United States Office of Water 

Environmental Protection 4603 April 2001 

Agency 

EPA-821-R-01-025 

Method 1623: Cryptosporidium and 

Giardia in Water by Filtration/IMS/FA 
Acknowledgments 

This method was prepared under the direction of William A. Telliard of the Engineering and Analysis 

Division within the U.S. Environmental Protection Agency (U.S. EPA) Office of Water. This document 

was prepared by DynCorp I&ET under a U.S. EPA Contract, with assistance from its subcontractor, 

Interface, Inc. 

The U.S. EPA Office of Water gratefully acknowledges the contributions of the following persons and 

organizations to the development of this method: 

Mike Arrowood, Centers for Disease Control, Division of Parasitic Diseases (MS-F13), 4770 Buford 

Highway, N.E., Atlanta, GA 30341-3724, USA 

Phil Berger, Office of Groundwater and Drinking Water, U.S. Environmental Protection Agency, 401 M 

Street, S.W., Washington, DC 20460, USA 

Jennifer Clancy, Clancy Environmental Consultants, Inc., P.O. Box 314, St. Albans, VT 05478, USA 

Kevin Connell, DynCorp I&ET, 6101 Stevenson Avenue, Alexandria, VA 22314, USA 

Ricardo DeLeon, Metropolitan Water District of Southern California, 700 Moreno Avenue, LaVerne, CA 

91760, USA 

Shirley Dzogan, EnviroTest Laboratories, 745 Logan Avenue, Winnipeg, Manitoba R3E 3L5, Canada 

Mary Ann Feige, Technical Support Center, Office of Ground Water and Drinking Water, U.S. 

Environmental Protection Agency, 26 W. Martin Luther King Drive, Cincinnati, OH 45268-1320, 

USA 

Colin Fricker, Thames Water Utilities, Manor Farm Road, Reading, Berkshire, RG2 0JN, England 

Carrie Hancock, CH Diagnostic & Consulting Service, Inc., 214 S.E. Nineteenth Street, Loveland, CO 

80537, USA 

Stephanie Harris, Region 10 Laboratory, U.S. Environmental Protection Agency, 7411 Beach Drive East, 

Port Orchard, WA 98366, USA 

Dale Rushneck, Interface, Inc., 3194 Worthington Avenue, Fort Collins, CO 80526, USA 

Frank Schaefer III, National Exposure Research Laboratory, U.S. Environmental Protection Agency, 26 

W. Martin Luther King Drive, Cincinnati, OH 45268-1320, USA 

Steve Schaub, Health and Ecological Criteria Division (4304), Office of Science and Technology, U.S. 

Environmental Protection Agency, 401 M Street, S.W., Washington, DC 20460, USA 

Ajaib Singh, City of Milwaukee Health Department, 841 North Broadway, Milwaukee, WI 53202, USA 

Huw Smith, Department of Bacteriology, Scottish Parasite Diagnostic Laboratory, Stobhill NHS Trust, 

Springburn, Glasgow, G21 3UW, Scotland 

Timothy Straub, Lockheed Martin, 7411 Beach Drive East, Port Orchard, WA 98366, USA 

William A. Telliard, Office of Science and Technology, U.S. Environmental Protection Agency, 401 M 

Street, S.W., Washington, DC 20460, USA 

Cryptosporidium cover photo courtesy of the U.S. Centers for Disease Control 

Giardia cover photo courtesy of CH Diagnostic & Consulting Service, Inc. 

Disclaimer 

This method has been reviewed by the U.S. EPA Office of Water and approved for publication. Mention 

of trade names or commercial products does not constitute endorsement or recommendation for use. 

i 
Questions regarding this method or its application should be addressed to: 

William A. Telliard
 


U.S. EPA Office of Water
 


Analytical Methods Staff
 


Mail Code 4303
 


Washington, DC 20460
 


Email: telliard.william@epa.gov
 


ii 
Introduction 

To support future regulation of protozoa in drinking water, the Safe Drinking Water Act Amendments of 

1996 require the U.S. Environmental Protection Agency (EPA) to evaluate the risk to public health posed 

by drinking water contaminants, including waterborne parasites, such as Cryptosporidium and Giardia. 

To implement these requirements, EPA must assess Cryptosporidium and Giardia occurrence in raw 

surface waters used as source waters for drinking water treatment plants. EPA Method 1623 was 

developed to support this assessment. 

EPA initiated an effort in 1996 to identify new and innovative technologies for protozoan monitoring 

and analysis. After evaluating potential alternatives to the then-current method through literature 

searches, discussions with research and commercial laboratories, and meetings with experts in the field, 

the Engineering and Analysis Division within the Office of Science and Technology within EPA's Office 

of Water developed draft Method 1622 for Cryptosporidium detection in December 1996. This 

Cryptosporidium-only method was validated through an interlaboratory study in August 1998, and was 

revised as a final, valid method for detecting Cryptosporidium in water in January 1999. 

Although development of an acceptable immunomagnetic separation system for Giardia lagged behind 

development of an acceptable system for Cryptosporidium, an acceptable system was identified in 

October 1998, and EPA validated a method for simultaneous detection of Cryptosporidium and Giardia 

in February 1999 and developed quality control (QC) acceptance criteria for the method based on this 

validation study. To avoid confusion with Method 1622, which already had been validated and was in 

use both domestically and internationally as a stand-alone Cryptosporidium-only detection method, EPA 

designated the new combined procedure EPA Method 1623. 

The interlaboratory validated versions of Method 1622 (January 1999; EPA-821-R-99-001) and Method 

1623 (April 1999; EPA-821-R-99-006) were used to analyze approximately 3,000 field and QC samples 

during the Information Collection Rule Supplemental Surveys (ICRSS) between March 1999 and 

February 2000. Method 1622 was used to analyze samples from March 1999 to mid-July 1999; Method 

1623 was used from mid-July 1999 to February 2000. The April 2001 revision of both methods include 

updated QC acceptance criteria based on analysis of the QC samples analyzed during the ICRSS. 

EPA Method 1623 is a performance-based method applicable to the determination of Cryptosporidium 

and Giardia in aqueous matrices. EPA Method 1623 requires filtration, immunomagnetic separation of 

the oocysts and cysts from the material captured, and an immunofluorescence assay for determination of 

oocyst and cyst concentrations, with confirmation through vital dye staining and differential interference 

contrast microscopy. 

The interlaboratory validation of EPA Method 1623 conducted by EPA used the Pall Gelman capsule 

filtration procedure, Dynal immunomagnetic separation (IMS) procedure, and Meridian sample staining 

procedure described in this document. Alternate procedures are allowed, provided that required quality 

control tests are performed and all quality control acceptance criteria in this method are met. 

Since the interlaboratory validation of EPA Method 1623, interlaboratory validation studies have been 

performed to demonstrate the equivalency of modified versions of the method using the following 

components: 

• Whatman Nuclepore CryptTestTM filter 

• IDEXX Filta-MaxTM filter 

• Waterborne Aqua-GloTM G/C Direct FL antibody stain 

• Waterborne Crypt-a-GloTM and Giardi-a-GloTM antibody stains 

iii 
The validation studies for these modified versions of the method met EPA’s performance-based 

measurement system Tier 2 validation for nationwide use (see Section 9.1.2 for details), and have been 

accepted by EPA as equivalent in performance to the original version of the method validated by EPA. 

The equipment and reagents used in these modified versions of the method are noted in Sections 6 and 7 

of the method; the procedures for using these equipment and reagent options are available from the 

manufacturers. 

Because this is a performance-based method, other alternative components not listed in the method may 

be available for evaluation and use by the laboratory. Confirming the acceptable performance of a 

modified version of the method using alternate components in a single laboratory does not require that 

an interlaboratory validation study be conducted. However, method modifications validated only in a 

single laboratory have not undergone sufficient testing to merit inclusion in the method. Only those 

modified versions of the method that have been demonstrated as equivalent at multiple laboratories on 

multiple water sources through a Tier 2 interlaboratory study will be cited in the method. 

iv 
Table of Contents
 


1.0 Scope and Application ......................................................... 1
 


2.0 Summary of Method ........................................................... 1
 


3.0 Definitions .................................................................. 2
 


4.0 Contamination, Interferences, and Organism Degradation.............................. 2
 


5.0 Safety ...................................................................... 3
 


6.0 Equipment and Supplies ........................................................ 3
 


7.0 Reagents and Standards ........................................................ 7
 


8.0 Sample Collection and Storage .................................................. 10
 


9.0 Quality Control .............................................................. 11
 


10.0 Microscope Calibration and Analyst Verification ................................... 17
 


11.0 Oocyst and Cyst Suspension Enumeration and Spiking ............................... 22
 


12.0 Sample Filtration and Elution ................................................... 31
 


13.0 Sample Concentration and Separation (Purification) ................................. 33
 


14.0 Sample Staining ............................................................. 38
 


15.0 Examination ................................................................ 39
 


16.0 Analysis of Complex Samples .................................................. 41
 


17.0 Method Performance .......................................................... 42
 


18.0 Pollution Prevention .......................................................... 42
 


19.0 Waste Management ........................................................... 42
 


20.0 References .................................................................. 42
 


21.0 Tables and Figures ........................................................... 43
 


Table 1. Method Holding Times ........................................... 43
 


Table 2. Tier 1 and Tier 2 Validation/Equivalency Demonstration Requirements .... 44
 


Table 3. Quality Control Acceptance Criteria for Cryptosporidium ............... 44
 


Table 4. Quality Control Acceptance Criteria for Giardia ....................... 45
 


Table 5. Distribution of Matrix Spike Recoveries from Multiple Samples 
 


Collected from 87 Source Waters During the ICR Supplemental Surveys . . . 45
 

Figure 1. Hemacytometer Platform Ruling ................................... 46
 

Figure 2. Manner of Counting Oocysts and Cysts in 1 Square mm. ................ 47
 

Figure 3. Laboratory Filtration System ...................................... 48
 

Figure 4. Methods for Scanning a Well Slide ................................. 49
 

22.0 Glossary of Definitions and Purposes ............................................. 50
 


v
Method 1623: Cryptosporidium and Giardia in Water 

by Filtration/IMS/FA 

1.0 Scope and Application 

1.1	 	 This method is for determination of the identity and concentration of Cryptosporidium (CAS 

Registry number 137259-50-8) and Giardia (CAS Registry number 137259-49-5) in water by 

filtration, immunomagnetic separation (IMS), and immunofluorescence assay (FA) microscopy. 

Cryptosporidium and Giardia may be confirmed using 4',6-diamidino-2-phenylindole (DAPI) 

staining and differential interference contrast (DIC) microscopy. The method has been validated 

in surface water, but may be used in other waters, provided the laboratory demonstrates that the 

method’s performance acceptance criteria are met. 

1.2	 	 This method is designed to meet the survey and monitoring requirements of the U.S. 

Environmental Protection Agency (EPA). It is based on laboratory testing of recommendations 

by a panel of experts convened by EPA. The panel was charged with recommending an 

improved protocol for recovery and detection of protozoa that could be tested and implemented 

with minimal additional research. 

1.3	 	 This method will not identify the species of Cryptosporidium or Giardia or the host species of 

origin, nor can it determine the viability or infectivity of detected oocysts and cysts. 

1.4	 	 This method is for use only by persons experienced in the determination of Cryptosporidium 

and Giardia by filtration, IMS, and FA. Experienced persons are defined in Section 22.2 as 

analysts. Laboratories unfamiliar with analyses of environmental samples by the techniques in 

this method should gain experience using water filtration techniques, IMS, fluorescent antibody 

staining with monoclonal antibodies, and microscopic examination of biological particulates 

using bright-field and DIC microscopy. 

1.5	 	 Any modification of the method beyond those expressly permitted is subject to the application 

and approval of alternative test procedures under 40 CFR Part 141.27. 

2.0 Summary of Method 

2.1	 	 A water sample is filtered and the oocysts, cysts, and extraneous materials are retained on the 

filter. Although EPA has only validated the method using laboratory filtration of bulk water 

samples shipped from the field, field-filtration also can be used. 

2.2 Elution and separation 

2.2.1	 	 Materials on the filter are eluted and the eluate is centrifuged to pellet the oocysts and 

cysts, and the supernatant fluid is aspirated. 

2.2.2	 	 The oocysts and cysts are magnetized by attachment of magnetic beads conjugated to 

anti-Cryptosporidium and anti-Giardia antibodies. The magnetized oocysts and cysts 

are separated from the extraneous materials using a magnet, and the extraneous 

materials are discarded. The magnetic bead complex is then detached from the oocysts 

and cysts. 

2.3 Enumeration 

2.3.1	 	 The oocysts and cysts are stained on well slides with fluorescently labeled monoclonal 

antibodies and 4',6-diamidino-2-phenylindole (DAPI). The stained sample is examined 

using fluorescence and differential interference contrast (DIC) microscopy. 

1 April 2001 
Method 1623 - Cryptosporidium and Giardia 

2.3.2	 	 Qualitative analysis is performed by scanning each slide well for objects that meet the 

size, shape, and fluorescence characteristics of Cryptosporidium oocysts or Giardia 

cysts. Potential oocysts or cysts are confirmed through DAPI staining characteristics 

and DIC microscopy. Oocysts and cysts are identified when the size, shape, color, and 

morphology agree with specified criteria and examples in a photographic library. 

2.3.3	 	 Quantitative analysis is performed by counting the total number of objects on the slide 

confirmed as oocysts or cysts. 

2.4	 	 Quality is assured through reproducible calibration and testing of the filtration, immunomagnetic 

separation (IMS), staining, and microscopy systems. Detailed information on these tests is 

provided in Section 9.0. 

3.0 Definitions 

3.1	 	 Cryptosporidium is defined as a protozoan parasite potentially found in water and other media. 

The six species of Cryptosporidium and their potential hosts are C. parvum (mammals, 

including humans); C. baileyi and C. meleagridis (birds); C. muris (rodents); C. serpentis 

(reptiles); and C. nasorum (fish). 

3.2	 	 Giardia is defined as a protozoan parasite potentially found in water and other media. The two 

species of Giardia and their potential hosts are G. intestinalis (humans) and G. muris (mice). 

3.3 Definitions for other terms used in this method are given in the glossary (Section 22.0). 

4.0 Contamination, Interferences, and Organism Degradation 

4.1	 	 Turbidity caused by inorganic and organic debris can interfere with the concentration, 

separation, and examination of the sample for Cryptosporidium oocysts and Giardia cysts. In 

addition to naturally-occurring debris, such as clays and algae, chemicals, such as iron and alum 

coagulants and polymers, may be added to finished waters during the treatment process, which 

may result in additional interference. 

4.2	 	 Organisms and debris that autofluoresce or demonstrate non-specific fluorescence, such as algal 

and yeast cells, when examined by epifluorescent microscopy, may interfere with the detection 

of oocysts and cysts and contribute to false positives by immunofluorescence assay (FA). 

4.3	 	 Solvents, reagents, labware, and other sample-processing hardware may yield artifacts that may 

cause misinterpretation of microscopic examinations for oocysts and cysts. All materials used 

shall be demonstrated to be free from interferences under the conditions of analysis by running a 

method blank (negative control sample) initially and a minimum of every week or after changes 

in source of reagent water. Specific selection of reagents and purification of solvents and other 

materials may be required. 

4.4	 	 Interferences co-extracted from samples will vary considerably from source to source, 

depending on the water being sampled. Experience suggests that high levels of algae, bacteria, 

and other protozoa can interfere in the identification of oocysts and cysts (Reference 20.1). 

4.5	 	 Freezing samples, filters, eluates, concentrates, or slides may interfere with the detection and/or 

identification of oocysts and cysts. 

4.6	 	 All equipment should be cleaned according to manufacturers’ instructions. Disposable supplies 

should be used wherever possible. 

April 2001 2 
Method 1623 - Cryptosporidium and Giardia 

5.0 Safety 

5.1	 	 The biohazard associated with, and the risk of infection from, oocysts and cysts is high in this 

method because live organisms are handled. This method does not purport to address all of the 

safety problems associated with its use. It is the responsibility of the laboratory to establish 

appropriate safety and health practices prior to use of this method. In particular, laboratory staff 

must know and observe the safety procedures required in a microbiology laboratory that handles 

pathogenic organisms while preparing, using, and disposing of sample concentrates, reagents 

and materials, and while operating sterilization equipment. 

5.2	 	 The toxicity or carcinogenicity of each compound or reagent used in this method has not been 

precisely determined; however, each chemical compound should be treated as a potential health 

hazard. Exposure to these compounds should be reduced to the lowest possible level. The 

laboratory is responsible for maintaining a current awareness file of Occupational Safety and 

Health Administration regulations regarding the safe handling of the chemicals specified in this 

method. A reference file of material safety data sheets should be made available to all personnel 

involved in these analyses. Additional information on laboratory safety can be found in 

References 20.2 through 20.5. 

5.3	 	 Samples may contain high concentrations of biohazards and toxic compounds, and must be 

handled with gloves and opened in a biological safety cabinet to prevent exposure. Reference 

materials and standards containing oocysts and cysts must also be handled with gloves and 

laboratory staff must never place gloves in or near the face after exposure to solutions known or 

suspected to contain oocysts and cysts. Do not mouth-pipette. 

5.4	 	 Laboratory personnel must change gloves after handling filters and other contaminant-prone 

equipment and reagents. Gloves must be removed or changed before touching any other 

laboratory surfaces or equipment. 

5.5	 	 Centers for Disease Control (CDC) regulations (42 CFR 72) prohibit interstate shipment of more 

than 4 L of solution known to contain infectious materials. State regulations may contain similar 

regulations for intrastate commerce. Unless the sample is known or suspected to contain 

Cryptosporidium, Giardia, or other infectious agents (e.g., during an outbreak), samples should 

be shipped as noninfectious and should not be marked as infectious. If a sample is known or 

suspected to be infectious, and the sample must be shipped to a laboratory by a transportation 

means affected by CDC or state regulations, the sample should be shipped in accordance with 

these regulations. 

6.0 Equipment and Supplies 

NOTE: Brand names, suppliers, and part numbers are for illustrative purposes only. 

No endorsement is implied. Equivalent performance may be achieved using apparatus 

and materials other than those specified here, but demonstration of equivalent 

performance that meets the requirements of this method is the responsibility of the 

laboratory. 

6.1	 	 Sample collection equipment for shipment of bulk water samples for laboratory filtration. 

Collapsible LDPE cubitainer for collection of 10-L bulk sample(s)—Cole Parmer cat. no. U06100-30 or equivalent. Fill completely to ensure collection of a full 10-L sample. Discard after 

one use. 

3 April 2001 
Method 1623 - Cryptosporidium and Giardia 

6.2	 	 Equipment for sample filtration. Three options have been demonstrated to be acceptable for use 

with Method 1623. Other options may be used if their acceptability is demonstrated according to 

the procedures outlined in Section 9.1.2. 

6.2.1	 	 Cubitainer spigot to facilitate laboratory filtration of sample (for use with any filtration 

option)—Cole Parmer cat. no. U-06061-01, or equivalent. 

6.2.2	 	 EnvirochekTM sampling capsule equipment requirements for use with the procedure 

described in Section 12.0. The version of the method using this filter was validated 

using 10-L sample volumes; alternate sample volumes may be used, provided the 

laboratory demonstrates acceptable performance on initial and ongoing spiked reagent 

water and source water samples (Section 9.1.2). 

6.2.2.1	 	 Sampling capsule—EnvirochekTM, Pall Gelman Laboratory, Ann Arbor, 

MI, product 12110 

6.2.2.2 Laboratory shaker with arms for agitation of sampling capsules 

6.2.2.2.1	 	 Laboratory shaker—Lab-Line model 3589, VWR 

Scientific cat. no. 57039-055, Fisher cat. no. 14260-11, 

or equivalent 

6.2.2.2.2	 	 Side arms for laboratory shaker—Lab-Line Model 3587-

4, VWR Scientific cat. no. 57039-045, Fisher cat. no. 

14260-13, or equivalent 

6.2.3	 	 CrypTestTM capsule filter equipment requirements. Follow the manufacturer’s 

instructions when using this filtration option. The version of the method using this filter 

was validated using 10-L sample volumes; alternate sample volumes may be used, 

provided the laboratory demonstrates acceptable performance on initial and ongoing 

spiked reagent water and matrix samples (Section 9.1.2). 

6.2.3.1	 	 Capsule filter—CrypTestTM, Whatman Inc, Clifton, NJ, product no. 

610064 

6.2.3.2	 	 Cartridge housing—Ametek 5-in. clear polycarbonate, Whatman cat. no. 

71503, or equivalent 

6.2.3.3 Ultrasonic bath—VWR Model 75T#21811-808, or equivalent 

6.2.3.4 Laboratory tubing—Tygon formula R-3603, or equivalent 

6.2.4	 	 Filta-MaxTM foam filter equipment requirements. Follow the manufacturer’s 

instructions when using this filtration option. The version of the method using this 

filter was validated using 50-L sample volumes; alternate sample volumes may be used, 

provided the laboratory demonstrates acceptable performance on initial and ongoing 

spiked reagent water and matrix samples (Section 9.1.2). 

6.2.4.1	 	 Foam filter—Filta-MaxTM, IDEXX, Westbrook, ME. Filter module and 

membrane: product code FMC 10601; filter membranes (100 pack), 

product code FMC 10800 

NOTE: Check at least one filter per batch to ensure that the filters have not been 

affected by improper storage or other factors that could result in brittleness or other 

problems. At a minimum confirm that the test filter expands properly in water before 

using the batch or shipping filters to the field. 

April 2001 4 
Method 1623 - Cryptosporidium and Giardia 

6.2.4.2	 	 Filter processing equipment—Filta-Max starter kit, IDEXX, Westbrook, 

ME, cat. no. FMC 11002. Includes all equipment required to run and 

process Filta-Max filter modules (manual wash station (FMC 10102) 

including plunger head (FMC 12001), elution tubing set (FMC 10301), 

vacuum set (FMC 10401), filter housing (FMC 10501), and magnetic 

stirrer (FMC 10901). 

6.3 Ancillary sampling equipment 

6.3.1	 	 Tubing—Glass, polytetrafluoroethylene (PTFE), high-density polyethylene (HDPE), or 

other tubing to which oocysts and cysts will not easily adhere—Tygon formula R-3603, 

or equivalent. If rigid tubing (glass, PTFE, HDPE) is used and the sampling system uses 

a peristaltic pump, a minimum length of compressible tubing may be used in the pump. 

Before use, the tubing must be autoclaved, thoroughly rinsed with detergent solution, 

followed by repeated rinsing with reagent water to minimize sample contamination. 

Alternately, decontaminate using hypochlorite solution, sodium thiosulfate, and 

multiple reagent water rinses; dispose of tubing when wear is evident. Dispose of 

tubing after one use whenever possible. 

6.3.2	 	 Flow control valve—0.5 gpm (0.03 L/s), Bertram Controls, Plast-O-Matic cat. no. 

FC050B1⁄2-PV, or equivalent; or 0.4- to 4-Lpm flow meter with valve—Alamo Water 

Treatment, San Antonio, TX, cat. no. R5310, or equivalent 

6.3.3 Centrifugal pump—Grainger, Springfield, VA, cat. no. 2P613, or equivalent 

6.3.4	 	 Flow meter—Sameco cold water totalizer, E. Clark and Associates, Northboro, MA, 

product no. WFU 10.110, or equivalent 

6.4 Equipment for spiking samples in the laboratory 

6.4.1 10-L carboy with bottom delivery port (1⁄2")—Cole-Palmer cat. no. 06080-42, or 

equivalent; calibrate to 10.0 L and mark level with waterproof marker 

6.4.2 Stir bar—Fisher cat. no. 14-511-93, or equivalent 

6.4.3 Stir plate—Fisher cat. no. 14-493-120S, or equivalent 

6.4.4	 	 Hemacytometer—Neubauer type, Hauser Scientific, Horsham, PA, cat. no. 3200 or 

1475, or equivalent 

6.4.5	 	 Hemacytometer coverslip—Hauser Scientific, cat. no. 5000 (for hemacytometer cat. no. 

3200) or 1461 (for hemacytometer cat. no 1475), or equivalent 

6.4.6 Lens paper without silicone—Fisher cat. no. 11-995, or equivalent 

6.4.7 Polystyrene or polypropylene conical tubes with screw caps—15- and 50-mL 

6.4.8 Equipment required for enumeration of spiking suspensions using membrane filters 

6.4.8.1	 	 Glass microanalysis filter holder—25-mm-diameter, with fritted glass 

support, Fisher cat. no. 09-753E, or equivalent. Replace stopper with 

size 8, one-hole rubber stopper, Fisher Cat. No. 14-135M, or equivalent. 

6.4.8.2	 	 Three-port vacuum filtration manifold and vacuum source—Fisher Cat. 

No. 09-753-39A, or equivalent 

6.4.8.3	 	 Cellulose acetate support membrane—1.2-μm-pore-size , 25-mmdiameter, Fisher cat. no. A12SP02500, or equivalent 

6.4.8.4	 	 Polycarbonate track-etch hydrophilic membrane filter—1-μm-pore-size , 

25-mm-diameter, Fisher cat. no. K10CP02500, or equivalent 

6.4.8.5 100 × 15 mm polystyrene petri dishes (bottoms only) 

6.4.8.6 60 × 15 mm polystyrene petri dishes 

5 April 2001 
Method 1623 - Cryptosporidium and Giardia 

6.4.8.7 Glass microscope slides—1 in. × 3 in or 2 in. × 3 in.
 


6.4.8.8 Coverslips—25 mm2
 


6.5 Immunomagnetic separation (IMS) apparatus
 


6.5.1 Sample mixer—Dynal Inc., Lake Success, NY, cat. no. 947.01, or equivalent
 


6.5.2	 	 Magnetic particle concentrator for 10-mL test tubes—Dynal MPC-1® , cat. no. 120.01,
 


or equivalent
 


6.5.3	 	 Magnetic particle concentrator for microcentrifuge tubes—Dynal MPC-M®, cat. no.
 


120.09, or equivalent
 


6.5.4	 	 Flat-sided sample tubes—16 × 125 mm Leighton-type tubes with 60 × 10 mm flat-sided
 


magnetic capture area, Dynal L10, cat. no. 740.03, or equivalent
 


6.6 Powder-free latex gloves—Fisher cat no. 113945B, or equivalent
 


6.7 Graduated cylinders, autoclavable—10-, 100-, and 1000-mL
 


6.8 Centrifuges
 


6.8.1	 	 Centrifuge capable of accepting 15- to 250-mL conical centrifuge tubes and achieving
 


1500 × G—International Equipment Company, Needham Heights, MA, Centrifuge Size
 


2, Model K with swinging bucket, or equivalent
 


6.8.2 Centrifuge tubes—Conical, graduated, 1.5-, 50-, and 250-mL
 


6.9 Microscope
 


6.9.1	 	 Epifluorescence/differential interference contrast (DIC) with stage and ocular
 


micrometers and 20X (N.A.=0.4) to 100X (N.A.=1.3) objectives—ZeissTM Axioskop,
 


OlympusTM BH, or equivalent
 


6.9.2	 	 Excitation/band-pass filters for immunofluorescence assay (FA)—ZeissTM 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
 


6.9.3	 	 Excitation/band-pass filters for DAPI—Filters cited below (Chroma Technology,
 


Brattleboro, VT), or equivalent
 


Microscope 

model Fluoro-chrome Excitation filter (nm) 

Dichroic beamsplitting mirror 

(nm) 

Barrier or 

suppression 

filter (nm) 

Chroma 

catalog 

number 

ZeissTM -

Axioskop DAPI (UV) 340-380 400 420 CZ902 

ZeissTM -IM35 DAPI (UV) 340-380 400 420 CZ702 

OlympusTM BH 

DAPI (UV) 340-380 400 420 11000 

Filter holder 91002 

OlympusTM BX 

DAPI (UV) 340-380 400 420 11000 

Filter holder 91008 

OlympusTM 

IMT2 

DAPI (UV) 340-380 400 420 11000 

Filter holder 91003 

6.10 Ancillary equipment for microscopy
 


6.10.1	 	 Well slides— Spot-On well slides, Dynal cat. no. 740.04; treated, 12-mm diameter well
 


slides, Meridian Diagnostics Inc., Cincinnati, OH, cat. no. R2206; or equivalent
 


6.10.2 Glass coverslips—22 × 50 mm
 


April 2001 6 
Method 1623 - Cryptosporidium and Giardia 

6.10.3 Nonfluorescing immersion oil
 


6.10.4 Micropipette, adjustable:	 	 0- to 10-μL with 0- to 10-μL tips
 


10- to 100-μL , with 10- to 200-μL tips
 


100- to 1000-μL with 100- to 1000-μL tips
 


6.10.5 Forceps—Splinter, fine tip
 


6.10.6 Forceps—Blunt-end
 


6.10.7 Desiccant—DrieriteTM Absorbent, Fisher cat. no. 07-577-1A, or equivalent
 


6.10.8	 	 Humid chamber—A tightly sealed plastic container containing damp paper towels on
 


top of which the slides are placed
 


6.11 Pipettes—Glass or plastic
 


6.11.1 5-, 10-, and 25-mL
 


6.11.2 Pasteur, disposable
 


6.12 Balances
 


6.12.1 Analytical—Capable of weighing 0.1 mg
 


6.12.2 Top loading—Capable of weighing 10 mg
 


6.13 pH meter
 


6.14 Incubator—Fisher Scientific IsotempTM, or equivalent
 


6.15 Vortex mixer—Fisons Whirlmixer, or equivalent
 


6.16	 	 Vacuum source—Capable of maintaining 25 in. Hg, equipped with shutoff valve and vacuum
 


gauge
 


6.17 Miscellaneous labware and supplies
 


6.17.1 Test tubes and rack
 


6.17.2 Flasks—Suction, Erlenmeyer, and volumetric, various sizes
 


6.17.3 Beakers—Glass or plastic, 5-, 10-, 50-, 100-, 500-, 1000-, and 2000-mL
 


6.17.4 Lint-free tissues
 


6.18	 	 10- to 15-L graduated container—Fisher cat. no. 02-961-50B, or equivalent; calibrate to 9.0, 9.5,
 


10.0, 10.5, and 11.0 L and mark levels with waterproof marker
 


6.19	 	 Filters for filter-sterilizing reagents—Sterile Acrodisc, 0.45 μm , Gelman Sciences cat no. 4184, or
 


equivalent
 


7.0 Reagents and Standards
 


7.1	 	 Reagents for adjusting pH
 


7.1.1 Sodium hydroxide (NaOH)—ACS reagent grade, 6.0 N and 1.0 N in reagent water
 


7.1.2 Hydrochloric acid (HCl)—ACS reagent grade, 6.0 N, 1.0 N, and 0.1 N in reagent water
 


NOTE: Due to the low volumes of pH-adjusting reagents used in this method, and the 

impact that changes in pH have on the immunofluorescence assay, the laboratory 

should purchase standards at the required normality directly from a vendor. Normality 

should not be adjusted by the laboratory. 

7.2 Solvents—Acetone, glycerol, ethanol, and methanol, ACS reagent grade
 


7.3	 	 Reagent water—Water in which oocysts and cysts and interfering materials and substances,
 


including magnetic minerals, are not detected by this method
 


7 April 2001 
Method 1623 - Cryptosporidium and Giardia 

7.4 Reagents for eluting filters 

7.4.1 Reagents for eluting EnvirochekTM sampling capsules (Section 6.2.2) 

7.4.1.1	 	 Laureth-12—PPG Industries, Gurnee, IL, cat. no. 06194, or equivalent. 

Store Laureth-12 as a 10% solution in reagent water. Weigh 10 g of 

Laureth-12 and dissolve using a microwave or hot plate in 90 mL of 

reagent water. Dispense 10-mL aliquots into sterile vials and store at 

room temperature for up to 2 months, or in the freezer for up to a year. 

7.4.1.2	 	 1 M Tris, pH 7.4—Dissolve 121.1 g Tris (Fisher cat. no. BP152) in 700 

mL of reagent water and adjust pH to 7.4 with 1 N HCl or NaOH. Dilute 

to a final 1000 mL with reagent water and adjust the final pH. Filtersterilize through a 0.2-μm membrane into a sterile plastic container and 

store at room temperature. 

7.4.1.3	 	 0.5 M EDTA, 2 Na, pH 8.0—Dissolve 186.1 g ethylenediamine 

tetraacetic acid, disodium salt dihydrate (Fisher cat. no. S311) in 800 mL 

and adjust pH to 8.0 with 6.0 N HCl or NaOH. Dilute to a final volume 

of 1000 mL with reagent water and adjust to pH 8.0 with 1.0 N HCl or 

NaOH. 

7.4.1.4 Antifoam A—Sigma Chemical Co. cat. no. A5758, or equivalent 

7.4.1.5	 	 Preparation of elution buffer solution—Add the contents of a preprepared Laureth-12 vial (Section 7.4.1.1) to a 1000-mL graduated 

cylinder. Rinse the vial several times to ensure the transfer of the 

detergent to the cylinder. Add 10 mL of Tris solution (Section 7.4.1.2), 2 

mL of EDTA solution (Section 7.4.1.3), and 150 μL Antifoam A 

(Section 7.4.1.4). Dilute to 1000 mL with reagent water. 

7.4.2	 	 Reagents for eluting CrypTestTM capsule filters (Section 6.2.3). To 900 mL of reagent 

water add 8.0 g NaCl, 0.2 g KH2PO4, 2.9 g Na2HPO4 (12H2O) 0.2 g KCl, 0.2 g sodium 

lauryl sulfate (SDS), 0.2 mL Tween 80, and 0.02 mL Antifoam A (Sigma Chemical Co. 

cat. no. A5758, or equivalent). Adjust volume to 1 L with reagent water and adjust pH 

to 7.4 with 1 N NaOH or HCl. 

7.4.3 Reagents for eluting Filta-MaxTM foam filters (Section 6.2.4) 

7.4.3.1	 	 Phosphate buffered saline (PBS), pH 7.4—Sigma Chemical Co. cat. no. 

P-3813, or equivalent. Alternately, prepare PBS by adding the following 

to 1 L of reagent water: 8 g NaCl; 0.2 g KCl; 1.15 g Na2HPO4, anhydrous; 

and 0.2 g KH2PO4. 

7.4.3.2 Tween 20—Sigma Chemical Co. cat. no. P-7949, or equivalent 

7.4.3.3 High-vacuum grease—BDH/Merck. cat. no. 636082B, or equivalent 

7.4.3.4	 	 Preparation of PBST elution buffer. Add the contents of one sachet of 

PBS to 1.0 L of reagent water. Dissolve by stirring for 30 minutes. Add 

100 μL of Tween 20. Mix by stirring for 5 minutes. 

7.5	 	 Reagents for immunomagnetic separation (IMS)—Dynabeads® GC-Combo, Dynal cat. nos. 

730.02, 730.12, or equivalent 

7.6	 	 Direct antibody labeling reagents for detection of oocysts and cysts. Store reagents at 0 oC to 8 oC 

and return promptly to this temperature after each use. Do not allow any of the reagents to 

freeze. The reagents should be protected from exposure to light. Diluted, unused working 

reagents should be discarded after 48 hours. Discard reagents after the expiration date is reached. 

The labeling reagents in Sections 7.6.1-7.6.3 have been approved for use with this method. 

April 2001 8 
Method 1623 - Cryptosporidium and Giardia 

7.6.1	 	 Merifluor Cryptosporidium/Giardia, Meridian Diagnostics cat. no. 250050, Cincinnati, 

OH, or equivalent 

7.6.2	 	 Aqua-GloTM G/C Direct FL, Waterborne cat. no. A100FLR, New Orleans, LA, or 

equivalent 

7.6.3	 	 Crypt-a-GloTM and Giardi-a-GloTM, Waterborne cat. nos. A400FLR and A300FLR, 

respectively, New Orleans, LA, or equivalent 

NOTE: If a laboratory will use multiple types of labeling reagents, the laboratory 

must demonstrate acceptable performance through an initial precision and recovery 

test (Section 9.4) for each type, and must perform positive and negative staining 

controls for each batch of slides stained using each product. However, the laboratory 

is not required to analyze additional ongoing precision and recovery samples or 

method blank samples for each type. 

7.6.4	 	 Diluent for labeling reagents—Phosphate buffered saline (PBS), pH 7.4—Sigma 

Chemical Co. cat. no. P-3813, or equivalent. Alternately, prepare PBS by adding the 

following to 1 L of reagent water: 8 g NaCl; 0.2 g KCl; 1.15 g Na2HPO4, anhydrous; and 

0.2 g KH2PO4. Filter-sterilize (Section 6.19) or autoclave. Discard if growth is detected 

or after 6 months, whichever comes first. 

7.7 4',6-diamidino-2-phenylindole (DAPI) stain—Sigma Chemical Co. cat. no. A5758, or equivalent 

7.7.1	 	 Stock solution—Dissolve 2 mg/mL DAPI in absolute methanol. Prepare volume 

consistent with minimum use. Store at 0 o

C to 8 oC in the dark. Do not allow to freeze. 

Discard unused solution when positive staining control fails. 

7.7.2	 	 Staining solution (1/5000 dilution in PBS [Section 7.6.4])—Add 10 μL of 2 mg/mL 

DAPI stock solution to 50 mL of PBS. Prepare daily. Store at 0 o

C to 8 o C in the dark 

except when staining. Do not allow to freeze. The solution concentration may be 

increased up to 1μg /mL if fading/diffusion of DAPI staining is encountered, but the 

staining solution must be tested first on expendable environmental samples to confirm 

that staining intensity is appropriate. 

7.8 Mounting medium 

7.8.1	 	 DABCO/glycerol mounting medium (2%)—Dissolve 2 g of DABCO (Sigma Chemical 

Co. cat no. D-2522, or equivalent) in 95 mL of warm glycerol/PBS (60% glycerol, 40% 

PBS [Section 7.6.4]). After the DABCO has dissolved completely, adjust the solution 

volume to 100 mL by adding an appropriate volume of glycerol/PBS solution. 

Alternately, dissolve the DABCO in 40 mL of PBS, then add azide (1 mL of 100X, or 

10% solution), then 60 mL of glycerol. 

7.8.2 Mounting medium supplied with Merifluor direct labeling kit (Section 7.6.1) 

7.9	 	 Clear fingernail polish or clear fixative, PGC Scientifics, Gaithersburg, MD, cat. no. 60-4890, or 

equivalent 

7.10 Oocyst and cyst suspensions for spiking 

7.10.1	 	 Enumerated spiking suspensions prepared by flow cytometer—not heat-fixed or 

formalin fixed: Wisconsin State Laboratory of Hygiene Flow Cytometry Unit or 

equivalent 

7.10.2 Materials for manual enumeration of spiking suspensions 

7.10.2.1	 	 Purified Cryptosporidium oocyst stock suspension for manual 

enumeration—not heat-fixed or formalin-fixed: Sterling Parasitology 

Laboratory, University of Arizona, Tucson, or equivalent 

9 April 2001 
Method 1623 - Cryptosporidium and Giardia 

7.10.2.2	 	 Purified Giardia cyst stock suspension for manual enumeration—not 

heat-fixed or formalin-fixed: Waterborne, Inc., New Orleans, LA; 

Hyperion Research, Medicine Hat, Alberta, Canada; or equivalent 

7.10.2.3	 	 Tween-20, 0.01%—Dissolve 1.0 mL of a 10% solution of Tween-20 in 1 

L of reagent water 

7.10.2.4	 	 Storage procedure—Store oocyst and cyst suspensions at 0 o C to 8 o C, 

until ready to use; do not allow to freeze 

7.11	 	 Additional reagents for enumeration of spiking suspensions using membrane filtration (Section 

11.3.6)—Sigmacote® Sigma Company Product No. SL-2, or equivalent 

8.0 Sample Collection and Storage 

8.1	 	 Samples are collected as bulk samples and shipped to the laboratory for processing through the 

entire method, or are filtered in the field and shipped to the laboratory for processing from 

elution (Section 12.2.6) onward. Samples must be shipped via overnight service on the day they 

are collected. Chill samples as much as possible between collection and shipment by storing in a 

refrigerator or pre-icing the sample in a cooler. If the sample is pre-iced before shipping, replace 

with fresh ice immediately before shipment. Samples should be shipped at 0 o C to 8 o C, unless the 

time required to chill the sample to 8 o C would prevent the sample from being shipped overnight 

for receipt at the laboratory the day after collection. Samples must not be allowed to freeze. 

Upon receipt, the laboratory should record the temperature of the samples and store them 

refrigerated at 0 o C to 8 o C until processed. Results from samples shipped overnight to the 

laboratory and received at >8 o C should be qualified by the laboratory. 

NOTE: See transportation precautions in Section 5.5. 

8.2	 	 Sample holding times. Sample processing should be completed as soon as possible by the 

laboratory. The laboratory should complete sample filtration, elution, concentration, purification, 

and staining the day the sample is received wherever possible. However, the laboratory is 

permitted to split up the sample processing steps if processing a sample completely in one day is 

not possible. If this is necessary, sample processing can be halted after filtration, application of 

the purified sample onto the slide, or staining. Table 1, in Section 21.0 provides a breakdown of 

the holding times for each set of steps. Sections 8.2.1 through 8.2.4 provide descriptions of these 

holding times. 

8.2.1	 	 Sample collection and filtration. Sample elution must be initiated within 96 hours of 

sample collection (if shipped to the laboratory as a bulk sample) or filtration (if filtered 

in the field). 

8.2.2	 	 Sample elution, concentration, and purification. The laboratory must complete the 

elution, concentration, and purification (Sections 12.2.6 through 13.3.3.11) in one work 

day. It is critical that these steps be completed in one work day to minimize the time that 

any target organisms present in the sample sit in eluate or concentrated matrix. This 

process ends with the application of the purified sample on the slide for drying. 

8.2.3	 	 Staining. The sample must be stained within 72 hours of application of the purified 

sample to the slide. 

8.2.4	 	 Examination. Although immunofluorescence assay (FA) and 4',6-diamidino-2-

phenylindole (DAPI) and differential interference contrast (DIC) microscopy 

examination and confirmation should be performed immediately after staining is 

complete, laboratories have up to 7 days from completion of sample staining to 

complete the examination and confirmation of samples. However, if fading/diffusion of 

FITC or DAPI staining is noticed, the laboratory must reduce this holding time. In 

April 2001 10 
Method 1623 - Cryptosporidium and Giardia 

addition the laboratory may adjust the concentration of the DAPI staining solution 

(Sections 7.7.2) so that fading/diffusion does not occur. 

8.5	 	 Spiking suspension enumeration holding times. Flow-cytometer-sorted spiking suspensions 

(Sections 7.10.1 and 11.2) used for spiked quality control (QC) samples (Section 9) must be used 

within the expiration date noted on the suspension. Laboratories should use flow-cytometersorted spiking suspensions containing live organisms within two weeks of preparation at the flow 

cytometry laboratory. Manually enumerated spiking suspensions must be used within 24 hours of 

enumeration of the spiking suspension if the hemacytometer chamber technique is used (Section 

11.3.4); or within 24 hours of application of the spiking suspension to the slides if the well slide 

or membrane filter enumeration technique is used (Sections 11.3.5 and 11.3.6). 

9.0 Quality Control 

9.1	 	 Each laboratory that uses this method is required to operate a formal quality assurance (QA) 

program (Reference 20.6). The minimum requirements of this program consist of an initial 

demonstration of laboratory capability through performance of the initial precision and recovery 

(IPR) test (Section 9.4), analysis of spiked samples to evaluate and document data quality, and 

analysis of standards and blanks as tests of continued performance. Laboratory performance is 

compared to established performance criteria to determine if the results of analyses meet the 

performance characteristics of the method. 

9.1.1	 	 A test of the microscope used for detection of oocysts and cysts is performed prior to 

examination of slides. This test is described in Section 10.0. 

9.1.2	 	 In recognition of advances that are occurring in analytical technology, the laboratory is 

permitted to modify certain method procedures to improve recovery or lower the costs 

of measurements, provided that all required quality control (QC) tests are performed 

and all QC acceptance criteria are met. Method procedures that can be modified include 

front-end techniques, such as filtration or immunomagnetic separation (IMS). The 

laboratory is not permitted to use an alternate determinative technique to replace 

immunofluorescence assay in this method (the use of different determinative techniques 

are considered to be different methods, rather than modified version of this method). 

However, the laboratory is permitted to modify the immunofluorescence assay 

procedure, provided that all required QC tests are performed (Section 9.1.2.1) and all 

QC acceptance criteria are met (see guidance on the use of multiple labeling reagents in 

Section 7.6). 

9.1.2.1 Method modification validation/equivalency demonstration requirements 

9.1.2.1.1
	 Method modifications at a single laboratory. Each 

time a modification is made to this method for use in a 

single laboratory, the laboratory is required to validate 

the modification according to Tier 1 of EPA’s 

performance-based measurement system (PBMS) (Table 

2 and Reference 20.7) to demonstrate that the 

modification produces results equivalent or superior to 

results produced by this method as written. Briefly, each 

time a modification is made to this method, the 

laboratory is required to demonstrate acceptable 

modified method performance through the IPR test 

(Section 9.4). IPR results must meet the QC acceptance 

criteria in Tables 3 and 4 in Section 21.0, and should be 

comparable to previous results using the unmodified 

procedure. Although not required, the laboratory also 

should perform a matrix spike/matrix spike duplicate 

(MS/MSD) test to demonstrate the performance of the 

11 April 2001 
Method 1623 - Cryptosporidium and Giardia 

modified method in at least one real-world matrix 

before analyzing field samples using the modified 

method. The laboratory is required to perform MS 

samples using the modified method at the frequency 

noted in Section 9.1.8. 

9.1.2.1.2	 	 Method modifications for nationwide approval. If the 

laboratory or a manufacturer seeks EPA approval of a 

method modification for nationwide use, the laboratory 

or manufacturer must validate the modification 

according to Tier 2 of EPA’s PBMS (Table 2 and 

Reference 20.7). Briefly, at least three laboratories must 

perform IPR tests (Section 9.4) and MS/MSD (Section 

9.5) tests using the modified method, and all tests must 

meet the QC acceptance criteria specified in Tables 3 

and 4 in Section 21.0. Upon nationwide approval, 

laboratories electing to use the modified method still 

must demonstrate acceptable performance in their own 

laboratory according to the requirements in Section 

9.1.2.1.1. 

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	 

9.1.2.2.2	 

9.1.2.2.3 

9.1.2.2.4	 

9.1.2.2.5	 

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. 
 

A listing of the analyte(s) measured (Cryptosporidium 
 

and Giardia). 
 

A narrative stating reason(s) for the modification. 
 

Results from all QC tests comparing the modified 
 

method to this method, including: 
 

(a) IPR (Section 9.4) 
 

(b) MS/MSD (Section 9.5) 
 

(c) Analysis of method blanks (Section 9.6) 
 

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) 

(b) 

(c) 

(d) 

(e) 

(f) 

(g) 

(h) 

Sample numbers and other identifiers
 


Source of spiking suspensions, as well as lot
 


number and date received (Section 7.10)
 


Spike enumeration date and time
 


All spiking suspension enumeration counts and
 


calculations (Section 11.0)
 


Sample spiking dates and times
 


Volume filtered (Section 12.2.5.2)
 


Filtration and elution dates and times
 


Pellet volume, resuspended concentrate volume,
 


resuspended concentrate volume transferred to
 


IMS, and all calculations required to verify the
 


percent of concentrate examined (Section 13.2)
 


April 2001 12 
Method 1623 - Cryptosporidium and Giardia 

(i) 	 Purification completion dates and times (Section
 

3.3.3.11)
 

(j) 	 Staining completion dates and times (Section
 

14.10)
 

(k) Staining control results (Section 15.2.1)
 

(l) 	 All required examination information (Section
 

15.2.2)
 

(m) 	 Examination completion dates and times
 

(Section 15.2.4)
 

(n) Analysis sequence/run chronology
 

(o) 	 Lot numbers of elution, IMS, and staining
 

reagents
 

(p) 	 Copies of bench sheets, logbooks, and other
 

recordings of raw data
 

(q) 	 Data system outputs, and other data to link the
 

raw data to the results reported
 

9.1.3	 	 The laboratory shall spike a separate sample aliquot from the same source to monitor 

method performance. This MS test is described in Section 9.5.1. 

9.1.4	 	 Analysis of method blanks is required to demonstrate freedom from contamination. 

The procedures and criteria for analysis of a method blank are described in Section 9.6. 

9.1.5	 	 The laboratory shall, on an ongoing basis, demonstrate through analysis of the ongoing 

precision and recovery (OPR) sample that the analysis system is in control. These 

procedures are described in Section 9.7. 

9.1.6	 	 The laboratory shall maintain records to define the quality of data that are generated. 

Development of accuracy statements is described in Sections 9.5.1.4 and 9.7.3. 

9.1.7	 	 The laboratory shall analyze one method blank (Section 9.6) and one OPR sample 

(Section 9.7) each week during which samples are analyzed if 20 or fewer field samples 

are analyzed during this period. The laboratory shall analyze one laboratory blank and 

one OPR sample for every 20 samples if more than 20 samples are analyzed in a week. 

9.1.8	 	 The laboratory shall analyze one MS sample (Section 9.5.1) when samples are first 

received from a utility for which the laboratory has never before analyzed samples. The 

MS analysis is performed on an additional (second) sample sent from the utility. If the 

laboratory routinely analyzes samples from 1 or more utilities, 1 MS analysis must be 

performed per 20 field samples. For example, when a laboratory receives the first 

sample from a given site, the laboratory must obtain a second aliquot of this sample to 

be used for the MS. When the laboratory receives the 21st sample from this site, a 

separate aliquot of this 21st sample must be collected and spiked. 

9.2 Micropipette calibration 

9.2.1	 	 Micropipettes must be sent to the manufacturer for calibration annually. Alternately, a 

qualified independent technician specializing in micropipette calibration can be used. 

Documentation on the precision of the recalibrated micropipette must be obtained from 

the manufacturer or technician. 

9.2.2	 	 Internal and external calibration records must be kept on file in the laboratory’s QA 

logbook. 

9.2.3	 	 If a micropipette calibration problem is suspected, the laboratory shall tare an empty 

weighing boat on the analytical balance and pipette the following volumes of reagent 

water into the weigh boat using the pipette in question: 100% of the maximum 

dispensing capacity of the micropipette, 50% of the capacity, and 10% of the capacity. 

Ten replicates should be performed at each weight. Record the weight of the water 

13 April 2001 
Method 1623 - Cryptosporidium and Giardia 

(assume that 1.00 mL of reagent water weighs 1.00 g) and calculate the relative standard 

deviation (RSD) for each. If the weight of the reagent water is within 1% of the desired 

weight (mL) and the RSD of the replicates at each weight is within 1%, then the pipette 

remains acceptable for use. 

9.2.4	 	 If the weight of the reagent water is outside the acceptable limits, consult the 

manufacturer’s instruction manual troubleshooting section and repeat steps described in 

Section 9.2.3. If problems with the pipette persist, the laboratory must send the pipette 

to the manufacturer for recalibration. 

9.3	 	 Microscope adjustment and certification: Adjust the microscope as specified in Section 10.0. All 

of the requirements in Section 10.0 must be met prior to analysis of IPRs, blanks, OPRs, field 

samples, and MS/MSDs. 

9.4	 	 Initial precision and recovery (IPR)—To establish the ability to demonstrate control over the 

analytical system and to generate acceptable precision and recovery, the laboratory shall perform 

the following operations: 

9.4.1	 	 Using the spiking procedure in Section 11.4 and enumerated spiking suspensions 

(Section 7.10.1 or Section 11.3), spike, filter, elute, concentrate, separate (purify), stain, 

and examine four reagent water samples spiked with 100 to 500 oocysts and 100 to 500 

cysts. If more than one process will be used for filtration and/or separation of samples, 

a separate set of IPR samples must be prepared for each process. 

NOTE: IPR tests must be accompanied by analysis of a method blank (Section 9.6). 

9.4.2	 	 Using results of the four analyses, calculate the average percent recovery and the 

relative standard deviation (RSD) of the recoveries for Cryptosporidium and for 

Giardia. The RSD is the standard deviation divided by the mean times 100. 

9.4.3	 	 Compare RSD and the mean with the corresponding limits for initial precision and 

recovery in Tables 3 and 4 in Section 21.0. If the RSD and the mean meet the 

acceptance criteria, system performance is acceptable and analysis of blanks and 

samples may begin. If the RSD or the mean falls outside the range for recovery, system 

performance is unacceptable. In this event, correct the problem and repeat the test 

(Section 9.4.1). 

9.5 Matrix spike (MS) and matrix spike duplicate (MSD): 

9.5.1	 	 Matrix spike—The laboratory shall spike and analyze a separate field sample aliquot to 

determine the effect of the matrix on the method’s oocyst and cyst recovery. The MS 

shall be analyzed according to the frequency in Section 9.1.8. 

9.5.1.1	 	 Analyze an unspiked field sample according to the procedures in 

Sections 12.0 to 15.0. Using the spiking procedure in Section 11.4 and 

enumerated spiking suspensions (Section 7.10.1 or Section 11.3), spike, 

filter, elute, concentrate, separate (purify), stain, and examine a second 

field sample aliquot with the number of organisms used in the IPR or 

OPR tests (Sections 9.4 and 9.7). 

9.5.1.2	 	 For each organism, calculate the percent recovery (R) using the 

following equation. 

R =100 x 

Nsp - Ns 

T 

where 

R is the percent recovery 

Nsp is the number of oocysts or cysts detected in the spiked 

sample 

April 2001 14 
Method 1623 - Cryptosporidium and Giardia 

Ns is the number of oocysts or cysts detected in the unspiked
 


sample
 


T is the true value of the oocysts or cysts spiked
 


9.5.1.3	 	 Compare the recovery for each organism with the corresponding limits 

in Tables 3 and 4 in Section 21.0. 

NOTE: Some sample matrices may prevent the acceptance criteria in Tables 3 and 4 

from being met. An assessment of the distribution of MS recoveries across 430 MS 

samples from 87 sites during the ICR Supplemental Surveys is provided in Table 5. 

9.5.1.4	 	 As part of the QA program for the laboratory, method precision for 

samples should be assessed and records maintained. After the analysis 

of five samples for which the spike recovery for each organism passes 

the tests in Section 9.5.1.3, the laboratory should calculate the average 

percent recovery (P) and the standard deviation of the percent recovery 

(sr). Express the precision assessment as a percent recovery interval 

from P - 2 sr to P + 2 sr for each matrix. For example, if P = 80% and sr 

= 30%, the accuracy interval is expressed as 20% to 140%. The precision 

assessment should be updated regularly across all MS samples and 

stratified by MS samples for each source. 

9.5.2	 	 Matrix spike duplicate—MSD analysis is required as part of nationwide approval of a 

modified version of this method to demonstrate that the modified version of this 

method produces results equal or superior to results produced by the method as written 

(Section 9.1.2.1.2). At the same time the laboratory spikes and analyzes the second field 

sample aliquot in Section 9.5.1.1, the laboratory shall spike and analyze a third, 

identical field sample aliquot. 

NOTE: Matrix spike duplicate samples are only required for Tier 2 validation studies. 

They are recommended for Tier 1 validation, but not required. 

9.5.2.1	 	 For each organism, calculate the percent recovery (R) using the equation 

in Section 9.5.1.2. 

9.5.2.2	 	 Calculate the mean of the number of oocysts or cysts in the MS and 

MSD (Xmean) (= [MS+MSD]/2). 

9.5.2.3	 	 Calculate the relative percent difference (RPD) of the recoveries using 

the following equation: 

RPD =100 

NMS- NMSD 

Xmean 

where 

RPD is the relative percent difference 

NMS is the number of oocysts or cysts detected in the MS 

NMSD is the number of oocysts or cysts detected in the MSD 

Xmean is the mean number of oocysts or cysts detected in the MS 

and MSD 

9.5.2.4	 	 Compare the mean MS/MSD recovery and RPD with the corresponding 

limits in Tables 3 and 4 in Section 21.0 for each organism. 

9.6	 	 Method blank (negative control sample, laboratory blank): Reagent water blanks are analyzed to 

demonstrate freedom from contamination. Analyze the blank immediately prior to analysis of the 

IPR test (Section 9.4) and OPR test (Section 9.7) and prior to analysis of samples for the week to 

demonstrate freedom from contamination. 

15 April 2001 
Method 1623 - Cryptosporidium and Giardia 

9.6.1	 	 Filter, elute, concentrate, separate (purify), stain, and examine at least one reagent water 

blank per week (Section 9.1.7) according to the procedures in Sections 12.0 to 15.0. If 

more than 20 samples are analyzed in a week, process and analyze one reagent water 

blank for every 20 samples. 

9.6.2	 	 If Cryptosporidium oocysts, Giardia cysts, 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 or cysts is assumed to be contaminated and should be recollected, if 

possible. Any method blank in which oocysts or cysts 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 11.4 and enumerated spiking suspensions (Section 

7.10.1 or Section 11.3), filter, elute, concentrate, separate (purify), stain, and examine at least one 

reagent water sample spiked with 100 to 500 oocysts and 100 to 500 cysts each week to verify all 

performance criteria. The laboratory must analyze one OPR sample for every 20 samples if more 

than 20 samples are analyzed in a week. If multiple method variations are used, separate OPR 

samples must be prepared for each method variation. Adjustment and/or recalibration of the 

analytical system shall be performed until all performance criteria are met. Only after all 

performance criteria are met may samples be analyzed. 

9.7.1 Examine the slide from the OPR prior to analysis of samples from the same batch. 

9.7.1.1	 	 Using 200X to 400X magnification, more than 50% of the oocysts or 

cysts must appear undamaged and morphologically intact; otherwise, the 

analytical process is damaging the organisms. Determine the step or 

reagent that is causing damage to the organisms. Correct the problem 

and repeat the OPR test. 

9.7.1.2	 	 Identify and enumerate each organism using epifluorescence 

microscopy. The first three presumptive Cryptosporidium oocysts and 

three Giardia cysts identified in the OPR sample must be examined 

using FITC, DAPI, and DIC, as per Section 15.2, and the detailed 

characteristics (size, shape, DAPI category, and DIC category) reported 

on the Cryptosporidium and Giardia report form, as well as any 

additional comments on organism appearance, if notable. 

9.7.2 For each organism, calculate the percent recovery (R) using the following equation: 

N 

R = 100 x 

T 

where:
 


R = the percent recovery
 


N = the number of oocysts or cysts detected
 


T = the number of oocysts or cysts spiked
 


9.7.3	 	 Compare the recovery with the limits for ongoing precision and recovery in Tables 3 

and 4 in Section 21.0. If the recovery meets the acceptance criteria, system performance 

is acceptable and analysis of blanks and samples may proceed. If, however, the 

recovery falls outside of the range given, system performance is unacceptable. In this 

event, there may be a problem with the microscope or with the filtration or separation 

systems. Troubleshoot the problem using the procedures at Section 9.7.4 as a guide. 

After assessing the issue, reanalyze the OPR sample. All samples must be associated 

with an OPR that passes the criteria in Section 21.0. Samples that are not associated 

with an acceptable OPR must be flagged accordingly. 

April 2001 16 
Method 1623 - Cryptosporidium and Giardia 

9.7.4	 	 Troubleshooting. If an OPR sample has failed, and the cause of the failure is not 

known, the laboratory generally should identify the problem working backward in the 

analytical process from the microscopic examination to filtration. 

9.7.4.1	 	 Microscope system and antibody stain: To determine if the failure of 

the OPR test is due to changes in the microscope or problems with the 

antibody stain, re-examine the positive staining control (Section 15.2.1), 

check Köhler illumination, and check the fluorescence of the 

fluorescein-labeled monoclonal antibodies (Mabs) and 4',6-diamidino-2-

phenylindole (DAPI). If results are unacceptable, re-examine the 

previously-prepared positive staining control to determine whether the 

problem is associated with the microscope or the antibody stain. 

9.7.4.2	 	 Separation (purification) system: To determine if the failure of the 

OPR test is attributable to the separation system, check system 

performance by spiking a 10-mL volume of reagent water with 100 - 500 

oocysts and cysts and processing the sample through the IMS, staining, 

and examination procedures in Sections 13.3 through 15.0. 

9.7.4.3	 	 Filtration/elution/concentration system: If the failure of the OPR test 

is attributable to the filtration/elution/concentration system, check system 

performance by processing spiked reagent water according to the 

procedures in Section 12.2 through 13.2.2.1, and filter, stain, and 

examine the sample concentrate according to Section 11.3.6. 

9.7.5	 	 The laboratory should add results that pass the specifications in Section 9.7.3 to initial 

and previous ongoing data and update the QC chart to form a graphic representation of 

continued laboratory performance. The laboratory should develop a statement of 

laboratory accuracy (reagent water, raw surface water) by calculating the average 

percent recovery (R) and the standard deviation of percent recovery (sr). Express the 

accuracy as a recovery interval from R - 2 sr to R + 2 sr. For example, if R = 95% and sr 

= 25%, the accuracy is 45% to 145%. 

9.8	 	 The laboratory should periodically analyze an external QC sample, such as a performance 

evaluation or standard reference material, when available. The laboratory also should 

periodically participate in interlaboratory comparison studies using the method. 

9.9	 	 The specifications contained in this method can be met if the analytical system is under control. 

The standards used for initial (Section 9.4) and ongoing (Section 9.7) precision and recovery 

should be identical, so that the most precise results will be obtained. The microscope in 

particular will provide the most reproducible results if dedicated to the settings and conditions 

required for the determination of Cryptosporidium and Giardia by this method. 

9.10	 	 Depending on specific program requirements, field replicates may be collected to determine the 

precision of the sampling technique, and duplicate spiked samples may be required to determine 

the precision of the analysis. 

10.0 Microscope Calibration and Analyst Verification 

10.1	 	 In a room capable of being darkened to near-complete darkness, assemble the microscope, all 

filters, and attachments. The microscope should be placed on a solid surface free from vibration. 

Adequate workspace should be provided on either side of the microscope for taking notes and 

placement of slides and ancillary materials. 

10.2	 	 Using the manuals provided with the microscope, all analysts must familiarize themselves with 

operation of the microscope. 

10.3 Microscope adjustment and calibration (adapted from Reference 20.6) 

17 April 2001 
Method 1623 - Cryptosporidium and Giardia 

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 and 

cysts will not be possible. Consequently, it is imperative that all portions 

of the microscope from the light sources to the oculars are properly 

adjusted. 

10.3.1.2	 	 While microscopes from various vendors are configured somewhat 

differently, they all operate on the same general physical principles. 

Therefore, slight deviations or adjustments may be required to make the 

procedures below work for a particular instrument. 

10.3.1.3	 	 The sections below assume that the mercury bulb has not exceeded time 

limits of operation, that the lamp socket is connected to the lamp house, 

and that the condenser is adjusted to produce Köhler illumination. 

10.3.1.4	 	 Persons with astigmatism should always wear contact lenses or glasses 

when using the microscope. 

CAUTION: In the procedures below, do not touch the quartz portion of the 

mercury bulb with your bare fingers. Finger oils can cause rapid degradation of the 

quartz and premature failure of the bulb. 

WARNING: Never look at the ultraviolet (UV) light from the mercury lamp, lamp 

house, or the UV image without a barrier filter in place. UV radiation can cause 

serious eye damage. 

10.3.2	 	 Epifluorescent mercury bulb adjustment: The purpose of this procedure is to ensure 

even field illumination. This procedure must be followed when the microscope is first 

used, when replacing bulbs, and if problems such as diminished fluorescence or 

uneven field illumination are experienced. 

10.3.2.1	 	 Remove the diffuser lens between the lamp and microscope or swing it 

out of the transmitted light path. 

10.3.2.2	 	 Using a prepared microscope slide, adjust the focus so the image in the 

oculars is sharply defined. 

10.3.2.3 Replace the slide with a business card or a piece of lens paper. 

10.3.2.4	 	 Close the field diaphragm (iris diaphragm in the microscope base) so 

only a small point of light is visible on the card. This dot of light 

indicates the location of the center of the field of view. 

10.3.2.5	 	 Mount the mercury lamp house on the microscope without the UV 

diffuser lens in place and turn on the mercury bulb. 

10.3.2.6	 	 Remove the objective in the light path from the nosepiece. A primary 

(brighter) and secondary image (dimmer) of the mercury bulb arc 

should appear on the card after focusing the image with the appropriate 

adjustment. 

10.3.2.7	 	 Using the lamp house adjustments, adjust the primary and secondary 

mercury bulb images so they are side by side (parallel to each other) 

with the transmitted light dot in between them. 

April 2001 18 
Method 1623 - Cryptosporidium and Giardia 

10.3.2.8 Reattach the objective to the nosepiece. 

10.3.2.9	 	 Insert the diffuser lens into the light path between the mercury lamp 

house and the microscope. 

10.3.2.10	 	 Turn off the transmitted light and replace the card with a slide of 

fluorescent material. Check the field for even fluorescent illumination. 

Adjustment of the diffuser lens probably will be required. Additional 

slight adjustments as in Section 10.3.2.7 above may be required. 

10.3.2.11	 	 Maintain a log of the number of hours the UV bulb has been used. 

Never use the bulb for longer than it has been rated. Fifty-watt bulbs 

should not be used longer than 100 hours; 100-watt bulbs should not be 

used longer than 200 hours. 

10.3.3	 	 Transmitted bulb adjustment: The purpose of this procedure is to center the filament 

and ensure even field illumination. This procedure must be followed when the bulb is 

changed. 

10.3.3.1	 	 Remove the diffuser lens between the lamp and microscope or swing it 

out of the transmitted light path. 

10.3.3.2	 	 Using a prepared microscope slide and a 40X (or similar) objective, 

adjust the focus so the image in the oculars is sharply defined. 

10.3.3.3	 	 Without the ocular or Bertrand optics in place, view the pupil and 

filament image at the bottom of the tube. 

10.3.3.4	 	 Focus the lamp filament image with the appropriate adjustment on the 

lamp house. 

10.3.3.5	 	 Similarly, center the lamp filament image within the pupil with the 

appropriate adjustment(s) on the lamp house. 

10.3.3.6	 	 Insert the diffuser lens into the light path between the transmitted lamp 

house and the microscope. 

10.3.4	 	 Adjustment of the interpupillary distance and oculars for each eye: These adjustments 

are necessary so that eye strain is reduced to a minimum, and must be made for each 

individual using the microscope. Section 10.3.4.2 assumes use of a microscope with 

both oculars adjustable; Section 10.3.4.3 assumes use of a microscope with a single 

adjustable ocular. The procedure must be followed each time an analyst uses the 

microscope. 

10.3.4.1 Interpupillary distance 

10.3.4.1.1	 	 Place a prepared slide on the microscope stage, turn on 

the transmitted light, and focus the specimen image 

using the coarse and fine adjustment knobs. 

10.3.4.1.2	 	 Using both hands, move the oculars closer together or 

farther apart until a single circle of light is observed 

while looking through the oculars with both eyes. Note 

interpupillary distance. 

10.3.4.2	 	 Ocular adjustment for microscopes capable of viewing a photographic 

frame through the viewing binoculars: This procedure assumes both 

oculars are adjustable. 

10.3.4.2.1	 	 Place a card between the right ocular and eye keeping 

both eyes open. Adjust the correction (focusing) collar 

on the left ocular by focusing the left ocular until it 

19 April 2001 
Method 1623 - Cryptosporidium and Giardia 

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 coarse or fine 

adjustment. 

10.3.5	 	Calibration of an ocular micrometer: This section assumes that a reticle has been 

installed in one of the oculars by a microscopy specialist and that a stage micrometer is 

available for calibrating the ocular micrometer (reticle). Once installed, the ocular 

reticle should be left in place. The more an ocular is manipulated the greater the 

probability is for it to become contaminated with dust particles. This calibration should 

be done for each objective in use on the microscope. If there is a top lens on the 

microscope, the calibration procedure must be done for the respective objective at each 

top lens setting. The procedure must be followed when the microscope is first used and 

each time the objective is changed. 

10.3.5.1	 	 Place the stage micrometer on the microscope stage, turn on the 

transmitted light, and focus the micrometer image using the coarse and 

fine adjustment knobs for the objective to be calibrated. Continue 

adjusting the focus on the stage micrometer so you can distinguish 

between the large (0.1 mm) and the small (0.01 mm) divisions. 

10.3.5.2	 	 Adjust the stage and ocular with the micrometer so the 0 line on the 

ocular micrometer is exactly superimposed on the 0 line on the stage 

micrometer. 

10.3.5.3	 	 Without changing the stage adjustment, find a point as distant as 

possible from the two 0 lines where two other lines are exactly 

superimposed. 

10.3.5.4	 	 Determine the number of ocular micrometer spaces as well as the 

number of millimeters on the stage micrometer between the two points 

of superimposition. For example: Suppose 48 ocular micrometer spaces 

equal 0.6 mm. 

10.3.5.5 Calculate the number of mm/ocular micrometer space. For example: 

0.6 mm 0.0125 mm 

= 48 ocular micrometer spaces ocular micrometer space 

April 2001 20 
Method 1623 - Cryptosporidium and Giardia 

10.3.5.6	 	 Because most measurements of microorganisms are given in μm rather 

than mm, the value calculated above must be converted to μm by 

multiplying it by 1000 μm /mm. For example: 

0.0125 mm 1,000 μm 12.5 μm 

x = 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 

no. 

Objective 

power Description 

No. of ocular 

micrometer 

spaces 

No. of stage 

micrometer 

mm1 

μm/ocular 

micrometer 

space2 

1 10X N.A.3= 

2 20X N.A.= 

3 40X N.A.= 

4 100X N.A.= 

1

100 μm /mm 2

(Stage micrometer length in mm × (1000 μm /mm)) ÷ no. ocular micrometer 

spaces 

3

N.A. refers to numerical aperature. The numerical aperature value is engraved 

on the barrel of the objective. 

10.3.6 Köhler illumination: This section assumes that Köhler illumination will be established 

for only the 100X oil DIC objective that will be used to identify internal morphological 

characteristics in Cryptosporidium oocysts and Giardia cysts. If more than one 

objective is to be used for DIC, then each time the objective is changed, Köhler 

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 Köhler 

illumination is not properly established, then DIC will not work to its maximal 

potential. These steps need to become second nature and must be practiced regularly 

until they are a matter of reflex rather than a chore. The procedure must be followed 

each time an analyst uses the microscope and each time the objective is changed. 

10.3.6.1 Place a prepared slide on the microscope stage, place oil on the slide, 

move the 100X oil objective into place, turn on the transmitted light, and 

focus the specimen image using the coarse and fine adjustment knobs. 

10.3.6.2 At this point both the radiant field diaphragm in the microscope base 

and the aperture diaphragm in the condenser should be wide open. Now 

close down the radiant field diaphragm in the microscope base until the 

lighted field is reduced to a small opening. 

10.3.6.3 Using the condenser centering screws on the front right and left of the 

condenser, move the small lighted portion of the field to the center of 

the visual field. 

10.3.6.4 Now look to see whether the leaves of the iris field diaphragm are 

sharply defined (focused) or not. If they are not sharply defined, then 

they can be focused distinctly by changing the height of the condenser 

up and down with the condenser focusing knob while you are looking 

through the binoculars. Once you have accomplished the precise 

21 April 2001 
Method 1623 - Cryptosporidium and Giardia 

focusing of the radiant field diaphragm leaves, open the radiant field 

diaphragm until the leaves just disappear from view. 

10.3.6.5	 	 The aperture diaphragm of the condenser is now adjusted to make it 

compatible with the total numerical aperture of the optical system. This 

is done by removing an ocular, looking into the tube at the rear focal 

plane of the objective, and stopping down the aperture diaphragm iris 

leaves until they are visible just inside the rear plane of the objective. 

10.3.6.6	 	 After completing the adjustment of the aperture diaphragm in the 

condenser, return the ocular to its tube and proceed with the adjustments 

required to establish DIC 

10.4	 	 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 and Giardia cysts by FA and 4',6-

diamidino-2-phenylindole (DAPI) that the analysts (Section 22.2) determine are 

accurate (Section 15.2). 

10.4.2	 	 Similarly, take color photographs of interfering organisms and materials by FA and 

DAPI that the analysts believe are not Cryptosporidium oocysts or Giardia cysts. 

Quantify the size, shape, microscope settings, and other characteristics that can be used 

to differentiate oocysts and cysts from interfering debris and that will result in positive 

identification of DAPI positive or negative organisms. 

10.5	 	 Verification of performance: Until standard reference materials, such as National Institute of 

Standards and Technology standard reference materials, are available that contain a reliable 

number of DAPI positive or negative oocysts and cysts, this method shall rely upon the ability of 

the analyst for identification and enumeration of oocysts and cysts. 

10.5.1	 	 At least monthly when microscopic examinations are being performed, the laboratory 

shall prepare a slide containing 40 to 100 oocysts and 40 to 100 cysts. More than 50% of 

the oocysts and cysts must be DAPI positive. 

10.5.2	 	 Each analyst shall determine the total number of oocysts and cysts and the number that 

are DAPI positive or negative using the slide prepared in Section 10.5.1. 

10.5.3	 	 The total number and the number of DAPI positive or negative oocysts and cysts 

determined by each analyst (Section 10.5.2.) must be within ±10% of each other. If the 

number is not within this range, the analysts must identify the source of any variability 

between analysts’ examination criteria, prepare a new slide, and repeat the performance 

verification (Sections 10.5.1 to 10.5.2). 

10.5.4	 	 Document the date, name(s) of analyst(s), number of total, DAPI positive or negative 

oocysts and cysts determined by the analyst(s), whether the test was passed/failed and 

the results of attempts before the test was passed. 

10.5.5	 	 Only after an analyst has passed the criteria in Section 10.5.3, may oocysts and cysts in 

QC samples and field samples be identified and enumerated. 

11.0 Oocyst and Cyst Suspension Enumeration and Spiking 

11.1	 	 This method requires routine analysis of spiked QC samples to demonstrate acceptable initial 

and ongoing laboratory and method performance (initial precision and recovery samples 

[Section 9.4], matrix spike and matrix spike duplicate samples [Section 9.5], and ongoing 

precision and recovery samples [Section 9.7]). The organisms used for these samples must be 

enumerated to calculate recoveries and precision. EPA recommends that flow cytometry be used 

for this enumeration, rather than manual techniques. Flow cytometer–sorted spikes generally are 

characterized by a relative standard deviation of ≤ 2.5%, versus greater variability for manual 

April 2001 22 
Method 1623 - Cryptosporidium and Giardia 

enumeration techniques (Reference 20.8). Guidance on preparing spiking suspensions using a 

flow cytometer is provided in Section 11.2. Manual enumeration procedures are provided in 

Section 11.3. The procedure for spiking bulk samples in the laboratory is provided in Section 

11.4. 

11.2	 	 Flow cytometry enumeration guidelines. Although it is unlikely that many laboratories 

performing Method 1623 will have direct access to a flow cytometer for preparing spiking 

suspensions, flow-sorted suspensions are available from commercial vendors and other sources 

(Section 7.10.1). The information provided in Sections 11.2.1 through 11.2.4 is simply meant as 

a guideline for preparing spiking suspensions using a flow cytometer. Laboratories performing 

flow cytometry must develop and implement detailed standardized protocols for calibration and 

operation of the flow cytometer. 

11.2.1	 	 Spiking suspensions should be prepared using unstained organisms that have not been 

heat-fixed or formalin-fixed. 

11.2.2	 	 Spiking suspensions should be prepared using Cryptosporidium parvum oocysts <3 

months old, and Giardia intestinalis cysts <2 weeks old. 

11.2.3	 	Initial calibration. Immediately before sorting spiking suspensions, an initial 

calibration of the flow cytometer should be performed by conducting 10 sequential 

sorts directly onto membranes or well slides. The oocyst and cyst levels used for the 

initial calibration should be the same as the levels used for the spiking suspensions. 

Each initial calibration sample should be stained and manually counted microscopically 

and the manual counts used to verify the accuracy of the system. The relative standard 

deviation (RSD) of the 10 counts should be ≤ 2.5%. If the RSD is > 2.5%, the 

laboratory should perform the initial calibration again, until the RSD of the 10 counts is 

≤ 2.5%. In addition to counting the organisms, the laboratory also should evaluate the 

quality of the organisms using DAPI and DIC to confirm that the organisms are in good 

condition. 

11.2.4	 	 Ongoing calibration. When sorting the spiking suspensions for use in QC samples, the 

laboratory should perform ongoing calibration samples at a 10% frequency, at a 

minimum. The laboratory should sort the first run and every eleventh sample directly 

onto a membrane or well slide. Each ongoing calibration sample should be stained and 

manually counted microscopically and the manual counts used to verify the accuracy of 

the system. The mean of the ongoing calibration counts also should be used as the 

estimated spike dose, if the relative standard deviation (RSD) of the ongoing calibration 

counts is ≤ 2.5%. If the RSD is > 2.5%, the laboratory should discard the batch. 

11.2.5	 	 Method blanks. Depending on the operation of the flow cytometer, method blanks 

should be prepared and examined at the same frequency as the ongoing calibration 

samples (Section 11.2.4). 

11.2.6	 	 Holding time criteria. Flow-cytometer-sorted spiking suspensions (Sections 7.10.1 and 

11.2) used for spiked quality control (QC) samples (Section 9) must be used within the 

expiration date noted on the suspension. Laboratories should use flow-cytometer-sorted 

spiking suspensions containing live organisms within two weeks of preparation at the 

flow cytometry laboratory. 

11.3	 	 Manual enumeration procedures. Two sets of manual enumerations are required per 

organism before purified Cryptosporidium oocyst and Giardia cyst stock suspensions (Sections 

7.9.2.1 and 7.9.2.2) received from suppliers can be used to spike samples in the laboratory. First, 

the stock suspension must be diluted and enumerated (Section 11.3.3) to yield a suspension at 

the appropriate oocyst or cyst concentration for spiking (spiking suspension). Then, 10 aliquots 

of spiking suspension must be enumerated to calculate a mean spike dose. Spiking suspensions 

can be enumerated using hemacytometer chamber counting (Section 11.3.4), well slide counting 

(Section 11.3.5), or membrane filter counting (Section 11.3.6). 

23 April 2001 
Method 1623 - Cryptosporidium and Giardia 

11.3.1	 	 Precision criteria. The relative standard deviation (RSD) of the calculated mean spike 

dose for manually enumerated spiking suspensions must be ≤16% for Cryptosporidium 

and ≤19% for Giardia before proceeding (these criteria are based on the pooled RSDs 

of 105 manual Cryptosporidium enumerations and 104 manual Giardia enumerations 

submitted by 20 different laboratories under the EPA Protozoa Performance Evaluation 

Program). 

11.3.2	 	 Holding time criteria. Manually enumerated spiking suspensions must be used within 

24 hours of enumeration of the spiking suspension if the hemacytometer chamber 

technique is used (Section 11.3.4); or within 24 hours of application of the spiking 

suspension or membrane filter to the slides if the well slide or membrane filter 

enumeration technique is used (Sections 11.3.5 and 11.3.6). 

11.3.3 Enumerating and diluting stock suspensions 

11.3.3.1	 	 Purified, concentrated stock suspensions (Sections 7.10.2.1 and 

7.10.2.2) must be diluted and enumerated before the diluted suspensions 

are used to spike samples in the laboratory. Stock suspensions should be 

diluted with reagent water/Tween-20, 0.01% (Section 7.10.2.3), to a 

concentration of 20 to 50 organisms per large hemacytometer square 

before proceeding to Section 11.3.3.2. 

11.3.3.2	 	 Apply a clean hemacytometer coverslip (Section 6.4.5) to the 

hemacytometer and load the hemacytometer chamber with 10 μL of 

vortexed suspension per chamber. If this operation has been properly 

executed, the liquid should amply fill the entire chamber without 

bubbles or overflowing into the surrounding moats. Repeat this step 

with a clean, dry hemacytometer and coverslip if loading has been 

incorrectly performed. See Section 11.3.3.13, below, for the 

hemacytometer cleaning procedure. 

11.3.3.3	 	 Place the hemacytometer on the microscope stage and allow the oocysts 

or cysts to settle for 2 minutes Do not attempt to adjust the coverslip, 

apply clips, or in any way disturb the chamber after it has been filled. 

11.3.3.4 Use 200X magnification. 

11.3.3.5 Move the chamber so the ruled area is centered underneath it. 

11.3.3.6	 	 Move the objective close to the coverslip while watching it from the side 

of the microscope, rather than through the microscope. 

11.3.3.7 Focus up from the coverslip until the hemacytometer ruling appears. 

11.3.3.8	 	 At each of the four corners of the chamber is a 1-square-mm area 

divided into 16 squares in which organisms are to be counted (Figure 1). 

Beginning with the top row of four squares, count with a hand-tally 

counter in the directions indicated in Figure 2. Avoid counting 

organisms twice by counting only those touching the top and left 

boundary lines. Count each square millimeter in this fashion. 

11.3.3.9	 	 Use the following formula to determine the number of organisms per 

mL of suspension: 

number of organismscounted 10 dilutionfactor 1000 mm3 

x x x = number of mm2 counted 1mm 1 1mL number of organisms mL 

11.3.3.10 Record the result on a hemacytometer data sheet. 

April 2001 24 
Method 1623 - Cryptosporidium and Giardia 

11.3.3.11	 	 A total of six different hemacytometer chambers must be loaded, 

counted, and averaged for each suspension to achieve optimal counting 

accuracy. 

11.3.3.12	 	 Based on the hemacytometer counts, the stock suspension should be 

diluted to a final concentration of between 8000 and 12,000 organisms 

per mL (80 to 120 organisms per 10 μL ); however, ranges as great as 

5000 to 15,000 organisms per mL (50 to 150 organisms per 10 μL ) can 

be used. 

NOTE: If the diluted stock suspensions (the spiking suspensions) will be enumerated 

using hemacytometer chamber counts (Section 11.3.4) or membrane filter counts 

(Section 11.3.6), then the stock suspensions should be diluted with 0.01% Tween-20. If 

the spiking suspensions will be enumerated using well slide counts (Section 11.3.3), 

then the stock suspensions should be diluted in reagent water.. 

To calculate the volume (in μL ) of stock suspension required per mL of 

reagent water (or reagent water/Tween-20, 0.01%), use the following 

formula: 

required number of organisms x 1000 μL

volume of stock suspension ( μL ) required = number of organisms / mL of stock suspension 

If the volume is less than 10 μL , an additional dilution of the stock 

suspension is recommended before proceeding. 

To calculate the dilution factor needed to achieve the required number 

of organisms per 10 μL , use the following formula: 

number of organisms required x 10 μL total volume ( μL ) = predicted number of organisms per 10 μL (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 (μL ) = 

total volume ( μL ) - stock suspension volume required ( μL ) 

11.3.3.13	 	 After each use, the hemacytometer and coverslip must be cleaned 

immediately to prevent the organisms and debris from drying on it. 

Since this apparatus is precisely machined, abrasives cannot be used to 

clean it, as they will disturb the flooding and volume relationships. 

11.3.3.13.1	 	 Rinse the hemacytometer and cover glass first with tap 

water, then 70% ethanol, and finally with acetone. 

11.3.3.13.2	 	 Dry and polish the hemacytometer chamber and cover 

glass with lens paper. Store it in a secure place. 

11.3.3.14	 	 Several factors are known to introduce errors into hemacytometer 

counts, including: 

• Inadequate mixing of suspension before flooding the chamber 

25 April 2001 
Method 1623 - Cryptosporidium and Giardia 

• 	 Irregular filling of the chamber, trapped air bubbles, dust, or oil 

on the chamber or coverslip 

• 	 Total number of organisms counted is too low to provide 

statistical confidence in the result 

• Error in recording tally 

• 	 Calculation error; failure to consider dilution factor, or area 

counted 

• 	 Inadequate cleaning and removal of organisms from the 

previous count 

• 	 Allowing filled chamber to sit too long, so that the chamber 

suspension dries and concentrates. 

11.3.4 Enumerating spiking suspensions using a hemacytometer chamber 

NOTE: Spiking suspensions enumerated using a hemacytometer chamber must be 

used within 24 hours of enumeration. 

11.3.4.1	 	 Vortex the tube containing the spiking suspension (diluted stock 

suspension; Section 11.3.3) for a minimum of 2 minutes. Gently invert 

the tube three times. 

11.3.4.2	 	 To an appropriate-size beaker containing a stir bar, add enough spiking 

suspension to perform all spike testing and the enumeration as 

described. The liquid volume and beaker relationship should be such 

that a spinning stir bar does not splash the sides of the beaker, the stir 

bar has unimpeded rotation, and there is enough room to draw sample 

from the beaker with a 10-μL micropipette without touching the stir bar. 

Cover the beaker with a watch glass or petri dish to prevent evaporation 

between sample withdrawals. 

11.3.4.3	 	 Allow the beaker contents to stir for a minimum of 30 minutes before 

beginning enumeration. 

11.3.4.4	 	 While the stir bar is still spinning, remove a 10-μL aliquot and carefully 

load one side of the hemacytometer. Count all organisms on the 

platform, at 200X magnification using phase-contrast or darkfield 

microscopy. The count must include the entire area under the 

hemacytometer, not just the four outer 1-mm2

 squares. Repeat this 

procedure nine times. This step allows confirmation of the number of 

organisms per 10 μL (Section 11.3.3.12). Based on the 10 counts, 

calculate the mean, standard deviation, and RSD of the counts. Record 

the counts and the calculations on a spiking suspension enumeration 

form. The relative standard deviation (RSD) of the calculated mean 

spike dose must be ≤16% for Cryptosporidium and ≤19% for Giardia 

before proceeding. If the RSD is unacceptable, or the mean number is 

outside the expected range, add additional oocysts from stock 

suspension or dilute the contents of the beaker appropriately with 

reagent water. Repeat the process to confirm counts. Refer to Section 

11.3.3.14 for factors that may introduce errors. 

April 2001 26 
Method 1623 - Cryptosporidium and Giardia 

11.3.5 Enumerating spiking suspensions using well slides 

NOTE: Spiking suspensions enumerated using well slides must be used within 24 

hours of application of the spiking suspension to the slides. 

11.3.5.1	 	 Remove well slides from cold storage and lay the slides on a flat surface 

for 15 minutes to allow them to warm to room temperature. 

11.3.5.2	 	 Vortex the tube containing the spiking suspension (diluted stock 

suspension; Section 11.3.3) for a minimum of 2 minutes. Gently invert 

the tube three times. 

11.3.5.3	 	 Remove a 10-μL aliquot from the spiking suspension and apply it to the 

center of a well. 

11.3.5.4	 	 Before removing subsequent aliquots, cap the tube and gently invert it 

three times to ensure that the oocysts or cysts are in suspension. 

11.3.5.5	 	 Ten wells must be prepared and counted, and the counts averaged, to 

sufficiently enumerate the spike dose. Air-dry the well slides. Because 

temperature and humidity varies from laboratory to laboratory, no 

minimum time is specified. However, the laboratory must take care to 

ensure that the sample has dried completely before staining to prevent 

losses during the rinse steps. A slide warmer set at 35 o C to 42 o C also 

can be used. 

11.3.5.6 Positive and negative controls must be prepared. 

11.3.5.6.1	 	 For the positive control, pipette 10 μL of positive 

antigen or 200 to 400 intact oocysts or cysts to the center 

of a well and distribute evenly over the well area. 

11.3.5.6.2	 	 For the negative control, pipette 50 μL of PBS onto the 

center of a well and spread it over the well area with a 

pipette tip. 

11.3.5.6.3 Air-dry the control slides. 

11.3.5.7	 	 Apply 50- μL of absolute methanol to each well containing the dried 

sample and allow to air-dry for 3 to 5 minutes. 

11.3.5.8	 	 Follow the manufacturer’s instructions (Section 7.6) in applying the 

stain to the slide. 

11.3.5.9	 	 Place the slides in a humid chamber in the dark and incubate at room 

temperature for approximately 30 minutes. The humid chamber consists 

of a tightly sealed plastic container containing damp paper towels on top 

of which the slides are placed. 

11.3.5.10	 	 Apply one drop of wash buffer (prepared according to the 

manufacturer’s instructions [Section 7.6]) to each well. Tilt each slide on 

a clean paper towel, long edge down. Gently aspirate the excess 

detection reagent from below the well using a clean Pasteur pipette or 

absorb with a paper towel or other absorbent material. Avoid disturbing 

the sample. 

NOTE: If using the Merifluor stain (Section 7.6.1), do not allow slides to dry 

completely. 

11.3.5.11 Add mounting medium (Section 7.8) to each well. 

27 April 2001 
Method 1623 - Cryptosporidium and Giardia 

11.3.5.12	 	 Apply a cover slip. Use a tissue to remove excess mounting fluid from 

the edges of the coverslip. Seal the edges of the coverslip onto the slide 

using clear nail polish. 

11.3.5.13	 	 Record the date and time that staining was completed. If slides will not 

be read immediately, store in a humid chamber in the dark at 0 o C to 8 o C 

until ready for examination. 

11.3.5.14	 	 After examination of the 10 wells, calculate the mean, standard 

deviation, and RSD of the 10 replicates. Record the counts and the 

calculations on a spiking suspension enumeration form. The relative 

standard deviation (RSD) of the calculated mean spike dose must be 

≤16% for Cryptosporidium and ≤19% for Giardia before proceeding. If 

the RSD is unacceptable, or the mean number is outside the expected 

range, add additional oocysts from stock suspension or dilute the 

contents of the beaker appropriately with reagent water. Repeat the 

process to confirm counts. 

11.3.6 Enumeration of spiking suspensions using membrane filters 

NOTE: Spiking suspensions enumerated using membrane filters must be used within 

24 hours of application of the filters to the slides. 

11.3.6.1	 	 Precoat the glass funnels with Sigmacote® by placing the funnel in a 

large petri dish and applying 5-mL of Sigmacoat® to the funnel opening 

using a pipette and allowing it to run down the inside of the funnel. 

Repeat for all funnels to be used. The pooled Sigmacoat® may be 

returned to the bottle for re-use. Place the funnels at 35 o C or 41 o C for 

approximately 5 minutes to dry. 

11.3.6.2 Place foil around the bottoms of the 100 × 15 mm petri dishes. 

11.3.6.3	 	 Filter-sterilize (Section 6.19) approximately 10 mL of PBS pH 7.2 

(Section 7. 9. 4). Dilute detection reagent (Section 7.7) as per 

manufacturer’s instructions using sterile PBS. Multiply the anticipated 

number of filters to be stained by 100 mL to calculate total volume of 

stain required. Divide the total volume required by 5 to obtain the 

microliters of antibody necessary. Subtract the volume of antibody from 

the total stain volume to obtain the required microliters of sterile PBS to 

add to the antibody. 

11.3.6.4	 	 Label the tops of foil-covered, 60 × 15 mm petri dishes for 10 spiking 

suspensions plus positive and negative staining controls and multiple 

filter blanks controls (one negative control, plus a blank after every five 

sample filters to control for carry-over). Create a humid chamber by 

laying damp paper towels on the bottom of a stain tray (the inverted 

foil-lined petri dishes will protect filters from light and prevent 

evaporation during incubation). 

11.3.6.5	 	 Place a decontaminated and cleaned filter holder base (Section 6.4.8.1) 

into each of the three ports of the vacuum manifold (Section 6.4.8.2). 

11.3.6.6	 	 Pour approximately 10 mL of 0.01% Tween 20 into a 60 × 15 mm petri 

dish. 

11.3.6.7	 	 Using forceps, moisten a 1.2- μm cellulose-acetate support membrane 

(Section 6.4.8.3) in the 0.01% Tween 20 and place it on the fritted glass 

support of one of the filter bases. Moisten a polycarbonate filter (Section 

April 2001 28 
Method 1623 - Cryptosporidium and Giardia 

6.4.8.4) the same way and position it on top of the cellulose-acetate 

support membrane. Carefully clamp the glass funnel to the loaded filter 

support. Repeat for the other two filters. 

11.3.6.8	 	 Add 5 mL of 0.01% Tween 20 to each of the three filtration units and 

allow to stand. 

11.3.6.9	 	 Vortex the tube containing the spiking suspension (diluted stock 

suspension; Section 11.3.3) for a minimum of 2 minutes. Gently invert 

the tube three times. 

11.3.6.10	 	 Using a micropipettor, sequentially remove two, 10-μL aliquots from the 

spiking suspension and pipet into the 5 mL of 0.01% Tween 20 standing 

in the unit. Rinse the pipet tip twice after each addition. Apply 10 μL of 

0.01% Tween 20 to the third unit to serve as the negative control. Apply 

vacuum at 2" Hg and allow liquid to drain to miniscus, then close off 

vacuum. Pipet 10 mL of reagent water into each funnel and drain to 

miniscus, closing off the vacuum. Repeat the rinse and drain all fluid, 

close off the vacuum. 

11.3.6.11	 	 Pipet 100 mL of diluted antibody to the center of the bottom of a 60 × 15 

mm petri dish for each sample. 

11.3.6.12	 	 Unclamp the top funnel and transfer each cellulose acetate support 

membrane/ polycarbonate filter combination onto the drop of stain 

using forceps (apply each membrane/filter combination to a different 

petri dish containing stain). Roll the filter into the drop to exclude air. 

Place the small petri dish containing the filter onto the damp towel and 

cover with the corresponding labeled foil-covered top. Incubate for 

approximately 45 minutes at room temperature. 

11.3.6.13	 	 Reclamp the top funnels, apply vacuum and rinse each three times, each 

time with 20 mL of reagent water. 

11.3.6.14	 	 Repeat Sections 11.3.6.4 through 11.3.6.10 for the next three samples (if 

that the diluted spiking suspension has sat less than 15 minutes, reduce 

the suspension vortex time to 60 seconds). Ten, 10-μL spiking 

suspension aliquots must be prepared and counted, and the counts 

averaged, to sufficiently enumerate the spike dose. Include a filter blank 

sample at a frequency of every five samples; rotate the position of filter 

blank to eventually include all three filter placements. 

11.3.6.15	 	 Repeat Sections 11.3.6.4 through 11.3.6.10 until the 10-μL spiking 

suspensions have been filtered. The last batch should include a 10-μL 

0.01 Tween 20 blank control and 20 μL of positive control antigen as a 

positive staining control. 

11.3.6.16	 	 Label slides. After incubation is complete, for each sample, transfer the 

cellulose acetate filter support and polycarbonate filter from drop of 

stain and place on fritted glass support. Cycle vacuum on and off briefly 

to remove excess fluid. Peel the top polycarbonate filter off the 

supporting filter and place on labeled slide. Discard cellulose acetate 

filter support. Mount and apply coverslips to the filters immediately to 

avoid drying. 

11.3.6.17 To each slide, add 20 μL of mounting medium (Section 7.8). 

11.3.6.18	 	 Apply a coverslip. Seal the edges of the coverslip onto the slide using 

clear nail polish. (Sealing may be delayed until cover slips are applied to 

all slides.) 

29 April 2001 
Method 1623 - Cryptosporidium and Giardia 

11.3.6.19	 	 Record the date and time that staining was completed. If slides will not 

be read immediately, store sealed slides in a closed container in the dark 

at 0 o C to 8 oC until ready for examination. 

11.3.6.20	 	 After examination of the 10 slides, calculate the mean, standard 

deviation, and RSD of the 10 replicates. Record the counts and the 

calculations on a spiking suspension enumeration form. The relative 

standard deviation (RSD) of the calculated mean spike dose must be 

≤16% for Cryptosporidium and ≤19% for Giardia before proceeding. If 

the RSD is unacceptable, or the mean number is outside the expected 

range, add additional oocysts from stock suspension or dilute the 

contents of the beaker appropriately with reagent water. Repeat the 

process to confirm counts. 

11.3.6.21	 	 If oocysts or cysts are detected on the filter blanks, modify the rinse 

procedure to ensure that no carryover occurs and repeat enumeration. 

11.4 Procedure for spiking samples in the laboratory with enumerated spiking suspensions. 

11.4.1 Arrange a bottom-dispensing container to feed the filter. 

11.4.2	 	 For initial precision and recovery (Section 9.4) and ongoing precision and recovery 

(Section 9.7) samples, fill the container with a volume of reagent water equal to the 

volume of the field samples analyzed in the analytical batch. For matrix spike samples 

(Section 9.5), fill the container with the field sample to be spiked. Continuously mix the 

sample (using a stir bar and stir plate for smaller-volume samples and alternate means 

for larger-volume samples). 

11.4.3	 	 Vortex the spiking suspension(s) (Section 11.2 or Section 11.3) for a minimum of 2 

minutes. 

11.4.3.1	 	 For flow cytometer–enumerated suspensions (where the entire volume 

of a spiking suspension tube will be used): 

11.4.3.1.1 

11.4.3.1.2 

11.4.3.1.3 

11.4.3.1.4 

11.4.3.1.5 

11.4.3.1.6 

Add 400 μL of Antifoam A to 100 mL of reagent water, 

and mix well to emulsify. 

Add 500 μL of the diluted antifoam to the tube 

containing the spiking suspension and vortex for 2 

minutes. 

Pour the suspension into the sample container. 

Add 20 mL of reagent water to the empty tube, cap, 

vortex 10 seconds to rinse, and add the rinsate to the 

carboy. 

Repeat this rinse using another 20 mL of reagent water. 

Record the estimated number of organisms spiked, the 

date and time the sample was spiked, and the sample 

volume spiked on a bench sheet. Proceed to Section 

11.4.4. 

11.4.3.2 For manually enumerated spiking suspensions: 

11.4.3.2.1	 	 Rinse a pipette tip with 0.01% Tween-20 once, then 

rinse with the well-mixed spiking suspension a 

minimum of five times before pulling an aliquot to be 

used to spike the container. 

April 2001 30 
Method 1623 - Cryptosporidium and Giardia 

11.4.3.2.2	 	 Add the spiking suspension(s) to the carboy, delivering 

the aliquot below the surface of the water. 

11.4.3.2.3	 	 Record the estimated number of organisms spiked, the 

date and time the sample was spiked, and the sample 

volume spiked on a bench sheet. Proceed to Section 

11.4.4 

11.4.4 Allow the spiking suspensions to mix for approximately 1 minute in the container. 

11.4.5	 	 Turn on the pump and allow the flow rate to stabilize. Set flow at the rate designated 

for the filter being used. As the carboy is depleted, check the flow rate and adjust if 

necessary. 

11.4.6	 	 When the water level approaches the discharge port of the carboy, tilt the container so 

that it is completely emptied. At that time, turn off the pump and add sufficient reagent 

water to the container to rinse. Swirl the contents to rinse down the sides. 

11.4.7	 	 Turn on the pump. Allow all of the water to flow through the filter and turn off the 

pump. 

12.0 Sample Filtration and Elution 

12.1	 	 A water sample is filtered according to the procedures in Section 12.2. Alternate procedures may 

be used if the laboratory first demonstrates that the alternate procedure provides equivalent or 

superior performance per Section 9.1.2. 

NOTE: Sample elution must be initiated within 96 hours of sample collection (if 

shipped to the laboratory as a bulk sample) or filtration (if filtered in the field). 

12.2	 	 Capsule filtration (adapted from Reference 20.9). This procedure was validated using 10-L 

sample volumes. Alternate sample volumes may be used, provided the laboratory demonstrates 

acceptable performance on initial and ongoing spiked reagent water and source water samples 

(Section 9.1.2). 

NOTE: The filtration procedures specified in Section 12.2.1 - 12.2.5.3 are specific to 

laboratory filtration of a bulk sample, and reflect the procedures used during the 

interlaboratory validation of this method (Reference 20.10). These procedures may 

require modification if samples will be filtered in the field. 

12.2.1 Flow rate adjustment 

12.2.1.1	 	 Connect the sampling system, minus the capsule, to a carboy filled with 

reagent water (Figure 3). 

12.2.1.2 Turn on the pump and adjust the flow rate to 2.0 L/min. 

12.2.1.3	 	 Allow 2 to 10 L of reagent water to flush the system. Adjust the pump 

speed as required during this period. Turn off the pump when the flow 

rate has been adjusted. 

12.2.2	 	Install the capsule filter in the line, securing the inlet and outlet ends with the 

appropriate clamps/fittings. 

12.2.3	 	Record the sample number, sample turbidity (if not provided with the field sample), 

sample type, and sample filtration start date and time on a bench sheet. 

12.2.4 Filtration 

12.2.4.1	 	 Connect the sampling system to the field carboy of sample water, or 

transfer the sample water to the laboratory carboy used in Section 

31 April 2001 
Method 1623 - Cryptosporidium and Giardia 

12.2.1.1. If the sample will be filtered from a field carboy, a spigot 

(Section 6.2.1) can be used with the carboy to facilitate sample filtration. 

NOTE: If the bulk field sample is transferred to a laboratory carboy, the laboratory 

carboy must be cleaned and disinfected before it is used with another field sample. 

12.2.4.2	 	 Place the drain end of the sampling system tubing into an empty 

graduated container with a capacity of 10 to 15 L, calibrated at 9.0, 9.5, 

10.0, 10.5, and 11.0 L (Section 6.18). This container will be used to 

determine the sample volume filtered. Alternately, connect a flow meter 

(Section 6.3.4) downstream of the filter, and record the initial meter 

reading. 

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, gently shaking or 

tapping the capsule, if necessary. Turn on the pump to start water 

flowing through the filter. Verify that the flow rate is 2 L/min. 

12.2.4.4	 	 After all of the sample has passed through the filter, turn off the pump. 

Allow the pressure to decrease until flow stops. (If the sample was 

filtered in the field, and excess sample remains in the filter upon receipt 

in the laboratory, pull the remaining sample volume through the filter 

before eluting the filter [Section 12.2.6].) 

12.2.5 Disassembly 

12.2.5.1	 	 Disconnect the inlet end of the capsule filter assembly while maintaining 

the level of the inlet fitting above the level of the outlet fitting to prevent 

backwashing and the loss of oocysts and cysts from the filter. Restart the 

pump and allow as much water to drain as possible. Turn off the pump. 

12.2.5.2	 	 Based on the water level in the graduated container or meter reading, 

record the volume filtered on the bench sheet to the nearest quarter liter. 

Discard the contents of the graduated container. 

12.2.5.3 Loosen the outlet fitting, then cap the inlet and outlet fittings. 

12.2.6 Elution 

NOTE: The laboratory must complete the elution, concentration, and purification 

(Sections 12.2.6 through 13.3.3.11) in one work day. It is critical that these steps be 

completed in one work day to minimize the time that any target organisms present in 

the sample sit in eluate or concentrated matrix. This process ends with the application 

of the purified sample on the slide for drying. 

12.2.6.1 Setup 

12.2.6.1.1	 	 Assemble the laboratory shaker with the clamps aligned 

vertically so that the filters will be aligned horizontally. 

Extend the clamp arms to their maximum distance from 

the horizontal shaker rods to maximize the shaking 

action. 

12.2.6.1.2	 	 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. 

12.2.6.1.3	 	 Designate at least one 250-mL conical centrifuge tube 

for each sample and label with the sample number. 

April 2001 32 
Method 1623 - Cryptosporidium and Giardia 

12.2.6.2	 	 Elution 

12.2.6.2.1 

12.2.6.2.2 

12.2.6.2.3 

12.2.6.2.4 

12.2.6.2.5 

12.2.6.2.6 

12.2.6.2.7 

12.2.6.2.8 

Record the elution date and time on the bench sheet. 

Using a ring stand or other means, clamp each capsule 

in a vertical position with the inlet end up. Remove the 

inlet cap and allow the liquid level to stabilize. 

Pour elution buffer through the inlet fitting. Sufficient 

elution buffer must be added to cover the pleated white 

membrane with buffer solution. Replace the inlet cap 

and clamp the cap in place. 

Securely clamp the capsule in one of the clamps on the 

laboratory shaker with the bleed valve positioned at the 

top on a vertical axis (in the 12 o'clock position). Turn 

on the shaker and set the speed to maximum 

(approximately 900 rpm). Agitate the capsule for 

approximately 5 minutes. Time the agitation using a lab 

timer, rather than the timer on the shaker to ensure 

accurate time measurement. 

Remove the filter from the shaker, remove the inlet cap, 

and pour the contents of the capsule into the 250-mL 

conical centrifuge tube. 

Clamp the capsule vertically with the inlet end up and 

add sufficient volume of elution buffer through the inlet 

fitting to cover the pleated membrane. Replace the inlet 

cap. 

Return the capsule to the shaker with the bleed valve 

positioned at the 4 o'clock position. Turn on the shaker 

and agitate the capsule for approximately 5 minutes. 

Remove the filter from the shaker, but leave the elution 

buffer in the capsule. Re-clamp the capsule to the 

shaker at the 8 o’clock position. Turn on the shaker and 

agitate the capsule for a final 5 minutes. 

Remove the filter from the shaker and pour the contents 

into the 250-mL centrifuge tube. Rinse down the inside 

of the capsule filter walls with reagent water or elution 

buffer using a squirt bottle inserted in the inlet end of 

the capsule. Invert the capsule filter over the centrifuge 

tube and ensure that as much of the eluate as possible 

has been transferred. 

12.2.7 Proceed to Section 13.0 for concentration and separation (purification). 

13.0 Sample Concentration and Separation (Purification) 

13.1	 	 During concentration and separation, the filter eluate is concentrated through centrifugation, and 

the oocysts and cysts in the sample are separated from other particulates through 

immunomagnetic separation (IMS). Alternate procedures and products may be used if the 

laboratory first demonstrates equivalent or superior performance as per Section 9.1.2. 

33 April 2001 
Method 1623 - Cryptosporidium and Giardia 

13.2 Adjustment of pellet volume 

13.2.1	 	Centrifuge the 250-mL centrifuge tube containing the capsule filter eluate at 1500 × G 

for 15 minutes. Allow the centrifuge to coast to a stop—do not use the brake. Record 

the pellet volume (volume of solids) on the bench sheet. 

NOTE: Recoveries may be improved if centrifugation force is increased to 2000 × G. 

However, do not use this higher force if the sample contains sand or other gritty 

material that may degrade the condition of any oocysts and/or cysts in the sample. 

13.2.2	 	 Using a Pasteur pipette, carefully aspirate the supernatant to 5 mL above the pellet. 

Extra care must be taken to avoid aspirating oocysts and cysts during this step, 

particularly if the sample is reagent water (e.g. initial or ongoing precision and recovery 

sample). 

13.2.3	 	If the packed pellet volume is ≤ 0.5 mL, vortex the tube vigorously until pellet is 

completely resuspended. Swirl the centrifuge tube gently to reduce any foaming after 

vortexing. Record the resuspended pellet volume on the bench sheet. Proceed to 

Section 13.3. 

NOTE: Extra care must be taken with samples containing sand or other gritty 

material when vortexing to ensure that the condition of any oocysts and/or cysts in the 

sample is not compromised. 

13.2.4	 	If the packed pellet volume is > 0.5 mL, the concentrate needs to be separated into 

multiple subsamples (a subsample is equivalent to no greater than 0.5 mL of packed 

pellet material, the recommended maximum amount of particulate material to process 

through the subsequent purification and examination steps in the method). Use the 

following formula to determine the total volume required in the centrifuge tube before 

separating the concentrate into two or more subsamples: 

total volume (mL) required = pellet volume x 5 mL 0.5 mL 

(For example, if the packed pellet volume is 1.2 mL, the total volume required is 12 

mL.) Add reagent water to the centrifuge tube to bring the total volume to the level 

calculated above. Vortex the tube vigorously for 10 to 15 seconds to completely 

resuspend the pellet. Record the resuspended pellet volume on the bench sheet. 

NOTE: Extra care must be taken with samples containing sand or other gritty 

material when vortexing to ensure that the condition of any oocysts in the sample is not 

compromised. 

13.2.4.1	 	 Analysis of entire sample. If analysis of the entire sample is required, 

determine the number of subsamples to be processed independently 

through the remainder of the method: 

13.2.4.1.1	 	 Calculate number of subsamples: Divide the total 

volume in the centrifuge tube by 5 mL and round up to 

the nearest integer (for example, if the resuspended 

volume in Section 13.2.4 is 12 mL, then the number of 

subsamples would be 12 mL / 5 mL = 2.4, rounded = 3 

subsamples). 

13.2.4.1.2	 	 Determine volume of resuspended concentrate per 

subsample. Divide the total volume in the centrifuge 

tube by the calculated number of subsamples (for 

April 2001 34 
Method 1623 - Cryptosporidium and Giardia 

13.2.4.1.3
 


example, if the resuspended volume in Section 13.2.4 is 

12 mL, then the volume to use for each subsample = 12 

mL / 3 subsamples = 4 mL). 

Process subsamples through IMS. Proceed to Section 

13.3, and transfer aliquots of the resuspended 

concentrate equivalent to the volume in the previous 

step to multiple, flat-sided sample tubes in Section 

13.3.2.1. Process the sample as multiple, independent 

subsamples from Section 13.3 onward, including the 

preparation and examination of separate slides for each 

aliquot. Record the volume of resuspended concentrate 

transferred to IMS on the bench sheet (this will be equal 

to the volume recorded in Section 13.2.4). Also record 

the number of subsamples processed independently 

through the method on the bench sheet. 

13.2.4.2	 	 Analysis of partial sample. If not all of the concentrate will be 

examined, proceed to Section 13.3, and transfer one or more 5-mL 

aliquots of the resuspended concentrate to one or more flat-sided sample 

tubes in Section 13.3.2.1. Record the volume of resuspended 

concentrate transferred to IMS on the bench sheet. To determine the 

volume analyzed, calculate the percent of the concentrate examined 

using the following formula: 

percent examined = total volume of resuspended concentrate transferred to IMS x 100% total volume of resuspended concentrate in Section 13.2.4 

Then multiply the volume filtered (Section 12.2.5.2) by this percentage 

to determine the volume analyzed. 

13.3 IMS procedure (adapted from Reference 20.11) 

NOTE: The IMS procedure should be performed on a bench top with all materials at 

room temperature, ranging from 15 o

C to 25 oC. 

13.3.1 Preparation and addition of reagents 

13.3.1.1	 	 Prepare a 1X dilution of SL-buffer-A from the 10X SL-buffer-A (clear, 

colorless solution) supplied. Use reagent water (demineralized; Section 

7.3) as the diluent. For every 1 mL of 1X SL-buffer-A required, take 100 

μL of 10X SL-buffer-A and make up to 1 mL with the diluent water. A 

volume of 1.5 mL of 1X SL-buffer-A will be required per sample or 

subsample on which the Dynal IMS procedure is performed. 

13.3.1.2	 	 For each sample or subsample (Section 13.2) to be processed through 

IMS, add 1 mL of the 10X SL-buffer-A (supplied—not the diluted 1X 

SL-buffer-A) to a flat-sided tube (Section 6.5.4). 

13.3.1.3	 	 For each subsample, add 1 mL of the 10X SL-buffer-B (supplied— 

magenta solution) to the flat-sided tube containing the 10X SL-buffer-A. 

13.3.2 Oocyst and cyst capture 

13.3.2.1	 	 Use a graduated, 10-mL pipette that has been pre-rinsed with elution 

buffer to transfer the water sample concentrate from Section 13.2 to the 

flat-sided tube(s) containing the SL-buffer. If all of the concentrate is 

used, rinse the centrifuge tube twice with reagent water and add the 

rinsate to the flat-sided tube containing the concentrate (or to the tube 

35 April 2001 
Method 1623 - Cryptosporidium and Giardia 

containing the first subsample, if multiple subsamples will be 

processed). Each of the two rinses should be half the volume needed to 

bring the total volume in the flat-sided sample tube to 10 mL. (For 

example, if 5 mL was transferred after resuspension of the pellet, the 

centrifuge tube would be rinsed twice with 2.5 mL of reagent water to 

bring the total volume in the flat-sided tube to 10 mL.) Visually inspect 

the centrifuge tube after completing the transfer to ensure that no 

concentrate remains. If multiple subsamples will be processed, bring the 

volume in the remaining flat-sided tubes to 10 mL with reagent water. 

Label the flat-sided tube(s) with the sample number (and subsample 

letters). 

13.3.2.2	 	 Vortex the Dynabeads®Crypto-Combo vial from the IMS kit for 

approximately 10 seconds to suspend the beads. Ensure that the beads 

are fully resuspended by inverting the sample tube and making sure that 

there is no residual pellet at the bottom. 

13.3.2.3	 	 Add 100 μL of the resuspended Dynabeads®Crypto-Combo (Section 

13.3.2.2) to the sample tube(s) containing the water sample concentrate 

and SL-buffer. 

13.3.2.4	 	 Vortex the Dynabeads®Giardia-Combo vial from the IMS kit for 

approximately 10 seconds to suspend the beads. Ensure that the beads 

are fully resuspended by inverting the tube and making sure that there is 

no residual pellet at the bottom. 

13.3.2.5	 	 Add 100 μL of the resuspended Dynabeads®Giardia-Combo (Section 

13.3.2.4) to the sample tube(s) containing the water sample concentrate, 

Dynabeads®Crypto-Combo, and SL-buffer. 

13.3.2.6	 	 Affix the sample tube(s) to a rotating mixer and rotate at approximately 

18 rpm for 1 hour at room temperature. 

13.3.2.7	 	 After rotating for 1 hour, remove each sample tube from the mixer and 

place the tube in the magnetic particle concentrator (MPC-1) with flat 

side of the tube toward the magnet. 

13.3.2.8	 	 Without removing the sample tube from the MPC-1, place the magnet 

side of the MPC-1 downwards, so the tube is horizontal and the flat side 

of the tube is facing down. 

13.3.2.9	 	 Gently rock the sample tube by hand end-to-end through approximately 

90o

, tilting the cap-end and base-end of the tube up and down in turn. 

Continue the tilting action for 2 minutes with approximately one tilt per 

second. 

13.3.2.10	 	 Ensure that the tilting action is continued throughout this period to 

prevent binding of low-mass, magnetic or magnetizable material. If the 

sample in the MPC-1 is allowed to stand motionless for more than 10 

seconds, repeat Section 13.3.2.9 before continuing to Section 13.3.2.11. 

13.3.2.11	 	 Return the MPC-1 to the upright position, sample tube vertical, with cap 

at top. Immediately remove the cap and, keeping the flat side of the tube 

on top, pour off all of the supernatant from the tube held in the MPC-1 

into a suitable container. Do not shake the tube and do not remove the 

tube from MPC-1 during this step. 

13.3.2.12	 	 Remove the sample tube from the MPC-1 and resuspend the sample in 

1-mL 1X SL-buffer-A (prepared from 10X SL-buffer-A 

April 2001 36 
Method 1623 - Cryptosporidium and Giardia 

stock—supplied). Mix very gently to resuspend all material in the tube. 

Do not vortex. 

13.3.2.13	 	 Quantitatively transfer (transfer followed by two rinses) all the liquid 

from the sample tube to a labeled, 1.5-mL microcentrifuge tube. Use 1 

mL of 1X SL-buffer-A to perform the first rinse and 0.5 mL of reagent 

water for the second rinse. Liberally rinse down the sides of the 

Leighton tube before transferring. Allow the flat-sided sample tube to sit 

for a minimum of 1 minute after transfer of the second rinse volume, 

then use a pipette to collect any residual volume that drips down to the 

bottom of the tube to ensure that as much sample volume is recovered 

as possible. Ensure that all of the liquid and beads are transferred. 

13.3.2.14	 	 Place the microcentrifuge tube into the second magnetic particle 

concentrator (MPC-M), with its magnetic strip in place. 

13.3.2.15	 	 Without removing the microcentrifuge tube from MPC-M, gently 

rock/roll the tube through 180o by hand. Continue for approximately 1 

minute with approximately one 180o roll/rock per second. At the end of 

this step, the beads should produce a distinct brown dot at the back of 

the tube. 

13.3.2.16	 	 Immediately aspirate the supernatant from the tube and cap held in the 

MPC-M. If more than one sample is being processed, conduct three 90o 

rock/roll actions before removing the supernatant from each tube. Take 

care not to disturb the material attached to the wall of the tube adjacent 

to the magnet. Do not shake the tube. Do not remove the tube from 

MPC-M while conducting these steps. 

13.3.3 Dissociation of beads/oocyst/cyst complex 

NOTE: Two acid dissociations are required. 

13.3.3.1 Remove the magnetic strip from the MPC-M. 

13.3.3.2	 	 Add 50 μL of 0.1 N HCl, then vortex at the highest setting for 

approximately 50 seconds. 

NOTE: The laboratory should use 0.1-N standards purchased directly from a vendor, 

rather than adjusting the normality in-house. 

13.3.3.3	 	 Place the tube in the MPC-M without the magnetic strip in place and 

allow to stand in a vertical position for at least 10 minutes at room 

temperature. 

13.3.3.4 Vortex vigorously for approximately 30 seconds. 

13.3.3.5	 	 Ensure that all of the sample is at the base of the tube. Place the 

microcentrifuge tube in the MPC-M. 

13.3.3.6	 	 Replace magnetic strip in MPC-M and allow the tube to stand 

undisturbed for a minimum of 10 seconds. 

13.3.3.7 Prepare a well slide for sample screening and label the slide. 

13.3.3.8	 	 Add 5 μL of 1.0 N NaOH to the sample wells of two well slides (add 10 

μL to the sample well of one well slide if the volume from the two 

required dissociations will be added to the same slide). 

37 April 2001 
Method 1623 - Cryptosporidium and Giardia 

NOTE: The laboratory should use 1.0-N standards purchased directly from a vendor 

rather than adjusting the normality in-house. 

13.3.3.9	 	 Without removing the microcentrifuge tube from the MPC-M, transfer 

all of the sample from the microcentrifuge tube in the MPC-M to the 

sample well with the NaOH. Do not disturb the beads at the back wall of 

the tube. Ensure that all of the fluid is transferred. 

13.3.3.10	 	 Do not discard the beads or microcentrifuge tube after transferring the 

volume from the first acid dissociation to the well slide. Perform the 

steps in Sections 13.3.3.1 through 13.3.3.9 a second time. The volume 

from the second dissociation can be added to the slide containing the 

volume from the first dissociation, or can be applied to a second slide. 

NOTE: If one slide is used, exert extra care when using Dynal Spot-On slides to 

ensure that the sample stays within the smaller-diameter wells on these slides. 

13.3.3.11 Record the date and time the purified sample was applied to the slide(s). 

13.3.3.12	 	 Air-dry the sample on the well slide(s). Because temperature and 

humidity varies from laboratory to laboratory, no minimum time is 

specified. However, the laboratory must take care to ensure that the 

sample has dried completely before staining to prevent losses during the 

rinse steps. A slide warmer set at 35 o C to 42 o C also can be used. 

14.0 Sample Staining 

NOTE: The sample must be stained within 72 hours of application of the purified 

sample to the slide. 

14.1 Prepare positive and negative controls. 

14.1.1	 	 For the positive control, pipette 10 μL of positive antigen or 200 to 400 intact oocysts 

and 200 to 400 cysts to the center of a well. 

14.1.2	 	 For the negative control, pipette 50 μL of 150 mM PBS (Section 7.6.4) into the center of 

a well and spread it over the well area with a pipette tip. 

14.1.3 Air-dry the control slides (see Section 13.3.3.12 for guidance). 

14.2	 	 Apply 50-μL of absolute methanol to each well containing the dried sample and allow to air-dry 

for 3 to 5 minutes. 

14.3 Follow manufacturer’s instructions in applying stain to slide. 

14.4	 	 Place the slides in a humid chamber in the dark and incubate at room temperature for 

approximately 30 minutes. The humid chamber consists of a tightly sealed plastic container 

containing damp paper towels on top of which the slides are placed. 

14.5	 	 Apply one drop of wash buffer (prepared according to the manufacturer’s instructions [Section 

7.6]) to each well. Tilt each slide on a clean paper towel, long edge down. Gently aspirate the 

excess detection reagent from below the well using a clean Pasteur pipette or absorb with paper 

towel or other absorbent material placed at edge of slide. Avoid disturbing the sample. 

NOTE: If using the Merifluor stain (Section 7.6.1), do not allow slides to dry 

completely. 

April 2001 38 
Method 1623 - Cryptosporidium and Giardia 

14.6	 	 Apply 50 μL of 4',6-diamidino-2-phenylindole (DAPI) staining solution (Section 7.7.2) to each 

well. Allow to stand at room temperature for a minimum of 1 minute. (The solution 

concentration may be increased up to 1 μg /mL if fading/diffusion of DAPI staining is 

encountered, but the staining solution must be tested first on expendable environmental samples 

to confirm that staining intensity is appropriate.) 

14.7	 	 Apply one drop of wash buffer (prepared according to the manufacturer’s instructions [Section 

7.6]) to each well. Tilt each slide on a clean paper towel, long edge down. Gently aspirate the 

excess DAPI staining solution from below the well using a clean Pasteur pipette or absorb with 

paper towel or other absorbent material placed at edge of slide. Avoid disturbing the sample. 

NOTE: If using the Merifluor stain (Section 7.6.1), do not allow slides to dry 

completely. 

14.8 Add mounting medium (Section 7.8) to each well. 

14.9	 	 Apply a cover slip. Use a tissue to remove excess mounting fluid from the edges of the 

coverslip. Seal the edges of the coverslip onto the slide using clear nail polish. 

14.10	 	Record the date and time that staining was completed on the bench sheet. If slides will not be 

read immediately, store in a humid chamber in the dark at 0 o

C to 8 oC until ready for 

examination. 

15.0 Examination 

NOTE: Although immunofluorescence assay (FA) and 4',6-diamidino-2-phenylindole 

(DAPI) and differential interference contrast (DIC) microscopy examination and 

confirmation should be performed immediately after staining is complete, laboratories 

have up to 7 days from completion of sample staining to complete the examination and 

confirmation of samples. However, if fading/diffusion of FITC or DAPI staining is 

noticed, the laboratory must reduce this holding time. In addition the laboratory may 

adjust the concentration of the DAPI staining solution (Sections 7.7.2) so that 

fading/diffusion does not occur. 

15.1	 	 Scanning technique: Scan each well in a systematic fashion. An up-and-down or a side-to-side 

scanning pattern may be used (Figure 4). 

15.2	 	 Examination using immunofluorescence assay (FA), 4',6-diamidino-2-phenylindole (DAPI) 

staining characteristics, and differential interference contrast (DIC) microscopy. The minimum 

magnification requirements for each type of examination are noted below. 

NOTE: All shape and measurements must be determined using 1000X magnification 

and reported to the nearest 0.5 μm . 

Record examination results for Cryptosporidium oocysts on a Cryptosporidium report form; 

record examination results for Giardia cysts on a Giardia report form. All oocysts and cysts that 

meet the criteria specified in Sections 15.2.2 and 15.2.3, less atypical organisms specifically 

identified as non-target organisms by DIC or DAPI (e.g. possessing spikes, stalks, appendages, 

pores, one or two large nuclei filling the cell, red fluorescing chloroplasts, crystals, spores, etc), 

must be reported. 

15.2.1 Positive and negative staining control. 

15.2.1.1	 	 Each analyst must characterize a minimum of three Cryptosporidium 

oocysts and three Giardia cysts on the positive staining control slide 

before examining field sample slides. This characterization must be 

performed by each analyst during each microscope examination session. 

39 April 2001 
Method 1623 - Cryptosporidium and Giardia 

FITC examination must be conducted at a minimum of 200X total 

magnification, DAPI examination must be conducted at a minimum of 

400X, and DIC examination must be conducted at a minimum of 1000X. 

Size, shape, and DIC and DAPI characteristics of the three 

Cryptosporidium oocysts and Giardia cysts must be recorded by the 

analyst on a microscope log. The analyst also must indicate on each 

sample report form whether the positive staining control was acceptable. 

15.2.1.2	 	 Examine the negative staining control to confirm that it does not contain 

any oocysts or cysts (Section 14.1). Indicate on each sample report form 

whether the negative staining control was acceptable. 

15.2.1.3	 	 If the positive staining control contains oocysts and cysts within the 

expected range and at the appropriate fluorescence for both FA and 

DAPI, and the negative staining control does not contain any oocysts or 

cysts (Section 14.1), proceed to Sections 15.2.2 and 15.2.3. 

15.2.2 Sample examination—Cryptosporidium 

15.2.2.1	 	 FITC examination (the analyst must use a minimum of 200X total 

magnification). Use epifluorescence to scan the entire well for applegreen fluorescence of oocyst and cyst shapes. When brilliant applegreen fluorescing ovoid or spherical objects 4 to 6 μm in diameter are 

observed with brightly highlighted edges, increase magnification to 

400X and switch the microscope to the UV filter block for DAPI 

(Section 15.2.2.2), then to DIC (Section 15.2.2.3). 

15.2.2.2	 	 DAPI examination (the analyst must use a minimum of 400X total 

magnification). Using the UV filter block for DAPI, the object will 

exhibit one of the following characteristics: 

(a) Light blue internal staining (no distinct nuclei) with a green rim 

(b) Intense blue internal staining 

(c) Up to four distinct, sky-blue nuclei 

Record oocysts in category (a) as DAPI negative; record oocysts in 

categories (b) and (c) as DAPI positive. 

15.2.2.3	 	 DIC examination (the analyst must use a minimum of 1000X total 

magnification). Using DIC, look for external or internal morphological 

characteristics atypical of Cryptosporidium oocysts (e.g., spikes, stalks, 

appendages, pores, one or two large nuclei filling the cell, red 

fluorescing chloroplasts, crystals, spores, etc.) (adapted from Reference 

20.6). If atypical structures are not observed, then categorize each applegreen fluorescing object as: 

(a) An empty Cryptosporidium oocyst 

(b) A Cryptosporidium oocyst with amorphous structure 

(c) A Cryptosporidium oocyst with internal structure (one to four 

sporozoites/oocyst) 

Using 1000X total magnification, record the shape, measurements (to the 

nearest 0.5 μm ), and number of sporozoites (if applicable) for each 

apple-green fluorescing object meeting the size and shape characteristics. 

Although not a defining characteristic, surface oocyst folds may be 

observed in some specimens. 

NOTE: All measurements must be made at 1000X magnification. 

April 2001 40 
Method 1623 - Cryptosporidium and Giardia 

15.2.3 Sample examination—Giardia 

15.2.3.1	 	 FITC examination (the analyst must use a minimum of 200X total 

magnification). When brilliant apple-green fluorescing round to oval 

objects (8 - 18 μm long by 5 - 15 μm wide) are observed, increase 

magnification to 400X and switch the microscope to the UV filter block 

for DAPI (Section 15.2.3.2) then to DIC (Section 15.2.3.3). 

15.2.3.2	 	 DAPI examination (the analyst must use a minimum of 400X total 

magnification). Using the UV filter block for DAPI, the object will 

exhibit one or more of the following characteristics: 

(a) Light blue internal staining (no distinct nuclei) and a green rim 

(b) Intense blue internal staining 

(c) Two to four sky-blue nuclei 

Record cysts in category (a) as DAPI negative; record cysts in categories 

(b) and (c) as DAPI positive. 

15.2.3.3	 	 DIC examination (the analyst must use a minimum of 1000X total 

magnification). Using DIC, look for external or internal morphological 

characteristics atypical of Giardia cysts (e.g., spikes, stalks, appendages, 

pores, one or two large nuclei filling the cell, red fluorescing 

chloroplasts, crystals, spores, etc.) (adapted from Reference 20.6). If 

atypical structures are not observed, then categorize each object meeting 

the criteria specified in Sections 15.2.3.1 - 15.2.3.3 as one of the 

following, based on DIC examination: 

(a) An empty Giardia cyst 

(b) A Giardia cyst with amorphous structure 

(c) A Giardia cyst with one type of internal structure (nuclei, 

median body, or axonemes), or 

(d) A Giardia cyst with more than one type of internal structure 

Using 1000X total magnification, record the shape, measurements (to the 

nearest 0.5 μm ), and number of nuclei and presence of median body or 

axonemes (if applicable) for each apple-green fluorescing object 

meeting the size and shape characteristics. 

NOTE: All measurements must be made at 1000X magnification. 

15.2.4 Record the date and time that sample examination was completed on the report form. 

15.2.5 Report Cryptosporidium and Giardia concentrations as oocysts/L and cysts/L. 

16.0 Analysis of Complex Samples 

16.1	 	 Some samples may contain high levels (>1000/L) of oocysts and cysts and/or interfering 

organisms, substances, or materials. Some samples may clog the filter (Section 12.0); others will 

not allow separation of the oocysts and cysts from the retentate or eluate; and others may contain 

materials that preclude or confuse microscopic examination. 

16.2	 	 If the sample holding time has not been exceeded and a full-volume sample cannot be filtered, 

dilute an aliquot of sample with reagent water and filter this smaller aliquot (Section 12.0). This 

dilution must be recorded and reported with the results. 

16.3	 	 If the holding times for the sample and for microscopic examination of the cleaned up 

retentate/eluate have been exceeded, the site should be re-sampled. If this is not possible, the 

results should be qualified accordingly. 

41 April 2001 
Method 1623 - Cryptosporidium and Giardia 

17.0 Method Performance 

17.1	 	 Method acceptance criteria are shown in Tables 3 and 4 in Section 21.0. The initial and ongoing 

precision and recovery criteria are based on the results of spiked reagent water samples analyzed 

during the Information Collection Rule Supplemental Surveys (Reference 20.12). The matrix 

spike and matrix spike duplicate criteria are based on spiked source water data generated during 

the interlaboratory validation study of Method 1623 involving 11 laboratories and 11 raw surface 

water matrices across the U.S. (Reference 20.10). 

NOTE: Some sample matrices may prevent the MS acceptance criteria in Tables 3 

and 4 to be met. An assessment of the distribution of MS recoveries across 430 MS 

samples from 87 sites during the ICR Supplemental Surveys is provided in Table 5. 

18.0 Pollution Prevention 

18.1	 	 The solutions and reagents used in this method pose little threat to the environment when 

recycled and managed properly. 

18.2	 	 Solutions and reagents should be prepared in volumes consistent with laboratory use to 

minimize the volume of expired materials to be disposed. 

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. An overview of these requirements can be found in the 

Environmental Management Guide for Small Laboratories (EPA 233-B-98-001). 

19.2	 	 Samples, reference materials, and equipment known or suspected to have viable oocysts or cysts 

attached or contained must be sterilized prior to disposal. 

19.3	 	 For further information on waste management, consult The Waste Management Manual for 

Laboratory Personnel and Less is Better: Laboratory Chemical Management for Waste 

Reduction, both available from the American Chemical Society's Department of Government 

Relations and Science Policy, 1155 16th Street N.W., Washington, D.C. 20036. 

20.0 References 

20.1	 	 Rodgers, Mark R., Flanigan, Debbie J., and Jakubowski, Walter, 1995. Applied and 

Environmental Microbiology 61 (10), 3759-3763. 

20.2	 	 Fleming, Diane O., et al.(eds.), Laboratory Safety: Principles and Practices, 2nd edition.1995. 

ASM Press, Washington, DC 

20.3 "Working with Carcinogens," DHEW, PHS, CDC, NIOSH, Publication 77-206, (1977). 

20.4 "OSHA Safety and Health Standards, General Industry," OSHA 2206, 29 CFR 1910 (1976). 

20.5 "Safety in Academic Chemistry Laboratories," ACS Committee on Chemical Safety (1979). 

20.6	 	 ICR Microbial Laboratory Manual, EPA/600/R-95/178, National Exposure Research Laboratory, 

Office of Research and Development, U.S. Environmental Protection Agency, 26 Martin Luther 

King Drive, Cincinnati, OH 45268 (1996). 

20.7	 	 USEPA. EPA Guide to Method Flexibility and Approval of EPA Water Methods, EPA 821-D96-004. Office of Water, Engineering and Analysis Division, Washington, DC 20460 (1996). 

April 2001 42 
Method 1623 - Cryptosporidium and Giardia 

20.8	 	 Connell, K., C.C. Rodgers, H.L. Shank-Givens, J Scheller, M.L Pope, and K. Miller, 2000. 

Building a Better Protozoa Data Set. Journal AWWA, 92:10:30. 

20.9	 	 "EnvirochekTM Sampling Capsule," PN 32915, Gelman Sciences, 600 South Wagner Road, Ann 

Arbor, MI 48103-9019 (1996). 

20.10	 	USEPA. Results of the Interlaboratory Method Validation Study for Determination of 

Cryptosporidium and Giardia Using USEPA Method 1623, EPA-821-R-01-028. Office of Water, 

Office of Science and Technology, Engineering and Analysis Division, Washington, DC (2001). 

20.11	 	"Dynabeads® GC-Combo,” Dynal Microbiology R&D, P.O. Box 8146 Dep., 0212 Oslo, Norway 

(September 1998, Revision no. 01). 

20.12	 	USEPA. Implementation and Results of the Information Collection Rule Supplemental Surveys. 

EPA-815-R-01-003. Office of Water, Office of Ground Water and Drinking Water, Standards 

and Risk Management Division, Washington, DC (2001). 

20.13	 	Connell, K., J. Scheller, K. Miller, and C.C. Rodgers, 2000. Performance of Methods 1622 and 

1623 in the ICR Supplemental Surveys. Proceedings, American Water Works Association Water 

Quality Technology Conference, November 5 - 9, 2000, Salt Lake City, UT. 

21.0 Tables and Figures 

Table 1. Method Holding Times (See Section 8.2 for details) 

Sample Processing Step Maximum Allowable Time between Breaks 

Collection 

Filtration 

> Up to 96 hours are permitted between sample collection (if shipped to the laboratory as a 

bulk sample) or filtration (if filtered in the field) and initiation of elution 

Elution 

These steps must be completed in 1 working 

day 

Concentration 

Purification 

Application of purified sample to slide 

Drying of sample 

> Up to 72 hours are permitted from application of the purified sample to the slide to staining 

Staining 

> Up to 7 days are permitted between sample staining and examination 

Examination 

43 April 2001 
Method 1623 - Cryptosporidium and Giardia 

Table 2. Tier 1 and Tier 2 Validation/Equivalency Demonstration Requirements 

Test Description Tier 1 modification(1) Tier 2 modification(2) 

IPR 

(Section 9.4) 4 replicates of spiked 

reagent water 

Required. Must be accompanied by a 

method blank. Required per laboratory 

Method blank 

(Section 9.6) Unspiked reagent 

water Required Required per laboratory 

MS 

(Section 9.5.1) Spiked matrix water 

Required on each water to which the 

modification will be applied and on every 

20th sample of that water thereafter. Must be 

accompanied by an unspiked field sample 

collected at the same time as the MS sample 

Not required 

MS/MSD 

(Section 9.5) 

2 replicates of spiked 

matrix water 

Recommended, but not required. Must be 

accompanied by an unspiked field sample 

collected at the same time as the MS sample 

Required per laboratory. 

Each laboratory must 

analyze a different water. 

(1)	 	 If a modification will be used only in one laboratory, these tests must be performed and the results must 

meet all of the QC acceptance criteria in the method (these tests also are required the first time a laboratory 

uses the validated version of the method) 

(2)	 	 If nationwide approval of a modification is sought for one type of water matrix (such as surface water), a 

minimum of 3 laboratories must perform the tests and the results from each lab individually must meet all 

QC acceptance criteria in the method. If more than 3 laboratories are used in a study, a minimum of 75% of 

the laboratories must meet all QC acceptance criteria. 

NOTE: The initial precision and recovery and ongoing precision and recovery (OPR) 

acceptance criteria listed in Tables 3 and 4 are based on results from 293 

Cryptosporidium OPR samples and 186 Giardia OPR samples analyzed by six 

laboratories during the Information Collection Rule Supplemental Surveys (Reference 

20.12). The matrix spike acceptance criteria are based on data generated through 

interlaboratory validation of Method 1623 (Reference 20.10). 

Table 3. Quality Control Acceptance Criteria for Cryptosporidium 

Performance test Section Acceptance criteria 

Initial precision and recovery 

Mean recovery (percent) 

Precision (as maximum relative standard deviation) 

9.4 

9.4.2 

9.4.2 

24 - 100 

55 

Ongoing precision and recovery (percent) 9.7 11 - 100 

Matrix spike/matrix spike duplicate (for method modifications) 

Mean recovery1, 2 (as percent) 

Precision (as maximum relative percent difference) 

9.5 

9.5.2 

9.5.2 

13 - 111 

61 

(1)	 	 The acceptance criteria for mean MS/MSD recovery serves as the acceptance criteria for MS recovery 

during routine use of the method (Section 9.5.1). 

(2)	 	 Some sample matrices may prevent the acceptance criteria from being met. An assessment of the 

distribution of MS recoveries from multiple MS samples from 87 sites during the ICR Supplemental 

Surveys is provided in Table 5. 

April 2001 44 
Method 1623 - Cryptosporidium and Giardia 

Table 4. Quality Control Acceptance Criteria for Giardia 

Performance test Section Acceptance criteria 

Initial precision and recovery 

Mean recovery (percent) 

Precision (as maximum relative standard deviation) 

9.4 

9.4.2 

9.4.2 

24 - 100 

49 

Ongoing precision and recovery (percent) 9.7 14 - 100 

Matrix spike/matrix spike duplicate (for method modifications) 

Mean recovery* (as percent) 

Precision (as maximum relative percent difference) 

9.5 

9.5.2 

9.5.2 

15 - 118 

30 

(1)	 	 The acceptance criteria for mean MS/MSD recovery serves as the acceptance criteria for MS recovery 

during routine use of the method (Section 9.5.1). 

(2)	 	 Some sample matrices may prevent the acceptance criteria from being met. An assessment of the 

distribution of MS recoveries across multiple MS samples from 87 sites during the ICR Supplemental 

Surveys is provided in Table 5. 

Table 5. Distribution of Matrix Spike Recoveries from Multiple Samples Collected from 87 

Source Waters During the ICR Supplemental Surveys (Adapted from Reference 20.13) 

MS Recovery Range 

Percent of 430 Cryptosporidium MS 

Samples in Recovery Range 

Percent of 270 Giardia MS 

Samples in Recovery Range 

<10% 6.7% 5.2% 

>10% - 20% 6.3% 4.8% 

>20% - 30% 14.9% 7.0% 

>30% - 40% 14.2% 8.5% 

>40% - 50% 18.4% 17.4% 

>50% - 60% 17.4% 16.3% 

>60% - 70% 11.2% 16.7% 

>70% - 80% 8.4% 14.1% 

>80% - 90% 2.3% 6.3% 

>90% 0.2% 3.7% 

45 April 2001 
Method 1623 - Cryptosporidium and Giardia 

Figure 1.	 	 Hemacytometer Platform Ruling. Squares 1, 2, 3, and 4 

are used to count stock suspensions of Cryptosporidium

oocysts and Giardia cysts (after Miale, 1967) 

April 2001 46 
Method 1623 - Cryptosporidium and Giardia 

Figure 2.	 	 Manner of Counting Oocysts and Cysts in 1 Square mm. 

Dark organisms are counted and light organisms are

omitted (after Miale, 1967). 

47 April 2001 
Method 1623 - Cryptosporidium and Giardia

Figure 3. Laboratory Filtration System 

April 2001 48
Method 1623 - Cryptosporidium and Giardia 

Figure 4. Methods for Scanning a Well Slide 

49 April 2001 
Method 1623 - Cryptosporidium and Giardia 

22.0 Glossary of Definitions and Purposes 

These definitions and purposes are specific to this method but have been conformed to common 

usage as much as possible. 

22.1 Units of weight and measure and their abbreviations 

22.1.1 Symbols o

C degrees Celsius 

μL microliter 

< less than 

> greater than 

% percent 

22.1.2 Alphabetical characters 

cm centimeter 

g gram 

G acceleration due to gravity 

hr hour 

ID inside diameter 

in. inch 

L liter 

m meter 

mg milligram 

mL milliliter 

mm millimeter 

mM millimolar 

N normal; gram molecular weight of solute divided by hydrogen equivalent of 

solute, per liter of solution 

RSD relative standard deviation 

sr standard deviation of recovery 

X average percent recovery 

22.2 Definitions, acronyms, and abbreviations (in alphabetical order) 

Analyst—The analyst must have at least 2 years of college lecture and laboratory course work in 

microbiology or a closely related field. The analyst also must have at least 6 months of 

continuous bench experience with environmental protozoa detection techniques and IFA 

microscopy, and must have successfully analyzed at least 50 water and/or wastewater samples for 

Cryptosporidium and Giardia. Six months of additional experience in the above areas may be 

substituted for two years of college. 

Analyte—A protozoan parasite tested for by this method. The analytes in this method are 

Cryptosporidium and Giardia. 

Axoneme—An internal flagellar structure that occurs in some protozoa, such as Giardia, 

Spironucleous, and Trichonmonas. 

Cyst—A phase or a form of an organism produced either in response to environmental conditions 

or as a normal part of the life cycle of the organism. It is characterized by a thick and 

environmentally resistant cell wall. 

Flow cytometer—A particle-sorting instrument capable of counting protozoa. 

April 2001 50 
Method 1623 - Cryptosporidium and Giardia 

Immunomagnetic separation (IMS)—A purification procedure that uses microscopic, 

magnetically responsive particles coated with an antibodies targeted to react with a specific 

pathogen in a fluid stream. Pathogens are selectively removed from other debris using a magnetic 

field. 

Initial precision and recovery (IPR)—Four aliquots of spiking suspension analyzed to establish 

the ability to generate acceptable precision and accuracy. An IPR is performed prior to the first 

time this method is used and any time the method or instrumentation is modified. 

Laboratory blank—See Method blank 

Laboratory control sample (LCS)—See Ongoing precision and recovery (OPR) standard 

Matrix spike (MS)—A sample prepared by adding a known quantity of organisms to a specified 

amount of sample matrix for which an independent estimate of target analyte concentration is 

available. A matrix spike is used to determine the effect of the matrix on a method’s recovery 

efficiency. 

May—This action, activity, or procedural step is neither required nor prohibited. 

May not—This action, activity, or procedural step is prohibited. 

Median bodies—Prominent, dark-staining, paired organelles consisting of microtubules and 

found in the posterior half of Giardia. In G. intestinalis (from humans), these structures often 

have a claw-hammer shape, while in G. muris (from mice), the median bodies are round. 

Method blank—An aliquot of reagent water that is treated exactly as a sample, including 

exposure to all glassware, equipment, solvents, and procedures that are used with samples. The 

method blank is used to determine if analytes or interferences are present in the laboratory 

environment, the reagents, or the apparatus. 

Must—This action, activity, or procedural step is required. 

Negative control—See Method blank 

Nucleus—A membrane-bound organelle containing genetic material. Nuclei are a prominent 

internal structure seen both in Cryptosporidium oocysts and Giardia cysts. In Cryptosporidium 

oocysts, there is one nucleus per sporozoite. One to four nuclei can be seen in Giardia cysts. 

Oocyst—The encysted zygote of some sporozoa; e.g., Cryptosporidium. The oocyst is a phase or 

form of the organism produced as a normal part of the life cycle of the organism. It is 

characterized by a thick and environmentally resistant outer wall. 

Ongoing precision and recovery (OPR) standard—A method blank spiked with known quantities 

of analytes. The OPR is analyzed exactly like a sample. Its purpose is to assure that the results 

produced by the laboratory remain within the limits specified in this method for precision and 

recovery. 

Oocyst and cyst spiking suspension—See Spiking suspension 

Oocyst and cyst stock suspension—See Stock suspension 

51 April 2001 
Method 1623 - Cryptosporidium and Giardia 

Positive control—See Ongoing precision and recovery standard 

PTFE—Polytetrafluoroethylene 

Quantitative transfer—The process of transferring a solution from one container to another using 

a pipette in which as much solution as possible is transferred, followed by rinsing of the walls of 

the source container with a small volume of rinsing solution (e.g., reagent water, buffer, etc.), 

followed by transfer of the rinsing solution, followed by a second rinse and transfer. 

Reagent water—Water demonstrated to be free from the analytes of interest and potentially 

interfering substances at the method detection limit for the analyte. 

Reagent water blank—see Method blank 

Relative standard deviation (RSD)—The standard deviation divided by the mean times 100. 

RSD—See Relative standard deviation 

Should—This action, activity, or procedural step is suggested but not required. 

Spiking suspension—Diluted stock suspension containing the organism(s) of interest at a 

concentration appropriate for spiking samples. 

Sporozoite—A motile, infective stage of certain protozoans; e.g., Cryptosporidium. There are 

four sporozoites in each Cryptosporidium oocyst, and they are generally banana-shaped. 

Stock suspension—A concentrated suspension containing the organism(s) of interest that is 

obtained from a source that will attest to the host source, purity, authenticity, and viability of the 

organism(s). 

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