - Draft
10.3.4.2 Ocular adjustment for microscopes capable of viewing a photographic
frame through the viewing binoculars: This procedure assumes both
oculars are adjustable.
10.3.4.2.1 Place a card between the right ocular and eye keeping
both eyes open. Adjust the correction (focusing) collar on
the left ocular by focusing the left ocular until it reads the
same as the interpupillary distance. Bring an image
located in the center of the field of view into as sharp a
focus as possible.
10.3.4.2.2 Transfer the card to between the left eye and ocular.
Again keeping both eyes open, bring the same image into
as sharp a focus for the right eye as possible by adjusting
the ocular correction (focusing) collar at the top of the
right ocular.
10.3.4.3 Ocular adjustment for microscopes without binocular capability: This
procedure assumes a single focusing ocular. The following procedure
assumes that only the right ocular is capable of adjustment.
10.3.4.3.1 Place a card between the right ocular and eye keeping
both eyes open. Using the fine adjustment, focus the
image for the left eye to its sharpest point.
10.3.4.3.2 Transfer the card to between the left eye and ocular.
Keeping both eyes open, bring the image for the right eye
into sharp focus by adjusting the ocular collar at the top
of the ocular without touching the coexse or fine
adjustment.
10.3.5 Calibration of an ocular micrometer: This section assumes that a reticle has been installed
in one of the oculars by a microscopy specialist and that a stage micrometer is available
for calibrating the ocular micrometer (reticle). Once installed, the ocular reticle should be
left in place. The more an ocular is manipulated the greater the probability is for it to
become contaminated with dust particles. This calibration should be done for each
objective in use on the microscope. If there is a top lens on the microscope, the
calibration procedure must be done for the respective objective at each top lens setting.
The procedure must be followed when the microscope is first used and each time
objective is changed.
10.3.5.1 Place the stage micrometer on the microscope stage, turn on the
transmitted light, and focus the micrometer image using the coarse and
fine adjustment knobs for the objective to be calibrated. Continue
adjusting the focus on the stage micrometer so you can distinguish
between the large (0.1 mm) and the small (0.01 mm) divisions.
10.3.5.2 Adjust the stage and ocular with the micrometer so the 0 line on the
ocular micrometer is exactly superimposed on the 0 lime on the stage
micrometer.
10.3.5.3 Without changing the stage adjustment, find a point as distant as possible
from the two 0 lines where two other lines are exactly superimposed.
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Method 1622 -Draft
10.3.5.4
10.3.5.5
10.3.5.6
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.
Calculate the number of mm/ocular micrometer space. For example:
0.6 mm
0.0125 mm
48 ocular micrometer spaces ocular micrometer space
Because most measurements of microorganisms are given in /am rather
than mm, the value calculated above must be converted to ^m by
multiplying it by 1000 ^m/mm. For example:
0.0125 mm
1,000
12.5
ocular micrometer space mm ocular micrometer space
10.3.5.7
Follow the procedure below for each objective. Record the information as
shown in the example below and keep the information available at the
microscope.
Item
#
1
2
3
4
Obj.
Power
10X
20X
40X
100X
Description
N.A.C =
N.A.=
N.A. =
N.A.=
No. of Ocular
Microm.
Spaces
No. of
Stage
Microm.
mma
a 1000 ^m/mm
//m/Ocular
Micrometer
Spaceb
b (Stage Micrometer length in mm x (1,000 ^m/mm)) -r No. Ocular Micrometer Spaces
c N.A. stands for numerical aperture. The numerical aperture value is engraved on
the barrel of the objective.
10.3.6 Kohler illumination: This section assumes that Kohler illumination will be established for
only the 100X oil D.I.C. objective that will be used to identify internal morphological
characteristics in Cryptosporidium oocysts. If more than one objective is to be used for
D.I.C., then each time the objective is changed, Kohler illumination must be
reestablished for the new objective lens. Previous sections have adjusted oculars and light
sources. This section aligns and focuses the light going through the condenser underneath
the stage at the specimen to be observed. If Kohler illumination is not properly
19
December 1997 Draft
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Method 1622 - Draft
established, then D.I.C. will not work to its maximal potential. These steps need to
become second nature and must be practiced regularly until they are a matter of reflex
rather than a chore. The procedure must be followed each time an analyst uses the
microscope and each time the objective is changed.
10.3.6.1 Place a prepared slide on the microscope stage, place oil on the slide,
move the 100X oil objective into place, turn on the transmitted light, and
focus the specimen image using the coarse and fine adjustment knobs.
10.3.6.2 At this point both the radiant field diaphragm in the microscope base and
the aperture diaphragm in the condenser should be wide open. Now close
down the radiant field diaphragm in the microscope base until the lighted
field is reduced to a small opening.
10.3.6.3 Using the condenser centering screws on the front right and left of the
condenser, move the small lighted portion of the field to the center of the
visual field.
10.3.6.4 Now look to see whether the leaves of the iris field diaphragm are sharply
defined (focused) or not. If they are not sharply defined, then they can be
focused distinctly by changing the height of the condenser up and down
with the condenser focusing knob while you are looking through the
binoculars. Once you have accomplished the precise focusing of the
radiant field diaphragm leaves, open the radiant field diaphragm until the
leaves just disappear from view.
10.3.6.5 The aperture diaphragm of the condenser is adjusted now 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.
10.3.6.6 After completing the adjustment of the aperture diaphragm in the
condenser, return the ocular to its tube and proceed with the adjustments
required to establish D.I.C.
10.4 Protozoa libraries: Each laboratory is encouraged to develop libraries of photographs and drawings
for identification of protozoa.
10.4.1 Take color photographs of Cryptosporidium oocysts by FA and 4',6-d.iamidino-2-
phenylindole (DAPI) that the principal analyst/supervisor (Sections 1.5,10.5, and 15)
determines are true and accurate.
10.4.2 Similarly, take color photographs of interfering organisms and materials by FA and .DAPI
that the principal analyst/supervisor believes are not Cryptosporidium oocysts. Quantify
the size, shape, microscope settings, and other characteristics that can be used to
differentiate oocysts from interfering debris and that will result in positive identification
of DAPI + or - organisms.
10.5 Verification of performance: Until standard reference materials, such as National Institute of
Standards and Technology standard reference materials, are available that contain a reliable
number of DAPI + or - oocysts, this method shall rely upon the ability of the principal
analyst/supervisor for identification and enumeration of oocysts.
December 1997 Draft
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Method 1622-Draft
10.5.1
10.5.2
10.5.3
At least monthly during which microscopic examinations are to be performed, the
principal analyst/supervisor shall prepare a slide containing 40 to 100 oocysts. More than
50% of the oocysts must be DAPI +. The principal analyst/supervisor shall determine the
numbers of total oocysts by FA and number of oocysts that are DAPI + or -, using the
procedures in this method, and these numbers shall be known only to the principal
analyst/supervisor.
Each analyst shall determine the total number of oocysts and the number that are DAPI +
or -, using the slide provided by the principal analyst/supervisor (Section 10.5.1).
The total number and the number of DAPI + or - oocysts determined by each analyst
(Section 10.5.2.) must be within ±10% of the number determined by the principal
analyst/supervisor. If the number is not within this range, the principal analyst/ supervisor
and the analyst shall resolve how to identify and enumerate DAPI + or - oocysts, and the
principal analyst/supervisor shall prepare a new slide and the test (Sections 10.5.1 to
10.5.2) shall be repeated.
NOTE: If the laboratory has only a principal analyst/supervisor; i.e., the
laboratory is not of sufficient size to support one or more analysts, the
principal analyst/supervisor shall perform the identification and
enumeration of total, DAPI + and - oocysts on each day on which oocysts are to
be identified and enumerated in samples, and the reputation of the laboratory
shall rest with the principal analyst/supervisor.
10.5.4
10.5.5
Document the date, name of principal analyst/supervisor, name(s) of analyst(s), number
of total, DAPI + or - oocysts placed on the slide, number determined by the principal
analyst/supervisor, number determined by the analyses), whether the test was
passed/failed for each analyst, and the number of attempts prior to passage.
Only after an analyst has passed the criteria in Section 10.5.3, may oocysts in blanks,
standards, and samples be identified and enumerated.
11.0 Oocyst Suspension Enumeration and Spiking
11.1 Two sets of enumerations are required before purified oocyst stock suspensions received from
suppliers can be used to spike samples in the laboratory. First, the oocyst stock suspension must be
diluted and enumerated to yield an oocyst suspension at the appropriate concentration for spiking
(oocyst spiking suspension). Then, 10 aliquots of oocyst spiking suspension must be enumerated to
calculate a mean spike dose. Oocyst spiking suspensions can be enumerated using either
hemacytometer chamber counting or well-slide counting. The procedure for diluting and
enumerating purified oocyst stock suspensions is provided in Section 11.2. The two procedures for
enumerating oocyst spiking suspensions are provided in Sections 11.3 and 11.4. The procedure for
spiking 10-L carboys in the laboratory is provided in Section 11.5.
11.2 Enumerating purified stock oocyst suspension
11.2.1 Concentrated Cryptosporidium oocyst stock suspension must be diluted and enumerated
before the diluted suspension is used to spike samples in the laboratory. Stock suspension
should be diluted with reagent water/Tween 20, 0.01%, to a concentration of 20 to 50
oocysts per large hemacytometer square before proceeding to Section 11.2.2.
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December 1997 Draft
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Method 1622 - Draft
11.2.2
11.2.3
11.2.4
11.2.5
11.2.6
11.2.7
11.2.8
11.2.9
Apply a clean hemacytometer coverslip to the hemacytometer and load the
hemacytometer chamber with 10 /A. of vortexed oocyst suspension per chamber. If this
operation has been properly executed, the liquid should amply fill the entire chamber
without bubbles or overflowing into the surrounding moats. Repeat this step with a clean,
dry hemacytometer and coverslip if loading has been incorrectly performed. See Section
11.2.13, below, for the hemacytometer cleaning procedure.
Place the hemacytometer on the microscope stage and allow the oocysts to settle for 2
minutes Do not attempt to adjust the coverslip, apply clips, or in any way disturb the
chamber after it has been filled.
Use a magnification of 400X to 500X.
Move the chamber so the ruled area is centered underneath it.
Move the objective close to the coverslip while watching it from the side of the
microscope, rather than through the microscope.
Focus up from the coverslip until the hemacytometer ruling appears.
At each of the four corners of the chamber is a 1-square-mm area divided into 16 squares
in which oocysts are to be counted (Figure 1). Beginning with the topi row of four
squares, count with a hand-tally counter in the directions indicated in Figure 2. Avoid
counting oocysts twice by counting only those touching the top and left boundary lines.
Count each square millimeter in this fashion.
Use the following formula to determine the number of oocysts per mL of suspension:
number of oocysts counted 10
number of mm 2 counted 1mm
^dilution factor x1000 mm* =nL,mberof oocysts/mL
1
1 mL
11.2.10 Record the result on a hemacytometer data sheet.
11.2.11 A total of six different hemacytometer chambers must be loaded, counted, and averaged
for each oocyst suspension to achieve optimal counting accuracy.
11.2.12 Based on the hemacytometer counts, the stock suspension should be diluted to a final
concentration of between 8000 and 12,000 oocysts per mL (80 to 120 oocysts per 10
//L); however, ranges as great as 5000 to 15,000 oocysts per mL (50 to 150 oocysts per
10 //L) can be used.
NOTE: If the diluted stock suspension (the spiking suspension) will be
enumerated using hemacytometer chamber counts (Section 11.3), then the stock
suspension should be diluted with reagent water/Tween 20, 0.01%. If the
spiking suspension will be enumerated using well-slide counts (Section
11.4), or if both hemacytometer chamber counts and well-slide counts will be
used to enumerate the oocyst spiking suspension, then the stock suspension
should be diluted using reagent water only.
December 1997 Draft
22
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Method 1622 - Draft
To calculate the volume (in .//L) of stock oocyst suspension required per mL of reagent
water (or reagent water/Tween 20, 0.01%), use the following formula:
volume of stock suspension(pL) required = re^uired number of oocysts x WOO ul
number of oocysts I mL of stock suspension
If the volume is less than 10 pL, an additional dilution of the stock suspension is
recommended before proceeding.
To calculate the dilution factor needed to achieve the required number of oocysts per 10
fjL, use the following formula:
tote/ volume (fiL) = number of oocysts required xW(iL
predicted number of oocysts per 70/zL (80 to 120)
To calculate the volume of reagent water (or reagent water/Tween 20, 0.01 %) needed,
use the following formula:
reagent water volume (pL) =
total volume (u.1) - stock oocyst suspension volume required (u.L)
11.2.13 After each use, the hemacytometer and coverslip must be cleaned immediately to prevent
the oocysts and debris from drying on it. Since this apparatus is precisely machined,
abrasives cannot be used to clean it, as they will disturb the flooding and volume
relationships.
11.2.13.1 Rinse the hemacytometer and cover glass first with tap water, then 70%
ethanol, and finally with acetone.
11.2.13.2 Dry and polish the hemacytometer chamber and cover glass with lens
paper. Store it in a secure place.
11.2.14 Several factors are known to introduce errors into hemacytometer counts, including:
Inadequate suspension mixing before flooding the chamber
Irregular filling of the chamber, trapped air bubbles, dust, or oil on the chamber or
coverslip
Total number of oocysts counted is too low to provide statistical confidence in the
result
• Error in recording tally
Calculation error; failure to consider dilution factor, or area counted
Inadequate cleaning and removal of oocysts from the previous count
Allowing filled chamber to sit too long, so that the chamber suspension dries and
concentrates.
11.3 Enumerating the oocyst spiking suspension using a hemacytometer chamber
23
December 1997 Draft
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Method 1622 - Draft
11.3.1 Vortex the stock oocyst suspension for a minimum of 2 minutes before removing any
aliquots. To an appropriate-size beaker containing a stir bar, add enough oocyst spiking
suspension (diluted stock oocyst suspension; Section 11.2) to perform all spike testing
and the enumeration as described. The liquid volume and beaker relationship should be
such that a spinning stir bar does not splash the sides of the beaker, the stir bar has
unimpeded rotation, and there is enough room to draw sample from the; beaker with a 10-
A*L micropipette without touching the stir bar. Cover the beaker with a watch glass or
petri dish to prevent evaporation between sample withdrawals.
11.3.2 Allow the beaker contents to stir for a minimum of 30 minutes before beginning
enumeration.
11.3.3 While the stir bar is still spinning, remove a 10-^L aliquot and carefully load one side of
the hemacytometer. Count all organisms on the platform, at 200X magnification using
phase-contrast or darkfield microscopy. The count must include the entire area under the
hemacytometer, not just the four outer 1-square-mm squares. Repeat this procedure nine
times. This step allows confirmation of the number of oocysts per 10 //L (Section
11.2.12). If the mean number is outside the expected range, add additional oocysts from
stock suspension or dilute the contents of the beaker appropriately with reagent water.
Repeat the process to confirm counts. Refer to Section 11.2.14 for factors that may
introduce errors into the hemacytometer counts and use and care of the hemacytometer
and hemacytometer coverslip.
11.4 Enumerating the oocyst spiking suspension using well slides
11.4.1 Remove 12-mm-diameter well slides from the box in the refrigerator s.nd lay the slides on
a flat surface for 15 minutes to allow them to warm to room temperature.
11.4.2 Vortex the vial containing oocyst spiking suspension (diluted stock oocyst suspension;
Section 11.2) for approximately 30 seconds.
11.4.3 Remove a 10-fj.L aliquot from the oocyst spiking suspension and apply it to the center of
a well.
11.4.4 Before removing subsequent aliquots, cap the vial and gently invert it three times to
ensure oocysts are in suspension.
11.4.5 Ten wells must be counted, and the counts averaged, to sufficiently enumerate the spike
dose.
11.4.6 Positive and negative controls must be prepared.
11.4.6.1 For the positive control, pipette 10 /A. of positive antigen or 200 to 500
intact oocysts onto the center of a well and distribute positive antigen or
oocysts evenly over the well area.
11.4.6.2 For the negative control, pipette 75 ^L of 150 mM PBS onto the center of
a well and spread it over the well area with a pipette tip.
11.4.7 Place the well slides containing the samples in a 42°C incubator and evaporate to dryness
(approximately 1 to 2 hours).
11.4.8 Apply 50-AiL of absolute methanol to each well containing the dried sample and allow
the slide to air dry until the methanol has evaporated (approximately 3 to 5 minutes).
11.4.9 Follow manufacturer's instructions in preparing dilutions of anti-Cryptosporidium sp.
fluorescein-labeled monoclonal antibody (Mab) and overlay the 10 spike-dose wells, the
December 1997 Draft
24
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Method 1622 -Draft
positive-control well and the negative-control well with 50 fj.L of fluorescein-labeled
Mab. Place the slides in a humid chamber and incubate at 37 °C for approximately 30
minutes. The humid chamber consists of a tightly sealed plastic container containing
damp paper towels on which the slides are placed.
11.4.10 After 30 minutes, remove the slides from the humid chamber. Use a clean Pasteur pipette
tip attached to a vacuum source to gently aspirate the excess fluorescein-labeled Mab
from the side of each well. When performing this step, ensure that the vacuum source is
at the absolute minimum (<2 in Hg vac.) and ensure that the pipette tip does not scratch
the well surface.
11.4.11 Apply 65 to 75 ^L of 150 mM PBS, pH 7.2, to each of the 12 wells and allow to stand
for 1 to 5 minutes, then aspirate the excess PBS. When removing the excess PBS, ensure
that the pipette tip does not scratch the well surface. Repeat this washing procedure two
more times.
11.4.12 Apply a drop of reagent water to each well and allow to stand for approximately 1
minute, then aspirate the excess reagent water.
11.4.13 Place the slides containing the fluorescein-labeled Mab in a dry box and allow the slides
to stand in the dark for approximately 1 hour at room temperature. The dry box consists
of a tightly sealed plastic container with desiccant in the bottom. A paper towel must be
placed over the desiccant.
11.4.14 Apply 10 /J.L of mounting medium (60% glycerol, 40% 150 mM PBS) containing an
anti-fadant (2% DABCO, or equivalent) to the center of each well.
11.4.15 Place a 22 x 50 mm coverslip on each three-well microscope slide and gently depress the
coverslip at the edges. Use a tissue to remove excess mounting fluid from the edges of
the coverslip, then seal the edges of the coverslip onto the slide by using clear nail polish.
Store in a dry box, in the dark, until ready for enumeration.
Procedure for spiking samples in the laboratory with enumerated oocyst spiking suspension
11.5.1 Arrange the bottom-dispensing, 10-L carboy to gravity-feed a filter housing. Housing
outlet will feed self-priming centrifugal pump.
Install the capsule filter or the membrane disk filter in its appropriate housing. Place large
stir bar in the carboy. Fill the carboy with 10.0 L of reagent water. Place the carboy on
the stir plate. Turn the stirrer on so that the bar creates a vortex.
Vortex the tube containing the spiking suspension (Section 11.3 or 11.4) for
approximately 2 minutes, sonicate for approximately 2 minutes, and vortex again for
approximately 2 minutes. Rinse a pipette tip with Tween 20, 0.01% once, then a
minimum of five times with the spiking suspension prior to pulling an aliquot to be used
to spike the carboy.
Add the spiking suspension to the carboy, delivering the oocysts below the surface of the
reagent water. Allow the spiking suspension to mix for approximately 1 minute in the
carboy.
After adding the spiking suspension, rinse the tube that contained the spiking suspension
and pipette tip with reagent water, vortex the tube, and add the rinsate to the carboy.
Allow the rinsate approximately 1 minute to mix in the carboy.
Turn on the pump and allow the flow rate to stabilize. Set flow at the rate designated for
the filter under test. As the carboy is depleted, check the flow rate and adjust if necessary.
11.5
11.5.2
11.5.3
11.5.4
11.5.5
11.5.6
25
December 1997 Draft
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Method 1622 - Draft
11.5.7 When the water level approaches the discharge port of the carboy, turn off the stirrer and
tilt the carboy so that it is completely emptied. At that time, turn off the pump and add 1
L of reagent water to the carboy. Swirl the contents to rinse down the sides.
11.5.8 Turn on the pump. Allow the pump to pull all the water through the filter and turn off the
pump.
12.0 Sample Filtration and Elution
12.1 A capsule filter is used to filter the 10-L water sample received by the laboratory, according to the
procedures in Section 12.2. Alternate procedures, such as vortex-flow filtration (VFF) or
membrane disk filtration, may be used if the laboratory first demonstrates that the quality control
acceptance criteria listed in Table 1 can be met. Alternate filtration procedures are provided in
Section 12.3.
12.2 Capsule filtration (adapted from Reference 20.7)
12.2.1 Flow rate adjustment
12.2.1.1 Connect the sampling system, minus the capsule, to a ceirboy filled with
reagent water (Figure 3).
12.2.1.2 Turn on the pump and adjust the flow rate to 2.0 L/min.
12.2.1.3 Allow 2 to 10 L of reagent water to flush the system. Adjust the pump
speed as required during this period. Turn off the pump when the flow
rate has been adjusted.
12.2.2 Install the capsule filter in the line, securing the inlet and outlet ends with the appropriate
clamps/fittings.
12.2.3 Record the sample number, sample turbidity (if not provided with the field sample), and
the name of analyst filtering the sample on the bench sheet and on the capsule filter.
12.2.4 Filtration
12.2.4.1 Connect the sampling system to the field carboy of sample water, or
transfer the sample water to the lab carboy used in Section 12.2.1.
NOTE: If the field sample is transferred to a lab carboy, the lab 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 6.17). This container will be used to
determine the sample volume filtered.
12.2.4.3 Allow the carboy discharge tube and capsule to fill with sample water.
Vent residual air using the bleed valve/vent port. Turn on the pump to
start water flowing through the filter. Verify that the flow rate is 2 L/min.
12.2 A A After the sample has passed through the filter, turn off the pump. Allow
the pressure to decay until flow stops.
12.2.4.5 Based on the water level in the graduated container (Section 12.2.4.2),
record the volume filtered on the bench sheet to the nearest quarter liter.
Discard the contents of the graduated container.
December 1997 Draft
26
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Method 1622-Draft
12.2.5
Disassembly
12.2.5.1
12.2.6
12.2.5.2
Elution
12.2.6.1
12.2.6.2
Disconnect the inlet end of the capsule filter assembly while maintaining
the level of the inlet fitting above the level of the outlet fitting to prevent
backwashing and the loss of oocysts from the filter. Restart the pump and
allow as much water to drain as possible. Turn off the pump and record
the meter reading and volume filtered on the bench sheet and on the label
Loosen the outlet fitting, then cap the inlet and outlet fittings.
Setup
12.2.6.1.1
12.2.6.1.2
12.2.6.1.3
Elution
12.2.6.2.1
12.2.6.2.2
12.2.6.2.3
12.2.6.2.4
12.2.6.2.5
12.2.6.2.6
Assemble the wrist-action shaker with the clamps aligned
vertically so that the filters will be aligned horizontally.
Prepare sufficient elution buffer so that all samples to be
eluted that day can be eluted with the same batch of
buffer. Elution may require up to 275 mL of buffer per
sample.
Designate at least one 250-mL conical centrifuge tube for
each sample and label with the sample number.
Record the name of the analyst performing the elution on
the bench sheet. Using a ring stand or other means, clamp
each capsule in a vertical position with the inlet end up.
Remove the inlet cap and allow the liquid level to
stabilize.
Pour elution buffer through the inlet fitting. Sufficient
elution buffer must be added to cover the pleated white
membrane with buffer solution. Replace the inlet cap and
clamp the cap in place.
Securely clamp the capsule in one of the clamps on the
wrist-action 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 80% of maximum
(approximately 600 rpm). Agitate the capsule for
approximately 5 minutes.
Remove the filter from the shaker, remove the inlet cap,
and pour the contents of the capsule into the 250-mL
conical centrifuge tube.
Clamp the capsule vertically with the inlet end up and
add sufficient volume of elution buffer through the inlet
fitting to cover the pleated membrane. Replace the inlet
cap and clamp in place.
Return the capsule to its clamp on the shaker with the
bleed valve positioned on a horizontal axis (3 or 9 o'clock
position). Turn on the shaker and agitate the capsule for
27
December 1997 Draft
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Method 1622 - Draft
approximately 5 minutes. Add the contents of the capsule
to the centrifuge tube.
12.2.7 Proceed to Section 13 for concentration and separation (purification).
12.3 Alternate filtration procedures
NOTE: Alternate filtration procedures, such as vortex flow filtration
(Section 12.4) or membrane disk filtration procedure (Section 12.5) may be
used instead of capsule filtration only if the laboratory demonstrates that
the quality control acceptance criteria listed in Table 1 are met. The
procedures listed below have been tested on a preliminary basis, but have not
been fully validated.
12.4 Vortex-flow filtration (adapted from Reference 20.8) (alternate procedure requiring demonstration
of performance prior to use)
12.4.1 Integrity testing of seals
12.4.1.1 If the headset is assembled, proceed to 12.4.1.2. If the headset is
disassembled, refer to Appendix 1 of the instrument manual provided
with the vortex flow concentrator, and assemble the headset as indicated
(Figure 4). The seal faces should exhibit no scoring or uneven wear. If
they appear to be scored or overworn, replace both the upper and lower
seals. The bottom seal (around the shaft below the headset) should be
visibly compressed. There should be very little play in the headset. Turn
the magnet manually; bearings should turn smoothly without grinding.
12.4.1.2 Firmly insert the rubber stopper into the filter outlet opening on the
bottom of the drive shaft. Close inlet valve #2. Fill chamber with reagent
water. Assemble the headset, chamber, and triclover clamp. The
membrane is not used during the integrity test. Mount the assembled
headset apparatus in the bayonet mount of the drive unit.
12.4.1.3 Close the vent line. Connect the air feed line to the sample port. Open
inlet valve #2 and pressurize chamber to approximately 10 psig using the
regulator on the pressurized gas source.
12.4.1.4 Turn on the rotor.
12.4.1.5 Open the filtrate line and examine it for water passing through the filtrate
tubing. If water is not passing through, increase the chamber pressure to
approximately 20 psig. Re-examine the filtrate line for water. If water
does not enter the filtrate line at 20 psig, the quality of the seals is
acceptable. If water passes into filtrate line, the seals need to be replaced.
12.4.1.6 Close inlet line valve #2 and stop the rotor. Turn off the pressurized gas.
Slowly open the vent line valve and bleed excess pressure from the
chamber. Disconnect the air feed line and drain water from the chamber.
12.4.1.7 Remove headset from bayonet mount. Open triclover clamp and remove
chamber from shaft. Remove the rubber stopper.
12.4.1.8 If the integrity test failed, disassemble the headset and replace the seals
(Section 12.4.1.1). Seals do not need to be replaced if they were not
compromised at 20 psig.
12.4.1.9 Initial and date the maintenance sheet by the unit to indicate when the
integrity test was passed.
December 1997 Draft
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Method 1622 - Draft
12.4.2.2
12.4.2 Membrane cartridge installation
12.4.2.1 Examine the membrane cartridge to ensure that the rubber or cellophane
bumper is on the bottom edge of the cartridge and is not twisted or folded
over. Soak the membrane cartridge for 0.5 hours in reagent water.
Carefully mount the membrane cartridge onto the shaft. Do not touch the
membrane surface. You should feel a distinct "click" when the membrane
seals. Carefully remove the protective plastic sheet from the membrane.
Using caution to ensure that membrane surface is not damaged, insert the
membrane into the chamber and seat the headset on the O-ring seal.
Before tightening the triclover clamp and adding the magnet cap and ring,
ensure that the membrane is centered in the chamber (watch through
bottom of chamber while manually rotating drive magnet). If the
membrane appears to touch the sides of the chamber, remove the
membrane and apply gentle pressure to the bottom of membrane on the
side where gap is the greatest. Seal the chamber with the triclover clamp
and add the magnet cap and ring.
Mount the assembled concentration apparatus in the bayonet mount of the
drive unit and attach the tubing (Figure 5).
Priming the chamber
12.4.2.3
12.4.3
12.4.4
12.4.5
12.4.3.1
12.4.3.2
Verify that the pressure reservoir contains approximately 1 L of water.
Open inlet valve #1, open inlet valve #2, and the vent port, and close the
filtrate line valve. Set pump speed to minimum.
Begin pumping water from the pressure reservoir slowly, until water
begins to enter the chamber. When the water level reaches the top of the
chamber, turn off the pump, remove the chamber assembly from the
bayonet mount and tilt it to remove any air trapped under the membrane
cartridge. Reseat the chamber assembly and continue priming with the
pump until all air has cleared the chamber. There should be no more air
bubbles in the inlet line.
Tighten the clamp to close the vent port. Close inlet valve #2. Attach
pump tubing to filtrate port and open filtrate line valve. Connect vent
tubing to vent port.
Place the level sensor into filtrate tank (optional). Adjust the level sensor
in the tank to turn off the unit before the pressure reservoir empties (set to
approximately 9 L for a 10 L sample). Remove excess priming water from
the pressure reservoir. The apparatus is now ready for sample preparation.
Sample preparation
12.4.4.1
12.4.3.3
12.4.3.4
Quantitatively transfer a well-mixed 10-L water sample to be tested into
the pressure reservoir.
Add oocyst spiking suspension according to Sections 11.5.3 to 11.5.5, if a
spiked sample is to be filtered.
Connect the regulator and air feed line to pressure reservoir. Close the
reservoir lid.
Sample concentration
12.4.5.1 Open the inlet valves and the filtrate valve. Set the pump speed to
minimum. Turn on the pump and rotor.
12.4.4.2
12.4.4.3
29
December 1997 Draft
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Method 1622 - Draft
12.4.5.2 Pressurize the reservoir to approximately 6 psig using the regulator on the
pressurized gas source.
12.4.5.3 Increase the pump speed to approximately 10 L/hr (167 mL/min).
12.4.5.4 Measure the filtrate rate. If the rate exceeds 10 L/hr, decrease the pump
speed. If the rate is below 10 L/hr (only expected for the most turbid raw
water samples), increase the pump speed. Record the start time and initial
filtrate rate.
12.4.5.5 Concentrate the sample in this mode of operation until the level sensor is
tripped by the rising filtrate level. At this point, the pump and rotor will
stop automatically.
12.4.5.6 Restart the process by pressing the level sensor override switch. When the
last of the sample exits the pressure reservoir, air will begin to enter the
concentration chamber through the feed line. When the chamber volume
drops to approximately 10 mL (meniscus 5 to 6 cm above bottom of
chamber when rotor is on), immediately close inlet valve #2 and stop the
pump and rotor. Do not overconcentrate. Turn off the air flow to the
pressure reservoir and bleed off excess pressure from the reservoir.
(Removal of too much liquid from chamber while membrane rotates
could result in erratic rotation and possibly damage membrane or
chamber). Fill the reservoir with 1 L of reagent water v/hile rinsing-down
all interior surfaces of the reservoir. Run the reagent water through the
system.
12.4.6 Backflush and sample collection
12.4.6.1 Slowly open the clamp on the line from the vent port to bleed any residual
pressure from the separation chamber. Leave vent line open.
12.4.6.2 With the pump outlet tubing immersed in filtrate, reveirse direction on the
pump. Turn on the pump and rotor, and backflush until the concentrate
level reaches approximately 50% of the chamber volume. Turn off the
pump and rotor.
12.4.6.3 Close the vent line and the filtrate line. Disconnect the inlet tubing with
150 mL glass beaker in position for sample collection. Remove the
chamber and tilt it to drain the concentrate from sample port into a beaker.
Pour beaker contents into 250-mL conical centrifuge tube calibrated at 5,
10, and 100 mL and triple rinse beaker with reagent water. Open vent line
to facilitate drainage.
12.4.6.4 To ensure maximum oocyst recovery, spray approximately 5 mL of
reagent water from a laboratory wash bottle onto the chamber and
membrane to rinse the cartridge, housing, and immediate inlet line. Add
the rinse water to the final concentrate.
12.4.7 Specifications: The membrane must be examined following concentration and must
reveal no scoring, nicking, or tearing. If membrane is scored, nicked, or torn, oocyst
recoveries may be compromised.
12.4.8 Proceed to Section 13 for concentration and separation (purification),
12.4.9 Cleaning
12.4.9.1 Wash the pressure reservoir, separation chamber, and other non-
disposable parts with hot, soapy water. Rinse three times with reagent
water.
December 1997 Draft
30
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Method 1622 - Draft
12.4.9.2 Fill the reservoir with 2 L of hot, soapy hypochlorite solution (minimum
of 5%) while rinsing-down all interior surfaces of the reservoir. Run the
hypochlorite solution through the system without a membrane cartridge in
place. Use a pressurized gas source, but remove the peristaltic pump to
increase system flow rate.
12.4.9.3 Fill the reservoir with 1 L or reagent water while rinsing-down all interior
surfaces of the reservoir. Run the reagent water through the system
without a membrane cartridge in place. Repeat this rinsing process three
times.
12.5 Membrane disk filtration (alternate procedure requiring demonstration of performance prior to use)
12.5.1 Assembly of the membrane disk housing (Figure 6)
12.5.1.1 Clean and assemble the PTFE-coated support screen, PTFE gasket,
underdrain support, outlet fitting, and legs to the lower plate. Also clean
and assemble the inlet fitting, vent valve, and flow deflector to the upper
plate. Check to ensure that the support screen and gasket have no
scratches or damage that may prevent adequate seating of the housing.
Verify that the lower plate and PTFE support screen are dry. Center a
drain disk on the PTFE support screen.
Center a filter on the drain disk, with shinier side facing up, using forceps
or hands protected with non-powdered gloves. Using forceps or gloved
hands, smooth the filter until it is centered and lays flat on the drain disk.
Apply upper plate and press down firmly on lower assembly. Lift the
upper plate and examine the filter, which will adhere to upper plate
gasket. The filter should extend beyond gasket around entire perimeter.
12.5.1.4 Tighten a pair of opposing wingnuts simultaneously, then move to the
next pair. Several rotations of tightening may be necessary to completely
seat the filter housing.
Flow rate adjustment
12.5.2.1
12.5.1.2
12.5.1.3
12.5.2
Connect the sampling system minus the filter to a source of reagent water
(Figure 3).
Turn on the pump and adjust the flow rate to 2.0 L/min. A lesser flow rate
may be required for the membrane disk filter.
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 and flushing of the system is completed.
Install the membrane disk filter in the line, securing the inlet and outlet
ends with the appropriate clamps/fittings.
Record the sample number, sample turbidity (if not provided with the field sample), and
name of analyst collecting the sample the bench sheet and on labels to be attached to the
specimen cup and the 250-mL conical centrifuge tube that will hold the membrane disk
filter-sample.
12.5.4 Filtration
12.5.2.2
12.5.2.3
12.5.2.4
12.5.3
12.5.4.1
Connect the sampling system to the field carboy of sample water, or
transfer the sample water to the lab carboy used in Section 12.5.2.1
31
December 1997 Draft
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Method 1622 - Draft
NOTE: If the field sample is transferred to a lab carboy, the lab carboy
must be cleaned and disinfected before it is used with another field sample.
12.5.5
12.5.4.2
12.5.4.3
12.5.4.4
Disassembly
12.5.5.1
12.5.5.2
12.5.5.3
12.5.5.4
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 6.17). This container will be used to
determine the sample volume filtered.
Turn on the pump to start water flow through the filter. Vent residual air
using the bleed valve/vent port. Tilt the filter assembly back and forth
slowly in several directions to move residual air to the vent port, then vent
the air.
After the sample has passed through the filter, turn off the pump. Based
on the water level in the graduated container (Section 12.5.4.2), record
the volume filtered on the bench sheet to the nearest quarter liter. Discard
the contents of the graduated container.
Disconnect the inlet end of the membrane disk 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 ifrom the filter.
Connect a supply of compressed air or nitrogen at < 5 psig from a
cylinder, compressor, or portable pump to the inlet fitting and purge the
assembly of water. Continue to pump or purge until water no longer flows
from the assembly when it is rocked, but do not allow the assembly to dry.
Stop the pump or turn off the gas supply and disconnect the tubing from
the outlet fitting.
Disassemble the inlet plate and O-ring from the remainder of the
assembly. Be careful not to disturb the filter.
Using the blunt-end forceps, lift one edge of the filter and fold to the
opposite side to form a semi-circle. Fold again to form a quarter circle.
For 293-mm filters, fold again.
NOTE: Avoid touching the sample side of the filter. If sample side is
touched, rinse gloves with reagent water into the specimen cup to be used for
elution.
12.5.5.5 Pick up the folded filter with the forceps and place into a clean, 125-mL
or 250-mL specimen cup (142-mm or 293-mm filter, respectively). Using
a squirt bottle of reagent water, rinse the upper plate off into the specimen
cup. Add the minimum amount of reagent water to cover the filter. Cap
the cup.
12.5.6 Membrane disk filter elution—The oocysts are washed from the membrane disk filter by
hand-kneading and sonication
12.5.6.1 Estimate the volume of reagent water in the beaker arid add an aliquot of
elution buffer to equal 20% of that volume.
12.5.6.2 Holding the filter with gloved hands, gently knead the filter between the
thumb and forefinger in the solution to remove as much material as
possible. Unfold the filter and draw the filter through the thumb and
December 1997 Draft
32
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Method 1622 - Draft
forefinger to remove material that had been folded inside. Perform this
task for approximately 2 minutes.
Place the beaker containing the filter in the sonication bath and sonicate
for 1 to 2 minutes. Push the filter below the surface of the solution with a
disposable pipette.
After sonication, repeat the kneading process (Section 12.5.6.2). Squeeze
the excess solution from the membrane filter. Pour the solution into the
labeled 250-mL centrifuge tube. Rinse the beaker with 150 mM PBS and
add this rinsate to the sample.
If a high number of particles are observed in the eluent, repeat the elution
process (Section 12.5.6.1 to 12.5.6.4).
12.5.7 Proceed to Section 13 for concentration and separation (purification).
12.5.6.3
12.5.6.4
12.5.6.5
13.0 Sample Concentration and Separation (Purification)
13.1 During concentration and separation, the filter eluate is concentrated through centrifugation, and
the oocysts in the sample are separated from other particulates through Dynal immunomagnetic
separation (IMS). Alternate procedures, such as ImmuCell IMS, may be used if the laboratory first
demonstrates that the quality control acceptance criteria listed in Table 1 are met. Alternate
separation procedures are provided in Section 13.4.
13.2 Adjustment of pellet volume
13.2.1 Centrifuge the 250-mL centrifuge tube containing the VFF retentate or capsule or
membrane disk filter eluate at 1000 to 1100 x G for 10 to 15 minutes. Allow the
centrifuge to coast to a stop.
13.2.2 Record the initial pellet volume (volume of solids) and the date and time that
concentration was completed on the bench sheet. Using a Pasteur pipette, carefully
aspirate off the supernatant to just above the pellet.
13.2.2.1 If the pellet volume is less than or equal to 0.5 mL, add reagent water to
the centrifuge tube to bring the total volume to 10 mL. Vortex the tube for
10 to 15 seconds to resuspend the pellet. Proceed to Section 13.3 (Dynal
IMS) or Section 13.5 (ImmuCell IMS).
13.2.2.2 If the pellet volume is greater than 0.5 mL, use the following formula to
determine the total volume needed in the centrifuge tube to adjust the
pellet to 0.5 mL:
total volume (mL) required = pe"et V°'Ume xWmL
0.5 mL
Add reagent water to the centrifuge tube to bring the total volume to the
level calculated above. Vortex the tube for 10 to 15 seconds to resuspend
the pellet. Record this resuspended volume on the bench sheet. Using a
Pasteur pipette, reduce the resuspended solution in the tube to a final
volume of 10 mL (which will contain 0.5 mL of solids). Proceed to
Section 13.3 (Dynal IMS) or Section 13.4 for alternate separation
procedures.
13.3 Dynal IMS procedure (adapted from Reference 20.9)
13.3.1 Preparation of reagents
13.3.1.1 Prepare a IX dilution of SL-buffer-A from the 10 X SL-buffer-A (clear,
colorless solution) supplied. Use reagent water (demineralized; Section
33
December 1997 Draft
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Method 1622 - Draft
13.3.1.2
13.3.1.3
13.3.2 Oocyst capture
13.3.2.1
13.3.2.2
13.3.2.3
13.3.2.4
13.3.2.5
13.3.2.6
13.3.2.7
13.3.2.8
13.3.2.9
13.3.2.10
13.3.2.11
13.3.2.12
13.3.2.13
7.3) as the diluent. For every 1 mL of IX SL-buffer-A required, take 100
fjL of 10 X SL-buffer-A and make up to 1 mL with the diluent water. 1
mL of IX SL-buffer-A will be required per sample or subsample on
which the Dynal IMS procedure is performed.
To a flat-sided sample tube (125 x 16 mm flat-sided Leighton tube) add 1
mL of the 10 X SL-buffer-A (supplied—not the diluted IX SL-buffer-A).
Add 1 mL of the 10 X SL-buffer-B (supplied—magenta solution) to the
sample tube containing the 10 X SL-buffer-A.
Transfer the water sample concentrate from Section 13.2.2 to the sample
tube containing the SL-buffer. Label the tube with the sample number.
Cap the tube and vortex the Dynabeads® anti-Cryptosporidium for
approximately 10 seconds to suspend the beads. Ensure that the beads are
fully resuspended by inverting the tube and making sure that there is no
residual pellet at the bottom.
Add 100 //L of the resuspended beads (Section 13.3.2,2) to the sample
tube containing the water sample concentrate and SL-buffer.
Affix the sample tube to a rotating mixer and rotate at approximately 25
rpm for 1 hour.
After rotating for 1 hour, remove the tube from mixer and place in the
magnetic particle concentrator (MPC-1) with flat side of tube toward the
magnet.
Without removing the tube from the MPC-1, place the: magnet side of the
MPC-1 downwards, so the tube is horizontal and the flat side of the
Leighton tube is facing down.
Gently rock the tube by hand end-to-end through approximately 90°,
tilting cap-end and base-end of the tube up and down in turn. Continue
the tilting action for 2 minutes with approximately one tilt per second.
Ensure that the tilting action is continued throughout this period to
prevent binding of low-mass material that is magnetic or magnetizable. If
the sample in the MPC-1 is allowed to stand motionless for more than 10
seconds, repeat Section 13.3.2.7 before continuing procedure.
Return the MPC-1 to the upright position, tube vertical, with cap at top.
Immediately remove cap and decant (pour off) all the supernatant from
the tube held in the MPC-1 into a suitable container. Do not shake the
tube and do not remove the tube from MPC-1 during this step.
Remove the tube from the MPC-1 and resuspend the sample in 1-mL IX
SL-buffer-A (prepared from 10X SL-buffer-A stock supplied). Mix very
gently to resuspend all material in the tube. Do not vortex.
Quantitatively transfer all the liquid from the sample tube to a labeled,
1.5-mL microcentrifuge tube.
Place the microcentrifuge in the second magnetic particle concentrator
(MPC-M), with magnetic strip in place.
Without removing the microcentrifuge tube from MPC-M gently rock/roll
the tube through 180° by hand (see Figure 1 in information provided with
IMS kit). Continue for approximately 1 minute with approximately one
December 1997 Draft
34
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Method 1622 - Draft
13.3.2.14
180° roll/rock per second. At the end of this step, the beads and oocysts
should produce a well-formed brown dot on the back of the tube.
Immediately aspirate the supernatant from the tube and cap held in the
MPC-M. If more than one sample is being processed, conduct three 180°
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 tube from MPC-M
while conducting these steps.
13.3.3 Dissociation of beads/oocyst complex
13.3.3.1 Remove the magnetic strip from the MPC-M.
13.3.3.2 Add 100 /^.L of 0.1 N HC1, then vortex for 5 seconds.
13.3.3.3
13.3.3.4
13.3.3.5
13.3.3.6
13.3.3.7
13.3.3.8
Incubate for 5 minutes at room temperature.
Vortex for 5 seconds
Replace magnetic strip in MPC-M and replace tube in MPC-M. Gently
rock the tube end to end through approximately 90°, tilting cap-end and
base-end of the tube up and down in turn. Continue the tilting action for
30 seconds with approximately one tilt per second.
Remove 12-mm-diameter well slides from the box in the refrigerator and
lay the slides on a flat surface for 15 minutes to allow them to warm to
room temperature.
Add 10 [du of 1.0 N NaOH to a sample well.
Transfer all of the sample from the microcentrifuge tube in the MPC-M to
the sample well with the NaOH. Do not disturb the beads at the back wall
of the tube.
13.3.3.9 Proceed to Section 14 for sample staining.
13.4 Alternate separation procedures
NOTE: Alternate separation procedures, such as ImmuCell IMS (Section 13.5)
may be used instead ofDynal IMS only if the laboratory demonstrates that the
quality control acceptance criteria listed in Table 1 are met. The procedure
listed below has been tested on a preliminary basis, but has not been fully
validated.
13.5
ImmuCell IMS procedure (adapted from Reference 20.10) (alternate procedure requiring
demonstration of performance prior to use)
13.5.1 Oocyst capture procedure
13.5.1.1
13.5.1.2
13.5.1.3
13.5.1.4
13.5.1.5
Transfer the 10-mL sample (Section 13.2.2) into a 50 mL polypropylene
conical tube. Packed pellet volume must not exceed 0.5 mL.
Add 100 AiL of IMS Reagent A. and 0.5 mL of 20X PBS. Vortex gently.
Add 50 {J.L of Cryptosporidium Magnetic Beads to the sample in the 50-
mL conical tube and rotate end-over-end for 60 minutes at room
temperature.
Add 20 mL IMS Reagent B to the bead suspension.
Place 100-mm petri dish onto magnet. Place the magnet onto standard
orbital shaker and begin rotating at 75 rpm, or agitate on mini-orbital
shaker at medium speed. Pour suspension into petri dish and agitate for at
35
December 1997 Draft
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Method 1622 - Draft
13.5.1.6
least 2 minutes. Magnetic beads should be attached to dish around the
circumference of the magnetic disk, while debris remains in suspension.
Turn off the orbital shaker. Immediately remove as much supernatant as
possible from the edge of the petri dish using a 25 mL pipette and discard.
While the petri dish remains on the magnet, gently tilt the dish and
carefully remove remaining supernatant, and discard.
NOTE: Do not remove any of the magnetic bead pellet when removing the
supernatant.
13.5.1.7 Remove the petri dish from the magnet. Use 1 mL of IMS Reagent B
(diluted to IX) to rinse the pellet from the petri dish. Pipette reagent over
beads repeatedly to ensure ALL beads are rinsed from the dish. Remove
bead suspension and place in 1.5-mL microcentrifuge tube.
13.5.1.8 Place the microcentrifuge tube containing the bead suspension in the tube
slot in the magnet. Magnetically separate beads from Reagent B for 2
minutes at room temperature.
13.5.1.9 While the tube remains in the slot, completely remove supernatant with 1-
mL micropipette. Remove the tube from the slot.
13.5.2 Release of oocysts from magnetic beads
13.5.2.1 Add 50-^L 0.1 N HCL to the tube containing beads.
13.5.2.2 Vortex the tube for 10 seconds on medium setting. Let the tube stand for
5 minutes. Vortex for 10 seconds again.
13.5.2.3 Place the tube in the tube slot in the magnetic device. Magnetically
separate for 2 minutes at room temperature.
13.5.2.4 During magnetic separation, remove 12-mm-diameter well slides from the
box in the refrigerator and lay the slides on a flat surface for 15 minutes to
allow them to warm to room temperature. Prepare one well of a well slide
by adding 5 //L of 1.0 N NaOH.
13.5.2.5 Transfer 50 fjL from the sample tube onto the well of the slide prepared
in Section 13.5.2.4.
13.5.2.6 Perform a second acid treatment of the magnetic beads to ensure efficient
release by repeating Sections 13.5.2.1 to 13.5.2.5. This results in two
wells of the well slide containing sample for analysis.
13.5.2.7 Proceed to Section 14 for sample staining.
14.0 Sample Staining
14.1 Prepare positive and negative controls. For the positive control, pipette 10 ^L of positive antigen
or 200 to 500 intact oocysts to the center of a well. For the negative control, pipette 75 /J.L of 150
mM PBS into the center of a well and spread it over the well area with a pipette tip.
14.2 Place the well slides containing the samples in a 42°C incubator and evaporate to dryness
(approximately 1 to 2 hours).
14.3 Apply 50-fjL of absolute methanol to each well containing the dried sample and allow to air dry
for 3 to 5 minutes.
14.4 Follow manufacturer's instructions in preparing dilutions of anti-Cryptosporidium sp. fluorescein-
labeled monoclonal antibody (Mab) and overlay the sample well, the positive-control well, and the
negative-control well with 50 fjL of fluorescein-labeled Mab. Place the slides in a humid chamber
December 1997 Draft
36
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Method 1622 - Draft
and incubate at 37 °C for approximately 30 minutes. The humid chamber consists of a tightly
sealed plastic container containing damp paper towels on which the slides are placed.
After 30 minutes, remove the slides and use a clean Pasteur pipette tip attached to a vacuum source
to gently aspirate excess fluorescein-labeled Mab from the side of each well. When performing this
step, ensure that the vacuum source is at a minimum (<2 in Hg vac.) and ensure that the pipette tip
does not scratch the well surface.
Apply 65 to 75 //L of 150 mM PBS, pH 7.2, to each well and allow to stand for 1 to 5 minutes,
then aspirate the excess PBS. When removing the excess PBS, ensure that the pipette tip does not
scratch the well surface. Repeat this washing procedure two more times.
Apply 50 yuL of 4',6-diamidino-2-phenylindole (DAPI) solution (1/5000 dilution in PBS, prepared
daily by adding 10 (j.L of 2 mg/mL DAPI [Section 7.8.2] to 50 mL of 150 mM PBS) to each well.
Allow to stand at room temperature for approximately 2 minutes.
Remove the excess DAPI solution by aspiration.
Apply 65 to 75 fjL. of 150 mM PBS, pH 7.2, to each well and allow to stand for 1 to 5 minutes,
then aspirate the excess PBS. When removing the excess PBS, ensure that the pipette tip does not
scratch the well surface. Repeat this washing procedure two more times.
Apply a drop of reagent water to each well and allow to stand for approximately 1 minute, then
aspirate the excess reagent water.
Place the slide in a dry box and allow the slides to stand in the dark for approximately 1 hour at
room temperature. The dry box consists of a tightly sealed plastic container with desiccant in the
bottom. A paper towel must be placed over the desiccant.
Apply 10 pL of mounting medium (60% glycerol, 40% 150 mM PBS) containing an anti-fadant
(2% DABCO, or equivalent) to the center of each well.
Place a 22 x 50 mm coverslip on each three-well microscope slide and gently depress the coverslip
at the edges. Use a tissue to remove excess mounting fluid from the edges of the coverslip and then
seal the edges of the coverslip onto the slide using clear nail polish. Record the date and time that
staining was completed on the bench sheet. Store slides in a dry box, in the dark, until ready for
examination.
15.0 Examination
Scanning technique: Scan each well in a systematic fashion. An up-and-down or a side-to-side
scanning pattern may be used (Figure 7).
Examination and confirmation using immunofluorescence assay (FA), 4',6-diamidino-2-
phenylindole (DAPI) staining characteristics, and differential interference contrast (D.I.C.)
microscopy. Record examination and confirmation results on the Cryptosporidium report form.
15.2.1 If the positive control contains oocysts within the expected range and at the appropriate
fluorescence for both FA and DAPI, and the negative control does not contain any
oocysts (Section 14.1), use epifluorescence to scan the entire coverslip for each sample at
not less than 200X total magnification for apple-green fluorescence of oocyst shapes.
15.2.2 When brilliant apple-green fluorescing ovoid or spherical objects 4 to 6 /^m in diameter
are observed with brightly highlighted edges, switch the microscope to the UV filter
block for DAPI, then to D.I.C.
14.5
14.6
14.7
14.8
14.9
14.10
14.11
14.12
14.13
15.1
15.2
37
December 1997 Draft
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Method 1622 - Draft
(b)
(c)
1 5.2.2.1 Using the UV filter block for DAPI, the object will exhibit one of the
following characteristics if it is a Cryptosporidium oocyst:
(a) Up to four distinct, sky-blue nuclei within a single oocyst
(b) Oocysts with intense blue internal staining
(c) Oocysts with light blue internal staining (no distinct nuclei)
(a) and (b) are recorded as DAPI +; (c) is recorded as DAPI -.
1 5.2.2.2 Using D.I.C., look for external or internal morphological characteristics
atypical of Cryptosporidium oocysts (e.g., spikes, stalks, appendages,
pores, one or two large nuclei filling the cell, red fluorescing chloroplasts,
crystals, spores, etc.) (adapted from Reference 20.6).
1 5.2.2.2.1 If atypical structures are not observed, then categorize
each apple-green fluorescing object as:
(a) An empty Cryptosporidium oocyst
A Cryptosporidium oocyst with amorphous
structure
A Cryptosporidium oocyst with internal structure
(one to four sporozoites/oocyst)
Record the shape and measurements to the nearest 0.5
jum at 1000X total magnification for each such object.
Although not a defining characteristic, surface oocyst
folds may be observed in some specimens.
1 5.2.2.2.2 For each oocyst, record the number of sporozoites
observed. Cryptosporidium oocysts with sporozoites
must be confirmed by a principal analyst/supervisor.
Record the date and time that sample examination and
confirmation was completed on the report form.
16.0 Analysis of Complex Samples
16.1 Some samples may contain high levels (>1000/L) of oocysts and/or interfering organisms,
substances, or materials. Some samples will not filter (Section 12); others will not allow separation
of the oocysts from the retentate or eluate; and others may contain materials that preclude or
confuse microscopic examination. In these cases, dilute the original sample and filter and analyze
10 L of the diluted sample. If the sample is diluted at any step during analysis, the laboratory must
record the original and final volumes and the volume analyzed.
1 6.2 If the sample holding time has not been exceeded and a full 10-L sample cannot be filtered, dilute
an aliquot of sample to 10 L with reagent water and filter this smaller aliquot (Section 12). This
dilution must be recorded and reported with the results.
1 6.3 If the holding times for the sample and for microscopic examination of the cleaned up
retentate/eluate have been exceeded, the site must be re-sampled.
17.0 Method Performance
1 7.1 Expected method performance data are shown in Table 1 . These data are based on two single-
laboratory validation studies.
17.2 Results from collaborative field and laboratory studies will be used to calculate statements of
overall single-operator precision and bias and interlaboratory precision and bias. These statements
will be presented in tables added to this method.
December 1997 Draft
38
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Method 1622 - Draft
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.
Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize
the volume of expired materials to be disposed.
18.2
19.0 Waste Management
19.1 It is the laboratory's responsibility to comply with all federal, state, and local regulations governing
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.
Samples, reference materials, and equipment known or suspected to have viable oocysts attached
or contained must be sterilized prior to disposal.
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.
19.2
19.3
20.0
20.1
20.2
20.3
20.4
20.5
20.6
20.7
20.8
20.9
20.10
References
Rodgers, Mark R., Flanigan, Debbie J., and Jakubowski, Walter, Applied and Environmental
Microbiology 6J.(10), 3759-3763 (October 1995).
Fleming, Diane O., et al. (eds.), Laboratory Safety: Principles and Practices, 2nd edition. 1995.
ASM Press, Washington, DC
"Working with Carcinogens," DHEW, PHS, CDC, NIOSH, Publication 77-206, (Aug 1977).
"OSHA Safety and Health Standards, General Industry," OSHA 2206, 29 CFR 1910 (Jan 1976).
"Safety in Academic Chemistry Laboratories," ACS Committee on Chemical Safety (1979).
ICR Microbial Laboratory Manual, EPA/600/R-95/178, National Exposure Research Laboratory,
Office of Research and Development, U.S. Environmental Protection Agency, 26 Martin Luther
King Drive, Cincinnati, OH 45268.
"Envirochek™ Sampling Capsule," PN 32915, Gelman Sciences, 600 South Wagner Road, Ann
Arbor, MI 48103-9019 (September 1996).
"Concentration of Water by Vortex Flow Filtration," Document Number WDX-MAN/DOC-120,
Effective 5/8/97, ImmuCell Corporation, 56 Evergreen Drive, Portland, Maine 04103 (May 1997).
"Dynabeads® anti-Cryptosporidium Prototype Procedure, Second Revision," Dynal Microbiology
R&D, P.O. Box 8146 Dep., 0212 Oslo, Norway (May 1997).
"Cryptosporidium-Giardia Combo IMS Procedure," Effective 10/1/97, ImmuCell Corporation, 56
Evergreen Drive, Portland, Maine 04103 (October 1997).
39
December 1997 Draft
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Method 1622 - Draft
21.0 Tables and Figures
NOTE: All acceptance criteria listed in Table 1 were generated through two,
single-laboratory validation studies using capsule filtration andDynal
immunomagnetic separation.
Table 1. Quality control acceptance criteria for performance tests for
Performance test
Initial precision and recovery (Section 9.4)
Precision (as maximum relative standard deviation)
Recovery (percent)
Ongoing precision and recovery (Section 9.7) (percent)
Matrix spike recovery (Section 9.5) (percent)
Acceptance criteria
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15-88
14-95
8-127
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1 mm
1/5 mm
D
B
Figure 1. Hemacytometer platform ruling. Squares 1, 2, 3, and 4 are
used to count stock suspensions of Cryptosporidium
oocysts (after Miale, 1967)
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O
_CL
O
D
D
O
O
Figure 2. Manner of counting Cryptosporidium oocysts in 1 square
mm. Dark oocysts are counted and light oocysts are
omitted, (after Miale, 1967)
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capsule
filter
sample
membrane
disk filter
flow controller
drain
Figure 3.
Laboratory filtration system for capsule filter or membrane
disk filter
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I
Method 1622 - Draft
cap ring
magnet cap
nut
washer
magnet-
thin o-ring
shaft carrier
bearings housing
thick o-ring
upper rubber seal
headcap
driveshaft
lower rubber seal
driveshaft o-ring
Figure 4. Vortex-flow filter concentrator drive assembly
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sample
feed line
pressurized
gas source
level
sensor
beaker
Figure 5. Vortex-flow filter system
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inlet
vent valve
upper plate
diffuser
filter —
drain disk
support screen
PTFE gasket
support
lower plate
outlet
Figure 6. Membrane disk filter assembly
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Figure 7. Methods for scanning a well slide
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22.0 Glossary of Definitions and Purposes
These definitions and purposes are specific to this method but have been conformed to common
usage as much as possible.
22.1 Units of weight and measure and their abbreviations
22.1.1 Symbols
°C degrees Celsius
//L 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
min minute
mL milliliter
mm millimeter
mM milliMole
N normal; gram molecular weight of solute divided by hydrogen equivalent of
solute, per liter of solution
OD outside diameter
psig pounds-per-square-inch gauge
qt quart
s second
sr standard deviation of recovery
v/v volume per unit volume
w/v weight per unit volume
X average percent recovery
22.2 Definitions, acronyms, and abbreviations (in alphabetical order).
Analyst—The analyst must have two years of college lecture and laboratory course work in
microbiology or a closely related field. The analyst also must have at least 6 months bench
experience, must have at least 3 months experience with FA techniques, and must have
successfully analyzed at least 50 water and/or wastewater samples for Cryptosporidium. Six
months of additional experience in the above areas may be substituted for two years of college. In
addition, the analyst must have analyzed a minimum of one PE sample for C,ryptosporidium, and
results must have fallen with acceptance limits. The analyst must also demonstrate acceptable
performance during an on-site evaluation.
Analyte—A protozoan parasite tested for by this method. The analyte in this method is
Cryptosporidium.
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Field blank—An aliquot of reagent water or other reference matrix that is placed in a sample
container in the laboratory or the field, and treated as a sample in all respects, including exposure
to sampling site conditions, storage, and all analytical procedures. The purpose of the field blank is
to determine if the field or sample transporting procedures and environments have contaminated
the sample.
Immunomagnetic separation (IMS)—A purification method that uses microscopic, magnetically
responsive particles coated with an antibodies targeted to react with a specific pathogen in a fluid
stream. Pathogens are selectively removed from other debris using a magnetic field.
Initial precision and recovery (IPR)—four aliquots of oocyst spiking suspension analyzed to
establish the ability to generate acceptable precision and accuracy. An IPR is performed prior to
the first time this method is used and any time the method or instrumentation is modified.
Laboratory blank—See Method blank.
Laboratory control sample (LCS)—See Ongoing precision and recovery (OPR) standard.
Laboratory reagent blank—See Method blank.
Matrix spike (MS)—A sample prepared by adding a known quantity of organisms to a specified
amount of sample matrix for which an independent estimate of target analyte concentration is
available. A matrix spike is used to determine the effect of the matrix on a method's recovery
efficiency.
May—This action, activity, or procedural step is neither required nor prohibited.
May not—This action, activity, or procedural step is prohibited.
Method blank—An aliquot of reagent water that is treated exactly as a sample including exposure
to all glassware, equipment, solvents, 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.
Minimum level (ML)—The level at which the entire analytical system must give a recognizable
signal and acceptable calibration point for the analyte. It is equivalent to the concentration of the
lowest calibration standard, assuming that all method-specified sample weights, volumes, and
concentration and separation procedures have been employed.
Must—This action, activity, or procedural step is required.
Negative control—See Method blank
Nucleus—A membrane-bound organelle containing genetic material. Nuclei are a prominent
internal structure seen both in Cryptosporidium oocysts. In Cryptosporidium oocysts, there is one
nucleus per sporozoite.
Oocyst—The encysted zygote of some sporozoa; e.g., Cryptosporidium. The oocyst is a phase or
form of the organism produced as a normal part of the life cycle of the organism. It is characterized
by a thick and environmentally resistant outer wall.
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Ongoing precision and recovery standard (OPR)—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.
Positive control—See Ongoing precision and recovery standard
Preferred—Optional
Preparation blank—See Method blank.
Primary dilution standard—A solution containing the specified analytes that is purchased or
prepared from stock solutions and diluted as needed to prepare calibration solutions and other
solutions.
Principal analyst/supervisor—The principal analyst/supervisor must be an experienced
microbiologist with at least a B.A./B.S. in microbiology or a closely related field. The principal
analyst also must have at least 1 year of continuous bench experience with immunofluorescent
antibody (FA) techniques and microscopic identification and have analyzed at least 100 water
and/or wastewater samples for Cryptosporidium. The principal analyst/ supervisor must
demonstrate acceptable performance during an on-site evaluation.
PTFE—Polytetrafluoroethylene
Quality control check sample (QCS)—A sample containing all or a subset of l:he analytes at known
concentrations. The QCS is obtained from a source external to the laboratory or is prepared from a
source of standards different from the source of calibration standards. It is used to check laboratory
performance with test materials prepared external to the normal preparation process.
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), 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 arid potentially
interfering substances at the method detection limit for the analyte.
Relative standard deviation (RSD)—The standard deviation times 100 divided by the mean.
RSD—See Relative standard deviation.
Should—This action, activity, or procedural step is suggested but not required.
Sporozoite—A motile, infective stage of certain protozoans; e.g., Cryptosporidium. There are four
sporozoites in each Cryptosporidium oocyst, and they are generally banana-shaped.
Stock suspension—A suspension containing an analyte that is prepared using a reference material
traceable to EPA, the National Institute of Science and Technology (NIST), or a source that will
attest to the purity and authenticity of the reference material.
Technician—The technician filters samples using VFF and filters, performs centrifugation,
elution, concentration, and purification using MS, and places the purified retentates on slides for
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microscopic examination, but does not perform microscopic protozoan detection and
identification. The technician must have at least three months of experience in filter extraction and
processing of protozoa samples.
Vortex-flow filtration (VFF)—Filtration technology in which water is passed through a cylindrical
membrane filter rotating at high speed within an outer jacket. Rotation of the membrane creates
waves of turbulence, known as Taylor's vortices, which continuously "scrub" the surface of the
membrane, preventing blockages.
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