Method 1623: Cryptosporidium and


Giardia in Water by Filtration/IMS/FA 


December 2005

Office of Water (4607) 

EPA 815-R-05-002 

http://www.epa.gov/microbes/ 

December 2005 

Printed on Recycled Paper 
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 CSC under a U.S. EPA contract, with assistance from its subcontractor, Interface, Inc. 

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

organizations to the development of this method: 

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

4770 Buford Highway, N.E., Atlanta, GA 30341-3724, USA 

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

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

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

Kevin Connell, CSC, 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 (retired), 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 Moulton (Hancock), 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 

Stephanie Harris,Manchester Laboratory, U.S. Environmental Protection Agency, Region 10, 7411 

Beach Drive East, Port Orchard, WA 98366, USA 

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

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

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

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

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

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

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

Springburn, Glasgow, G21 3UW, Scotland 

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

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

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

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

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

i 
Disclaimer 

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

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

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

Carrie Moulton 

Coordinator, Laboratory Quality Assurance Program for the Analysis of Cryptosporidium 

U.S. Environmental Protection Agency


Office of Ground Water and Drinking Water


Technical Support Center, MC140


26 West Martin Luther King Drive


Cincinnati, OH 45268-1320


(513) 569-7919


(513) 569-7191 (fax)


moulton.carrie@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. 

Method Development and Validation 

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

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

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

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

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

Cryptosporidium-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. 

Changes in the April 2001 Versions of the Methods 

Both methods were revised in April 2001, after completion of the ICRSS and multiple meetings with 

researchers and experienced laboratory staff to discuss potential method updates. Changes incorporated in 

the April 2001 revisions of the methods (EPA-821-R-01-025 and EPA-821-R-01-026) included the 

following: 

C Nationwide approval of modified versions of the methods using the following components: 

(a) Whatman Nuclepore CrypTestTM filter 

(b) IDEXX Filta-Max® filter 

(c) Waterborne Aqua-GloTM G/C Direct FL antibody stain 

(d) Waterborne Crypt-a-GloTM and Giardi-a-GloTM antibody stains 

C Clarified sample acceptance criteria 

C Modified capsule filter elution procedure 

C Modified concentrate aspiration procedure 

iii 
C Modified IMS acid dissociation procedure 

C Updated QC acceptance criteria for IPR and OPR tests 

C Addition of a troubleshooting section for QC failures 

C Modified holding times 

C Inclusion of flow cytometry–sorted spiking suspensions 

Changes in the June 2003 Versions of the Methods 

Both methods were revised again in June 2003 to support proposal of EPA’s Long Term 2 Enhanced 

Surface Water Treatment Rule. Changes incorporated into the December 2002 versions include: 

•	 Nationwide approval of a modified version of the methods using the Pall Gelman EnvirochekTM 

HV filter 

•	 Removal of Whatman Nuclepore CrypTestTM filter from the methods as a result of discontinuation 

of the product by the manufacturer 

•	 Nationwide approval of the use of BTF EasySeedTM irradiated oocysts and cysts for use in routine 

quality control (QC) samples 

•	 Minor clarifications and corrections 

•	 Rejection criteria for sample condition upon receipt 

•	 Guidance on measuring sample temperatures 

•	 Clarification of QC sample requirements and use of QC sample results 

•	 Guidance on minimizing carry-over debris onto microscope slides after IMS 

Changes in the December 2005 Versions of the Methods 

Both methods were revised again in 2005 to support promulgation of EPA’s Long Term 2 Enhanced 

Surface Water Treatment Rule. Changes incorporated into the June 2003 versions include: 

•	 Nationwide approval of the use of portable continuous-flow centrifugation as a modified version 

of the method. The product met all method acceptance criteria for Cryptosporidium using 50-L 

source water samples (but not Giardia, however, individual laboratories are permitted to 

demonstrate acceptable performance for Giardia in their laboratory). 

•	 Addition of BTF EasyStainTM monoclonal antibody stain as an acceptable reagent for staining in 

Methods 1622/1623. The product was validated through an interlaboratory validation study using 

the Pall EnvirochekTM HV filter. 

•	 Clarification of the analyst verification procedure 

•	 Clarification of sample condition criteria upon receipt 

Performance-Based Method Concept and Modifications Approved for Nationwide Use 

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

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

oocysts and cysts from the material captured, and enumeration of the target organisms based on the results 

of immunofluorescence assay, 4',6-diamidino-2-phenylindole (DAPI) staining results, and differential 

interference contrast microscopy. 

iv 
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 (no longer available) 

• IDEXX Filta-Max® filter 

• Pall Gelman EnvirochekTM HV filter 

• Portable Continuous-Flow Centrifugation (PCFC) 

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

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

• BTF EasyStainTM antibody stain 

• BTF EasySeedTM irradiated oocysts and cysts for use in routine QC samples 

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. 

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

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

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

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

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

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

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

v 
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 ......................................................... 4


7.0 Reagents and Standards ......................................................... 8


8.0 Sample Collection and Storage ................................................... 11


9.0 Quality Control ............................................................... 13


10.0 Microscope Calibration and Analyst Verification .................................... 21


11.0 Oocyst and Cyst Suspension Enumeration and Sample Spiking ......................... 28


12.0 Sample Filtration and Elution .................................................... 36


13.0 Sample Concentration and Separation (Purification) .................................. 46


14.0 Sample Staining .............................................................. 51


15.0 Examination ................................................................. 52


16.0 Analysis of Complex Samples ................................................... 54


17.0 Method Performance ........................................................... 55


18.0 Pollution Prevention ........................................................... 55


19.0 Waste Management ............................................................ 55


20.0 References ................................................................... 55


21.0 Tables and Figures ............................................................ 57


22.0 Glossary of Definitions and Purposes .............................................. 66


vi 
Method 1623: Cryptosporidium and Giardia in Water 

by Filtration/IMS/FA 

1.0	 Scope and Application 

1.1	 This method is for the detection of Cryptosporidium (CAS Registry number 137259-50-8) and 

Giardia (CAS Registry number 137259-49-5) in water by concentration, immunomagnetic 

separation (IMS), and immunofluorescence assay (FA) microscopy. Cryptosporidium and Giardia 

may be verified 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 identifies the genera, Cryptosporidium or Giardia, but not the species. The method 

cannot determine the host species of origin, nor can it determine the viability or infectivity of 

detected oocysts and cysts. 

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

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

principal analysts. Laboratories unfamiliar with analyses of environmental samples by the 

techniques in this method 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 may be used. 

2.2	 Elution and separation 

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

cysts, and the supernatant fluid is aspirated. 

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

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

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

discarded. The magnetic bead complex is then detached from the oocysts and cysts. 

2.3	 Enumeration 

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

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

using fluorescence and differential interference contrast (DIC) microscopy. 

1	 December 2005 
Method 1623 - C ryptosporidium and G iardia 

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

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

cysts. 

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

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 a genus of protozoan parasites potentially found in water and other media. 

The recent taxonomy of the genus Cryptosporidium includes the following species and their 

potential hosts: C. hominis (humans; formerly C. parvum genotype I; Reference 20.1); C. parvum 

(bovine and other mammals including humans; formerly genotype II;); C. baileyi and C. 

meleagridis (birds); C. muris (rodents); C. canis (dogs); C. felis (cats); C. serpentis (reptiles); and 

C. nasorum (fish). Cryptosporidium oocysts are defined in this method as objects exhibiting 

brilliant apple green fluorescence under UV light (FA-positive), typical size (4 to 6 μm) and shape 

(round to oval), and no atypical characteristics by FA, DAPI fluorescence, or DIC microscopy. 

Examination and characterization using fluorescence (FITC and DAPI stain) and DIC microscopy 

are required for exclusion of atypical organisms (e.g., those possessing spikes, stalks, appendages, 

pores, one or two large nuclei filling the cell, red fluorescing chloroplasts, crystals, spores, etc.). 

3.2	 Giardia is a genus of protozoan parasites potentially found in water and other media. The recent 

taxonomy of the genus Giardia includes the following species and their potential hosts: G. 

lamblia (also called G. intestinalis or G. duodenalis; humans and other mammals); G. muris 

(rodents); G. agilis (amphibians); G. psittaci and G. ardeae (birds). Recent molecular studies 

suggest the division of G. lamblia into multiple genotypes (Reference 20.2). Giardia cysts are 

defined in this method as objects exhibiting brilliant apple green fluorescence under UV light 

(FA-positive), typical size (8 to 18 μm long by 5 to 15 μm wide) and shape (oval to round), and no 

atypical characteristics by FA, DAPI fluorescence, or DIC microscopy. Examination and 

characterization by fluorescence (FITC and DAPI stain) and DIC microscopy are required for 

exclusion of atypical organisms (e.g., those possessing spikes, stalks, appendages, pores, one or 

two large nuclei filling the cell, red fluorescing chloroplasts, crystals, spores, etc.). 

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

4.0	 Contamination, Interferences, and Organism Degradation 

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

and examination of the sample for Cryptosporidium oocysts and Giardia cysts. In addition to 

naturally-occurring debris, e.g. clays and algae, chemicals, e.g. iron, alum coagulants and 

polymers added to source waters during the treatment process may result in additional 

interference. 

4.2	 Organisms and debris that autofluoresce or demonstrate non-specific immunofluorescence, such 

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

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

(Reference 20.3). 

December 2005	 2 
Method 1623 - C ryptosporidium and G iardia 

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

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

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

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

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

materials may be required. 

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

identification of oocysts and cysts. 

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

should be used wherever possible. 

5.0	 Safety 

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

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

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

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

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

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

materials, and while operating sterilization equipment. 

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

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

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

laboratory is responsible for maintaining current knowledge 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 20.4 

through 20.7. 

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

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

handled with gloves and laboratory staff must never place gloves in or near the face after exposure 

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

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

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

surfaces or equipment. 

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

than 4 L of solution known to contain infectious materials (see 

http://www.cdc.gov/od/ohs/biosfty/shipregs.htm for details). State regulations may contain similar 

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

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

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

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

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

these regulations. 

3	 December 2005 
Method 1623 - C ryptosporidium and G iardia 

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. 

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

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

the procedures outlined in Section 9.1.2. 

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

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

6.2.2	 Original EnvirochekTM sampling capsule or EnvirochekTM HV sampling capsule 

equipment requirements (for use with the procedure described in Section 12.2). The 

versions of the method using these filters were validated using 10-L and 50-L sample 

volumes, respectively. Alternate sample volumes may be used, provided the laboratory 

demonstrates acceptable performance on initial and ongoing spiked reagent water and 

source water samples (Section 9.1.2). 

6.2.2.1 Sampling capsule 

6.2.2.1.1	 EnvirochekTM, Pall Corporation, Ann Arbor, MI, part no. 

12110 (individual filter) and or part no.12107 (box of 25 

filters) (www.pall.com or (800) 521-1520 ext. 2) 

6.2.2.1.2	 EnvirochekTM HV, Pall Corporation, Ann Arbor, MI, part 

no. 12099 (individual filter) or part no.12098 (box of 25 

filters) (www.pall.com or (800) 521-1520 ext. 2) 

6.2.2.2 Laboratory shaker with arms for agitation of sampling capsules 

6.2.2.2.1	 Laboratory shaker—Lab-Line model 3589 (available 

through VWR Scientific cat. no. 57039-055), Pall 

Corporation part no. 4821, Fisher cat. no. 14260-11, or 

equivalent 

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

4 (available through VWR Scientific cat. no. 57039-045), 

Fisher cat. no. 14260-13, or equivalent 

6.2.3	 Filta-Max® foam filter equipment requirements (for use with the procedure described in 

Section 12.3). The version of the method using this filter was validated using 50-L 

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). 

December 2005	 4 
Method 1623 - C ryptosporidium and G iardia 

6.2.3.1	 Foam filter—Filta-Max®, IDEXX, Westbrook, ME. Filter module cat. 

no. FMC 10603 

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

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

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

using the batch or shipping filters to the field. 

6.2.3.2	 Filter processing equipment—Filta-Max® starter kit, IDEXX, 

Westbrook, ME, cat. no. FMC 11002. Starter kit includes manual wash 

station with clamp set (FMC 10101 or 10106) including plunger head 

(FMC 12001), tubing set (FMC 10307), vacuum set (FMC 10401), MKII 

filter housing with hose-tail fittings (FMC 10504) and green housing 

tools (FMC 10506). In addition, processing requires magnetic stirrer 

(FMC 10901) and filter membranes, 100 pk, (FMC 10800). 

6.2.4	 Portable Continuous-Flow Centrifuge (PCFC) requirements (for use with procedures 

described in Section 12.4). The version of the method using this technique was validated 

for Cryptosporidium in 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). Individual laboratories are also 

permitted to demonstrate acceptable performance for Giardia in their laboratory. The 

technique is based on technology from Haemonetics Corporation, Braintree, MA. 

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 after one use whenever possible or when wear is 

evident. 

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	 Pump— peristaltic, centrifugal, impeller, or diaphragm pump; MasterFlex I/P® 

EasyLoad® peristaltic pump (Cole-Parmer cat. No. EW-77963-10) with 77601-10 

pumphead, 77410-00 drive unit, and 06429-73 Tygon LFL tubing; Dayton, model 

number 3YU61 (available through Grainger), Jabsco Flexible Impeller Pump (ColeParmer cat. No. EW-75202-00); Simer, model number M40; or equivalent. It is 

recommended that the pump be placed on the effluent side of the filter, when possible, to 

reduce the risk of contamination and the amount of tubing replaced or cleaned. 

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

product no. WFU 10.110; Omega flow meter, Stamford, CT, model FTB4105; or 

equivalent. Alternatively, use a graduated carboy(s) (See Section 6.18) 

6.4 Equipment for spiking samples in the laboratory 

6.4.1 Collapsible 10-L LDPE cubitainer with cubitainer spigot—Cole Parmer cat. no. U06100-30 or equivalent and Cole Parmer cat. no. U-06061-01, or equivalent. Discard 

after one use to eliminate possible contamination. Alternatively, use clean, 10-L carboy 

5	 December 2005 
Method 1623 - C ryptosporidium and G iardia 

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. 14-513-66, or equivalent


6.4.3	 Stir plate—Fisher cat. no. 11-510-49S, S50461HP, or equivalent


6.4.4	 Hemacytometer—Neubauer type, Hausser Scientific, Horsham, PA, product no. 3200 or


1475, or equivalent


6.4.5	 Hemacytometer coverslip—Hausser Scientific, product 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-mm-


diameter, 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


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


MPC®-6, cat. No 120.02, or equivalent


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


120.09 (no longer available); Dynal MPC®-S, cat. no. 120.20, 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. Hoffman Modulation Contrast optics may be equivalent. 

December 2005	 6 
Method 1623 - C ryptosporidium and G iardia 

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

ZeissTM -

Axioskop

ZeissTM -IM35

OlympusTM BH

OlympusTM BX

OlympusTM 

IMT2

Fluoro-chrome

DAPI (UV)

DAPI (UV)

DAPI (UV)

DAPI (UV)

DAPI (UV)

Excitation 

filter (nm)

Dichroic 

beam- splitting 

mirror (nm) 

340-380 400

340-380 400

340-380 400

Filter holder

340-380 400

Filter holder

340-380 400

Filter holder

Barrier or 

suppression 

filter (nm)

420

420

420

420

420 

Chroma 

catalog 

number

CZ902

CZ702

11000

91002

11000

91008

11000

91003

6.10	 Ancillary equipment for microscopy 

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

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

6.10.2	 Glass coverslips—22 × 50 mm 

6.10.3	 Nonfluorescing immersion oil—Type FF, Cargille cat. no. 16212, or equivalent 

6.10.4	 Micropipette, adjustable: 0- to 10-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 

7	 December 2005 
Method 1623 - C ryptosporidium and G iardia 

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, Pall Corporation, 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 must 

purchase standards at the required normality directly from a vendor. Normality must 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. See Reference 20.8 (Section 9020) 

for reagent water requirements. 

7.4	 Reagents for eluting filters 

NOTE: Laboratories should store prepared eluting solution for no more than 1 week or 

when noticeably turbid, whichever comes sooner. 

7.4.1	 Reagents for eluting EnvirochekTM and EnvirochekTM HV 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. Alternatively, use prepared TRIS, Sigma 

T6066 or equivalent. 

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

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

of reagent water and adjust pH to 8.0 with 6.0 N 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. Alternatively, use prepared EDTA, Sigma E5134 or 

equivalent. 

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 

December 2005	 8 
Method 1623 - C ryptosporidium and G iardia 

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 Filta-Max® foam filters (Section 6.2.3) 

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

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

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

and 0.2 g KH2PO4. 

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

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

7.4.2.4	 Preparation of PBST elution buffer. Add 100 μL of Tween® 20 to 

prepared PBS (Section 7.4.2.1). Alternatively, add the contents of one 

packet 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. 

7.4.3	 Reagents for Portable Continuous-Flow Centrifuge (Section 6.2.4) 

7.4.3.1	 Sodium dodecyl sulfate—Sigma Chemical Co. cat. no. 71730 or 

equivalent 

7.4.3.2	 TWEEN 80— Sigma Chemical Co. cat. no. P1754 or equivalent 

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

7.4.3.4	 Preparation of concentrated elution buffer. Add above reagents to obtain 

a final concentration of 1% sodium dodecyl sulfate, 0.01% TWEEN 80, 

and 0.001% Antifoam A in concentrated sample volume of ~250mL 

7.5	 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 between 1°C 

and 10°C and return promptly to this temperature after each use. Do not allow any of the reagents 

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

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

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

7.6.1	 MeriFluor® Cryptosporidium/Giardia, Meridian Diagnostics cat. no. 250050, 

Cincinnati, OH, or equivalent 

7.6.2	 Aqua-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 

7.6.4	 EasyStainTMC&G, BTF Pty Limited, Sydney, Australia or equivalent 

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

demonstrate acceptable performance through an initial precision and recovery test 

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

each batch of slides stained using each product. However, the laboratory is not required 

to analyze additional ongoing precision and recovery samples or method blank samples 

for each type. The performance of each labeling reagent used also should be monitored in 

each source water type. 

7.6.5	 Diluent for labeling reagents—Phosphate buffered saline (PBS) (Section 7.4.2). 

9	 December 2005 
Method 1623 - Cryptosporidium and Giardia 

7.7	 4',6-diamidino-2-phenylindole (DAPI) stain—Sigma Chemical Co. cat. no. D9542, or equivalent 

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

consistent with minimum use. Store between 1°C and 10°C in the dark. Do not allow to 

freeze. Discard unused solution when positive staining control fails or after specified 

time determined by laboratory. 

7.7.2	 Staining solution—Follow antibody kit manufacturer’s instructions. Add 10 μL of 2 

mg/mL DAPI stock solution to 50 mL of PBS for use with Aqua-GloTM G/C Direct FL or 

MeriFluor® Cryptosporidium/Giardia. Add 50 μL of 2 mg/mL DAPI stock solution to 

50 mL of PBS for use with EasyStainTM. Prepare working solution daily and store 

between 1°C and 10°C (do not allow to freeze). DAPI is light sensitive; therefore, store 

in the dark except when staining. The DAPI concentration may be increased if 

fading/diffusion of DAPI staining is encountered, but the staining solution must be tested 

first on expendable environmental samples to confirm that staining intensity is 

appropriate. 

7.8	 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). 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® Cryptosporidium/Giardia, Meridian 

Diagnostics cat. no. 250050, or equivalent (Section 7.6.1) 

7.8.3	 Mounting medium supplied with Aqua-GloTM G/C Direct FL kit, Waterborne cat. no. 

A100FLR, cat. no. M101, or equivalent (Section 7.6.2) 

7.8.4	 Mounting medium supplied with EasyStainTMC&G, BTF Pty Limited or equivalent 

(Section 7.6.4) 

7.8.5	 Elvanol or equivalent permanent, non-fade archiving mounting medium 

7.9	 Clear fingernail polish or clear fixative, PGC Scientifics, Gaithersburg, MD, cat. no. 60-4890-00, 

or equivalent 

7.10	 Oocyst and cyst suspensions for spiking 

7.10.1 Enumerated spiking suspensions prepared by flow cytometer—not formalin fixed. 

7.10.1.1 Live, flow cytometer–sorted oocysts and cysts—Wisconsin State 

Laboratory of Hygiene Flow Cytometry Unit ([608] 224-6260), or 

equivalent 

7.10.1.2 Irradiated, flow cytometer–sorted oocysts and cysts—flow 

cytometer–sorted oocysts and cysts—BTF EasySeedTM 

(contact@btfbio.com), or equivalent 

7.10.2	 Materials for manual enumeration of spiking suspensions 

7.10.2.1 Purified Cryptosporidium oocyst stock suspension for manual 

enumeration—not formalin-fixed: Sterling Parasitology Laboratory, 

University of Arizona, Tucson, or equivalent 

7.10.2.2 Purified Giardia cyst stock suspension for manual enumeration—not 

formalin-fixed: Waterborne, Inc., New Orleans, LA; Hyperion Research, 

Medicine Hat, Alberta, Canada; or equivalent 

December 2005	 10 
Method 1623 - C ryptosporidium and G iardia 

7.10.2.3	 Tween® 20 , 0.01%—Dissolve 1.0 mL of a 10% solution of Tween® 20 

in 1 L of reagent water 

7.10.3 Storage procedure—Store oocyst and cyst suspensions between 1°C and 10°C, until 

ready to use; do not allow to freeze 

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

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

8.0 Sample Collection and Storage 

8.1 Sample collection, shipment, and receipt 

8.1.1	 Sample collection. Samples are collected as bulk samples and shipped to the laboratory 

on ice for processing through the entire method, or are filtered in the field and shipped to 

the laboratory on ice for processing from elution (Section 12.2.6) onward. 

8.1.2	 Sample shipment. Ambient water samples are dynamic environments and, depending on 

sample constituents and environmental conditions, Cryptosporidium oocysts or Giardia 

cysts present in a sample can degrade, potentially biasing analytical results. Samples 

should be chilled to reduce biological activity, and preserve the state of source water 

samples between collection and analysis. Samples analyzed by an off-site laboratory 

should be shipped on ice via overnight service on the day they are collected. 

NOTE: See transportation precautions in Section 5.5. 

8.1.2.1	 If samples are collected early in the day, chill samples by storing in a 

refrigerator between 1°C and 10°C or pre-icing the sample in a cooler. If 

the sample is pre-iced before shipping, replace with fresh ice immediately 

before shipment. 

8.1.2.2	 If samples are collected later in the day, these samples may be chilled 

overnight in a refrigerator between 1°C and 10°C. This should be 

considered for bulk water samples that will be shipped off-site, as this 

minimizes the potential for water samples collected during the summer to 

melt the ice in which they are packed and arrive at the laboratory at 

>20°C. 

8.1.2.3	 If samples are shipped after collection at >20°C with no chilling, the 

sample will not maintain the temperature during shipment at #20°C. 

8.1.2.4	 Public water systems shipping samples to off-site laboratories for analysis 

should include in the shipping container a means for monitoring the 

temperature of the sample during shipping to verify that the sample did 

not freeze or exceed 20°C. Suggested approaches for monitoring sample 

temperature during shipping are discussed in Section 8.1.4. 

8.1.3	 Sample receipt. Upon receipt, the laboratory must record the sample temperature. 

Samples that were not collected the same day they were received, and that are received at 

>20°C or frozen, or samples that the laboratory has determined exceeded >20°C or froze 

during shipment, must be rejected. After receipt, samples must be stored at the laboratory 

between 1°C and 10°C, and not frozen, until processed. 

8.1.4	 Suggestions on measuring sample temperature. Given the importance of maintaining 

sample temperatures for Cryptosporidium and Giardia determination, laboratories 

performing analyses using this method must establish acceptance criteria for receipt of 

samples transported to their laboratory. Several options are available to measure sample 

temperature upon receipt at the laboratory and, in some cases, during shipment: 

8.1.4.1	 Temperature sample. One option, for filtered samples only (not for 10-L 

bulk samples), is for the sampler to fill a small, inexpensive sample bottle 

11	 December 2005 
Method 1623 - Cryptosporidium and Giardia 

with water and pack this “temperature sample” next to the filtered 

sample. The temperature of this extra sample volume is measured upon 

receipt to estimate the temperature of the filter. Temperature sample 

bottles are not appropriate for use with bulk samples because of the 

potential effect that the difference in sample volume may have in 

temperature equilibration in the sample cooler. Example product: Cole 

Parmer cat. no. U-06252-20. 

8.1.4.2	 Thermometer vial. A similar option is to use a thermometer that is 

securely housed in a liquid-filled vial. Unlike temperature samples, the 

laboratory does not need to perform an additional step to monitor the 

temperature of the vial upon receipt, but instead just needs to read the 

thermometer. The thermometer vial is appropriate for use with filtered 

samples not bulk samples. Example product: Eagle-Picher Sentry 

Temperature Vial 3TR-40CS-F or 3TR-40CS. 

8.1.4.3	 iButton. Measures the sample temperature during shipment and upon 

receipt. An iButton is a small, waterproof device that contains a 

computer chip that can be programmed to record temperature at different 

time intervals. The information is then downloaded from the iButton 

onto a computer. The iButton should be placed in a temperature sample, 

rather than placed loose in the cooler, or attached to the sample container. 

This option is appropriate for use with both filtered and bulk samples. 

Information on Thermocron® iButtons is available from 

http://www.ibutton.com/. Distributors include http://www.pointsix.com/, 

http://www.rdsdistributing.com, and http://www.scigiene.com/. 

8.1.4.4	 Stick-on temperature strips. Another option is for the laboratory to 

apply a stick-on temperature strip to the outside of the sample container 

upon receipt at the laboratory. This option does not measure temperature 

as precisely as the other options, but provides an indication of sample 

temperature to verify that the sample temperature is acceptable. This 

option is appropriate for use with both filtered and bulk samples. Example 

product: Cole Parmer cat. no. U-90316-00. 

8.1.4.5	 Infrared thermometers. A final option is to measure the temperature of 

the surface of the sample container or filter using an infrared 

thermometer. The thermometer is pointed at the sample, and measures the 

temperature without coming in contact with the sample volume. This 

option is appropriate for use with both filtered and bulk samples. Example 

product: Cole Parmer cat. no. EW-39641-00. 

As with other laboratory equipment, all temperature measurement devices must be 

calibrated routinely to ensure accurate measurements. See the EPA Manual for the 

Certification of Laboratories Analyzing Drinking Water (Reference 20.9) for more 

information. 

8.2	 Sample holding times. Samples must be processed or examined within each of the holding times 

specified in Sections 8.2.1 through 8.2.4. Sample processing should be completed as soon as 

possible by the laboratory. The laboratory should complete sample filtration, elution, 

concentration, purification, and staining the day the sample is received wherever possible. 

However, the laboratory is permitted to split up the sample processing steps if processing a 

sample completely in one day is not possible. If this is necessary, sample processing can be halted 

after filtration, application of the purified sample onto the slide, or staining. Table 1, in Section 

21.0 provides a breakdown of the holding times for each set of steps. Sections 8.2.1 through 8.2.4 

provide descriptions of these holding times. 

December 2005	 12 
Method 1623 - Cryptosporidium and Giardia 

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

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

in the field). 

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

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

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

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

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

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

sample to the slide. 

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

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

examination and characterization should be performed immediately after staining is 

complete, laboratories have up to 168 hours (7 days) from the completion of sample 

staining to perform the examination and verification of samples. However, if 

fading/diffusion of FITC or DAPI staining is noticed, the laboratory must reduce this 

holding time. In addition the laboratory may adjust the concentration of the DAPI 

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

8.3 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. Manually enumerated spiking suspensions 

must be used within 24 hours of enumeration of the spiking suspension if the hemacytometer 

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

suspension to the slides if the well slide or membrane filter enumeration technique is used 

(Sections 11.3.5 and 11.3.6). Oocyst and cyst suspensions must be stored between 1°C and 10°C, 

until ready to use; do not allow to freeze. 

9.0 Quality Control 

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

program that addresses and documents data quality, instrument and equipment maintenance and 

performance, reagent quality and performance, analyst training and certification, and records 

storage and retrieval. General requirements and recommendations for QA and quality control 

(QC) procedures for microbiology laboratories are provided in References 20.8, 20.9, 20.10. The 

minimum analytical requirements of this program consist of an initial demonstration of laboratory 

capability (IDC) through performance of the initial precision and recovery (IPR) test (Section 

9.4), and ongoing demonstration of laboratory capability and method performance through the 

matrix spike (MS) test (Section 9.5.1), the method blank test (Section 9.6), the ongoing precision 

and recovery (OPR) test (Section 9.7), staining controls (Section 14.1 and 15.2.1), and analyst 

verification tests (Section 10.6). 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 frontend 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). 

13 December 2005 
Method 1623 - C ryptosporidium and G iardia 

However, the laboratory is permitted to modify the immunofluorescence assay 

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

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

Section 7.6). 

NOTE: Method modifications should be considered only to improve method 

performance, reduce cost, or reduce sample processing time. Method modifications that 

reduce cost or sample processing time, but that result in poorer method performance 

should not be used. 

9.1.2.1	 Method modification validation/equivalency demonstration requirements 

9.1.2.1.1	 Method modifications at a single laboratory. Each 

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

single laboratory, the laboratory must, at a minimum, 

validate the modification according to Tier 1 of EPA’s 

performance-based measurement system (PBMS) (Table 

2) to demonstrate that the modification produces results 

equivalent or superior to results produced by this method 

as written. Briefly, each time a modification is made to 

this method, the laboratory is required to demonstrate 

acceptable modified method performance through the 

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

acceptance criteria in Tables 3 and 4 in Section 21.0, and 

should be comparable to previous results using the 

unmodified procedure. Although not required, the 

laboratory also should perform a matrix spike/matrix 

spike duplicate (MS/MSD) test to demonstrate the 

performance of the modified method in at least one realworld 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. If the modified method involves 

changes that cannot be adequately evaluated through 

these tests, additional tests may be required to 

demonstrate acceptability. 

9.1.2.1.2	 Method modifications for nationwide approval. If the 

laboratory or a manufacturer seeks EPA approval of a 

method modification for nationwide use, the laboratory 

or manufacturer must, at a minimum, validate the 

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

2). Briefly, at least three laboratories must perform IPR 

tests (Section 9.4) and MS/MSD (Section 9.5) tests using 

the modified method, and all tests must meet the QC 

acceptance criteria specified in Tables 3 and 4 in Section 

21.0. Upon nationwide approval, laboratories electing to 

use the modified method still must demonstrate 

acceptable performance in their own laboratory according 

to the requirements in Section 9.1.2.1.1. If the modified 

method involves changes that cannot be adequately 

evaluated through these tests, additional tests may be 

required to demonstrate acceptability. 

9.1.2.2	 The laboratory is required to maintain records of modifications made to 

this method. These records include the following, at a minimum: 

December 2005	 14 
Method 1623 - Cryptosporidium and Giardia 

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)	 Sample numbers and other identifiers 

(b)	 Source of spiking suspensions, as well as lot 

number and date received (Section 7.10) 

(c)	 Spike enumeration date and time 

(d)	 All spiking suspension enumeration counts and 

calculations (Section 11.0) 

(e)	 Sample spiking dates and times 

(f)	 Volume filtered (Section 12.2.5.2) 

(g)	 Filtration and elution dates and times 

(h)	 Pellet volume, resuspended concentrate volume, 

resuspended concentrate volume transferred to 

IMS, and all calculations required to verify the 

percent of concentrate examined (Section 13.2) 

(i)	 Purification completion dates and times (Section 

13.3.3.11) 

(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. The frequency of the MS test is described in Section 9.1.8 and the 

procedures are described in Section 9.5.1. 

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

frequency of the analysis of method blanks is described in Section 9.1.7 and the 

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

15	 December 2005 
Method 1623 - Cryptosporidium and Giardia 

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. Frequency of 

OPR samples is described in Section 9.1.7 and the 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 9.7.6. 

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

(Section 9.7) each week (7 day or 168 hours time period which begins with processing 

the OPR) in 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 one week (7 day or 168 

hours) period. 

9.1.8	 The laboratory shall analyze MS samples (Section 9.5.1) at a minimum frequency of 1 

MS sample per 20 field samples from each source analyzed. The laboratory should 

analyze an MS sample when samples are first received from a PWS for which the 

laboratory has never before analyzed samples to identify potential method performance 

issues with the matrix (Section 9.5.1; Tables 3 and 4). If an MS sample cannot be 

analyzed on the first sampling event, the first MS sample should be analyzed as soon as 

possible to identify potential method performance issues with the matrix. 

9.2 Micropipette calibration 

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

qualified independent technician specializing in micropipette calibration can be used, or 

the calibration can be performed by the laboratory, provided the laboratory maintains a 

detailed procedure that can be evaluated by an independent auditor. Documentation on 

the precision of the recalibrated micropipette must be obtained from the manufacturer or 

technician. 

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

logbook. 

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

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

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

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

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

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

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

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

remains acceptable for use. 

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

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

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

the manufacturer for recalibration. 

9.3 Microscope adjustment and calibration —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, method 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, 

December 2005	 16 
Method 1623 - Cryptosporidium and Giardia 

and examine the four reagent water samples spiked with ~100-500 oocysts and ~100-500 

cysts. 

9.4.1.1	 The laboratory is permitted to analyze the four spiked reagent samples on 

the same day or on as many as four different days (provided that the 

spiked reagent samples are analyzed consecutively), and also may use 

different analysts and/or reagent lots for each sample (however, the 

procedures used for all analyses must be identical). Laboratories should 

note that the variability of four measurements performed on multiple days 

or using multiple analysts or reagent lots may be greater than the 

variability of measurements performed on the same day with the same 

analysts and reagent lots. As a result, the laboratory is at a greater risk of 

generating unacceptable IPR results if the test is performed across 

multiple days, analysts, and /or reagent lots. 

9.4.1.2	 If more than one modification will be used for filtration and/or separation 

of samples, a separate set of IPR samples must be prepared for each 

modification. 

9.4.1.3	 The set of four IPR samples must be accompanied by analysis of an 

acceptable method blank (Section 9.6). 

9.4.2	 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 counted


T = the number of oocysts or cysts spiked


This calculation assumes that the total volume spiked was processed and examined. 

9.4.3	 Using percent recovery (R) of the four analyses, calculate the mean percent recovery and 

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

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

9.4.4	 Compare the mean and RSD to the corresponding method performance acceptance 

criteria for initial precision and recovery in Tables 3 and 4 in Section 21.0. If the mean 

and RSD for recovery meet the acceptance criteria, system performance is acceptable 

and analysis of blanks and samples may begin. If the mean or RSD falls outside the range 

for recovery, system performance is unacceptable. In this event, trouble-shoot the 

problem by starting at the end of the method (see guidance in Section 9.7.5), correct the 

problem and repeat the IPR test (Section 9.4.1). 

9.4.5	 Examine and document the IPR slides following the procedure in Section 15.0. The first 

three Cryptosporidium oocysts and first three Giardia cysts identified in each IPR 

sample must be characterized (size, shape, DAPI category, and DIC category) and 

documented on the examination form, as well as any additional comments on organisms 

appearance, if notable. 

9.4.6	 Using 200X to 400X magnification, more than 50% of the oocysts or cysts must appear 

undamaged and morphologically intact; otherwise, the organisms in the spiking 

suspension may be of unacceptable quality or the analytical process may be damaging 

the organisms. If the quality of the organisms on the IPR test slides is unacceptable, 

examine the spiking suspension organisms directly (by centrifuging, if possible, to 

concentrate the organisms in a volume that can be applied directly to a slide). If the 

17	 December 2005 
Method 1623 - C ryptosporidium and G iardia 

unprocessed organisms appear undamaged and morphologically intact under DIC, 

determine the step or reagent that is causing damage to the organisms. Correct the 

problem (see Section 9.7.5) and repeat the IPR test. 

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

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

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

field sample must be that was collected from the same sampling location as split samples 

or as samples sequentially collected immediately after one another. The MS sample 

volume analyzed must be within 10% of the field sample volume. The MS shall be 

analyzed according to the frequency in Section 9.1.8. 

9.5.1.1	 Analyze an unspiked field sample according to the procedures in Sections 

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

spiking suspensions (Section 7.10.1 or Section 11.3), spike, filter, elute, 

concentrate, separate (purify), stain, and examine a second field sample 

aliquot with a similar number of organisms as that used in the IPR or 

OPR tests (Sections 9.4 and 9.7). 

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

equation. 

Nsp - Ns 

R = 100 x 

T 

N

where 

R is the percent recovery 

sp is the number of oocysts or cysts counted in the spiked sample 

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

sample 

T is the true value of the oocysts or cysts spiked 

9.5.1.3	 Compare the recovery for each organism with the acceptance criteria 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, the laboratory should calculate the mean percent recovery 

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

). Express the 

precision assessment as a percent recovery interval from P ! 2 s to P + 2 r

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 Tier 2 or 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. 

December 2005	 18 
Method 1623 - C ryptosporidium and G iardia 

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. 

(X

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

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

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

following equation: 

| NMS - NMSD |

RPD = 100 x 

XMEAN 

X

N

N

where 

RPD is the relative percent difference 

MS is the number of oocysts or cysts counted in the MS 

MSD is the number of oocysts or cysts counted in the MSD 

mean is the mean number of oocysts or cysts counted in the MS 

and MSD 

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

criteria 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 routinely 

analyzed to demonstrate freedom from contamination. Analyze the blank immediately after 

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

for the week to demonstrate freedom from contamination. 

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

method blank per week (Section 9.1.7) according to the procedures in Sections 12.0 to 

15.0. A method blank must be analyzed each week (7 day or 168 hours time period that 

begins with processing the OPR) in which samples are analyzed if 20 or fewer field 

samples are analyzed during this period. If more than 20 samples are analyzed in a week 

(7 days or 168 hours), process and analyze one reagent water method blank for every 20 

samples. 

9.6.2	 Actions 

9.6.2.1	 If Cryptosporidium oocysts, Giardia cysts, or potentially interfering 

organisms or materials that may be misidentified as oocysts or cysts are 

not found in the method blank, the method blank test is acceptable and 

analysis of samples may proceed. 

9.6.2.2	 If Cryptosporidium oocysts, Giardia cysts (as defined in Section 3), or 

any potentially interfering organism or materials that may be 

misidentified as oocysts or cysts are found in the method blank, the 

method blank test is unacceptable. Any field sample in a batch associated 

with an unacceptable method blank is assumed to be contaminated and 

should be recollected. Analysis of additional samples is halted until the 

source of contamination is eliminated, the method blank test is performed 

again, and no evidence of contamination is detected. 

9.7	 Ongoing precision and recovery (OPR; positive control sample; laboratory control 

sample)—Using the spiking procedure in Section 11.4 and enumerated spiking suspensions 

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

least one reagent water sample spiked with ~100 to 500 oocysts and ~100 to 500 cysts each week 

19	 December 2005 
Method 1623 - Cryptosporidium and Giardia 

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 should samples be analyzed. 

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

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

cysts must appear undamaged and morphologically intact; otherwise, the 

organisms in the spiking suspension may be of unacceptable quality or 

the analytical process may be damaging the organisms. Examine the 

spiking suspension organisms directly (by centrifuging, if possible, to 

concentrate the organisms in a volume that can be applied directly to a 

slide). If the organisms appear undamaged and morphologically intact 

under DIC, 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 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 acceptance criteria for ongoing precision and recovery in 

Tables 3 and 4 in Section 21.0. 

9.7.4	 Actions 

9.7.4.1 If the recoveries for Cryptosporidium and Giardia meet the acceptance 

criteria, system performance is acceptable and analysis of samples may 

proceed. 

9.7.4.2	 If the recovery for Cryptosporidium or Giardia falls outside of the 

criteria, system performance is unacceptable. Any sample in a batch 

associated with an unacceptable OPR sample is unacceptable. Analysis of 

additional samples is halted until the analytical system is brought under 

control. Troubleshoot the problem using the procedures at Section 9.7.5 

as a guide. After assessing the issue, perform another OPR test and verify 

that Cryptosporidium and Giardia recoveries meet the acceptance 

criteria. 

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

the laboratory generally should identify the problem working backward in the analytical 

process from the microscopic examination to filtration. 

December 2005	 20 
Method 1623 - Cryptosporidium and Giardia 

9.7.5.1 Quality of spiked organisms. Examine the spiking suspension organisms 

directly (by centrifuging, if possible, to concentrate the organisms in a 

volume that can be applied directly to a slide). If the organisms appear 

damaged under DIC, obtain fresh spiking materials. If the organisms 

appear undamaged and morphologically intact, determined whether the 

problem is associated with the microscope system or antibody stain 

(Section 9.7.5.2). 

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

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

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

check Köhler illumination, and check the fluorescence of the fluoresceinlabeled monoclonal antibodies (Mabs) and 4',6-diamidino-2-phenylindole 

(DAPI). If results are unacceptable, re-examine a previously-prepared 

positive staining control to determine whether the problem is associated 

with the microscope or the antibody stain. 

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

OPR test is attributable to the separation system, check system 

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

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

and examination procedures in Sections 13.3 through 15.0. Recoveries 

should be greater than 70%. 

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

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

performance by processing spiked reagent water according to the 

procedures in Section 12.2 through 13.2.2, and filter, stain, and examine 

the sample concentrate according to Section 11.3.6. 

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

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

continued laboratory performance. The laboratory should develop a statement of 

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

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

). Express the accuracy as 

a recovery interval from R ! 2 s to R + 2 sr

. For example, if R = 95% and sr r = 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. 

21 December 2005 
Method 1623 - C ryptosporidium and G iardia 

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 20.10) 

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. 

December 2005	 22 
Method 1623 - Cryptosporidium and Giardia 

10.3.2.7 

10.3.2.8 

10.3.2.9 

10.3.2.10 

10.3.2.11 

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.

Reattach the objective to the nosepiece.

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


house and the microscope.


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.

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 

23	 December 2005 
Method 1623 - Cryptosporidium and Giardia 

the left ocular by focusing the left ocular until it reads the 

same as the interpupillary distance. Bring an image 

located in the center of the field of view into as sharp a 

focus as possible. 

10.3.4.2.2 Transfer the card to between the left eye and ocular. 

Again keeping both eyes open, bring the same image into 

as sharp a focus for the right eye as possible by adjusting 

the ocular correction (focusing) collar at the top of the 

right ocular. 

10.3.4.3 Ocular adjustment for microscopes without binocular capability: This 

procedure assumes a single focusing ocular. The following procedure 

assumes that only the right ocular is capable of adjustment. 

10.3.4.3.1 Place a card between the right ocular and eye keeping 

both eyes open. Using the fine adjustment, focus the 

image for the left eye to its sharpest point. 

10.3.4.3.2 Transfer the card to between the left eye and ocular. 

Keeping both eyes open, bring the image for the right eye 

into sharp focus by adjusting the ocular collar at the top 

of the ocular without touching the 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 

December 2005 24 
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


x


ocular micrometer space


1,000 μm 12.5 μm 

= 

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

1000 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 focusing of the 

25 December 2005 
Method 1623 - Cryptosporidium and Giardia 

radiant field diaphragm leaves, open the radiant field diaphragm until the 

leaves just disappear from view. 

10.3.6.5	 The aperture diaphragm of the condenser should now be adjusted to make


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


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


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

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

10.3.6.6	 After completing the adjustment of the aperture diaphragm in the


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

required to establish DIC. 

10.4 Microscope cleaning procedure


10.4.1 Use canned air to remove dust from the lenses, filters, and microscope body.


10.4.2 Use a Kimwipe-dampened with a microscope cleaning solution (MCS) (consisting of 2


parts 90% isoproponal and 1 part acetone) to wipe down all surfaces of the microscope 

body. Dry off with a clean, dry Kimwipe. 

10.4.3 Protocol for cleaning oculars and condenser


10.4.3.1	 Use a new, clean Q-tip dampened with MCS to clean each lense. Start at


the center of the lens and spiral the Q-tip outward using little to no


pressure. Rotate the Q-tip head while spiraling to ensure a clean surface is 

always contacting the lens. 

10.4.3.2	 Repeat the procedure using a new, dry Q-tip. 


10.4.3.3	 Repeat Sections 10.4.3.1 and 10.4.3.2.


10.4.3.4	 Remove the ocular and repeat the cleaning procedure on the bottom lens


of the ocular.


10.4.4 Protocol for cleaning objective lenses


10.4.4.1	 Wipe 100X oil objective with lens paper to remove the bulk of the oil


from the objective.


10.4.4.2	 Hold a new Q-tip dampened with MCS at a 45° angle on the objective


and twirl.


10.4.4.3	 Repeat Sections 10.4.4.2 with a new, dry Q-tip.


10.4.4.4	 Repeat Sections 10.4.4.2 and 10.4.4.3.


10.4.4.5	 Clean all objectives whether they are used or not.


10.4.5 Protocol for cleaning light source lens and filters


10.4.5.1	 Using a Kimwipe dampened with microscope cleaning solution, wipe off


the surface of each lens and filter.


10.4.5.2	 Repeat the procedure using a dry Kimwipe.


10.4.5.3	 Repeat Sections 10.4.5.1 and 10.4.5.2.


10.4.6 Protocol for cleaning microscope stage


10.4.6.1	 Using a Kimwipe dampened with microscope cleaning solution, wipe off


the stage and stage clip. Be sure to clean off any residual immersion oil or


fingernail polish. Remove the stage clip if necessary to ensure that it is 

thoroughly cleaned. 

10.4.7 Use 409 and a paper towel to clean the bench top surrounding the microscope.


10.4.8 Frequency


10.4.8.1	 Perform Sections 10.4.2, 10.4.3, 10.4.4, 10.4.5 and 10.4.7 after each


December 2005	 26 
Method 1623 - Cryptosporidium and Giardia 

microscope session. 

10.4.8.2	 Perform complete cleaning each week. 

10.5	 Protozoa libraries: Each laboratory is encouraged to develop libraries of photographs and 

drawings for identification of protozoa. 

10.5.1 Take color photographs of Cryptosporidium oocysts and Giardia cysts by FA, 4',6-

diamidino-2-phenylindole (DAPI), and DIC that the analysts (Section 22.2) determine 

are accurate (Section 15.2). 

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

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

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

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

identification of positive or negative organisms. 

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

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

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

the analyst for identification and enumeration of oocysts and cysts. The goal of analyst 

verification is to encourage comparison and discussion among analysts to continually refine the 

consistency of characterizations between analysts. 

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

shall prepare a slide containing 40 to 200 oocysts and 40 to 200 cysts. More than 50% of 

the oocysts and cysts must be DAPI positive and undamaged under DIC. 

10.6.2	 Each analyst shall determine the total number of oocysts and cysts detected by FITC on 

the entire slide meeting the criteria in 10.6.1. For the same 10 oocysts and 10 cysts, each 

analyst shall determine the DAPI category (DAPI negative, DAPI positive internal 

intense blue and DAPI positive number of nuclei) and the DIC category (empty, 

containing amorphous structures, or containing identifiable internal structures) of each. 

The DAPI/DIC comparisons may be performed on the slide prepared in 10.6.1, OPR 

slide, MS slide, or a positive staining control slide. 

10.6.3	 Requirements for laboratories with multiple analysts 

10.6.3.1	 The total number of oocysts and cysts determined by each analyst 

(Section 10.6.2.) 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 10.6.1 to 10.6.2). It is 

recommended that the DAPI and DIC categorization of the same 10 

oocysts and 10 cysts occur with all analysts at the same time, i.e. each 

analyst determines the categorizations independently, then the differences 

in the DAPI and DIC categorizations among analysts are discussed and 

resolved, and these resolutions documented. Alternatively, organism 

coordinates may be recorded for each analyst to locate and categorize the 

organisms at different times. Differences among analysts must be 

discussed and resolved. 

10.6.3.2	 Document the date, name(s) of analyst(s), number of total oocysts and 

cysts, and DAPI and DIC categories determined by the analyst(s), 

whether the test was passed/failed and the results of attempts before the 

test was passed. 

10.6.3.3	 Only after an analyst has passed the criteria in Section 10.6.3, may 

oocysts and cysts in QC samples and field samples be identified and 

enumerated. 

27	 December 2005 
Method 1623 - Cryptosporidium and Giardia 

10.6.4 Laboratories with only one analyst should maintain a protozoa library (Section 10.5) and 

compare the results of the examinations performed in Sections 10.6.1 and 10.6.2 to 

photographs of oocysts and cysts and interfering organisms to verify that examination 

results are consistent with these references. These laboratories also should perform 

repetitive counts of a single verification slide for FITC. These laboratories should also 

coordinate with other laboratories to share slides and compare counts. 

11.0 Oocyst and Cyst Suspension Enumeration and Sample 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) and monitor method performance. EPA recommends that flow 

cytometry be used for this enumeration, rather than manual techniques. Flow cytometer–sorted 

spikes generally are characterized by a relative standard deviation of #2.5%, versus greater 

variability for manual enumeration techniques (Reference 20.11). 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 

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 fluorescence 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 

December 2005 28 
Method 1623 - Cryptosporidium and Giardia 

samples (Section 11.2.4). 

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

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

expiration date noted on the suspension. The holding time specified by the flow 

cytometry laboratory should be determined based on a holding time study. 

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

before purified Cryptosporidium oocyst and Giardia cyst stock suspensions (Section 7.10.2) 

received from suppliers can be used to spike samples in the laboratory. First, the stock suspension 

must be diluted and enumerated (Section 11.3.3) to yield a suspension at the appropriate oocyst or 

cyst concentration for spiking (spiking suspension). Then, 10 aliquots of spiking suspension must 

be enumerated to calculate a mean spike dose. Spiking suspensions can be enumerated using 

hemacytometer chamber counting (Section 11.3.4), well slide counting (Section 11.3.5), or 

membrane filter counting (Section 11.3.6). 

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

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

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

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 the objective. 

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

of the microscope, rather than through the microscope. 

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

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

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

29	 December 2005 
Method 1623 - C ryptosporidium and G iardia 

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 μL 

of suspension: 

number of 

organisms counted 10 dilution factor 1 mm3 number of organisms 

× × × = 

number of mm2 

counted 1 mm 1 1 μL μL 

11.3.3.10	 Record the result on a hemacytometer data sheet. 

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

and averaged for each suspension to achieve optimal counting accuracy. 

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

diluted to a final concentration of between 8 to 12 organisms per μL; 

however, ranges as great as 5 to 15 organisms per 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 11.3.5), then the 

stock suspensions should be diluted in reagent water. 

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

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

formula: 

required number of organisms 

volume of stock suspension (μL) required = 

number of organisms/ μL 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:L 

total volume (:L) =

predicted number of organisms per 10:L (8 to 12) 

To calculate the volume of reagent water (or reagent water/Tween® 20 , 

0.01%) needed, use the following formula: 

reagent water volume (:L) = total volume (:L) - stock suspension volume required (:L)

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,

December 2005	 30 
Method 1623 - C ryptosporidium and G iardia 

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: 

C Inadequate mixing of suspension before flooding the chamber 

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

on the chamber or coverslip 

C Total number of organisms counted is too low to provide 

statistical confidence in the result 

C Error in recording tally 

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

counted 

C Inadequate cleaning and removal of organisms from the previous 

count 

C 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 

31	 December 2005 
Method 1623 - C ryptosporidium and G iardia 

additional oocysts from stock suspension or dilute the contents of the 

beaker appropriately with reagent water. Repeat the process to confirm 

counts. Refer to Section 11.3.3.14 for factors that may introduce errors. 

11.3.5 Enumerating spiking suspensions using well slides 

NOTE: Spiking suspensions enumerated using well slides must be used within 24 hours 

of application of the spiking suspension to the slides. 

11.3.5.1	 Prepare well slides for sample screening and label the slides. 

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°C to 42°C also can 

be used. 

11.3.5.6	 Positive and negative controls must be prepared. 

11.3.5.6.1 For the positive control, pipette 10 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	 Follow the manufacturer’s instructions (Section 7.6) in applying the stain 

to the slide. 

11.3.5.8	 Place the slides in a humid chamber in the dark and incubate according to 

manufacturer’s directions. The humid chamber consists of a tightly sealed 

plastic container containing damp paper towels on top of which the slides 

are placed. 

11.3.5.9	 Apply one drop of wash buffer (prepared according to the manufacturer’s 

instructions [Section 7.6]) to each well. Tilt each slide on a clean paper 

towel, long edge down. Gently aspirate the excess detection reagent from 

below the well using a clean Pasteur pipette or absorb with a paper towel 

or other absorbent material. Avoid disturbing the sample. 

NOTE: If using the MeriFluor® Cryptosporidium/Giardia stain (Section 7.6.1), do not 

allow slides to dry completely. 

11.3.5.10	 Add mounting medium (Section 7.8) to each well. 

December 2005	 32 
Method 1623 - C ryptosporidium and G iardia 

11.3.5.11	 Apply a cover slip. Use a tissue to remove excess mounting fluid from the 

edges of the coverslip. Seal the edges of the coverslip onto the slide using 

clear nail polish. 

11.3.5.12	 Record the date and time that staining was completed. If slides will not be 

read immediately, store in a humid chamber in the dark between 1°C and 

10°C until ready for examination. 

11.3.5.13	 After examination of the 10 wells, calculate the mean, standard deviation, 

and RSD of the 10 replicates. Record the counts and the calculations on a 

spiking suspension enumeration form. The relative standard deviation 

(RSD) of the calculated mean spike dose must be #16% for 

Cryptosporidium and #19% for 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°C or 41°C for 

approximately 5 minutes to dry. 

11.3.6.2	 Place foil around the bottoms of the 100 × 15 mm petri dishes. 

11.3.6.3	 Filter-sterilize (Section 6.19) approximately 10 mL of PBS (Section 

7.4.2.1). Dilute detection reagent (Section 7.6) 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 foillined 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 6.4.8.4) the same way and position it on top of the cellulose33 December 2005 
Method 1623 - Cryptosporidium and Giardia 

11.3.6.8 

11.3.6.9 

11.3.6.10 

11.3.6.11 

11.3.6.12 

11.3.6.13 

11.3.6.14 

11.3.6.15 

11.3.6.16 

11.3.6.17 

11.3.6.18 

acetate support membrane. Carefully clamp the glass funnel to the loaded


filter support. Repeat for the other two filters.

Add 5 mL of 0.01% Tween® 20 to each of the three filtration units and


allow to stand.


Vortex the tube containing the spiking suspension (diluted stock


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


the tube three times.

Using a micropipettor, sequentially remove two, 10-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.

Pipet 100 mL of diluted antibody to the center of the bottom of a 60 × 15


mm petri dish for each sample.


Unclamp the top funnel and transfer each cellulose acetate support


membrane/ polycarbonate filter combination onto the drop of stain using


forceps (apply each membrane/filter combination to a different petri dish


containing stain). Roll the filter into the drop to exclude air. Place the


small petri dish containing the filter onto the damp towel and cover with


the corresponding labeled foil-covered top. Incubate for approximately 45


minutes at room temperature.

Reclamp the top funnels, apply vacuum and rinse each three times, each


time with 20 mL of reagent water.


Repeat Sections 11.3.6.4 through 11.3.6.10 for the next three samples (if


that the diluted spiking suspension has sat less than 15 minutes, reduce


the suspension vortex time to 60 seconds). Ten, 10-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.

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.

Label slides. After incubation is complete, for each sample, transfer the


cellulose acetate filter support and polycarbonate filter from drop of stain 

and place on fritted glass support. Cycle vacuum on and off briefly to 

remove excess fluid. Peel the top polycarbonate filter off the supporting 

filter and place on labeled slide. Discard cellulose acetate filter support. 

Mount and apply coverslips to the filters immediately to avoid drying.

To each slide, add 20 μL of mounting medium (Section 7.8). 

Apply a coverslip. Seal the edges of the coverslip onto the slide using 

clear nail polish. (Sealing may be delayed until cover slips are applied to 

all slides.) 

December 2005 34 
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 

between 1°C and 10°C until ready for examination. 

11.3.6.20	 After examination of the 10 slides, calculate the mean, standard 

deviation, and RSD of the 10 replicates. Record the counts and the 

calculations on a spiking suspension enumeration form. The relative 

standard deviation (RSD) of the calculated mean spike dose must be 

#16% for Cryptosporidium 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 disposable cubitainer or bottom-dispensing container to feed the filter or insert 

the influent end of the tube connected to the filter through the top of a carboy to allow 

siphoning of the sample. 

11.4.2	 For initial precision and recovery (Section 9.4) and ongoing precision and recovery 

(Section 9.7) samples, fill the container with 10 L of reagent water or a volume of 

reagent water equal to the volume of the field samples analyzed in the analytical batch. 

For matrix spike samples (Section 9.5), fill the container with the field sample to be 

spiked. Continuously mix the sample (using a stir bar and stir plate for smaller-volume 

samples and alternate means for larger-volume samples). 

11.4.3 Follow the procedures in Section 11.4.3.1 or manufacturer’s instructions for flow 

cytometer–enumerated suspensions and the procedures in Section 11.4.3.2 for manually 

enumerated suspensions. 

11.4.3.1	 For flow cytometer–enumerated suspensions (where the entire volume of 

a spiking suspension tube will be used): 

11.4.3.1.1	 Add 400 μL of Antifoam A to 100 mL of reagent water, 

and mix well to emulsify. 

11.4.3.1.2	 Add 500 μL of the diluted antifoam to the tube 

containing the spiking suspension and vortex for 30 

seconds. 

11.4.3.1.3	 Pour the suspension into the sample container. 

11.4.3.1.4	 Add 20 mL of reagent water to the empty tube, cap, 

vortex 10 seconds to rinse, and add the rinsate to the 

carboy. 

11.4.3.1.5	 Repeat this rinse using another 20 mL of reagent water. 

11.4.3.1.6	 Record the estimated number of organisms spiked, the 

date and time the sample was spiked, and the sample 

volume spiked on a bench sheet. Proceed to Section 

11.4.4. 

11.4.3.2	 For manually enumerated spiking suspensions: 

11.4.3.2.1	 Vortex the spiking suspension(s) (Section 11.2 or Section 

11.3) for a minimum of 30 seconds. 

11.4.3.2.2	 Rinse a pipette tip with 0.01% Tween® 20 once, then 

repeatedly pipette the well-mixed spiking suspension a 

35	 December 2005 
Method 1623 - C ryptosporidium and G iardia 

minimum of five times before withdrawing an aliquot to 

spike the sample. 

11.4.3.2.3 Add the spiking suspension(s) to the carboy, delivering 

the aliquot below the surface of the sample. 

11.4.3.2.4 Record the estimated number of organisms spiked, the 

date and time the sample was spiked, and the sample 

volume spiked on a bench sheet. Proceed to Section 

11.4.4 

11.4.4	 Allow the spiked sample 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 1-L PBST or 

reagent water to the 10-L carboy to rinse (5 L PBST or reagent water rinse to 50-L 

carboy). Swirl the contents to rinse down the sides. Additional rinses may be performed. 

11.4.7 Turn on the pump. Allow all of the water to flow through the filter and turn off the 

pump. 

11.4.8	 Proceed to filter disassembly. 

12.0 Sample Filtration and Elution 

12.1	 A water sample is filtered according to the procedures in Section 12.2, 12.3, or 12.4. 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 20.12). This procedure was validated using 10-L 

sample volumes (for the original Envirochek TM filter) and 50-L sample volumes (for the 

EnvirochekTM HV filter). Alternate sample volumes may be used, provided the laboratory 

demonstrates acceptable performance on initial and ongoing spiked reagent water and source 

water samples (Section 9.1.2). 

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	 Mix the sample well by shaking, add stir bar and place on stir plate. Turn 

on stir plate to lowest setting needed to keep sample thoroughly mixed. 

Connect the sampling system to the field carboy of sample water, or 

December 2005	 36 
Method 1623 - C ryptosporidium and G iardia 

transfer the sample water to the laboratory carboy used in Section 

12.2.1.1. If the sample will be filtered from a field carboy, a spigot 

(Section 6.2.1) can be used with the carboy to facilitate sample filtration. 

NOTE: If the bulk field sample is transferred to a laboratory carboy, the laboratory 

carboy must be cleaned and disinfected before it is used with another field sample. 

12.2.4.2	 Place the drain end of the sampling system tubing into an empty 

graduated container with a capacity of 10 to 15 L, calibrated at 9.0, 9.5, 

10.0, 10.5, and 11.0 L (Section 6.18). This container will be used to 

determine the sample volume filtered. Alternately, connect a flow meter 

(Section 6.3.4) downstream of the filter, and record the initial meter 

reading. 

12.2.4.3	 Allow the carboy discharge tube and capsule to fill with sample water by 

gravity. Vent residual air using the bleed valve/vent port, gently shaking 

or tapping the capsule, if necessary. Turn on the pump to start water 

flowing through the filter. Verify that the flow rate is 2 L/min. 

12.2.4.4	 After all of the sample has passed through the filter, turn off the pump. 

Allow the pressure to decrease until flow stops. (If the sample was 

filtered in the field, and excess sample remains in the filter capsule upon 

receipt in the laboratory, pull the remaining sample volume through the 

filter before eluting the filter [Section 12.2.6].) 

12.2.4.5	 Turn off stir plate; add 1 L PBST or reagent water rinse (to 10-L carboy) 

or 5 L PBST or reagent water rinse (to 50-L carboy). Swirl or shake the 

carboy to rinse down the side walls. 

12.2.4.6	 Reconnect to pump, turn on pump and allow pump to pull all water 

through filter; turn off pump. 

12.2.5 Disassembly 

12.2.5.1	 Disconnect the inlet end of the capsule filter assembly while maintaining 

the level of the inlet fitting above the level of the outlet fitting to prevent 

backwashing and the loss of oocysts and cysts from the filter. Restart the 

pump and allow as much water to drain as possible. Turn off the pump. 

12.2.5.2	 Based on the water level in the graduated container and 1⁄2-L hash marks 

or meter reading, record the volume filtered on the bench sheet to the 

nearest quarter liter. Discard the contents of the graduated container. 

12.2.5.3 Loosen the outlet fitting, then cap the inlet and outlet fittings. 

12.2.6 Elution 

NOTE: The laboratory must complete the elution, concentration, and purification 

(Sections 12.2.6 through 13.3.3.11) in one 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. 

37 December 2005
Method 1623 - Cryptosporidium and Giardia 

12.2.6.1.2	 Prepare sufficient quantity of elution buffer to elute all 

samples that are associated with the OPR/MB which used 

that batch of elution buffer. Elution may require up to 

275 mL of buffer per sample. 

12.2.6.1.3	 Designate at least one 250-mL conical centrifuge tube for 

each sample and label with the sample number. 

12.2.6.2 Elution 

12.2.6.2.1	 Record the elution date and time on the bench sheet. 

Using a ring stand or other means, clamp each capsule in 

a vertical position with the inlet end up. 

12.2.6.2.2	 Remove the inlet cap, pour elution buffer through the 

inlet fitting, and allow the liquid level to stabilize. 

Sufficient elution buffer must be added to cover the 

pleated white membrane with buffer solution or elution 

buffer may be measured to ensure the use of one 250-mL 

centrifuge tube. Replace the inlet cap. 

12.2.6.2.3	 Securely clamp the capsule in one of the clamps on the 

laboratory shaker with the bleed valve positioned at the 

top on a vertical axis (in the 12 o'clock position). Turn on 

the shaker and set the speed to maximum (approximately 

900 rpm or per manufacturer’s instructions). Agitate the 

capsule for approximately 5 minutes. Time the agitation 

using a lab timer, rather than the timer on the shaker to 

ensure accurate time measurement. 

12.2.6.2.4	 Remove the filter from the shaker, remove the inlet cap, 

and pour the contents of the capsule into the 250-mL 

conical centrifuge tube. 

12.2.6.2.5	 Clamp the capsule vertically with the inlet end up and 

add sufficient volume of elution buffer through the inlet 

fitting to cover the pleated membrane. Replace the inlet 

cap. 

12.2.6.2.6	 Return the capsule to the shaker with the bleed valve 

positioned at the 4 o'clock position. Turn on the shaker 

and agitate the capsule for approximately 5 minutes. 

12.2.6.2.7	 Remove the filter from the shaker, but leave the elution 

buffer in the capsule. Re-clamp the capsule to the shaker 

at the 8 o’clock position. Turn on the shaker and agitate 

the capsule for a final 5 minutes. 

12.2.6.2.8	 Remove the filter from the shaker and pour the contents 

into the 250-mL centrifuge tube. Rinse down the inside 

of the capsule filter walls with reagent water or elution 

buffer using a squirt bottle inserted in the inlet end of the 

capsule. Invert the capsule filter over the centrifuge tube 

and ensure that as much of the eluate as possible has been 

transferred. 

12.2.7 Proceed to Section 13.0 for concentration and separation (purification). 

12.3	 Sample filtration using the Filta-Max® foam filter. This procedure was validated using 50-L 

sample volumes. Alternate sample volumes may be used, provided the laboratory demonstrates 

December 2005	 38 
Method 1623 - C ryptosporidium and G iardia 

acceptable performance on initial and ongoing spiked reagent water and source water samples 

(Section 9.1.2). 

NOTE: The filtration procedures specified in Sections 12.3.1.2 - 12.3.1.6.3 are specific 

to laboratory filtration of a bulk sample. These procedures may require modification if 

samples will be filtered in the field. 

12.3.1 Filtration 

12.3.1.1	 Flow rate adjustment 

12.3.1.1.1	 Connect the sampling system, minus the filter housing, to 

a carboy filled with reagent water. 

12.3.1.1.2	 Place the peristaltic pump upstream of the filter housing. 

12.3.1.1.3	 Turn on the pump and adjust the flow rate to 1 to 4 L per 

minute. 

NOTE: A head pressure of 0.5 bar (7.5 psi) is required to create flow through the filter, 

and the recommended pressure of 5 bar (75 psi) should produce the flow rate of 3 to 4 L 

per minute. The maximum operating pressure of 8 bar (120 psi) should not be exceeded. 

12.3.1.1.4	 Allow 2 to 10 L of reagent water to flush the system. 

Adjust the pump speed as necessary during this period. 

Turn off the pump when the flow rate has been adjusted. 

12.3.1.2	 Place filter module into the filter housing bolt head down and secure lid, 

hand tighten housings, apply gentle pressure to create the seal between 

the module and the ‘O’ rings in the base and the lid of the housing. 

Excessive tightening is not necessary, and may shorten the life of the ‘O’ 

rings. Tools may be used to tighten housing to the alignment marks (refer 

to manufacturer’s instructions). ‘O’ rings should be lightly greased before 

use (refer to manufacturer’s instructions). 

12.3.1.3	 Install the filter housing in the line, securing the inlet and outlet ends with 

the appropriate clamps/fittings. Verify that the filter housing is installed 

so that the end closest to the screw top cap is the inlet and the opposite 

end is the outlet. 

12.3.1.4	 Record the sample number, sample turbidity (if not provided with the 

field sample), and the name of the analyst filtering the sample on a bench 

sheet. 

12.3.1.5	 Filtration 

12.3.1.5.1	 Connect the sampling system to the field carboy of 

sample water, or transfer the sample water to the 

laboratory carboy used in Section 12.3.1.1.1. If the 

sample will be filtered from a field carboy, a spigot can 

be used with the carboy to facilitate sample filtration. 

NOTE: If the bulk field sample is transferred to a laboratory carboy, the laboratory 

carboy must be cleaned and disinfected before it is used with another field sample. 

12.3.1.5.2
 Place the drain end of the sampling system tubing into an 

empty graduated container with a capacity greater than or 

equal to the volume to be filtered. This container will be 

used to determine the sample volume filtered. 

Alternately, connect a flow meter downstream of the 

filter, and record the initial meter reading. 

39	 December 2005 
Method 1623 - C ryptosporidium and G iardia 

12.3.1.5.3 Allow the carboy discharge tube and filter housing to fill 

12.3.1.5.4 

with sample water. Turn on the pump to start water 

flowing through the filter. Verify that the flow rate is 

between 1 and 4 L per min.

After all of the sample has passed through the filter, turn 

off the pump. Allow the pressure to decrease until flow 

12.3.1.6 Disassembly 

stops.

12.3.1.6.1 Disconnect the inlet end of the filter housing assembly 

12.3.1.6.2 

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.

Based on the water level in the graduated container or the

12.3.1.6.3 

meter reading, record the volume filtered on a bench 

sheet to the nearest quarter liter.

Loosen the outlet fitting, the filter housing should be 

sealed with rubber plugs. 

NOTE: Filters should be prevented from drying out, as this can impair their ability to 

expand when decompressed. 

12.3.2 Elution 

12.3.2.1	 The filter is eluted to wash the oocysts from the filter. This can be 

accomplished using the Filta-Max® wash station, which moves a plunger 

up and down a tube containing the filter and eluting solution (Section 

12.3.2.2), or a stomacher, which uses paddles to agitate the stomacher bag 

containing the foam filter in the eluting solution (Section 12.3.2.3). If the 

Filta-Max® automatic wash station is used please see the manufacturer’s 

operator’s guide for instructions on its use. If Filta-Max® Quick Connect 

kit is used please follow manufacturer’s instructions. 

12.3.2.2	 Filta-Max® wash station elution procedure 

12.3.2.2.1 First wash 

(a) Detach the removable plunger head using the tool 

provided, and remove the splash guard. 

(b) Place the filter membrane flat in the concentrator 

base with the rough side up. Locate the concentrator 

base in the jaws of the wash station and screw on the 

concentrator tube (the longer of the two tubes), 

creating a tight seal at the membrane. Take the 

assembled concentrator out of the jaws and place on 

the bench. 

(c) Replace the splash guard and temporarily secure it at 

least 15 cm above the end of the rack. Secure the 

plunger head with the tool provided ensuring that the 

lever is fully locked down. 

(d) Remove the filter module from the filter housing or 

transportation container. Pour excess liquid into the 

assembled concentrator, then rinse the housing or 

December 2005	 40 
Method 1623 - C ryptosporidium and G iardia 

container with PBST and add the rinse to the 

concentrator tube. Screw the filter module onto the 

base of the plunger. Locate the elution tube base in 

the jaws of the wash station and screw the elution 

tube (the shorter of the two tubes) firmly in place. 

(e) Pull the plunger down until the filter module sits at 

the bottom of the elution tube; the locking pin (at the 

top left of the wash station) should “click” to lock the 

plunger in position. 

(f) Remove the filter module bolt by turning the adapted 

allen key (provided) in a clockwise direction (as seen 

from above). Attach the steel tube to the elution tube 

base. 

(g) Add 600 mL of PBST to the assembled concentrator. 

If more than 50 mL of liquid has been recovered from 

the shipped filter module, reduce the volume of 

PBST accordingly. Screw the concentrator tube onto 

the base beneath the elution tube. Release the locking 

pin. 

NOTE: Gentle pressure on the lever, coupled with a pulling action on the locking pin 

should enable the pin to be easily released. 

(h) Wash the foam disks by moving the plunger up and 

down 20 times. Gentle movements of the plunger are 

recommended to avoid generating excess foam. 

NOTE: The plunger has an upper movement limit during the wash process to prevent it 

popping out of the top of the chamber. 

(i)	 Detach the concentrator and hold it such that the 

stainless steel tube is just above the level of the 

liquid. Purge the remaining liquid from the elution 

tube by moving the plunger up and down 5 times, 

then lock the plunger in place. To prevent drips, 

place the plug provided in the end of the steel tube. 

(j)	 Prior to the second wash the eluate from the first 

wash can be concentrated using the Filta-Max® 

apparatus according to Section 12.3.3.2.1 or the 

eluate can be decanted into a 2-L pooling beaker and 

set aside. 

12.3.2.2.2 Second wash 

(a) Add an additional 600 mL of PBST to the 

concentrator module, remove the plug from the end 

of the steel tube and screw the concentrator tube back 

onto the elution module base. Release the locking 

pin. 

(b) Wash the foam disks by moving the plunger up and 

down 10 times. Gentle movements of the plunger are 

recommended to avoid generating excess foam. 

41	 December 2005 
Method 1623 - Cryptosporidium and Giardia 

(c) The eluate can be concentrated using the Filta-Max® 

apparatus according to Section 12.3.3.2.2 or the 

eluate can be decanted into the 2-L pooling beaker 

containing the eluate from the first wash and 

concentrated using centrifugation, as described in 

Section 12.3.3.3. 

12.3.2.3	 Stomacher elution procedure 

12.3.2.3.1 First wash 

(a) Place the filter module in the stomacher bag then use 

the allen key to remove the bolt from the filter 

module, allowing the rings to expand. Remove the 

end caps from the stomacher bag and rinse with 

PBST into the stomacher bag. 

(b) Add 600 mL of PBST to stomacher bag containing 

the filter pads. Place bag in stomacher and wash for 5 

minutes on a normal setting. 

(c) Remove the bag from the stomacher and decant the 

eluate into a 2-L pooling beaker. 

12.3.2.3.2 Second wash 

(a) Add a second 600-mL aliquot of PBST to the 

stomacher bag. Place bag in stomacher and wash for 

5 minutes on a normal setting. Remove the bag from 

the stomacher and decant the eluate from the 

stomacher bag into the 2-L pooling beaker. Wring the 

stomacher bag by hand to remove eluate from the 

foam filter and add to the pooling beaker. Remove 

the foam filter from the bag and using a squirt bottle, 

rinse the stomacher bag with reagent water and add 

the rinse to the pooling beaker. 

(b) Proceed to concentration (Section 12.3.3). 

12.3.3 Concentration 

12.3.3.1	 The eluate can be concentrated using the Filta-Max® concentrator 

apparatus, which pulls most of the eluate through a membrane filter 

leaving the oocysts concentrated in a small volume of the remaining 

eluting solution (Section 12.3..2), or by directly centrifuging all of the 

eluting solution used to wash the filter (Section 12.3.2.3). 

12.3.3.2	 The Filta-Max® concentrator procedure 

12.3.3.2.1 Concentration of first wash 

(a) If the stomacher was used to elute the sample 

(Section 12.3.2.3), transfer 600 mL of eluate from the 

pooling beaker to the concentrator tube. Otherwise 

proceed to Step (b). 

(b) Stand the concentrator tube on a magnetic stirring 

plate and attach the lid (with magnetic stirrer bar). 

Connect the waste bottle trap and hand or electric 

vacuum pump to the valve on the concentrator base. 

Begin stirring and open the tap. Increase the vacuum 

using the hand pump. 

December 2005	 42 
Method 1623 - C ryptosporidium and G iardia 

NOTE: The force of the vacuum should not exceed 30 cmHg. 

12.3.3.2.2 

12.3.3.2.3 

(c) Allow the liquid to drain until it is approximately 

level with the middle of the stirrer bar then close the 

valve. Remove the magnetic stirrer, and rinse it with 

PBST or distilled water to recover all oocysts. Decant 

the concentrate into a 50-mL tube, then rinse the 

sides of the concentration tube and add the rinsate to 

the 50-mL tube.

Concentration of second wash 

(a) If the stomacher was used to elute the sample 

(Section 12.3.2.3), transfer the remaining 600 mL of 

eluate from the pooling beaker to the concentrator 

tube. Otherwise proceed to Step (b). 

(b) Add the concentrate, in the 50-mL tube, retained 

from the first concentration (Section 12.3.3.2.1 (c)) to 

the 600 mL of eluate from the second wash, then 

repeat concentration steps from Sections 12.3.3.2.1 

(b) and 12.3.3.2.1 (c). The final sample can be poured 

into the same 50-mL tube used to retain the first 

concentrate. Rinse the sides of the concentrator tube 

with PBST and add the rinse to the 50-mL tube. 

(c) Remove the magnetic stirrer. Insert the empty 

concentrator module into the jaws of the wash station 

and twist off the concentrator tube. 

(d) Transfer the membrane from the concentrator base to 

the bag provided using membrane forceps. 

Membrane elution. The membrane can be washed 

manually or using a stomacher: 

•	 Manual wash. Add 5 mL of PBST to the bag 

containing the membrane. Rub the surface of the 

membrane through the bag until the membrane 

appears clean. Using a pipette, transfer the eluate to a 

50-mL tube. Repeat the membrane wash with another 

5 mL of PBST and transfer the eluate to the 50-mL 

tube. (Optional: Perform a third wash using another 5 

mL of PBST, by hand-kneading an additional minute 

or placing the bag on a flat-headed vortexer and 

vortexing for one minute. Transfer the eluate to the 

50-mL tube.) 

NOTE: Mark the bag with an “X” to note which side of the membrane has the oocysts to 

encourage the hand-kneading to focus on the appropriate side of the membrane. 

•	 Stomacher wash. Add 5 mL of PBST to the bag 

containing the membrane. Place the bag containing 

the membrane into a small stomacher and stomach 

for 3 minutes. Using a pipette transfer the eluate to a 

50-mL tube. Repeat the wash two times using the 

stomacher and 5-mL aliquots of PBST. (Optional: 

43	 December 2005 
Method 1623 - C ryptosporidium and G iardia 

12.3.3.2.4


Perform a fourth wash using another 5 mL of PBST, 

by hand-kneading an additional minute or placing the 

bag on a flat-headed vortexer and vortexing for one 

minute. Transfer the eluate to the 50-mL tube.)

If the membrane filter clogs before concentration is 

complete, there are two possible options for completion 

of concentration. One option is replacing the membrane 

as often as necessary. Filter membranes may be placed 

smooth side up during the second concentration step. 

Another option is concentrating the remaining eluate 

using centrifugation. Both options are provided below. 

•	 Using multiple membranes. Disassemble the 

concentrator tube and pour any remaining eluate back 

into the pooling beaker. Remove the membrane using 

membrane forceps, placing it in the bag provided. 

Place a new membrane in the concentrator tube 

smooth side up, reassemble, return the eluate to the 

concentrator tube, rinse the pooling beaker and add 

rinse to the eluate, and continue the concentration. 

Replace the membrane as often as necessary. 

•	 Centrifuging remaining volume. Decant the 

remaining eluate into a 2-L pooling beaker. Rinse the 

sides of the concentrator tube and add to the pooling 

beaker. Remove the filter membrane and place it in 

the bag provided. Wash the membrane as described 

in Section 12.3.3.2.3, then concentrate the sample as 

described in Section 12.3.3.3.1. 

12.3.3.3	 If the Filta-Max® concentrator is not used for sample concentration, or if 

the membrane filter clogs before sample concentration is complete, then 

the procedures described in Section 12.3.3.3.1 should be used to 

concentrate the sample. If less than 50 mL of concentrate has been 

generated, the sample can be further concentrated, as described in Section 

12.3.3.3.2, to reduce the volume of sample to be processed through IMS. 

NOTE: The volume must not be reduced to less than 5 mL above the packed pellet. The 

maximum amount of pellet that should be processed through IMS is 0.5 mL. If the packed 

pellet is greater than 0.5 mL then the pellet may be subsampled as described in Section 

13.2.4. 

12.3.3.3.1 Centrifugation of greater than 50 mL of eluate 

(a) Decant the eluate from the 2-L pooling beaker into 

250-mL conical centrifuge tubes. Make sure that the 

centrifuge tubes are balanced. 

(b) Centrifuge the 250-mL centrifuge tubes containing 

the eluate at 1500 × G for 15 minutes. Allow the 

centrifuge to coast to a stop. 

(c) Using a Pasteur pipette, carefully aspirate off the 

supernatant to 5 mL above the pellet. If the sample is 

reagent water (e.g. initial or ongoing precision and 

recovery sample) extra care must be taken to avoid 

aspirating oocysts and cysts during this step. 

December 2005	 44 
Method 1623 - Cryptosporidium and Giardia 

(d) Vortex each 250-mL tube vigorously until pellet is 

completely resuspended. Swirl the centrifuge tube 

gently to reduce any foaming after vortexing. 

Combine the contents of each 250-mL centrifuge 

tube into a 50-mL centrifuge tube. Rinse each of the 

250-mL centrifuge tubes with PBST and add the 

rinse to the 50-mL tube. 

(e) Proceed to Section 12.3.3.3.2. 

12.3.3.3.2	 Centrifugation of less than 50 mL of eluate 

(a) Centrifuge the 50-mL centrifuge tube containing the 

combined concentrate at 1500 x G for 15 minutes. 

Allow the centrifuge to coast to a stop. Record the 

initial pellet volume (volume of solids) and the date 

and time that concentration was completed on a 

bench sheet. 

(b) Proceed to Section 13.0 for concentration and 

separation (purification). 

12.3.4 Maintenance and cleaning 

12.3.4.1 Maintenance of O-rings 

12.3.4.1.1	 Check all rubber O-rings for wear or deterioration prior 

to each use and replace as necessary. 

12.3.4.1.2	 Lubricate the plunger head O-ring inside and out with 

silicon before each use. 

12.3.4.1.3	 Lubricate all other O-rings (concentrator tube set, filter 

housing) regularly in order to preserve their condition. 

12.3.4.2 Cleaning 

12.3.4.2.1	 All components of the Filta-Max® system can be cleaned 

using warm water and laboratory detergent. After 

washing, rinse all components with oocyst and cyst free 

reagent water and dry them. All O-rings should be relubricated. Alternatively a mild (40°C) dishwasher cycle 

without bleach or rinse aid can be used. 

12.3.4.2.2	 To wash the detachable plunger head slide the locking 

pin out and wash the plunger head and locking pin in 

warm water and laboratory detergent. Rinse the plunger 

head and locking pin with oocyst and cyst free reagent 

water and dry. Lightly lubricate the locking pin and reassemble the plunger head. 

12.4	 Sample collection (filtration and concentration) using portable continuous-flow centrifugation. 

Please follow manufacturer’s instructions. This procedure was validated for the detection of 

Cryptosporidium using 50-L sample volumes. 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). Laboratories are permitted to demonstrate acceptable 

performance for Giardia in their individual laboratory. 

45	 December 2005 
Method 1623 - C ryptosporidium and G iardia 

13.0 Sample Concentration and Separation (Purification) 

13.1 During concentration and separation, the filter eluate is concentrated through centrifugation, and 

the oocysts and cysts in the sample are separated from other particulates through immunomagnetic 

separation (IMS). Alternate procedures and products may be used if the laboratory first 

demonstrates equivalent or superior performance as per Section 9.1.2. 

13.2 Adjustment of pellet volume 

13.2.1 Centrifuge the 250-mL centrifuge tube containing the capsule filter eluate at 1500 × 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 must be separated into 

multiple subsamples (a subsample is equivalent to no greater than 0.5 mL of packed 

pellet material, the recommended maximum amount of particulate material to process 

through the subsequent purification and examination steps in the method). Use the 

following formula to determine the total volume required in the centrifuge tube before 

separating the concentrate into two or more subsamples: 

pellet volume 

total volume (mL) required = x 5 mL 

0.5 mL

(For example, if the packed pellet volume is 1.2 mL, the total volume required is 12 mL.) 

Add reagent water to the centrifuge tube to bring the total volume to the level calculated 

above. 

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 

December 2005	 46 
Method 1623 - C ryptosporidium and G iardia 

13.2.4.1.2 

13.2.4.1.3 

subsamples would be 12 mL / 5 mL = 2.4, rounded = 3 subsamples).


Determine volume of resuspended concentrate per


subsample. Divide the total volume in the centrifuge


tube by the calculated number of subsamples (for


example, if the resuspended volume in Section 13.2.4 is


12 mL, then the volume to use for each subsample = 12


mL / 3 subsamples = 4 mL).

Process subsamples through IMS. Vortex the tube


vigorously for 10 to 15 seconds to completely resuspend


the pellet. Record the resuspended pellet volume on the


bench sheet. Proceed immediately to Section 13.3, and


transfer aliquots of the resuspended concentrate


equivalent to the volume in the previous step to multiple,


flat-sided sample tubes in Section 13.3.2.1. Process the


sample as multiple, independent subsamples from Section


13.3 onward, including the preparation and examination 

of separate slides for each aliquot. Record the volume of 

resuspended concentrate transferred to IMS on the bench 

sheet (this will be equal to the volume recorded in 

Section 13.2.4). Also record the number of subsamples 

processed independently through the method on the 

bench sheet.

13.2.4.2 Analysis of partial sample. If not all of the concentrate will be 

examined, vortex the tube vigorously for 10 to 15 seconds to completely 

resuspend the pellet. Record the resuspended pellet volume on the bench 

sheet. Proceed immediately to Section 13.3, and transfer one or more 5­

mL aliquots of the resuspended concentrate to one or more flat-sided 

sample tubes in Section 13.3.2.1. Record the volume of resuspended 

concentrate transferred to IMS on the bench sheet. To determine the 

volume analyzed, calculate the percent of the concentrate examined using 

the following formula: 

total volume of resuspended concentrate transferred to IMS 

percent examined = x 100% 

total volume of resuspended concentrate in Section 13.2.4 

Then multiply the volume filtered (Section 12.2.5.2) by this percentage to 

determine the volume analyzed. 

13.3 IMS procedure (adapted from Reference 20.13) 

NOTE: The IMS procedure should be performed on a bench top with all materials at 

room temperature, ranging from 15°C to 25°C. 

13.3.1 Preparation and addition of reagents 

13.3.1.1	 Prepare a 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, mix 100 

μL of 10X SL-buffer-A and 0.9 mL 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. 

47	 December 2005 
Method 1623 - Cryptosporidium and Giardia 

13.3.1.2 For each 10mL 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-buffers. If all of the concentrate is 

used, rinse the centrifuge tube twice with reagent water and add the 

rinsate to the flat-sided tube containing the concentrate (or to the tube 

containing the first subsample, if multiple subsamples will be processed). 

Each of the two rinses should be half the volume needed to bring the total 

volume in the flat-sided sample tube to 12 mL (including the buffers 

added in Sections 13.3.1.2 and 13.3.1.3). (For example, if the tube 

contained 1 mL of SL-buffer-A and 1 mL of SL-buffer-B, and 5 mL of 

sample was transferred after resuspension of the pellet, for a total of 7 

mL, the centrifuge tube would be rinsed twice with 2.5 mL of reagent 

water to bring the total volume in the flat-sided tube to 12 mL.) Visually 

inspect the centrifuge tube after completing the transfer to ensure that no 

concentrate remains. If multiple subsamples will be processed, bring the 

volume in the remaining flat-sided tubes to 12 mL with reagent water. 

Label the flat-sided tube(s) with the sample number (and subsample 

letters). 

13.3.2.2	 Vortex the Dynabeads®Crypto-Combo vial from the IMS kit for 

approximately 10 seconds to suspend the beads. Ensure that the beads are 

fully resuspended by 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-buffers. 

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-buffers. 

13.3.2.6	 Affix the sample tube(s) to a rotating mixer and rotate at approximately 

18 rpm for 1 hour at room temperature. 

13.3.2.7	 After rotating for 1 hour, remove each sample tube from the mixer and 

place the tube in the magnetic particle concentrator (MPC®-1 or MPC®-

6) with flat side of the tube toward the magnet. 

13.3.2.8 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 

90°, tilting the cap-end and base-end of the tube up and down in turn. 

December 2005	 48 
Method 1623 - C ryptosporidium and G iardia 

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, remove the flat-sided tube from the MPC®-1, shake the tube to 

resuspend all material, replace the sample tube in the MPC®-1 and repeat 

Section 13.3.2.9 before continuing to Section 13.3.2.11. 

13.3.2.11	 Return the MPC®-1 to the upright position, sample tube vertical, with 

cap at top. Immediately remove the cap and, keeping the flat side of the 

tube on top, pour off all of the supernatant from the tube held in the 

MPC®-1 into a suitable container. Do not shake the tube and do not 

remove the tube from MPC®-1 during this step. Allow more supernatant 

to settle; aspirate additional supernatant with pipette. 

13.3.2.12 Remove the sample tube from the MPC®-1 and resuspend the sample in 

0.5 mL 1X SL-buffer-A (prepared from 10X SL-buffer-A 

stock—supplied). Mix very gently to resuspend all material in the tube. 

Do not vortex. 

13.3.2.13	 Quantitatively transfer (transfer followed by two rinses) all the liquid 

from the sample tube to a labeled, 1.5-mL microcentrifuge tube. Use 0.5 

mL of 1X SL-buffer-A to perform the first rinse and 0.5 mL of 1X SLbuffer-A for the second rinse. 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 or MPC®-S), with its magnetic strip in place. 

13.3.2.15	 Without removing the microcentrifuge tube from MPC®-M, gently 

rock/roll the tube through 180° by hand. Continue for approximately 1 

minute with approximately one 180° roll/rock per second. At the end of 

this step, the beads should produce a distinct brown dot at the back of the 

tube. 

13.3.2.16	 Immediately aspirate the supernatant from the tube and cap held in the 

MPC®-M. If more than one sample is being processed, conduct three 90° 

rock/roll actions before removing the supernatant from each tube. Take 

care not to disturb the material attached to the wall of the tube adjacent to 

the magnet. Do not shake the tube. Do not remove the tube from MPC®-

M while conducting these steps. 

13.3.3 Dissociation of beads/oocyst/cyst complex 

NOTE: Two acid dissociations are required. 

13.3.3.1	 Remove the magnetic strip from the MPC®-M. 

13.3.3.2	 Add 50 μL of 0.1 N HCl, then vortex at the highest setting for 

approximately 50 seconds. 

NOTE: The laboratory must use 0.1-N standards purchased directly from a vendor, 

rather than adjusting the normality in-house. 

49	 December 2005 
Method 1623 - C ryptosporidium and G iardia 

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). 

NOTE: The laboratory must use 1.0-N standards purchased directly from a vendor 

rather than adjusting the normality in-house. 

13.3.3.9	 Without removing the microcentrifuge tube from the MPC®-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: The wells on Dynal Spot-On slides are likely to be too small to accommodate the 

volumes from both dissociations. 

13.3.3.11	 Record the date and time the purified sample was applied to the slide(s). 

13.3.3.12	 Air-dry the sample on the well slide(s). Because temperature and 

humidity vary from laboratory to laboratory, no minimum time is 

specified. However, the laboratory must take care to ensure that the 

sample has dried completely before staining to prevent losses during the 

rinse steps. A slide warmer set at 35°C to 42°C also can be used. 

13.3.4 Tips for minimizing carry-over of debris onto microscope slides after IMS 

•	 Make sure the resuspended pellet is fully homogenized before placing the tube in the 

MPC®-1 or MPC®-M to avoid trapping "clumps" or a dirty layer between the beads 

and the side of the tube. 

•	 When using the MPC®-1 magnet, make sure that the tube is snugged flat against the 

magnet. Push the tube flat if necessary. Sometimes the magnet is not flush with the 

outside of the holder and, therefore, the attraction between the beads and the magnet 

is not as strong as it should be. However, it can be difficult to determine this if you do 

not have more than one MPC®-1 to make comparisons. 

•	 After the supernatant has been poured off at Section 13.3.2.11, leave the tube in the 

MPC®-1 and allow time for any supernatant remaining in the tube to settle down to 

the bottom. Then aspirate the settled supernatant and associated particles from the 

bottom of the tube. The same can be done at Section 13.3.2.16 with the 

microcentrifuge tube. 

December 2005	 50 
Method 1623 - C ryptosporidium and G iardia 

•	 An additional rinse can also be performed at Section 13.3.2.11. After the supernatant 

has been poured off and any settled material is aspirated off the bottom, leave the tube 

in the MPC®-1 and add an additional 10 mL of reagent water or PBS to the tube and 

repeat Sections 13.3.2.9 and 13.3.2.11. Although labs have reported successfully 

using this technique to reduce carryover, because the attraction between the MPC®-1 

and the beads is not as great as the attraction between the MPC®-M and the beads, 

the chances would be greater for loss of cysts and oocysts doing the rinse at this step 

instead of at Section 13.3.2.16. 

•	 After the supernatant has been aspirated from the tube at Section 13.3.2.16, add 0.1 

mL of PBS, remove the tube from the MPC®-M, and resuspend. Repeat Sections 

13.3.2.15 and 13.3.2.16. 

•	 Use a slide with the largest diameter well available to spread out the sample as much 

as possible. 

14.0 Sample Staining 

NOTE: The sample must be stained within 72 hours of application of the purified sample 

to the slide. 

14.1	 Prepare positive and negative controls. 

14.1.1	 For the positive control, pipette 10 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 PBS (Section 7.4.2.1) 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). 

NOTE: If the laboratory has a large batch of slides that will be examined over several 

days, and is concerned that a single positive control may fade, due to multiple 

examinations, the laboratory should prepare multiple control slides with the batch of field 

slides and alternate between the positive controls when performing the positive control 

check. 

14.2	 Follow manufacturer’s instructions in applying stain to slides. 

14.3	 Place the slides in a humid chamber in the dark and incubate at room temperature for 

approximately 30 minutes. The humid chamber consists of a tightly sealed plastic container 

containing damp paper towels on top of which the slides are placed. 

14.4	 Remove slides from humid chamber and allow condensation to evaporate, if present. 

14.5	 Apply one drop of wash buffer (prepared according to the manufacturer’s instructions [Section 

7.6]) to each well. Tilt each slide on a clean paper towel, long edge down. Gently aspirate the 

excess detection reagent from below the well using a clean Pasteur pipette or absorb with paper 

towel or other absorbent material placed at edge of slide. Avoid disturbing the sample. 

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. 

51	 December 2005 
Method 1623 - C ryptosporidium and G iardia 

NOTE: If using the MeriFluor® Cryptosporidium/Giardia (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 between 1°C and 10°C 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 should be 

performed immediately after staining is complete, laboratories have up to 168 hours (7 

days) from completion of sample staining to complete the examination and verification of 

samples. However, if fading/diffusion of FITC or DAPI fluorescence is noticed, the 

laboratory must reduce this holding time. In addition the laboratory may adjust the 

concentration of the DAPI staining solution (Sections 7.7.2) 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 characterization (DAPI and DIC) and size measurements must be determined 

using 1000X magnification and reported to the nearest 0.5 μm. 

Record examination results for Cryptosporidium oocysts on a Cryptosporidium examination form; 

record examination results for Giardia cysts on a Giardia examination results form. All 

organisms that meet the criteria specified in Sections 15.2.2 and 15.2.3, less atypical organisms 

specifically identified as non-target organisms by DIC or DAPI (e.g. possessing spikes, stalks, 

appendages, pores, one or two large nuclei filling the cell, red fluorescing chloroplasts, crystals, 

spores, etc), must be reported. 

15.2.1	 Positive and negative staining control. Positive and negative staining controls must be 

acceptable before proceeding with examination of field sample slides. 

15.2.1.1	 Each analyst must characterize a minimum of three Cryptosporidium 

oocysts and three Giardia cysts on the positive staining control slide 

before examining field sample slides. This characterization must be 

performed by each analyst during each microscope examination session. 

FITC examination must be conducted at a minimum of 200X total 

magnification, DAPI examination must be conducted at a minimum of 

400X, and DIC examination and size measurements must be conducted at 

a minimum of 1000X. Size, shape, and DIC and DAPI characteristics of 

three Cryptosporidium oocysts and three Giardia cysts must be recorded 

by the analyst on a microscope log. The analyst also must indicate on 

each sample examination form whether the positive staining control was 

acceptable. 

December 2005	 52 
Method 1623 - Cryptosporidium and Giardia 

15.2.1.2 Examine the negative staining control to confirm that it does not contain 

any oocysts or cysts (Section 14.1). Indicate on each sample examination 

form whether the negative staining control was acceptable. 

15.2.1.3	 If the positive staining control contains oocysts and cysts within the 

expected range and at the appropriate fluorescence for both FA and 

DAPI, and the negative staining control does not contain any oocysts or 

cysts (Section 14.1), proceed to Sections 15.2.2 and 15.2.3. 

15.2.2 Sample examination—Cryptosporidium 

15.2.2.1	 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 apple-green 

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) at 1000X. 

15.2.2.2 DAPI fluorescence examination (the analyst must use a minimum of 

400X total magnification). Using the UV filter block for DAPI, the 

object will exhibit one of the following characteristics: 

(a) Light blue internal staining (no distinct nuclei) with a green rim 

(b) Intense blue internal staining 

(c) Up to four distinct, sky-blue nuclei 

Look for atypical DAPI fluorescence, e.g., more than four stained nuclei, 

size of stained nuclei, and wall structure and color. 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 [oil immersion lens]). Using DIC, look for external or 

internal morphological characteristics atypical of Cryptosporidium 

oocysts (e.g., spikes, stalks, appendages, pores, one or two large nuclei 

filling the cell, red fluorescing chloroplasts, crystals, spores, etc.) 

(adapted from Reference 20.10). If atypical structures are not observed, 

then categorize each apple-green fluorescing object as: 

(a) An empty Cryptosporidium oocyst 

(b) A Cryptosporidium oocyst with amorphous structure 

(c) A Cryptosporidium oocyst with internal structure (one to four 

sporozoites/oocyst) 

Using 1000X total magnification, record the shape, measurements (to the 

nearest 0.5 μm), and number of sporozoites (if applicable) for each applegreen fluorescing object meeting the size and shape characteristics. 

Although not a defining characteristic, surface oocyst folds may be 

observed in some specimens. 

15.2.2.4	 A positive result is a Cryptosporidium oocyst which exhibits typical IFA 

fluorescence, typical size and shape and exhibits nothing atypical on IFA, 

DAPI fluorescence, or DIC microscopy. A positive result must be 

characterized and assigned to one of the DAPI and DIC categories in 

Sections 15.2.2.2 and 15.2.2.3. 

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 ovoid 

53	 December 2005 
Method 1623 - Cryptosporidium and Giardia 

objects (8 - 18 μm long by 5 - 15 μm wide) are observed with brightly 

highlighted edges, increase magnification to 400X and switch the 

microscope to the UV filter block for DAPI (Section 15.2.3.2) then to 

DIC (Section 15.2.3.3) at 1000X. 

15.2.3.2 DAPI fluorescence examination (the analyst must use a minimum of 

400X total magnification). Using the UV filter block for DAPI, the object 

will exhibit one or more of the following characteristics: 

(a) Light blue internal staining (no distinct nuclei) and a green rim 

(b) Intense blue internal staining 

(c) Two to four sky-blue nuclei 

Look for atypical DAPI fluorescence, e.g., more than four stained nuclei, 

size of stained nuclei, and wall structure and color. Record cysts in 

category (a) as DAPI negative; record cysts in categories (b) and (c) as 

DAPI positive. 

15.2.3.3 DIC examination (the analyst must use a minimum of 1000X total 

magnification [oil immersion lens]). Using DIC microscopy, look for 

external or internal morphological characteristics atypical of Giardia 

cysts (e.g., spikes, stalks, appendages, pores, one or two large nuclei 

filling the cell, red fluorescing chloroplasts, crystals, spores, etc.) 

(adapted from Reference 20.10). If atypical structures are not observed, 

then categorize each object meeting the criteria specified in Sections 

15.2.3.1 through 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. 

15.2.3.4 A positive result is a Giardia cyst which exhibits typical IFA 

fluorescence, typical size and shape and exhibits nothing atypical on IFA, 

DAPI fluorescence, or DIC microscopy. A positive result must be 

characterized and assigned to one of the DAPI and DIC categories in 

Section 15.2.3.2 and 15.2.3.3. 

15.2.4 Record the date and time that sample examination was completed on the examination 

form. 

15.2.5 Report Cryptosporidium and Giardia concentrations as oocysts/L and cysts/L, 

respectively. 

15.2.6 Record analyst name 

16.0 Analysis of Complex Samples 

16.1 Some samples may contain high levels (>1000/L) of oocysts and cysts and/or interfering 

organisms, substances, or materials. Some samples may clog the filter (Section 12.0); others will 

not allow separation of the oocysts and cysts from the retentate or eluate; and others may contain 

materials that preclude or confuse microscopic examination. 

December 2005 54 
Method 1623 - C ryptosporidium and G iardia 

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. 

16.4 Some samples may adhere to the centrifuge tube walls. The use of siliconized or low-adhesion 

centrifuge tubes (Fisherbrand siliconized/low retention microcentrifuge tubes, 02-681-320 or 

equivalent) may reduce adhesion. Alternately, rinse centrifuge tubes with PBST elution buffer or 

Sigmacote® prior to use. 

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 20.11). 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 20.14). 

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

to be met. An assessment of the distribution of MS recoveries across 430 MS samples 

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

18.0 Pollution Prevention 

18.1 The solutions and reagents used in this method pose little threat to the environment when recycled 

and managed properly. 

18.2 Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize 

the volume of expired materials that need to be discarded. 

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 Morgan-Ryan, UM, A. Fall, L.A. Ward, N. Hijjawi, I. Sulaiman, R. Fayer, R.C.Thompson, M. 

Olson, A. Lal, L. Xiao. 2002. Cryptosporidium hominis n. sp. (Apicomplexa: Cryptosporidiidae 

from Homo sapiens). Journal Eukaryot Microbiol 49(6):433-450. 

20.2 Adam, R.D. 2001. Biology of Giardia lamblia. Clinical Microbiology Review 14(3):447-475. 

55 December 2005 
Method 1623 - Cryptosporidium and Giardia 

20.3	 Rodgers, Mark R., Flanigan, Debbie J., and Jakubowski, Walter, 1995. Applied and 

Environmental Microbiology 61 (10), 3759-3763. 

20.4	 Fleming, Diane O., et al.(eds.), Laboratory Safety: Principles and Practices, 2nd edition.1995. 

ASM Press, Washington, DC 

20.5	 "Working with Carcinogens," DHEW, PHS, CDC, NIOSH, Publication 77-206, (1977). 

20.6	 "OSHA Safety and Health Standards, General Industry," OSHA 2206, 29 CFR 1910 (1976). 

20.7	 "Safety in Academic Chemistry Laboratories," ACS Committee on Chemical Safety (1979). 

20.8	 APHA, AWWA, and WEF. 2005. Standard Methods for the Examination of Water and 

Wastewater; 21th Edition. American Public Health Association, American Water Works 

Association, Washington, D.C. 

20.9	 USEPA 2005. Manual for the Certification of Laboratories Analyzing Drinking Water; Criteria 

and Procedures; Quality Assurance. Fifth Edition. EPA 815-R-05-004. Office of Ground Water 

and Drinking Water, U.S. Environmental Protection Agency, 26 West Martin Luther King Drive, 

Cincinnati, OH 45268. 

20.10	 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). 

20.11	 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. 

20.12	 "EnvirochekTM Sampling Capsule," PN 32915, Gelman Sciences, 600 South Wagner Road, Ann 

Arbor, MI 48103-9019 (1996). 

20.13	 "Dynabeads® GC-Combo,” Dynal Microbiology R&D, P.O. Box 8146 Dep., 0212 Oslo, Norway 

(September 1998, Revision no. 01). 

20.14	 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). 

20.15	 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). 

20.16	 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. 

December 2005	 56 
Method 1623 - Cryptosporidium and Giardia 

21.0 Tables and Figures 

Table 1. Method Holding Times (See Section 8.2 for details) 

Sample Processing Step Maximum Allowable Time between Breaks 

(Samples should be processed as soon as possible) 

Collection 

Filtration 

' Up to 96 hours are permitted between sample collection (if shipped to the laboratory as a bulk 

sample) or filtration (if filtered in the field) and initiation of elution 

Elution 

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 

57 December 2005 
Method 1623 - C ryptosporidium and G iardia 

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 

20.15). The matrix spike acceptance criteria are based on data generated through 

interlaboratory validation of Method 1623 (Reference 20.14). 

December 2005	 58 
Method 1623 - Cryptosporidium and Giardia 

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 

9.4.3 

9.4.3 

24 - 100 

55 

Ongoing precision and recovery (percent) 9.7 11 - 100 

Matrix spike/matrix spike duplicate (for method modifications) 9.5 

Mean recovery1, 2 (as percent) 9.5.2.2 13 - 111 

Precision (as maximum relative percent difference) 9.5.2.3 61 

(1) The acceptance criteria for mean MS/MSD recovery serves as the acceptance criteria for MS recovery during 

routine use of the method (Section 9.5.1). 

(2) Some sample matrices may prevent the acceptance criteria from being met. An assessment of the distribution of 

MS recoveries from multiple MS samples from 87 sites during the ICR Supplemental Surveys is provided in Table 

5. 

Table 4. Quality Control Acceptance Criteria for Giardia 

Performance test Section Acceptance criteria 

Initial precision and recovery 

Mean recovery (percent) 

Precision (as maximum relative standard deviation) 

9.4 

9.4.3 

9.4.3 

24 - 100 

49 

Ongoing precision and recovery (percent) 9.7 14 - 100 

Matrix spike/matrix spike duplicate (for method modifications) 9.5 

Mean recovery1,2 (as percent) 9.5.2.2 15 - 118 

Precision (as maximum relative percent difference) 9.5.2.3 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. 

59 December 2005 
Method 1623 - Cryptosporidium and Giardia 

Table 5. Distribution of Matrix Spike Recoveries from Multiple Samples Collected from 87 Source Waters 

During the ICR Supplemental Surveys (Adapted from Reference 20.16) 

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% 

December 2005 60 
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) 

61 December 2005 
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). 

December 2005 62 
Method 1623 - Cryptosporidium and Giardia 

Figure 3a. Filtration Systems for EnvirochekTM or 

EnvirochekTMHV Capsule (unpressurized source - top, 

pressurized source - bottom) 

63 December 2005 
Method 1623 - Cryptosporidium and Giardia 

Figure 3b. Filtration Systems for Filta-Max® filters (unpressurized 

source - top, pressurized source - bottom)

December 2005 64 
Method 1623 - Cryptosporidium and Giardia 

Figure 4. Methods for Scanning a Well Slide 

65 December 2005 
Method 1623 - Cryptosporidium and Giardia 

22.0 Glossary of Definitions and Purposes 

These definitions and purposes are specific to this method but have been conformed to common 

usage as much as possible. 

22.1 Units of weight and measure and their abbreviations 

22.1.1 Symbols


°C degrees Celsius


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 

MCS microscope cleaning solution 

mg milligram 

mL milliliter 

mm millimeter 

mM millimolar 

N normal; gram molecular weight of solute divided by hydrogen equivalent of 

solute, per liter of solution 

RSD relative standard deviation 

sr standard deviation of recovery


X mean percent recovery


22.2 Definitions, acronyms, and abbreviations (in alphabetical order) 

Analyst—The analyst should have at least 2 years of college in microbiology or equivalent or 

closely related field. The analyst also should have a minimum of 6 months of continuous bench 

experience with Cryptosporidium and IFA microscopy. The analyst should have a minimum of 3 

months experience using EPA Method 1622 and/or EPA Method 1623 and should have 

successfully analyzed a minimum of 50 samples using EPA Method 1622 and/or EPA Method 

1623. 

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. 

December 2005 66 
Method 1623 - Cryptosporidium and Giardia 

Flow cytometer—A particle-sorting instrument capable of counting protozoa. 

Immunomagnetic separation (IMS)—A purification procedure that uses microscopic, magnetically 

responsive particles coated with an antibodies targeted to react with a specific pathogen in a fluid 

stream. Pathogens are selectively removed from other debris using a magnetic field. 

Initial precision and recovery (IPR)—Four aliquots of spiking suspension analyzed to establish 

the ability to generate acceptable precision and accuracy. An IPR is performed prior to the first 

time this method is used and any time the method or instrumentation is modified. 

Laboratory blank—See Method blank 

Laboratory control sample (LCS)—See Ongoing precision and recovery (OPR) standard 

Matrix spike (MS)—A sample prepared by adding a known quantity of organisms to a specified 

amount of sample matrix for which an independent estimate of target analyte concentration is 

available. A matrix spike is used to determine the effect of the matrix on a method’s recovery 

efficiency. 

May—This action, activity, or procedural step is neither required nor prohibited. 

May not—This action, activity, or procedural step is prohibited. 

Median bodies—Prominent, dark-staining, paired organelles consisting of microtubules and found 

in the posterior half of Giardia. In G. intestinalis (from humans), these structures often have a 

claw-hammer shape, while in G. muris (from mice), the median bodies are round. 

Method blank—An aliquot of reagent water that is treated exactly as a sample, including exposure 

to all glassware, equipment, solvents, and procedures that are used with samples. The method 

blank is used to determine if analytes or interferences are present in the laboratory environment, 

the reagents, or the apparatus. 

Must—This action, activity, or procedural step is required. 

Negative control—See Method blank 

Nucleus—A membrane-bound organelle containing genetic material. Nuclei are a prominent 

internal structure seen both in Cryptosporidium oocysts and Giardia cysts. In Cryptosporidium 

oocysts, there is one nucleus per sporozoite. One to four nuclei can be seen in Giardia cysts. 

Oocyst—The encysted zygote of some sporozoa; e.g., Cryptosporidium. The oocyst is a phase or 

form of the organism produced as a normal part of the life cycle of the organism. It is 

characterized by a thick and environmentally resistant outer wall. 

Ongoing precision and recovery (OPR) standard—A method blank spiked with known quantities 

of analytes. The OPR is analyzed exactly like a sample. Its purpose is to assure that the results 

produced by the laboratory remain within the limits specified in this method for precision and 

recovery. 

Oocyst and cyst spiking suspension—See Spiking suspension 

Oocyst and cyst stock suspension—See Stock suspension 

67 December 2005 
Method 1623 - Cryptosporidium and Giardia 

Positive control—See Ongoing precision and recovery standard 

Principal analyst—The principal analyst (may not be applicable to all monitoring programs) 

should have a BS/BA in microbiology or closely related field and a minimum of 1 year of 

continuous bench experience with Cryptosporidium and IFA microscopy. The principal analyst 

also should have a minimum of 6 months experience using EPA Method 1622 and/or EPA 

Method 1623 and should have analyzed a minimum of 100 samples using EPA Method 1622 

and/or EPA Method 1623. 

PTFE—Polytetrafluoroethylene 

Quantitative transfer—The process of transferring a solution from one container to another using 

a pipette in which as much solution as possible is transferred, followed by rinsing of the walls of 

the source container with a small volume of rinsing solution (e.g., reagent water, buffer, etc.), 

followed by transfer of the rinsing solution, followed by a second rinse and transfer. 

Reagent water—Water demonstrated to be free from the analytes of interest and potentially 

interfering substances at the method detection limit for the analyte. 

Reagent water blank—see Method blank 

Relative standard deviation (RSD)—The standard deviation divided by the mean times 100. 

RSD—See Relative standard deviation 

Should—This action, activity, or procedural step is suggested but not required. 

Spiking suspension—Diluted stock suspension containing the organism(s) of interest at a 

concentration appropriate for spiking samples. 

Sporozoite—A motile, infective stage of certain protozoans; e.g., Cryptosporidium. There are four 

sporozoites in each Cryptosporidium oocyst, and they are generally banana-shaped. 

Stock suspension—A concentrated suspension containing the organism(s) of interest that is 

obtained from a source that will attest to the host source, purity, authenticity, and viability of the 

organism(s). 

Technician—The technician filters samples, performs centrifugation, elution, concentration, and 

purification using IMS, and places purified samples on slides for microscopic examination, but 

does not perform microscopic protozoan detection and identification. No minimum education or 

experience requirements with Cryptosporidium and IFA microscopy apply to the technician. The 

technician should have at least 3 months of experience in filter extraction and processing of 

protozoa samples by EPA Method 1622/1623 and should have successfully processed a minimum 

of 50 samples using EPA Method 1622/1623. 

December 2005 68