______________________________________________________________________________________ 

MICROBIAL LABORATORY GUIDANCE MANUAL 

FOR THE FINAL LONG TERM 2 ENHANCED 

SURFACE WATER TREATMENT RULE 

Office of Water (4601M) 

 EPA 815-R06-006 

 February 2006 
U.S. Environmental Protection Agency 

Office of Water (4601M) 

1200 Pennsylvania Avenue NW 

Washington, DC 20460 

EPA 815-R-06-006 

http://www.epa.gov/safewater/disinfection/lt2/compliance.html 

February 2006 

 Printed on Recycled Paper 
Disclaimer 

The Standards and Risk Management Division, of the Office of Ground Water and Drinking Water, has 

reviewed and approved this guidance for publication. Neither the United States Government nor any of its 

employees, contractors, or their employees make any warranty, expressed or implied, or assumes any 

legal liability or responsibility for any third party’s use of or the results of such use of any information, 

apparatus, product, or process discussed in this report, or represents that its use by such party would not 

infringe on privately owned rights. Mention of trade names or commercial products does not constitute 

endorsement or recommendation for use. 

Contact for technical inquiries regarding Method 1623 and laboratory approval: 

 Carrie Moulton 

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 

Contact for technical inquires regarding sampling and analysis for E. coli: 

 Jennifer Best 

U.S. Environmental Protection Agency

Office of Ground Water and Drinking Water 

Technical Support Center, MC 140 

26 West Martin Luther King Drive 

Cincinnati, OH 45268 

 513-569-7012

 513-569-7191 (fax) 

best.jennifer@epa.gov 

All other inquiries should be addressed to: 

 Sean Conley 

U.S. Environmental Protection Agency

Office of Ground Water and Drinking Water 

Mail Code 4607M 

USEPA Headquarters, EPA East 

1200 Pennsylvania Ave., NW 

 Washington, DC 20460 

 202-564-1781

 202-564-3767 (fax) 

conley.sean@epa.gov 
TABLE OF CONTENTS 

Section 1: Introduction............................................................................................................................... 1

1.1 Background ................................................................................................................................................2

1.2 LT2 Rule Microbial Monitoring Requirements..........................................................................................2

1.3 Use of Cryptosporidium Data.....................................................................................................................4

1.3.1 Cryptosporidium Monitoring Sample Data...........................................................................................4

1.3.2 Cryptosporidium Matrix Spike Data.....................................................................................................5

1.4 Use of E. coli and Turbidity Data...............................................................................................................6

Section 2: General Microbial Laboratory Quality Assurance ................................................................ 7

2.1 Quality Assurance Plans .............................................................................................................................7

2.2 Sample Temperature Monitoring..............................................................................................................10

Section 3: Guidance for Cryptosporidium Laboratories ........................................................................ 13

3.1 LT2 Rule Cryptosporidium Sample Analysis Requirements....................................................................13

3.1.1 Approved Laboratories .......................................................................................................................13

3.1.2 Revised Cryptosporidium Method ......................................................................................................13

3.1.3 Minimum Sample Volume Analysis Requirements............................................................................14

3.1.4 Spiking Suspensions Requirements for Spiked Quality Control Samples ..........................................15

3.1.5 Acceptable Sample Results.................................................................................................................15

3.1.6 Cryptosporidium Oocyst Counts to Report.........................................................................................15

3.2 Laboratory Quality Assurance Evaluation Program .................................................................................16

3.2.1 Application .........................................................................................................................................16

3.2.2 Personnel Qualifications and Training................................................................................................17

3.2.3 Initial Proficiency Testing ..................................................................................................................18

3.2.4 On-Site Evaluation..............................................................................................................................19

3.2.5 Approval and Ongoing Proficiency Testing .......................................................................................19

3.2.6 Additional Training.............................................................................................................................20

3.2.7 Notifying Utilities of Change in Laboratory Status ............................................................................20

3.3 Cryptosporidium Method Quality Control ...............................................................................................20

3.3.1 Cryptosporidium Spiking Materials....................................................................................................22

3.3.2 Initial Precision and Recovery Test ....................................................................................................23

3.3.3 Method Blank Test .............................................................................................................................24

3.3.4 Ongoing Precision and Recovery Test................................................................................................24

3.3.5 Ongoing Precision and Recovery Control Charts ...............................................................................25

3.3.6 Quality Control Batches......................................................................................................................26

3.3.7 Holding Time Requirements...............................................................................................................26

3.3.8 Staining Controls ................................................................................................................................27

3.3.9 Examination Preparation.....................................................................................................................28

3.3.10 Verification of Analyst Performance .................................................................................................28

3.3.11 Acceptance Criteria for Receipt of Field Samples ..............................................................................29

3.3.12 Matrix Spike Samples .........................................................................................................................29

3.3.13 QC Guidance for Method Modifications and Use of Multiple Method Variations.............................31

3.3.14 Guidance on QC for Different Sample Volumes ................................................................................34

3.4 Sample Collection Procedures..................................................................................................................34

3.5 Recordkeeping..........................................................................................................................................36

3.6 Calculations for EPA Methods 1622/1623 ...............................................................................................39

3.6.1 Calculating Oocyst Concentrations.....................................................................................................39

3.6.2 Matrix Spike Recovery Calculations ..................................................................................................40

3.6.3 OPR Sample Calculations...................................................................................................................42

3.7 Electronic Data Reporting ........................................................................................................................42

3.7.1 Data Entry/Upload ..............................................................................................................................43

3.7.2 PWS Data Review ..............................................................................................................................44

i February 2006 
3.7.3 EPA/State Review ..............................................................................................................................45

3.8 Data Archiving .........................................................................................................................................45

3.8.1 Hardcopy Data ....................................................................................................................................45

3.8.2 Slides ..................................................................................................................................................45

3.9 Equipment, Supplies, Reagents, and Standards ........................................................................................46

Section 4: Guidance for E. coli Laboratories ......................................................................................... 47

4.1 Laboratory Certification Program.............................................................................................................48

4.2 Summary of LT2 Rule E. coli Methods....................................................................................................49

4.2.1 Most Probable Number (MPN) Methods............................................................................................49

4.2.2 Membrane Filtration (MF) Methods ...................................................................................................50

4.3 Quality Assurance/Quality Control for E. coli Analyses..........................................................................51

4.3.1 Quality Control Specifications Applicable to LT2 Samples ...............................................................52

4.3.2 Quality Control Specifications for E. coli Methods............................................................................52

4.4 Sample Collection Procedures..................................................................................................................53

4.5 Sample Volume and Dilution Guidance ...................................................................................................54

4.5.1 Sample Volume and Dilution Guidance for Multiple-Well Methods .................................................54

4.5.2 Sample Volume and Dilution Guidance for Multiple-Tube Methods.................................................54

4.5.3 Sample Volume and Dilution Guidance for Membrane Filtration......................................................54

4.6 E. coli Data Recording and Calculations ..................................................................................................55

4.6.1 Multiple-Well Data .............................................................................................................................56

4.6.2 Membrane Filtration Data...................................................................................................................58

4.6.3 Multiple-Tube Data.............................................................................................................................62

4.7 Electronic Data Reporting ........................................................................................................................65

4.7.1 Data Entry/Upload ..............................................................................................................................65

4.7.2 PWS Data Review ..............................................................................................................................66

4.7.3 EPA/State Review ..............................................................................................................................66

4.8 Data Archiving .........................................................................................................................................67

Section 5: References................................................................................................................................ 69

ii February 2006 
TABLES 

Table 1-1. Summary of LT2 Rule Monitoring Requirements .................................................................................3

Table 1-2. Bin Classifications for Filtered Systems................................................................................................4

Table 3-1. Suggested Laboratory QA Program Personnel Qualifications.............................................................17

Table 3-2. Summary of Routine QC Requirements ..............................................................................................22

Table 3-3. Method 1622/1623 Holding Times ......................................................................................................27

Table 3-4. Recommended QC for Different Types of Changes to EPA Method 1622/1623 ................................32

Table 3-5. Sample Receipt Data Elements to Record in the Laboratory...............................................................37

Table 3-6. Primary Data Elements to Record for Calculations .............................................................................38

Table 3-7. Key QC Data Elements to Record .......................................................................................................38

Table 3-8. LT2/Stage 2 Data Collection and Tracking System Data Entry, Review, and Transfer ......................43

Table 4-1. Approved E. coli Methods for LT2 Rule .............................................................................................48

Table 4-2. Incubation Time and Temperature Specifications for MPN Methods .................................................53

Table 4-3. Incubation Time and Temperature Specifications for Membrane Filter Methods ...............................53

Table 4-4. Recommended Minimum Record for Quanti-tray 2000® Colilert® and Colilert-18® Analyses .......56

Table 4-5. Recommended Data to Record for Quanti-Tray® Colilert® and Colilert-18® Analyses ...................57

Table 4-6. Recommended Data Elements for Record for Membrane Filtration Analyses ....................................59

Table 4-7. Recommended Data Elements to Record for 15-Tube MPN Methods (Colilert®) ............................62

Table 4-8. Recommended Data Elements to Record for 15-Tube Fermentation Methods ...................................63

Table 4-9. Examples of Different Combinations of Positive Tubes......................................................................64

FIGURES 

Figure 3-1. Process for Receiving and Monitoring Laboratory Approval ..............................................................21

Figure 3-2. Ongoing Precision and Recovery Control Chart Example...................................................................25

iii February 2006 
APPENDICES

Appendix A................................................................................................. Laboratory Biosafety Guidelines 

Appendix B ........................................................ EPA Method 1622 for Cryptosporidium (December 2005) 

Appendix C .................................... EPA Method 1623 for Cryptosporidium and Giardia (December 2005) 

Appendix D..............................Cryptosporidium Sample Results Acceptability Checklist for the LT2 Rule 

Appendix E .............................................................. Cryptosporidium Laboratory QA Program Application 

Appendix F ........................................................Cryptosporidium Laboratory QA Program Audit Checklist 

Appendix G......................................................................................... EPA Method 1622/1623 Bench Sheet 

Appendix H..........................................EPA Method 1622/1623 Cryptosporidium Slide Examination Form

iv February 2006 
ACRONYMS 

CFU Colony-forming unit 

CNFG Confluent growth 

DAPI 4, 6-diamidino-2-phenylindole 

DIC Differential interference contrast 

DCTS LT2/Stage 2 Data Collection and Tracking System 

EPA United States Environmental Protection Agency 

FA Fluorescence assay 

FITC Fluorescein isothiocyanate 

GWUDI Ground water under the direct influence [of surface water] 

ICR Information Collection Rule 

IDC Initial demonstration of capability 

IESWTR Interim Enhanced Surface Water Treatment Rule 

IFA Immunofluorescence assay 

IMS Immunomagnetic separation 

IPR Initial precision and recovery 

IPT Initial proficiency testing 

L Liter 

LT2 Rule Long Term 2 Enhanced Surface Water Treatment Rule 

LT2ESWTR Long Term 2 Enhanced Surface Water Treatment Rule 

mL Milliliter 

MPC Magnetic particle concentrator 

MPN Most probable number 

MS Matrix spike 

MS/MSD Matrix spike/matrix spike duplicate 

Fm Micrometer 

NA-MUG Nutrient agar (NA) with 4-methylumbelliferyl-beta-D-glucuronide (MUG) 

NELAC National Environmental Laboratory Accreditation Conference 

nm Nanometer 

NPDWR National Primary Drinking Water Regulations 

NTU Nephelometric turbidity unit 

OPR Ongoing precision and recovery 

OPT Ongoing proficiency testing 

PBMS Performance-based measurement system 

PT Proficiency testing 

PWS Public water system 

QA Quality assurance 

QAP Quality assurance plan 

QC Quality control 

RSD Relative standard deviation 

SDWA Safe Drinking Water Act 

SOP Standard operating procedure 

TNTC Too numerous to count 

UV Ultraviolet 

v February 2006 
SECTION 1: INTRODUCTION

The Long Term 2 Enhanced Surface Water Treatment Rule (LT2ESWTR or LT2 Rule; Reference 5.1) 

requires public water systems (PWSs) that use surface water or groundwater under the direct influence of 

surface water to monitor their source water (influent water prior to treatment plant) for Cryptosporidium, 

E. coli, and turbidity for a limited period [40 CFR § 141.701(a) and (c-h)]. In support of the monitoring 

requirements specified by the rule, three documents have been developed to provide guidance to the 

affected PWSs and the laboratories that support them: 

• Source Water Monitoring Guidance Manual for Public Water Systems for the Long Term 2 

Enhanced Surface Water Treatment Rule (LT2 Rule). This guidance manual for PWSs affected by 

the rule provides information on laboratory contracting, sample collection procedures, and data 

evaluation and interpretation. This guidance manual also provides information on grandfathering 

requirements for Cryptosporidium and E. coli data. 

• Microbial Laboratory Guidance Manual for the Long Term 2 Enhanced Surface Water Treatment 

Rule (LT2 Rule) (this document). The goal of this manual is to provide Cryptosporidium and E. 

coli laboratories analyzing samples in support of the LT2 Rule with guidance and detailed 

procedures for all aspects of microbial analyses under the rule to maximize data quality and 

consistency. 

• Users’ Manual for the LT2/Stage 2 Data Collection and Tracking System (DCTS). This manual 

provides PWSs and laboratories with instructions on the entry, review, and approval of electronic 

data using the LT2/Stage 2 DCTS, and for generating reports using the system. 

All of these manuals, as well as responses to frequently asked questions and an online microscopy module 

to assist analysts with identification of Cryptosporidium oocysts, are available at 

http://www.epa.gov/safewater/disinfection/lt2/compliance. 

This guidance document is provided to help implement the LT2 Rule. This guidance document does not, 

however, substitute for the LT2 Rule or the analytical methods approved for use under the rule. The 

material presented is intended solely for guidance and does not alter any regulatory or analytical method 

requirements not altered by the LT2 Rule itself. 

Sections 1 and 2 of the microbial laboratory LT2 manual provide LT2 background information and 

guidance on issues that apply to both Cryptosporidium and E. coli laboratories. Section 3 provides 

guidance specific to Cryptosporidium analyses for the LT2 Rule, and Section 4 provides guidance for E. 

coli analyses performed in support of the LT2 Rule. 

1 February 2006 
Section 1: Introduction 

1.1 Background 

The LT2 Rule is a National Primary Drinking Water Regulation that requires monitoring, reporting, and 

public notification for all PWSs that use surface water sources. The rule requires additional treatment 

techniques for some systems, based on Cryptosporidium monitoring results and the treatment currently 

provided (40 CFR § 141.711 ― 141.712). The LT2 Rule was developed to improve control of microbial 

pathogens in drinking water, including specifically the protozoan Cryptosporidium, and to address risk 

trade-offs between control of microbial pathogens and control of disinfection byproducts. 

1.2 LT2 Rule Microbial Monitoring Requirements 

Filtered PWSs serving a population of at least 10,000 people, and some filtered wholesale PWSs serving 

populations less than 10,000 people, are required to collect and analyze source water samples for 

Cryptosporidium, E. coli and turbidity for a minimum of 2 years [40 CFR § 141.701(a)(1)]. Unfiltered 

PWSs serving a population of at least 10,000 people, and some unfiltered wholesale PWSs serving 

populations less than 10,000 people, are required to collect and analyze source water samples for 

Cryptosporidium for a minimum of 2 years [40 CFR § 141.701(a)(2)]. Small, filtered systems (PWSs that 

serve fewer than 10,000 people) are required to monitor their source water for E. coli for a minimum of 1 

year. A subset of small filtered systems would then be required to collect and analyze source water 

samples for Cryptosporidium over a 1 or 2-year period if they exceed E. coli trigger levels [40 CFR § 

141.701(a)(3-4)]. Small, unfiltered systems are required to monitor their source water for 

Cryptosporidium instead of E. coli. [40 CFR 141.701(a)(6)]. 

Monitoring requirements for each system size, and the schedule for each stage of monitoring, are 

described in Table 1-1. Detailed guidance for sample collection during the LT2 Rule, and procedures for 

sample collection, documentation, and shipment, are provided in the Source Water Monitoring Guidance 

Manual for Public Water Systems for the Long Term 2 Enhanced Surface Water Treatment Rule (LT2 

Rule) (at http://www.epa.gov/safewater/disinfection/lt2/compliance). 

Cryptosporidium samples must be analyzed by a laboratory approved for analysis under the Laboratory 

Quality Assurance Evaluation Program for the Analysis of Cryptosporidium in Water (Section 3.2 of this 

manual, below) or a laboratory approved for Cryptosporidium analysis by an equivalent State laboratory 

certification program. However, at the time of publication, there were no equivalent State programs. 

Samples must be analyzed using EPA Method 1622/1623 [40 CFR § 141.704(a) and part 141.705] 

(References 5.2 and 5.3). 

E. coli samples must be analyzed by certified laboratories using methods approved under the LT2 Rule 

for surface water monitoring [40 CFR § 141.704(b)] (See Section 4 of this manual for details). Under the 

LT2 Rule, turbidity samples must be analyzed by a party approved by the State (usually a certified 

operator or a professional engineer) using methods approved in 141.74(a)(Reference 5.4)1)[40 CFR 

§141.704(c)]. 

2 February 2006 
Section 1: Introduction 

Table 1-1. Summary of LT2 Rule Monitoring Requirements (Initial Round of Monitoring) 

SCHEDULE Monitoring 

begins 

Monitoring 

duration 

Monitoring parameters and sample frequency 

requirements 

Cryptosporidium E. coli Turbidity 

SCHEDULE 1: 

Large systems 

serving ≥100,000 * 

October 1, 

2006 2 years a Minimum 

 1 sample/month b Minimum 1 sample/month c Minimum 1 sample/month c 

SCHEDULE 2: 

Large systems 

serving >50,000 

and <100,000 * 

April 1, 2007 2 years a Minimum 

 1 sample/month b Minimum 1 sample/month c Minimum 1 sample/month c 

SCHEDULE 3: 

Large systems 

serving >10,000 

and <50,000 * 

April 1, 2008 2 years a Minimum 

 1 sample/month b Minimum 

1 sample/month c Minimum 1 sample/month c 

SCHEDULE 4: 

Small filtered 

systems (serving 

fewer than 

10,000 * ) 

October 1, 

2008 1 year a,d See next row § Every two weeks N/A 

Small unfiltered 

systems (serving 

fewer than 10,000) 

and 

§

 Small filtered 

systems exceeding 

E. coli trigger 

levels d

 or that 

elect to proceed 

directly to 

Cryptosporidium 

monitoring or that 

fail to conduct E. 

coli monitoring 

April 1, 2010 

1 year b

, 2 Cryptosporidium 

samples per month, 

or 

2 years e

, 1 Cryptosporidium 

sample per month 

N/A N/A 

* Wholesale systems must comply with the requirements based on the population of the largest system in 

the combined distribution system [40 CFR § 141.700(b)(1)] 

a PWSs may be eligible to use (grandfather) data collected prior to the applicable monitoring start date if certain 

requirements are met [40 CFR § 141.707(a)(1)] b PWSs monitoring for Cryptosporidium may collect more than one sample per month if sampling is evenly spaced 

over the monitoring period [40 CFR § 141.701(a)(7)] c Unfiltered systems serving $10,000 are not required to perform E. coli monitoring or turbidity but to conduct source 

water monitoring that includes only Cryptosporidium sampling [40 CFR § 141.701(a)(2)] d Filtered systems serving fewer than 10,000 people must monitor for Cryptosporidium only if their E. coli annual 

mean concentrations is greater than 10 E. coli/100 mL for systems using lakes/reservoirs or is greater than 50 E. 

coli/100 mL for systems using flowing streams [40 CFR § 141.701(a)(4)] e Small systems collecting one sample per month for 2 years are still required, where applicable, to meet the 

treatment technique implementation deadlines in 40 CFR § 141.713 (c). The same treatment compliance dates 

apply to the PWS regardless of which Cryptosporidium sampling frequency is used (i.e., selecting the 2 year 

Cryptosporidium sampling frequency does not extend Cryptosporidium treatment compliance deadlines). 

N/A = Not applicable. No monitoring required. 

3 February 2006 
Section 1: Introduction 

1.3 Use of Cryptosporidium Data 

Two types of Cryptosporidium data are collected under the LT2 Rule: Cryptosporidium occurrence data 

from the analysis of field samples, and method performance data from the analysis of matrix spike (MS) 

samples. The use of occurrence data from field samples is discussed in Section 1.3.1; the use of method 

performance data from MS samples is discussed in Section 1.3.2. 

1.3.1 Cryptosporidium Monitoring Sample Data 

The concentration of Cryptosporidium oocysts in source water samples analyzed during the LT2 Rule will 

be used to calculate a mean Cryptosporidium concentration for a PWS and classify the PWSs into a 

treatment requirements “bin” (40 CFR § 141.710). These bin classifications are provided in Table 1-2. 

The treatment bin classification established for each PWS will be used to determine whether additional 

treatment is needed. PWSs in Bin 1 are not required to implement additional treatment. PWSs in Bins 2 - 

4 will be required to implement increasing levels of treatment and source water protection to address their 

higher risk for high Cryptosporidium source water concentrations. 

Table 1-2. Bin Classifications for Filtered Systems 

Average Sample Concentration Bin classification 

Cryptosporidium <0.075 oocyst/L or no monitoring 

requirement Bin 1 

0.075 oocysts/L Cryptosporidium < 1.0 oocysts/L Bin 2 

1.0 oocysts/L Cryptosporidium < 3.0 oocysts/L Bin 3 

Cryptosporidium  3.0 oocysts/L Bin 4 

PWSs that serve fewer than 10,000 people and NOT 

required to monitor for Cryptosporidium Bin 1 

The method used to average individual sample concentrations to determine a PWS’s bin classification 

depends on the number of samples collected and the length of the sampling period. 

For a filtered PWS monitoring for Cryptosporidium, bin classification would be based on the following 

calculations of bin concentration: 

• For PWSs that collect at least 48 samples during a two year monitoring period, the 

Cryptosporidium bin concentration is equal to the arithmetic mean of all sample concentrations 

[40 CFR § 141.710(b)(1)] 

• For PWSs that collect at least 24 samples, but not more than 47 samples, during a two year 

monitoring period, the Cryptosporidium bin concentration is equal to the highest arithmetic mean 

(average) of all sample concentrations in any 12 consecutive months in the monitoring period [40 

CFR § 141.710(b)(2)] 

• For PWSs serving fewer than 10,000 people and monitoring Cryptosporidium for only one year 

(i.e., collect 24 samples in 12 months), the bin concentration is based on the arithmetic mean of 

all sample concentrations [40 CFR § 141.710(b)(3)] 

4 February 2006 
Section 1: Introduction 

• For systems with plants operating only part of the year that monitor fewer than 12 months per 

year, the bin concentration is based on the highest arithmetic mean of all sample concentrations 

during any year of Cryptosporidium monitoring [40 CFR § 141.710(b)(4)] 

• For filtered PWSs in which sampling frequency varies, systems must first calculate a 

monthly average concentration for each month of monitoring. Systems must then use 

these monthly average concentrations, rather than individual sample concentrations, in 

the calculation of bin concentration as described in the four examples above [40 CFR § 

141.710(b)(5)] 

Unfiltered systems must calculate the arithmetic mean of all Cryptosporidium samples concentrations [40 

CFR § 141.712(a)(1)]. For unfiltered systems in which sampling frequency varies, systems must first 

calculate a monthly average for each month of monitoring. Systems must then use these monthly average 

concentrations, rather than individual sample concentrations, in the calculation of the mean 

Cryptosporidium level [40 CFR § 141.712(a)(3)] 

In all cases, the bin concentration is calculated using individual sample concentrations. These 

concentrations are calculated as “number of oocysts detected / volume (in L) analyzed.” Individual 

sample concentrations are not calculated as “oocysts detected / 10 L,” nor are bin concentrations 

calculated as the “sum of the oocysts detected / the sum of the volumes analyzed.” As a result, each 

sample has an equal weight on the final bin concentration. In cases where no oocysts are detected, the 

number of oocysts used to calculate the sample concentration is “0.” There should be no adjustment of 

the number of oocysts detected based on recovery or oocyst viability. 

1.3.2 Cryptosporidium Matrix Spike Data 

During LT2 Rule Cryptosporidium monitoring, PWSs are required to analyze, at a minimum, one MS 

sample for every 20 field samples from their source water [Section 9.1.8 of Method 1622/1623]. For all 

PWSs, the first MS sample should be collected and analyzed during the first sampling event under the 

monitoring program per Section 9.1.8 in EPA Method 1622.1623. Details on MS sample requirements are 

provided in Section 3.3.12. 

Based on this requirement, the following PWS categories must analyze at least two MS samples during 

LT2 Rule monitoring: 

• Large PWSs that perform monthly monitoring for two years (resulting in 24 samples) 

• Small PWSs that are triggered into Cryptosporidium monitoring and collect semi-monthly 

samples for one year or monthly samples for two years (resulting in 24 samples) 

For large PWSs that perform semi-monthly or more frequent monitoring for two years (resulting in 48 or 

more samples), a minimum of three MS samples should be collected and analyzed. If a PWS monitors 

more frequently or collects more than 60 samples, a minimum of four MS samples should be analyzed. 

Although MS sample results will not be used to adjust Cryptosporidium recoveries at any individual 

source water, the results will be used collectively to assess overall recovery and variability for EPA 

Method 1622/1623 in source water. 

5 February 2006 
Section 1: Introduction 

1.4 Use of E. coli and Turbidity Data 

E. coli and turbidity data are being collected by PWSs on Schedules 1-3 during LT2 Rule monitoring to 

confirm or refine the levels of E. coli that would indicate high Cryptosporidium concentrations in a source 

water. 

Small, filtered systems are permitted to monitor for E. coli, rather than more expensive Cryptosporidium 

analyses. Only those small filtered systems with E. coli levels that exceed the trigger level are required to 

monitor for Cryptosporidium to determine bin placement [40 CFR § 141.701 (a)(4)]. Based on the data 

from the Information Collection Rule (ICR) and Information Collection Rule Supplemental Survey 

(ICRSS), the following E. coli trigger levels were set: 

• A mean of 50 E. coli/100 mL for flowing stream–type source waters 

• A mean of 10 E. coli/100 mL for reservoir/lake source waters 

Small filtered PWSs that are monitoring for E. coli must collect at least one sample twice per month. 

Individual sample concentrations are then averaged to determine a mean E. coli level. A PWS’s mean E. 

coli level is compared to the E. coli trigger levels above to determine if the PWS is required to monitor 

for Cryptosporidium. 

The indicator data from large PWS monitoring will be reviewed and, if appropriate, guidance on alternate 

indicator trigger values will be issued to States prior to when small PWSs begin monitoring. States are 

allowed to approve alternative approaches to indicator monitoring for small PWSs [40 CFR § 

141.701(a)(5)]. 

6 February 2006 
SECTION 2: GENERAL MICROBIAL 

LABORATORY QUALITY ASSURANCE 

All laboratories analyzing Cryptosporidium and E. coli samples for the LT2 Rule should adhere to 

defined quality assurance (QA) procedures to ensure that analytical data generated under the rule are 

scientifically valid and are of known and acceptable quality. Detailed quality control (QC) requirements 

and recommendations specific to Cryptosporidium and E. coli analyses are discussed in Sections 3 and 4 

of this manual, respectively. Two QA issues that apply to both analyses—quality assurance plans and 

sample temperature monitoring—are discussed below, in Sections 2.1 and 2.2. 

2.1 Quality Assurance Plans 

As specified in both the Lab QA Program for Cryptosporidium laboratories 

(http://www.epa.gov/safewater/disinfection/lt2/, Section 3.2 of this manual, Reference 5.5) and the 

Laboratory Certification Manual (Chapter III, page III-4, Reference 5.6) for E. coli laboratories, each 

laboratory should operate a formal QA program and document the scope of this program through a QA 

plan. 

The laboratory’s QA plan should be a stand-alone document. However, some information can be 

incorporated into the document by reference, including laboratory standard operating procedures (SOPs), 

analytical methods, and quality control (QC) and calibration notebooks. Laboratories currently certified 

for coliform analysis under the drinking water laboratory certification program may use their current QA 

plan; however, this plan should be updated to address the specific requirements for LT2 Rule monitoring. 

Topics that should be addressed in the QA plan are outlined below. Details on LT2 Cryptosporidium 

requirements are provided in Section 3 of this manual; details on LT2 E. coli analyses are provided in 

Section 4. 

For Cryptosporidium laboratories, this QA plan should be available for review during a laboratory’s onsite audit, as part of the EPA’s Cryptosporidium Laboratory Quality Assurance Evaluation Program 

(Section 3.2 of this manual). For E. coli laboratories, this QA plan should be available for review during 

recertification audits as part of the National Primary Drinking Water Regulations. If the laboratory also 

performs turbidity testing, then the QA plan should address turbidity requirements. 

The following items should be addressed in each QA plan: 

1. Laboratory organization and responsibility 

• Include a chart showing the laboratory organization and line authority, including QA Managers 

• List the key individuals who are responsible for ensuring the production of valid measurements 

and the routine assessment of QC measurements 

• Specify who is responsible for internal audits and reviews of the implementation of the QA plan 

and its requirements 

2. Personnel 

• List each analyst’s academic background and water analysis experience 

• List each analyst’s training on the method 

• Describe training available to keep personnel up to date on methods and regulations 

7 February 2006 
Section 2: General Microbial Laboratory Quality Assurance 

3. Facilities 

• Arrangement and size of laboratory 

• Bench space 

• Storage space 

• Lighting 

• Air system 

• Lab reagent water system 

• Waste disposal system 

• Safety considerations. The laboratory should address biosafety in the laboratory when handling or 

processing Cryptosporidium samples and organism controls. Guidance on laboratory biosafety for 

Cryptosporidium is provided in Appendix A of this guidance manual. 

4. Field sampling procedures (with SOP used by laboratory or sent to PWS clients) 

• Describe how samples are collected, including sample containers, sample storage, transport times, 

and sample temperature 

• Describe sample identification and information recording system 

5. Laboratory sample handling procedures 

• Describe sample storage conditions 

• Describe the laboratory’s sample tracking system; specify procedures used to maintain the 

integrity of all samples, i.e., logging, tracking samples from receipt by laboratory through 

analysis to disposal 

• Describe sample acceptance criteria 

6. Equipment 

• Specifications for each piece of equipment used for Cryptosporidium, E. coli analyses, and/or 

turbidity 

• Calibration procedures, frequency, standards for each piece of equipment used for 

Cryptosporidium, E. coli analyses, and/or turbidity

• Quality control records for each piece of equipment used for Cryptosporidium, E. coli analyses, 

and/or turbidity 

• Preventative maintenance and schedules, documentation for each piece of equipment used for 

Cryptosporidium, E. coli analyses, and/or turbidity 

7. Supplies 

• Laboratory glassware and plastic ware acceptance conditions 

• Chemicals, reagents, dyes and culture media acceptance conditions 

• Chemicals, reagents, dyes, and culture media storage conditions 

• Filters acceptance conditions 

• Description of the laboratory’s reagent tracking system 

8. Laboratory practices (may reference SOP) 

• Preparation of reagent-grade water 

• Glassware washing and preparation 

• Sterilization procedures 

8 February 2006 
Section 2: General Microbial Laboratory Quality Assurance 

9. Analytical procedures 

• Describe all reference methods used 

• State that the analytical methods described in this manual will be followed 

• Identify available SOPs 

10. Quality control (QC) checks 

• Confirmation/ verification procedures, frequency 

• Sterility controls 

• Replicate analyses; frequency 

• QC samples, source; frequency 

• Positive and negative controls, proficiency testing (PT) samples, source; frequency 

• Spiked field samples 

• Between-analyst deviation 

11. Data reduction, verification, validation, and reporting 

• Data reduction (conversion of raw data to Cryptosporidium oocysts/L and/or E. coli/100 mL) 

• Procedures to ensure the accuracy of data transcription and calculations 

• Validation (ensuring that QC steps associated with a field result are acceptable) 

• Reporting, including procedures and format for reporting data to utilities/EPA 

12. Corrective actions 

• Define the laboratory response to unacceptable results from PT or QC samples and from internal 

QC checks 

• Identify persons with responsibility to take corrective action 

• Describe how the actions taken and the effectiveness of the actions taken will be documented 

13. Recordkeeping 

• Describe how records are to be maintained (e.g. electronically, hard copy, etc.) 

• Describe length of time records are to be kept (see archive requirements, Sections 3.8 and 4.8 of 

this manual) 

• State where records are to be stored 

The laboratory QA plan should be concise, but responsive to the above listed items. Additional guidance 

on developing QA plans is available in “Guidance on Quality Assurance Project Plans (QAPP) (G-5),” 

(EPA/240/R-02/009, December 2002), which is available as a download from 

http://www.epa.gov/quality/qa_docs.html#noneparqt. 

However, the goals of a lab QA plan in general are different from the goals of the Guidance on QAPP, 

and not all of the issues that should be addressed for laboratory QA during the LT2 Rule are covered by 

this guidance (i.e., laboratory sample handling and record keeping). However, some of the concepts 

presented in the QAPP guidance that typically are not included in laboratory QA plans may aid the 

laboratory in updating their QA plan to address specific LT2 requirements. 

9 February 2006 
Section 2: General Microbial Laboratory Quality Assurance 

2.2 Sample Temperature Monitoring 

Cryptosporidium oocysts present in a sample can degrade and E. coli present in a sample can grow or die 

off, biasing analytical results. Cryptosporidium and E. coli samples for LT2 Rule monitoring are stored 

and maintained between 1EC and 10EC to reduce biological activity. This is specified in Section 8.0 of 

the December 2005 versions of EPA Method 1622/1623 for Cryptosporidium samples and at 40 CFR § 

704(b)(3) and Chapter V, Section 6.3, of the Laboratory Certification Manual (Reference 5.6) for E. coli 

samples. 

Samples for all analyses should remain above freezing at all times. This is a requirement in Section 8.0 of 

the December 2005 versions of EPA Method 1622/1623. Although not a significant concern for 10 L 

water samples, this is a greater concern for Cryptosporidium filters and 120 or 250 mL E. coli samples 

that are shipped off-site with coolant materials, such as wet ice, blue ice, or gel packs. The sample 

collection protocols discussed in the Source Water Monitoring Guidance Manual for Public Water 

Systems for the Long Term 2 Enhanced Surface Water Treatment Rule (LT2 Rule) provide sample 

packing procedures for E. coli and Cryptosporidium samples. Utility personnel should follow these 

procedures to ensure that samples remain at acceptable temperatures during shipment. 

It is recommended that utilities practice the collection, packing, and shipping protocols prior to 

monitoring to ensure acceptable temperatures upon receipt at the laboratory. 

Because Cryptosporidium samples collected for the LT2 Rule must meet the QC criteria in the methods 

[40 CFR § 704 (a)], and because these QC criteria include receipt of samples at #20EC and not frozen, 

laboratories must reject LT2 Cryptosporidium samples that are received at >20EC or frozen unless the 

sample was collected the same day it was received. This is discussed further in Section 3.3.11 in this 

manual. In these cases, the PWS must re-collect and re-ship the sample. 

E. coli samples that are received at >10EC or frozen, or E. coli samples that the laboratory has determined 

exceeded 10EC or froze during shipment, must be rejected. After receipt, E. coli samples must be stored 

at the laboratory between 0EC and 10EC, and not frozen, until processed [40 CFR § 141.704(b)(3)]. 

Several options are available to measure sample temperature upon receipt at the laboratory and, in some 

cases, during shipment: 

• Temperature sample. One option, for Cryptosporidium filtered samples (not for 10 L bulk 

samples) and E. coli 120 and 250 mL samples, is for the PWS to fill a small, inexpensive sample 

bottle with water and pack this "temperature sample" next to the field sample. The temperature of 

this extra sample volume is measured upon receipt to estimate the temperature of the field 

sample. Temperature sample bottles are not appropriate for use with 10 L 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. C-06252-20 or equivalent. 

• Temperature vial. A similar option is to use a thermometer that is securely housed in a liquidfilled vial. Temperature vials are not appropriate for use with 10 L bulk samples for the reasons 

stated above. 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 reads the thermometer. 

Example product: Eagle-Picher Sentry Temperature Vial 3TR-40CS-F or 3TR-40CS or 

equivalent. 

10 February 2006 
Section 2: General Microbial Laboratory Quality Assurance 

• iButton. Another option for measuring the sample temperature during shipment and upon receipt 

is a Thermocron® iButton. An iButton is a small, waterproof device that contains a computer 

chip 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 in the cooler, 

rather than placed directly in the cooler, where it may be affected by close contact with the 

coolant. Again, this option is not appropriate for use with 10 L bulk samples. Example product: 

Thermocron® iButtons or equivalent. 

• 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 10 L bulk samples and field-filtered samples, but not for use with E. coli samples. 

Example product: Cole Parmer cat. no. C-90316-00. 

• Infrared thermometers. Another option is for the laboratory 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 10 L bulk samples and field-filtered samples. 

Example product: Cole Parmer cat. no. EW-35625-10. 

As with other laboratory equipment, all temperature measurement devices should be calibrated routinely 

to ensure accurate measurements. See the EPA Lab Certification Manual (Reference 5.6) for more 

information. 

All temperature readings should be documented and archived as required by the analytical methods. 

11 February 2006 
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12 February 2006 
SECTION 3: GUIDANCE FOR CRYPTOSPORIDIUM 

LABORATORIES 

Cryptosporidium analyses conducted in support of the LT2 Rule must be performed using EPA Method 

1622 or EPA Method 1623 [40 CFR § 141.704(a)]. Guidance on the use of these methods during the LT2 

Rule is provided in this section of the manual. 

3.1 LT2 Rule Cryptosporidium Sample Analysis Requirements 

LT2 Rule requirements of particular significance are summarized in Sections 3.1.1 through 3.1.6, below, 

and discussed in more detail along with guidance in the remainder of Section 3. 

3.1.1 Approved Laboratories 

Systems must have Cryptosporidium samples analyzed by a laboratory that has passed a quality assurance 

(QA) evaluation under EPA’s Laboratory Quality Assurance Evaluation Program for Analysis of 

Cryptosporidium in Water (Reference 5.5) or a laboratory that has been approved for Cryptosporidium 

analysis by an equivalent State laboratory approval program [40 CFR § 141.705(a)]. However, at the 

time of publication of this guidance document there were no equivalent State programs for approval of 

Cryptosporidium laboratories. Details on the elements of the Lab QA Program QA evaluation are 

provided in Section 3.2. It is suggested that each approved laboratory establish and maintain a 

relationship with another approved laboratory and develop a protocol to provide back-up analyses to 

clients if needed during LT2. A list of approved laboratories is posted at 

http://www.epa.gov/safewater/disinfection/lt2/. 

3.1.2 Revised Cryptosporidium Method 

The LT2 Rule requires that samples collected under the rule be analyzed using the December 2005 

version of Methods 1622/1623. EPA proposed the use of the April 2001 versions of EPA Methods 

1622/1623 in the LT2 Rule. However EPA requested and received comments on the use of updated 

versions to consolidate several method-related changes EPA believes are necessary to address LT2 Rule 

monitoring requirements. Methods 1622/1623 were revised and the December 2005 version of these 

methods (References 5.2 and 5.3) are included as Appendix B and Appendix C of this guidance manual. 

These changes include the following: 

• Increased flexibility in matrix spike (MS) and initial precision and recovery (IPR) requirements. 

The requirement that the laboratory must analyze an MS sample on the first sampling event for a 

new PWS has been changed to a recommendation; the revised method allows the IPR test to be 

performed across four different days, rather than restrict analyses to 1 day. 

• Clarification of some method procedures, including the spiking suspension vortexing procedure; 

the buffer volumes used during immunomagnetic separation (IMS); requiring (rather than 

recommending) that laboratories purchase HCl and NaOH standards at the normality specified in 

the method; and the use, or not, of methanol during slide staining in Section 14.2 of Method 

1622/1623 is per manufacturer’s instructions. 

13 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

• Addition of recommendations for minimizing carry-over of debris onto microscope slides after 

IMS and information on microscope cleaning. Clarification of the actions to take in the event of 

QC failures. 

• A change in the sample receipt temperature requirements to “#20EC, and not frozen,” and 

additional guidance on sample storage (between 1EC and 10EC) and shipping procedures based 

on time of day of collection. The revision includes suggested options for monitoring sample 

temperature during shipment and/or upon receipt at the laboratory. 

• Addition of the requirement for examination using differential interference contrast (DIC) 

microscopy to the analyst verification procedure. 

• Addition of an approved method modification using the Pall Gelman EnvirochekTM HV filter. 

This approval is based on an interlaboratory validation study demonstrating that three 

laboratories, each analyzing reagent water and a different source water, met all method 

acceptance criteria for Cryptosporidium (but not Giardia; however, individual laboratories are 

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

• Incorporation of detailed procedures for concentrating samples using an IDEXX Filta-Max® 

foam filter. (A method modification using this filter was already approved by EPA in the April 

2001 version of the methods.) 

• Addition of BTF EasySeedTM irradiated oocysts and cysts as acceptable materials for spiking 

routine QC samples. EPA approved the use of EasySeedTM based on side-by-side comparison 

tests of method recoveries using EasySeedTM and live, untreated organisms. 

• Removal of the Whatman Nuclepore CrypTestTM cartridge filter. Although a method modification 

using this filter was approved by EPA in the April 2001 versions of the methods, the filter is no 

longer available from the manufacturer, and so is no longer an option for sample filtration. 

• 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. 

• Addition of portable continuous-flow centrifugation (PCFC) as a filtration/concentration 

technique for the detection of Cryptosporidium in Methods 1622/1623. 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). 

3.1.3 Minimum Sample Volume Analysis Requirements 

Under LT2 Rule Cryptosporidium sample volume requirements [40 CFR § 141.704(a)(1)], PWSs are 

required to analyze, at a minimum, either 

• 10 L of sample, or 

• 2 mL of packed pellet volume, or 

• As much volume as two filters can accommodate before clogging (this condition applies only to 

filters that have been approved by EPA for nationwide use with EPA Method 1622/1623—the 

Pall Gelman EnvirochekTM and EnvirochekTM HV filters, or the IDEXX Filta-Max® foam filter) 

14 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

The LT2 Rule sample volume analysis requirement of 10 L (rather than 10.0 or 10.00 L) accommodates 

the potential for imprecisely filled sample containers or filters. Therefore, sample volumes of 9.5 L and 

higher would meet the LT Rule requirements. Sample volumes should be recorded to the nearest 0.25 L 

when using a graduated carboy instead of a flow meter to measure volume. 

Systems may analyze larger volumes than 10 L, and larger volumes analyzed should increase analytical 

sensitivity, provided method performance is acceptable. EPA prefers systems to analyze similar sample 

volumes throughout the monitoring period. However, data sets including different sample volumes will be 

accepted, provided the system analyzes the minimum sample volume requirements noted above. 

Matrix spike samples must be collected from the same location as the field sample and the volume 

analyzed must be within 10 percent of the volume analyzed for the field sample (Section 9.5 of Method 

1622/1623). It is suggested that the same volume of sample be collected for both the field and the matrix 

spike sample to ensure the volumes analyzed are within 10 percent. Additional guidance on matrix spike 

samples is provided in Section 3.3.12 of this manual. 

Additional guidance on sample volume and sample collection issues is provided in the Source Water 

Monitoring Guidance Manual for Public Water Systems for the Long Term 2 Enhanced Surface Water 

Treatment Rule (LT2 Rule), available for download from http://www.epa.gov/safewater/disinfection/lt2/ 

3.1.4 Spiking Suspensions Requirements for Spiked Quality Control 

Samples 

Flow cytometer–counted spiking suspensions must be used for ongoing precision and recovery (OPR) and 

matrix spike (MS) samples [40 CFR § 141.704(a)(3)]. The use of flow cytometer–counted spiking 

suspensions is a recommendation in EPA Method 1622/1623, and is a requirement in the LT2 Rule. 

Spiking suspensions are discussed in more detail in Section 3.3.1, below. 

3.1.5 Acceptable Sample Results 

Cryptosporidium sample results reported under the LT2 Rule must be generated at an approved laboratory 

and meet the quality control (QC) requirements specified in EPA Method 1622/1623. These requirements 

include, but are not limited to, sample temperature requirements, minimum frequencies for ongoing 

precision and recovery (OPR), method blank, and matrix spike samples; acceptable OPR and method 

blank results; holding time requirements; and staining control frequency and results. A checklist for these 

requirements is provided as Appendix D. Guidance on implementing Cryptosporidium method QC 

requirements is provided in Section 3.3, below. 

3.1.6 Cryptosporidium Oocyst Counts to Report 

Sample examination using EPA Method 1622/1623 includes an immunofluorescence assay using 

fluorescein isothiocyanate (FITC) as the primary antibody stain, 4',6-diamidino-2-phenylindole (DAPI) 

staining to detect nuclei, and differential interference contrast microscopy (DIC) to detect internal 

structures. Hoffman Modulation Contrast (HMC) optics may be a suitable alternative to DIC, provided 

that the laboratory demonstrates acceptable analyst performance and acceptable microscope capability to 

the technical auditor during the on-site laboratory audit conducted prior to laboratory approval through 

the Lab QA Program. Cryptosporidium oocysts to be reported using Section 15 of EPA Method 

1622/1623 are defined as the following: 

• Those determined by brilliant apple green fluorescence under UV light, size (4 to 6 Fm), and 

shape (round to oval) 

15 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

• Excluding any atypical organisms detected by FITC, DAPI, and DIC (e.g., those possessing 

spikes, stalks, appendages, pores, one or two large nuclei filling the cell, red fluorescing 

chloroplasts, crystals, spores, etc.) 

The oocyst counts for a sample, based on the above definition and appropriate magnification and 

examination procedures per Method 1622/1623, and the sample volume analyzed, will be used to 

calculate the oocyst concentration for each sample during the LT2 Rule. 

In a field sample, all organisms that meet the above definition must be counted and assigned to the 

appropriate categories. This reporting requirement cannot be met unless all Cryptosporidium-like 

organisms in a field sample are examined first under the FITC filter, then under the DAPI filter, and 

finally using DIC. If no organisms meet the above definition, the lab should report zero oocysts. The 

number listed in the summary row, “Total FA number”, on the Cryptosporidium examination results form 

and the volume examined (L) should be the minimum information reported to the utility (see Section 3.7 

of this manual for details regarding the LT2/Stage 2 Data Collection and Tracking System). 

3.2 Laboratory Quality Assurance Evaluation Program 

The objectives of the Lab QA Program are to evaluate laboratories’ competency to reliably measure for 

the occurrence of Cryptosporidium in surface water using EPA Method 1622/1623. Each laboratory 

participating in the program will be required to complete the following steps to be qualified through this 

program: 

• Complete an application (including a self-evaluation and initial demonstration of capability) 

• Perform initial proficiency testing (IPT) 

• Participate in an on-site evaluation 

• Perform ongoing proficiency testing (OPT) every four months 

Information on the Laboratory QA Program is available at http://www.epa.gov/safewater/disinfection/lt2/ 

and is summarized below, in Sections 3.2.1 - 3.2.7. 

3.2.1 Application 

Applications for the program (Appendix E) are available on the website, 

http://www.epa.gov/safewater/disinfection/lt2/, and may also be requested from the following address: 

Cryptosporidium Laboratory Quality Assurance Coordinator 

c/o CSC Water Programs 

6101 Stevenson Avenue 

Alexandria, VA 22304 

EPA reviews each application to verify that the laboratory has submitted the following information: 

• A completed self-evaluation checklist 

• Resumes of laboratory personnel 

• Standard operating procedures for each method version 

16 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

• Initial demonstration of capability (IDC) data, which consist of the following: 

- Acceptable initial precision and recovery (IPR) test results 

- Acceptable method blank result analyzed with IPR test 

- Acceptable matrix spike/matrix spike duplicate (MS/MSD) results with results from the 

unspiked matrix sample collected and analyzed at the same time 

• Table of contents from the laboratory’s quality assurance plan 

• Documentation of personnel training and list of samples analyzed and duration of time using the 

method 

• Example of client data reporting form 

• A statistical summary of percent recoveries for all OPR and MS samples analyzed over the past 

six months. 

Completed applications should be submitted to Cryptosporidium Laboratory Quality Assurance 

Coordinator, c/o CSC Water Programs, at the address listed above. 

3.2.2 Personnel Qualifications and Training 

As part of approval, EPA will determine whether laboratory personnel are qualified to analyze Cryptosporidium 

samples for LT2 Rule monitoring. Suggested personnel qualifications for the Lab QA Program are provided in 

Table 3-1. Each laboratory should have at least one principal analyst. 

Table 3-1. Suggested Laboratory QA Program Personnel Qualifications 

Position Education 

Experience with 

Crypto and IFA 

Microscopy 

Experience Using 

Method 1622/1623 

Number of 

Samples Analyzed 

Using Method 

1622/1623 

Principal Analyst 

BS/BA in 

Microbiology or 

closely related field 

1 year continuous 6 months 

100 (50 if approved 

as an analyst during 

Information 

Collection Rule 

[ICR]) 

Analyst 

2 years college in 

Microbiology or 

equivalent 

6 months continuous 3 months 

50 (25 if approved 

as an analyst during 

ICR) 

Technician No minimum 

requirement 

No minimum 

required 

3 months performing 

specific parts of 

procedures 

50 (25 if approved 

as an analyst during 

ICR) 

During the on-site evaluation (Section 3.2.4 of this manual), EPA will review laboratory records to verify 

that the personnel performing EPA Method 1622/1623 analyses are qualified to do the analyses required 

under LT2. For new staff that is added after the on-site evaluation, the laboratory should send a letter to 

EPA providing the following information on the new staff member: 

• Resume including education 

• Number of samples analyzed using EPA Method 1622/1623 

• Number of months of experience 

• Verification that analyst training followed the laboratory’s training SOP 

17 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

In addition, the following steps should be completed by new personnel as part of their training prior to 

analyzing samples for LT2 (specify in the laboratory’s training SOP): 

• Review laboratory SOPs for analysis of samples using Method 1622/1623 

• Observation of an experienced analyst performing the method 

• Performance of the method while being observed by an experienced analyst 

• Acceptable performance of a set of IPR samples using blind spikes 

• Analysis of as many MS/MSD samples as possible 

• Repeated study of microscopy module 

It is also helpful for trainees to examine and characterize oocysts that are live and inactivated to note the 

differences between the two states and variations which may occur in the staining and morphological 

quality of the oocysts. Comparing the quality of the organisms between live and inactivated oocysts may 

help define and troubleshoot any problems that may occur during processing and eliminate the possibility 

that the staining or morphological differences are due to the inactivation techniques used for the 

organisms. 

After the initial training is successfully completed, a trainee should analyze as many Ongoing Precision 

and Recovery (OPR) samples as possible. In addition, the trainee should analyze non LT2 samples using 

the same method to gain experience. To optimize the experience gained in the required 3 month period 

and 50 samples analyzed, the trainee should complete the analysis of 20 MS/MSD sets from a variety of 

source waters. At a minimum, it is recommended that samples analyzed to gain experience include 

different matrices that may demonstrate possible interferences with processing and/or examination. 

After the initial training is successfully completed, a trainee may assist with LT2 samples with an 

experienced analyst (i.e., both trainee and analyst may sign the laboratory bench sheet and slide 

examination form) to gain experience with a variety of source waters. It should be noted that any such 

assistance with processing should be supervised closely enough to proactively eliminate processing 

mistakes and preserve the analysis. The trainee may re-examine a slide after complete examination by an 

experienced analyst. The experienced analyst’s microscopy results should be used for reporting LT2 

results. When a trainee successfully completes the required experience and number of samples, 

documentation supporting the completion should be sent to the EPA and the trainee’s status will be 

changed to analyst or technician. The troubleshooting in Section 9.7.5 of Method 1622/1623 may be used 

during analyst training also. Proficiency of skill in photomicrography and development of an analyst 

photo library is encouraged. 

3.2.3 Initial Proficiency Testing 

After the laboratory’s application has been reviewed and accepted, EPA will send the laboratory a set of 

eight initial proficiency testing (IPT) samples, which consist of a suspension of Cryptosporidium oocysts 

in a concentrated matrix. Laboratories will resuspend these spikes in reagent water to produce simulated 

source water samples, and analyze the samples using the version of EPA Method 1622/1623 that the 

laboratory plans to use for routine Cryptosporidium analyses. 

Laboratory IPT data will be evaluated against the mean recovery and precision (as relative standard 

deviation [RSD]) criteria that EPA has established for IPT samples. If a laboratory fails the IPT criteria 

twice, EPA recommends that the laboratory 

• Receive additional training in performing the method (discussed further in Section 3.2.6 of this 

manual) 

18 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

• Repeat their IPR analyses until acceptable and submit the results to EPA 

If the laboratory does not pass the third IPT following additional training, they may consult with the EPA 

regarding the level and type of training undertaken. After satisfying these requirements, the laboratory 

may re-apply for IPT samples. 

3.2.4 On-Site Evaluation 

Each laboratory that passes the IPT is eligible to participate in an on-site evaluation next, which consists 

of two concurrently performed assessments: a data and QA evaluation and a technical evaluation. 

3.2.4.1 Data and QA Evaluation 

During the data and QA evaluation, laboratory documentation will be evaluated to verify compliance with 

QA program requirements. The evaluation will cover the following: 

• Equipment and personnel records 

• Data recording procedures, based on field sample data and quality control sample data 

• Quality control test frequency and acceptability 

• Quality assurance plans 

• Standard operating procedures 

To ensure consistency and thoroughness for all audits, the data auditor uses a detailed checklist 

(Appendix F) to evaluate specific factors under each of these categories. 

To prepare for the on-site evaluation, the laboratory can use the checklists provided with the program 

application to perform a self-audit. 

3.2.4.2 Technical Evaluation 

During the technical evaluation, laboratory sample processing and analysis using EPA Method 1622/1623 

will be evaluated. The laboratory will be assessed on its capabilities including the following: 

• Sample processing and analyses 

• Microscopy 

To ensure consistency and thoroughness for all audits, the technical auditor uses a detailed checklist 

(Appendix F) to evaluate specific factors under each of these categories. 

3.2.5 Approval and Ongoing Proficiency Testing 

Laboratories will be approved after they have submitted an acceptable application, passed the IPT and 

passed the on-site evaluation, as summarized in steps 1-3 in Figure 3-1. Laboratories that are approved 

will also receive a set of three ongoing proficiency testing (OPT) samples approximately every four 

months that should be analyzed in the same manner as the IPT samples. EPA will evaluate the precision 

and recovery data for OPT samples to determine if the laboratory continues to be qualified under the 

Laboratory QA Program. Laboratories must successfully evaluate the OPT samples to maintain EPA 

approval for the LT2. 

19 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

If a laboratory fails to meet the precision or recovery criteria for a set of OPT samples, the laboratory will 

be shipped a second set of samples. If the laboratory’s next set of OPT data are acceptable, no further 

action is required. 

If a laboratory fails the next set of OPT samples (two sets of OPTs in a row), it is recommended that the 

laboratory receive additional training and examine laboratory control charts. If the laboratory continues to 

fail OPT samples, EPA will reevaluate the laboratory’s approval for this program. 

3.2.6 Additional Training 

Additional training can be received at another approved laboratory or through training opportunities 

supported by the EPA. Laboratories also can consult universities, vendors, websites, and microscope 

service companies for possible training opportunities. EPA has produced an online microscopy module 

detailing the characterization of Giardia and Cryptosporidium. Viewing this module from the LT2 

website, http://www.epa.gov/safewater/disinfection/lt2/, is open to all analysts as a supplement to one-onone microscopy training. Practice samples may be performed including method blanks, OPR samples, 

MS samples, and non-LT2 samples. It is recommended that samples analyzed to gain additional practice 

include different matrices which may demonstrate possible interferences with processing and/or 

examination. 

3.2.7 Notifying Utilities of Change in Laboratory Status 

Two actions are necessary if a laboratory is disapproved: 

• The laboratory should notify clients 

• EPA will remove the laboratory from the approved list 

(http://www.epa.gov/saftewater/disinfection/lt2/index.html) 

If a laboratory receives notice that they have been “disapproved,” the laboratory will no longer be able to 

analyze samples under LT2 until they have been re-approved [141.705(a)]. The laboratory should 

immediately notify their clients of their status change and cease analyzing samples for LT2. As suggested 

in Section 3.1.1 of this manual, laboratories should have a protocol in place to divert samples to another 

approved laboratory in the event back-up analyses are needed. 

All samples being processed by the laboratory at the time of the disapproval are considered acceptable, 

provided all QC and holding time requirements (EPA Methods 1622 and 1623) are met. Analysis of 

these samples should be completed by the laboratory. However, no new LT2 sample analyses may be 

initiated unless/until the laboratory is re-approved. 

3.3 Cryptosporidium Method Quality Control 

During the LT2 Rule, Cryptosporidium samples must meet the quality control (QC) requirements listed in 

EPA Methods 1622/1623. The requirements discussed in this guidance manual are based on the 

December 2005 versions of EPA Methods 1622/1623 (References 5.2 and 5.3). Section 3.1 in this 

manual included QC for samples, Section 3.2 included QC for laboratories, and this section covers QC for 

the method. 

Sections 3.3.1 through 3.3.13 provide guidance on the implementation of the QC requirements specified 

in the December 2005 version of EPA Method 1622/1623. Routine QC requirements that must be verified 

internally by the laboratory before reporting LT2 Rule monitoring results are summarized in Table 3-2. 

QC guidance for method modifications and use of multiple method variations are covered in 3.3.14. This 

20 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

guidance is provided to help implement the QC requirements in the methods and does not substitute for, 

or alter, the method requirements. 

Figure 3-1. Process for Receiving and Monitoring Laboratory Approval 

21 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

Table 3-2. Summary of Routine QC Requirements 

QC sample 

or 

procedure Matrix 

Number 

of 

samples 

Frequency Purpose Control 

Charts 

IDC 

Reagent 

water 

and 

source 

water 

8 Once 

To demonstrate control over the 

analytical system; consists of IPR set, 

Method blank ,and MS/MSD as a 

requirement of Lab QA Program 

No IPR Reagent 

water 4 Initial use of method 

To establish initial control over the 

analytical system and demonstrate 

acceptable method performance 

(recovery and precision) 

Method 

Blank 

Reagent 

water 1 Each IPR and OPR set 

To demonstrate the absence of 

contamination throughout the 

analytical process 

OPR Reagent 

water 1 

At least each 

week samples 

are processed 

or every 20 

samples, 

whichever is 

more frequent 

To demonstrate ongoing control of 

the analytical system and verify 

continuing method performance 

(recovery and precision) 

Required 

MS Source 

water 2 

For each source 

water - initial 

sampling and 

every 20 

samples 

To determine the effect of the matrix 

on (oo)cyst recoveries; must be 

accompanied by an unspiked field 

sample collected at the same time as 

the MS sample 

Recommended 

Positive 

staining 

control 

none 1 

Process each 

time samples 

are stained; 

examine each 

microscope 

session 

To demonstrate ongoing control of 

the staining process and performance 

of reagents and microscope 

No 

Negative 

staining 

control 

none 1 

Each time 

samples are 

stained 

To demonstrate the absence of 

contamination through staining 

process 

Verification 

of Analyst 

Performance 

Reagent 

Water N/A Monthly Refine consistency of organism 

characterizations between analysts 

3.3.1 Cryptosporidium Spiking Materials 

During LT2 Cryptosporidium monitoring, laboratories must analyze samples spiked with 

Cryptosporidium oocysts to assess ongoing laboratory and method performance in accordance with 

method QC requirements. These ongoing spiked sample analyses include initial precision and recovery 

samples (IPRs), ongoing precision and recovery samples (OPRs), matrix spike samples, and positive 

staining controls (Sections 3.3.2, 3.3.4, 3.3.12, and 3.3.8 of this manual, respectively). Flow cytometer– 

counted spiking suspensions must be used for the IPR, OPR and matrix spike samples [40 CFR § 

141.704(a)(3)], and those suspensions must be used within the noted expiration (Section 8.3 of Method 

1622/1623). The laboratory should spike samples according to the procedures provided in Section 11.4 of 

EPA Method 1622/1623 or according to the procedures provided by the spiking suspension vendor. 

22 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

The commercial staining kits contain positive control organisms which may be used for routine positive 

staining control slides. These organisms are typically treated by various inactivation techniques and need 

to be evaluated for appropriate FITC fluorescence, DAPI-stained nuclei, and internal morphology. If an 

analyst notes that the appropriate features are not present, e.g., the majority of oocysts are DAPI negative 

or exhibit weak nuclei staining, it is suggested that a different vendor of positive control organism be 

utilized. Laboratories are encouraged to use the online microscopy module for comparison 

(http://www.epa.gov/safewater/disinfection/lt2/). Laboratories may also obtain positive staining control 

organisms from the vendors listed below and in Method 1622/1623 Section 7.10 which may be live or 

inactivated. 

Sources of flow cytometer–counted Cryptosporidium spiking suspensions for use with routine, spiked 

Cryptosporidium QC samples include the following: 

1. Wisconsin State Laboratory of Hygiene 

Flow Cytometry Unit 

http://www.slh.wisc.edu/ 

2601 Agriculture Drive 

Madison, WI 53718 

Phone: (608) 224-6260 

Fax: (608) 224-6213 

The Wisconsin State Laboratory of Hygiene prepares and distributes live Cryptosporidium 

parvum oocysts and Giardia intestinalis cysts that have not been treated to reduce viability. 

2. BioTechnology Frontiers (BTF) 

http://www.btfbio.com 

Unit 1, 35-41 Waterloo Road 

North Ryde NSW 2113 

Australia 

Phone: +61 2 8877 9150 

Fax: +61 2 8877 9101 

Email: contact@btfbio.com

BTF prepares and distributes Cryptosporidium parvum oocysts and Giardia intestinalis cysts that 

have been irradiated to inactivate the organisms. Note: Irradiated, flow cytometer–counted 

spiking suspensions may be used for routine laboratory QC samples, including initial precision 

and recovery (IPR) samples, ongoing precision and recovery (OPR) samples, and matrix spike 

(MS) samples. In accordance with EPA Method 1622/1623, irradiated organisms may not be used 

for interlaboratory validation studies performed to seek nationwide approval of modified versions 

of the methods. 

3.3.2 Initial Precision and Recovery Test 

The initial precision and recovery (IPR) test required by EPA Method 1622/1623 consists of four reagent 

water samples spiked with ~100 to 500 oocysts and is used to demonstrate acceptable performance with 

the method. Section 9 of EPA Method 1622/1623 also requires the IPR to be performed for each method 

modification (additional guidance on QC when using multiple method variations is provided in Section 

3.3.13 of this manual). 

The results of the four analyses are used to calculate the mean percent recovery and the relative standard 

deviation (RSD) of the recoveries for Cryptosporidium (Section 3.6.3 of this manual). For EPA Method 

23 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

1622/1623, the mean Cryptosporidium recovery should be from 24 percent to 100 percent and the RSD of 

the four recoveries should be less than or equal to 55 percent. Characterization of the first three 

Cryptosporidium oocysts and three Giardia cysts must be reported on the slide examination form for each 

IPR sample following Section 9.4.5 in EPA Method 1622/1623. 

3.3.3 Method Blank Test 

The method blank test required in Section 9.6 of EPA Method 1622/1623 consists of analysis of an 

unspiked reagent water sample to demonstrate freedom from contamination. The method requires that one 

method blank sample must be analyzed each week or every 20 field and matrix spike samples, whichever 

is more frequent (Section 9.1.7 in Method 1622/1623). A week is defined as any 168 hour (7 day) period 

that begins with the processing of the OPR. If more than one method variation will be used for filtration 

and/or another technique which exposes samples to different apparatus and/or reagents, a separate method 

blank is required for each variation. 

Method blank samples should be analyzed before any field samples in a batch are processed to verify 

acceptable performance. If one or more Cryptosporidium oocysts (as defined in Section 13 of Method 

1622/1623) are found in a blank, the method blank is unacceptable and analysis of additional samples is 

halted until the source of contamination is eliminated and a blank shows no evidence of contamination. 

Troubleshooting the problem and repeating the method blank to bring the analytical system under control 

is required before proceeding with sample analysis. If the repeated method blank is acceptable and field 

samples can be processed within holding times, no replacement samples are necessary. 

Note: If oocysts are detected in the method blank, analysis of additional samples is halted until 

the source of contamination is eliminated. 

3.3.4 Ongoing Precision and Recovery Test 

The ongoing precision and recovery (OPR) in Section 9.7 of EPA Method 1622/1623 entails analysis of a 

reagent water sample spiked with ~100 to 500 oocysts to demonstrate ongoing acceptable performance. 

One OPR sample should be analyzed each week or every 20 field and MS samples, whichever is more 

frequent (Section 9.1.7 of Method 1622/1623). A week is defined as any 168 hour (7 day) period that 

begins with the processing of the OPR (Section 9.1.7 of Method 1622/1623). If more than one method 

variation will be used for filtration and/or another technique, a separate OPR may be required for each 

variation. 

OPR samples should be analyzed before any field samples in a batch are processed to verify acceptable 

performance. OPR Cryptosporidium recovery should be from 11 percent to 100 percent to be considered 

acceptable (Section 9.7.3 of Method 1622/1623). Characterization of the first three Cryptosporidium 

oocysts and three Giardia cysts must be reported on the slide examination form as per Section 9.7.1.2 of 

EPA Method 1622/1623. If the OPR Cryptosporidium recovery is not acceptable, no samples may be 

processed. Troubleshooting the problem and repeating the OPR to bring the analytical system under 

control is required before proceeding with sample analysis. If the repeated OPR is acceptable and field 

samples can be processed within holding times, no replacement samples are necessary. 

Note: Ongoing precision and recovery results should be 11 percent to 100 percent. 

24 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

3.3.5 Ongoing Precision and Recovery Control Charts 

As noted in Section 9.7.6 of the December 2005 version of EPA Method 1622/1623, laboratories should 

maintain a quality control (QC) chart of OPR recoveries, graphically displaying the results of continuing 

performance. The control chart should be developed using the most recent 20 to 30 test results. 

The control chart is developed by plotting percent recovery of each OPR sample over time (Figure 3.2). 

Based on the mean of the recoveries (0 ) on the chart, the upper and lower control limits should be 

established as follows 

• Upper control limit = 0 + 2 standard deviations 

• Lower control limit = 0 - 2 standard deviations 

After each 5 to 10 new recovery measurements, new control limits should be recalculated using the most 

recent 20 to 30 data points. Control charts can be used to track the laboratory’s performance and 

determine if any trends in recovery are occurring. Control charts can also be used to compare 

performance of different method variations, different analysts, and/or other changes implemented by the 

laboratory. If recovery measurements fall outside the control limits, laboratories should take corrective 

action, investigating potential causes of the outlying result. The troubleshooting guidance for OPR 

failures provided in Section 9.7.5 of EPA Method 1622/1623 is also useful for investigating the cause of 

acceptable, but outlying, OPR measurements identified through the use of control charts. 

Figure 3-2. Ongoing Precision and Recovery Control Chart Example 

100% 

90% 

80% 

70% 

60% 

50% 

40% 

30% 

20% 

10% 

0% 

01/04/2001 02/01/2001 03/01/2001 03/29/2001 04/26/2001 05/24/2001 06/21/2001 07/19/2001 

(%) 

Recovery 

Mean + 2 Standard Deviations 

Mean - 2 Standard Deviations 

O

PR 

Reco

very 

25 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

3.3.6 Quality Control Batches 

All LT2 Cryptosporidium samples must be associated with an acceptable OPR and method blank sample 

as stated in Section 9 of the EPA Method 1622/1623. LT2 samples are associated with QC samples 

through a “QC batch.” A QC batch consists of an OPR and method blank and a maximum of 20 field and 

MS samples that are eluted, concentrated, and purified in the same week as the OPR and method blank 

samples using the same reagents (e.g., eluting solution). A week is defined as any 168 hour (7-day) period 

that begins with the processing of the OPR. If more than 20 field and MS samples are processed in a 

week, the OPR and method blank samples are associated with the field and MS samples which are eluted, 

concentrated, and purified using the same reagents as the OPR and MB and processed prior to the next 

OPR/MB. The next QC batch may begin concurrently with the processing of the last of the 20 samples 

associated with the previous OPR/MB. A field sample and its associated MS sample should be analyzed 

in the same “QC batch”. QC samples do not need to be analyzed necessarily during weeks in which no 

field samples are analyzed unless analytical practice would be beneficial for the laboratory. 

3.3.7 Holding Time Requirements 

During Cryptosporidium analyses for the LT2 Rule, sample processing should be completed as soon as 

possible by the laboratory. The laboratory should complete sample filtration (if sample is received in 

bulk), elution, concentration, purification, and staining the day the sample is received whenever possible. 

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

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

application of the purified sample onto the slide, or staining. 

The following holding times must be met for samples analyzed by EPA Method 1622/1623 during the 

LT2 Rule: 

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

collection whether shipped to the laboratory as a bulk sample or filtered in the field. 

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

concentration, purification, and application of the sample to the slide 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. Follow the stain manufacturer’s 

instructions regarding the drying procedure to use. Drying options include: on the lab bench, on 

the lab bench with air gently moving around the slides, on a slide warmer between 35EC and 

42EC, in a humid chamber or incubator between 35EC and 42EC, or in the refrigerator. The slides 

must be completely dried before staining and stored to maintain the dried state until stained. 

Storage of dried slides consists of storing on the lab benchtop (protected), in a desiccator box in 

the refrigerator, or in a desiccator box on the lab bench (if the lab is humid). 

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

slide including drying time. 

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

(DAPI) and differential interference contrast (DIC) microscopy examination and confirmation is 

ideally performed immediately after staining is complete, laboratories have up to 168 hours (7 

days) from completion of sample staining to complete the examination and confirmation of 

samples. However, if fading/diffusion of FITC or DAPI staining is noticed, the laboratory should 

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

staining solution so that fading/diffusion does not occur per Section 14.6 in Method 1622/1623. 

26 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

The laboratory also may evaluate the use of another mounting medium (alternatives are provided 

in Section 3.8.2 of this manual, below). 

LT2 Rule requirement: Each sample must meet the QC criteria for the methods. Per EPA 

Method 1622/1623, samples must be processed or examined 

within each of the holding times specified in Section 8.2 of the 

method. 

A breakdown of the holding times for each set of steps is provided in Table 3-3 

Table 3-3. Method 1622/1623 Holding Times (adapted from Table 1 EPA Method 1622/1623) 

Sample Processing Step Maximum Allowable Time between Breaks 

(samples should be processed as soon as possible) 

Collection Up to 96 hours are permitted between sample collection and 

initiation of elution 

Filtration 

Elution These steps must be completed in 1 working day 

Concentration 

Purification 

Application to slide 

Staining Up to 72 hours are permitted from application of the purified 

sample to the slide to staining 

Examination Up to 168 hours (7 days) are permitted between sample staining 

and examination 

3.3.8 Staining Controls 

Positive staining controls are used to verify that the FITC and DAPI stains are fluorescing appropriately. 

Positive staining controls are prepared by applying 200 to 400 intact oocysts to a slide and staining the 

slide with the same reagents and staining procedure used to stain field samples. The analyst examines 

several fields of view to verify that the stain is fluorescing at the appropriate intensity and uniformity. 

Each analyst must characterize a minimum of 3 Cryptosporidium oocysts on the positive staining control 

slide before examining field sample slides per Section 15.2.1.1 in Method 1622/1623. Control slides and 

sample slides should be read on the same day. If sample slides from the same staining batch are read over 

multiple days, the control slide should be rechecked each day before examination of the sample slides. 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 at the same time with the batch of field slides and alternate between the positive controls 

when performing the positive control check. 

Negative staining controls are used to verify that no oocysts or interfering particulates are present. 

Negative staining controls are prepared by staining and examining a slide with phosphate buffered saline 

solution. 

The analyst should indicate on each Cryptosporidium slide examination form whether the positive 

staining control and negative staining control were acceptable. 

27 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

LT2 Rule requirement: Each sample must meet the QC criteria for the methods. Per EPA 

Method 1622/1623, positive and negative staining controls must 

be acceptable (Section 15.2.1). 

3.3.9 Examination Preparation 

To help the analyst identify the target analyte during field sample slide examination, each analyst must 

characterize a minimum of three Cryptosporidium oocysts on the positive staining control slide before 

examining field sample slides. This characterization must be performed by each analyst during each 

microscope examination session. FITC examination must be conducted at a minimum of 200X total 

magnification, DAPI examination must be conducted at a minimum of 400X, and DIC examination and 

size measurements must be conducted at a minimum of 1000X. 

Size, shape, and DIC and DAPI characteristics of the three Cryptosporidium oocysts must be recorded by 

the analyst in a microscope log (Section 15.2.1.1 of Method 1622/1623). 

3.3.10 Verification of Analyst Performance 

Analyst verifications are ongoing comparisons of slide counts and characterizations used to assess and 

maintain consistency in slide examination among analysts. The goal is to encourage comparison and 

discussion among analysts to continually refine their microscopy skills. At least monthly when 

microscopic examinations are being performed, the laboratory shall prepare at least one slide containing 

40 to 200 oocysts. More than 50 percent of the oocysts must be DAPI positive and undamaged under 

DIC. Another option is to order prepared slides from Wisconsin State Laboratory of Hygiene, Flow 

Cytometry Unit (http://www.slh.wisc.edu/, (608) 224-6260). 

For laboratories with multiple analysts, each analyst shall determine the DAPI category (DAPI negative, 

DAPI positive intense internal blue staining, and DAPI positive with number of nuclei) and the DIC 

category (empty, containing amorphous structures, or internal structure characterization) of the same 10 

selected oocysts. It is recommended that the DAPI and DIC categorization of the selected oocysts occur 

with all analysts at the same time, i.e. each analyst determines the category independently, then the 

differences in the DAPI and DIC categorizations among analysts are discussed and resolved, and these 

resolutions documented. This round-robin approach with all analysts may encourage further discussion 

among analysts and lead to more consistent characterizations. Alternatively, organism coordinates may be 

recorded for each analyst to locate and categorize the organisms at different times. Differences among 

analysts must still be discussed and resolved. 

Laboratories should be aware that both FITC and especially DAPI fluorescence may fade during the 

process of analyst verification. Therefore, DAPI comparisons should be performed first and the UV light 

shutter should remain closed except for a few seconds during observation by the analyst. Repeat 

comparisons with new FITC organisms, again taking care to keep the shutter closed whenever the oculars 

are not being used. 

Each analyst shall also determine the total number of oocysts by FITC fluorescence at 20X magnification 

for the entire slide. It is recommended that this count be performed last or on a separate slide than that 

used for the DAPI and DIC characterization so that fading will not influence counts. The total number of 

oocysts enumerated by each analyst must be within ±10 percent 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. 

28 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

Laboratories with only one analyst should maintain a protozoa library and compare the results of slide 

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

results are consistent with these references. These laboratories may also perform repetitive counts of a 

single verification slide for FITC and DAPI. These laboratories are encouraged to coordinate with other 

laboratories to share slides and compare counts. 

Analyst verification serves as an on-going and consistent training venue for new and experienced 

analysts. In addition to the monthly verification of analyst performance, other training options exist at 

universities, microscope service companies, and existing laboratories. EPA has produced an online 

microscopy module detailing the characterization of Giardia and Cryptosporidium. Viewing this module 

from the LT2 website, http://www.epa.gov/safewater/disinfection/lt2/, is open to all analysts as a 

supplement to one-on-one microscopy training. 

3.3.11 Acceptance Criteria for Receipt of Field Samples 

Cryptosporidium samples for LT2 Rule monitoring should be stored between 1EC and 10EC to reduce 

biological activity and so they cannot freeze. This is specified in Section 8.0 of the December 2005 

versions of EPA Method 1622/1623. Because Cryptosporidium samples collected for the LT2 Rule must 

meet the QC criteria in the methods, and because these QC criteria include receipt of samples at #20EC 

and not frozen, laboratories must reject LT2 Cryptosporidium samples that are received at >20EC or 

frozen. In these cases, the PWS must re-collect and re-ship the sample. 

Several options available to measure sample temperature upon receipt at the laboratory and, in some 

cases, during shipment, are provided in Section 2.2 of this manual. 

LT2 Rule requirement: Each sample must meet the QC criteria for the methods. Per EPA 

Method 1622/1623, samples not received on the day of collection 

must be received at the laboratory at ≤ 20°C and not frozen 

(Section 8.1 of the method).

3.3.12 Matrix Spike Samples 

The matrix spike (MS) in EPA Method 1622/1623 (Section 9.5.1 of the December 2005 version) entails 

analysis of an extra bulk water sample spiked with ~100 to 500 oocysts in the laboratory to determine the 

effect of the source water matrix on the method’s oocyst recovery. The laboratory should analyze an MS 

sample when samples are first received from a PWS location for which the laboratory has never before 

analyzed samples to identify potential method performance issues with the matrix. 

LT2 Rule requirement: Each sample must meet EPA Method 1622/1623 requirements [40 

CFR § 141.704(a)], which include the following: (1) The MS and 

field sample must be collected from the same sampling location by 

splitting the sample stream or collecting the samples sequentially; 

(2) The volume of the MS sample analyzed must be within 10 

percent of the volume of the field sample analyze); and (3) The 

MS and field sample must be analyzed by the same procedure 

(Section 9.5.1 of Method 1622/1623). 

29 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

3.3.12.1 Matrix Spike Frequency 

For all PWSs, the first MS sample should be collected and analyzed during the first sampling event under 

the monitoring program per EPA Method 1622/1623 (Section 9.1.8 of Method 1622/1623). If it is not 

possible to analyze an MS sample for the first sampling event, the first MS sample should be analyzed as 

soon as possible to identify potential method performance issues with the matrix. The laboratory and 

PWS should evaluate the MS recoveries, as well as other attributes of sample processing and 

examination, and work together to determine whether sample filtration and processing procedures are 

working acceptably or need to be re-evaluated. Matrix spike samples may be analyzed more frequently 

than one every 20 field samples to better characterize method performance in the matrix. 

Based on this requirement, the following PWS categories must analyze at least two MS samples during 

LT2 Rule monitoring: 

• Large PWSs that perform monthly monitoring for two years (resulting in 24 samples) 

• Small PWSs that are triggered into Cryptosporidium monitoring and collect semi-monthly 

samples for one year or monthly samples for two years (resulting in 24 samples) 

For large PWSs that perform semi-monthly or more frequent monitoring for two years (resulting in 48 or 

more samples), a minimum of three MS samples should be collected and analyzed. If a PWS monitors 

more frequently or collects more than 60 samples, a minimum of four MS samples must be analyzed. 

3.3.12.2 Matrix Spike Samples Associated with Field-Filtered Samples 

Matrix spike samples must be collected as bulk samples and spiked in the laboratory prior to filtration. 

The volume of the MS sample must be within 10 percent of the volume of the associated field sample. 

PWSs that field-filter 10 L samples may field filter the monitoring sample, but must collect and ship the 

10 L MS sample in bulk to the laboratory for spiking, filtering, and analysis. 

For PWSs that field-filter >10 L samples, all but 10 L of the MS sample may be filtered in the field. The 

remaining 10 L of source water for MS analysis may be collected in bulk and shipped to the laboratory. 

The laboratory will then spike the 10 L bulk sample and pump it through the filter containing the balance 

of the sample already filtered in the field. The associated monitoring sample must be collected as usual 

[40 CFR § 141.704(a)(2)(ii)]. 

3.3.12.3 Matrix Spike Control Charts 

As with the OPR samples and described in Section 9.5.1.4 of the December 2005 version of EPA 

Methods 1622/1623, laboratories should assess precision of MS recoveries. This can be accomplished by 

maintaining a control chart that graphically displays the results of continuing performance. It is suggested 

that the precision assessment be maintained across all MS samples as well as stratified by source. The 

control chart across all MS samples should be developed when at least 5 MS samples have been 

completed. It is the laboratory’s decision as to the maximum number of samples used to determine 

precision assessments. For individual sources completing only two MS samples, precision estimates 

should be calculated using percent difference instead of standard deviation. If more MS samples are 

completed per source, the laboratory should generate precision assessments using the mean and standard 

deviation. 

30 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

The control chart is developed by plotting percent recovery of each matrix spike sample versus time. Based on the 

mean of the recoveries (0) on the chart, the upper and lower control limits should be established as follows: 

• Upper control limit = 0 + 2 standard deviations 

• Lower control limit = 0 - 2 standard deviations 

An example of a control chart (using OPR data, not MS data) is provided in Figure 3-2. Control charts 

can be used to track the laboratory’s performance and determine if any trends in recovery are occurring. 

Control charts can also be used to compare performance of different method variations, different analysts, 

and other changes implemented by the laboratory along with performance in different matrices. If 

recovery measurements fall outside the control limits, laboratories may take corrective action, 

investigating potential causes of the outlying result. 

3.3.13 QC Guidance for Method Modifications and Use of Multiple Method 

Variations 

EPA Methods 1622/1623 are performance-based methods and, therefore, allow method modifications if a 

laboratory can meet applicable QC criteria (EPA Method 1622/1623 [Section 9.1.2]). Table 3-4 presents 

examples of changes to EPA Method 1622/1623 that EPA considers to be “routine,” “occasional,” and 

“substantive,” and what QC steps are necessary to demonstrate acceptability before implementing these 

changes. 

31 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

Table 3-4. Recommended QC for Different Types of Changes to EPA Method 1622/1623 

 “Routine” Changes 

Using new lots of the same method component, such as 

• new lots of filters 

• IMS kits 

• stains 

• other reagents including reagent water 

Verification of reagent 

acceptability, per routine 

OPRs/method blanks, is 

recommended. 

Changing to new equipment that meets existing specifications in the 

method (e.g., pumps or centrifuges) 

“Occasional” Changes 

Using different equipment, reagents or procedures for which specifications 

are not included in the method, such as 

• lab shaker 

• IMS magnets 

• Leighton tubes 

• slides 

• mounting medium 

• centrifuge speeds 

• slide drying procedures 

• staining procedure using the same brand of reagents 

• vortex speeds during IMS 

• microscope 

Initial precision and recovery, 

method blank, matrix spike/matrix 

spike duplicate, and unspiked 

field sample are recommended. 

“Substantive” Changes 

Changing to a different filter 

New demonstration of acceptable 

performance is required through 

initial precision and recovery, and 

method blank. In addition, matrix 

spike/matrix spike duplicate, and 

unspiked field sample is strongly 

recommended. 

Changing flow rate (e.g., using a flow rate of 4 L/min with the EnvirochekTM 

HV version of Method 1623, rather than the 2 L/min flow rate specified in 

the method) 

Changing sample volumes (e.g., processing 50 L samples rather than 10 L 

samples) 

Changing to a different Filta-Max® concentration or elution procedure: 

• Standard wash station and concentrator tube 

• Stomacher and concentrator tube 

• Stomacher and centrifugation 

Incorporating the use of multiple filter membranes in the Filta-Max® 

concentrator tube 

Changing to a different antibody staining kit 

Incorporating additional rinses and transfers to reduce carryover from IMS 

to the slide 

Changing vendors for spike organisms 

Changing laboratory space or location 

3.3.13.1 Making a Substantive Change to the Method 

Substantive changes to Method 1622/1623 as described in Table 3-4 essentially comprise a new variation 

on the method. A method variation is the complete set of sample processing components (including the 

filter, IMS, and stain) and sample processing procedures (including filtration, concentration, purification, 

and staining) used to process a water sample for examination. Per EPA Method 1622/1623 (Section 

9.1.2), if a laboratory intends to switch completely from one method variation to another, then the 

laboratory should demonstrate acceptable QC as outlined in Table 3-4 using the new method variation 

before implementing this procedure for the analysis of field samples. If the laboratory demonstrates 

32 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

acceptable initial laboratory performance and implements the new method variation and discontinues use 

of the old method variation, then the laboratory must demonstrate acceptable ongoing laboratory 

performance (through the OPR, method blank, and OPT tests) using the new variation. 

Guidance on requirements for initial and ongoing demonstrations of acceptable laboratory performance 

for different method variations is provided in Sections 3.3.14.2 of this manual. The examples of 

substantive changes given in Table 3-4 have been shown to be effective in multiple laboratories with 

different source water matrices. Guidance to demonstrate acceptability of substantive changes that are 

novel is discussed in Section 3.3.13.3 of this manual. 

3.3.13.2 Using Multiple Method Variations 

Per EPA Method 1622/1623 (Section 9.1.2), if a laboratory intends to use multiple method variations 

(that differ through a substantive change) concurrently, then the laboratory demonstrates acceptable initial 

laboratory performance (through the IDC test per the Lab QA Program) using each method variation 

before implementing this procedure for the analysis of LT2 Rule samples. In addition, the laboratory 

demonstrates acceptable ongoing laboratory performance (through the OPR, method blank, and OPT 

tests) for each substantive method change for all but the following method variations: 

• Antibody staining kits. If a laboratory alternates among more than one antibody staining kit, the 

laboratory performs positive and negative staining controls for each antibody kit for each batch of 

slides for which the kit is used and should alternate between the kits for ongoing demonstrations 

of acceptable laboratory performance. MS samples should be processed using the same method 

variation as the associated field sample, regardless of the method variation used to demonstrate 

ongoing acceptable laboratory performance. 

• Additional rinses and transfers. If the laboratory uses additional rinses and transfers for some 

samples to reduce carryover from IMS onto the slide, the laboratory should use this procedure 

(which may reduce recoveries) to demonstrate acceptable ongoing laboratory performance on the 

same percentage of OPR samples as percentage of field samples analyzed with this variation. MS 

samples should be processed using the same method variation as the associated field sample, 

regardless of the method variation used to demonstrate ongoing acceptable laboratory 

performance. 

• Multiple membranes for Filta-Max® concentration. If the laboratory uses multiple membrane 

filters in the Filta-Max® particle concentrator for some samples, the laboratory should use 

multiple memebrane filters to demonstrate acceptable ongoing laboratory performance on at least 

the same percentage of OPR samples as the percentage of field samples analyzed with multiple 

membranes. MS samples should be processed using the same method variation (and same 

number of membranes) as the associated field sample, regardless of the method variation used to 

demonstrate ongoing acceptable laboratory performance. 

• Multiple sample volumes. See discussion in Section 3.3.14. 

3.3.13.3 Substantive Change Acceptability through Alternate Test Procedure (ATP) 

or Tier 2 validation 

Novel changes with new kinds of equipment or reagents cannot be evaluated using the QC tests and 

criteria described above because experiments with more than one water matrix or a study designed to 

compare the new procedure with an established test procedure are necessary for maintaining the same 

level of data quality. EPA’s Microbiological ATP Protocol (Reference 5.7) describes a process for 

33 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

conducting QC acceptance criteria-based studies and for conducting side-by-side comparisons to 

demonstrate comparability with an EPA-approved method. For example, the ATP protocol would be 

useful for evaluating new elution systems or new staining or immunomagnetic separation techniques. 

The Tier 2 validation is used if nationwide approval of a modification is sought (Section 9.1.2.1.2 of 

Method 1622/1623). A Tier 2 validation includes analysis of an IPR set, method blank, MS/MSD, and 

field sample at a minimum of 3 laboratories and compares the results to the methods’ QC criteria. 

3.3.14 Guidance on QC for Different Sample Volumes 

A laboratory with multiple PWS clients representing a range of sample volumes is not responsible for 

performing QC tests at all of the volumes. However, if the laboratory does analyze both 10 L and 50 L 

sample volumes for clients—or any volumes in between—then the laboratory should demonstrate 

acceptable performance in a manner representative of the sample volumes they process. Guidance on 

initial and ongoing demonstrations of acceptable laboratory performance is provided below, in Sections 

3.3.14.1 through 3.3.14.3. 

3.3.14.1 Initial Precision and Recovery Tests for Different Sample Volumes 

A laboratory with multiple PWS clients representing a range of sample volumes should successfully 

perform the IPR and method blank test, as well as successfully analyze matrix spike/matrix spike 

duplicate (MS/MSD) and initial proficiency testing (IPT) samples (for the Lab QA Program's initial 

demonstration of capability [IDC]), at the largest (most challenging) volume. The laboratory should 

demonstrate acceptable performance for these tests using spikes of no greater than 500 oocysts. 

3.3.14.2 Ongoing Precision and Recovery Tests and Method Blank Tests for 

Different Sample Volumes 

A laboratory demonstrates ongoing acceptable performance at both extremes of the volume spectrum by 

performing OPRs and method blanks at a volume consistent with the highest sample volume submitted by 

clients (e.g. 50 L) as well as OPRs and method blanks at a volume consistent with the lowest sample 

volume submitted by clients (but not less than 10 L). Labs analyzing multiple sample volumes, should 

demonstrate acceptable performance in a manner representative of the sample volumes they process i.e. at 

the same percentage as volumes in field samples submitted to the laboratory. Frequency of OPRs with 

different volumes should be consistent with the frequency of samples with different volumes, e.g. if half 

of the samples received have 50 L volumes and half have 10 L volumes, then half of the OPRs should be 

performed with 50 L and half with 10 L. Laboratories should work with their PWS clients to attempt to 

schedule clients with different sample volume sizes for different periods during the week, so the field 

samples can be batched with QC samples of comparable volume. 

3.3.14.3 Ongoing Proficiency Tests for Different Sample Volumes 

Laboratories with multiple PWS clients representing a range of sample volumes are not required to 

analyze OPT samples at each sample volume. The laboratory should notify EPA of the sample volume 

most representative of the LT2 samples processed and perform the OPT test using this volume. 

3.4 Sample Collection Procedures 

Several options are available to the PWS for collecting untreated surface water samples for 

Cryptosporidium analysis, including the following. 

• On-site filtration of water samples from pressurized or unpressurized sources using the Pall 

Gelman EnvirochekTM or EnvirochekTM HV capsule filter. 

34 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

• On-site filtration of water samples from pressurized or unpressurized sources using the IDEXX 

Filta-Max® foam filter. 

• Collection of bulk water samples for shipment to the laboratory for filtration and analysis. 

Detailed procedures for each of these options, as well as packing and shipping the samples from the PWS 

to the Cryptosporidium analysis laboratory, are provided as appendices in the Source Water Monitoring 

Guidance Manual for Public Water Systems for the Long Term 2 Enhanced Surface Water Treatment 

Rule (LT2 Rule) (Reference 5.15). 

As noted in the PWS guidance manual, EPA recommends that the laboratory and PWS conduct at least 

one practice sampling and analysis event prior to starting official LT2 monitoring. Based on previous 

experiences in the Information Collection Rule (ICR) and ICR Supplemental Surveys, unanticipated 

problems are often encountered during the first sampling event, but are addressed in subsequent events. 

Rather than risking sampling problems during official LT2 monitoring, the PWS and laboratory can 

identify and resolve any problems by conducting the practice sampling and analysis. 

The contract laboratory is often involved with PWSs regarding sampling issues including scheduling, 

multiple sources, location, and replacement samples. These issues are detailed in the Source Water 

Monitoring Guidance Manual for Public Water Systems for the Long Term 2 Enhanced Surface Water 

Treatment Rule (LT2 Rule); however, an overview is below. 

• Scheduling: PWSs are encouraged to work with the contract laboratory to establish a schedule 

that will comply with LT2 Rule requirements and is mutually acceptable to the PWS and the 

laboratory. 

• Sampling Location: PWSs are required to collect source water samples for the LT2 Rule from 

the plant intake prior to chemical treatment, unless approved by the State to collect the source 

water sample after chemical treatment [40 CFR § 141.703(b)(2)]. Systems that recycle filter 

backwash water must collect source water samples prior to the point of filter backwash water 

addition [40 CFR § 141.703(c)]. All Cryptosporidium, E. coli, and turbidity source water samples 

collected under LT2 Rule requirements should be collected from the same appropriate sampling 

location. 

• Multiple Sources: The use of multiple sources during monitoring must be consistent with routine 

operational practice [40 CFR § 141.703(e)]. If there is a tap prior to treatment where sources are 

combined, the sample must be collected there. If not, the PWS must pursue one of the following 

options: 

- collect manually from each source prior to treatment and composite into one sample in the 

same proportion as used by the plant at the time of collection 

- collect manually from each source prior to treatment, analyze separately, and calculate a 

weighted average of the analysis results. 

• Replacement Samples: Certain situations may dictate the need for a replacement sample, i.e. the 

PWS is unable to report a valid Cryptosporidium analytical result for a scheduled sampling date. 

The following list details some of the possible situations: 

- sample not collected during required time frame due to extreme conditions or situations that 

may pose a danger to the sampler 

- sample is lost or contaminated 

35 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

- laboratory exceeds analytical method holding time 

- sample receipt temperature fails criteria 

- volume requirements not met (field and/or MS sample) 

- QC samples fail acceptance criteria 

- problems encountered during processing 

- failure of an approved laboratory to analyze the sample 

The PWS must submit an explanation for the delayed sampling date to the EPA/State concurrent 

with the shipment of the replacement sample to the laboratory. The system must collect a 

replacement sample as close to the required date as feasible but within 21 days of being notified 

by the laboratory that a result cannot be reported for that date [40 CFR § 141.702(b)(2)]. 

3.5 Recordkeeping 

An effective record keeping system provides information on sample collection and preservation, 

analytical methods, raw data, calculations, reported results, and a record of persons responsible for 

sampling and analyses. For EPA Methods 1622/1623, original data, including microscope examination 

counts and notes, must be recorded. The data may be recorded on bench sheets (Appendix G) and slide 

examination forms (Appendix H). 

Data should be recorded in ink and a single line drawn through any change with an initialed, dated 

correction entered next to it. Data files may also be microfiche or electronic. Electronic data should be 

backed up by a protected tape or disk or hard copy. Under the LT2 Rule, monitoring data (both initial and 

second round of monitoring) must be kept until 3 years after bin classification for filtered systems or 

determination of mean Cryptosporidium levels for unfiltered systems [40 CFR § 141.722(a)]. Although it 

is the PWS’s responsibility to meet LT2 Rule data storage requirements for compliance monitoring 

samples, the PWS may contract this work to the laboratory. 

As laboratories perform Cryptosporidium analyses during the LT2 Rule, the following data recording 

practices should be followed: 

• Record sample identification information, including sample collection and receipt dates and 

conditions 

• Record all raw data (primary measurements) used to calculate final concentrations of oocysts/L 

for each sample 

• Record the date and time of each method step associated with a holding time to verify that all 

method holding times have been met 

• Record the name of the analyst performing each method step to verify that only qualified 

technicians and analysts are performing the method 

The minimum data elements that should be recorded for Cryptosporidium samples during the LT2 are 

discussed in detail below. These data elements are critical to ensuring that final sample concentrations can 

be verified using primary data and are necessary to demonstrate that all method-specified holding times 

were met. Standardized bench sheets and Cryptosporidium examination results forms are available for 

download as Appendices G and H on the LT2 website 

http://www.epa.gov/safewater/disinfection/lt2/compliance_mlmanual.html. 

36 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

Sampling records provided by the PWS with the sample should include the following information, at a 

minimum: 

• Public water system name and ID number* 

• Facility name and number* 

• Sample Collection Point Name and ID 

• Date and start/stop times of collection* 

• Sampler’s name and phone number (or alternate contact for laboratory if problems are 

encountered) 

• Source water temperature and turbidity 

• Volume filtered information (if the sample was filtered in the field) 

• Whether the filter clogged (if the sample was filtered in the field) 

• Analyses requested (e.g. routine field sample analysis or field sample + MS analysis) 

* Note: These three elements are used to identify the LT2 sample for tracking sample collection, 

analysis, reporting, and use. 

Detailed guidance on sample collection data recording, as well as forms and sample collection and 

shipping procedures can be found in the Source Water Monitoring Guidance Manual for Public Water 

Systems for the Long Term 2 Enhanced Surface Water Treatment Rule (LT2 Rule). This manual is 

available for download from http://www.epa.gov/safewater/disinfection/lt2/compliance.html. 

Upon receipt of the sample at the laboratory, laboratory personnel should record, at a minimum, the 

information in Table 3-5. Laboratories should immediately notify utilities of any deficiencies requiring a 

resample. 

Table 3-5. Sample Receipt Data Elements to Record in the Laboratory 

Public water system name and ID 

Facility name and ID 

Sample collection point name and ID 

Turbidity at the collection point taken immediately after sample collection 

Date and time of sample collection (start and stop times if field filtered) 

Date and time of sample receipt by laboratory 

Volume filtered (if sample is filtered in the field) 

Name of laboratory person receiving the sample 

Temperature of sample upon receipt 

Any deficiencies (deficiencies may include but are not limited to: exceeded sample holding time, transport 

temperature exceeded 20EC, or sample leaked during transport)

Laboratories analyzing samples for Cryptosporidium using EPA Method 1622/1623 in support of the LT2 

Rule should record the primary elements required to calculate the final concentrations and percent 

recoveries for matrix spike (MS), ongoing precision and recovery (OPR), and proficiency test samples. 

These primary data elements are provided in Table 3-6, and should be recorded on the EPA Method 

1622/1623 bench sheet (Appendix G) and slide examination form (Appendix H). 

37 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

Table 3-6. Primary Data Elements to Record for Calculations 

Estimated number of oocysts spiked (MS and OPR samples), based on information provided by the flow-cytometry 

laboratory with the spiking suspension 

Sample volume spiked, in L (MS, OPR and proficiency test samples)

Sample volume filtered, to nearest 1/4 L 

Number of filters used (if the filter clogged)

Pellet volume after concentration, to the nearest 0.1 mL

Total volume of resuspended concentrate, in mL

Volume of the resuspended concentrate transferred to IMS, in mL

Number of subsamples analyzed

Total number of oocysts detected in the sample 

To determine that all method QC requirements were met and that the samples were analyzed by qualified 

personnel according to the requirements of the Laboratory QA Program for the Analysis of 

Cryptosporidium the laboratory should record the elements in Appendices G and H. These appendices 

include, but are not limited to, the key QC data elements in Table 3-7. 

Table 3-7. Key QC Data Elements to Record 

Elution date and time (must be within 96 hours of sample collection) 

Slide preparation date and time (must be completed in same working day as elution) 

Sample staining date and time (must be completed within 72 hours of slide preparation) 

Sample examination date and time (must be completed within 168 hours (7 days) of sample staining) 

Person (PWS or Lab employee) performing filtration 

Analyst performing elution 

Analyst performing IMS 

Analyst performing sample staining 

Analyst performing sample examination 

Results of the positive and negative staining controls 

The laboratory should also record any additional information that will support the results obtained or 

allow problems with sample results and laboratory performance to be identified. This additional 

information includes the following: 

• Information on the version of EPA Method 1622/1623 used to perform the analysis including 

filter type, elution procedure, concentration procedure, IMS system used, detection kit used, and 

source of oocysts for spiking suspensions 

• Lot numbers of reagents and materials used during the analysis, including the filter, elution 

buffer, IMS system, detection kit, and spiking suspension 

• FITC, DAPI, and DIC information of all oocysts detected in the field samples using the slide 

examination form 

38 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

This information should be recorded on the EPA Method 1622/1623 bench sheet (Appendix G) and 

Cryptosporidium slide examination form (Appendix H), as appropriate. 

Size, shape, and DIC and DAPI characteristics of the three Cryptosporidium oocysts on the positive 

staining control slide (Section 15.2.1.1 of Method 1622/1623) must be recorded by the analyst on a 

microscope log. 

3.6 Calculations for EPA Methods 1622/1623 

During LT2 Rule monitoring, field sample results will be reported using the LT2/Stage 2 Data Collection 

and Tracking System (DCTS) described in Section 3.7 of this manual. The DCTS will reduce the data 

elements entered by the laboratory to yield final sample results in oocysts/L. The DCTS will also verify 

that LT2 Rule Cryptosporidium sample volume analysis requirements were met and calculate MS 

recoveries. Sections 3.6.1 through 3.6.3 are guidance for laboratories to perform the same type of 

calculations as the DCTS for oocyst concentrations, matrix spike recoveries, and OPR recoveries. The 

laboratory may choose to report the total oocysts and volume analyzed, along with the oocysts/L, and 

other primary data elements listed in Table 3-6 to the PWS. The PWS may then use that report during the 

DCTS data review process discussed in Section 3.7.2 of this manual. For QC samples, the laboratory 

calculates recoveries for OPR and MS samples to report to their PWS and to maintain QC control charts 

as discussed in Sections 3.3.5 and 3.3.12.3 also in this manual. 

3.6.1 Calculating Oocyst Concentrations 

To calculate the concentration of Cryptosporidium in a sample, reported as oocysts/L, the following 

information is needed: 

• Number of oocysts detected in the sample (recorded as a primary measurement from the slide 

examination form) 

• Volume analyzed 

Using these two data elements, the final concentration should be calculated as: 

oocysts detected in the sample 

final concentration (oocysts/L) = 

volume analyzed (L) 

If 100% of the sample volume filtered is examined, then the volume analyzed equals the volume filtered. 

This applies whether one filter or more than one filter was used; if more than one filter was used, and all 

of the volume filtered through the multiple filters is processed through the remainder of the method, then 

the volume examined is simply the sum of the volumes filtered through each of the filters used. 

If <100% of the volume filtered was processed through the remainder of the method, then additional 

calculations are needed to determine the volume analyzed. This is discussed in Section 3.6.1.1 through 

3.6.1.3 of this manual. 

3.6.1.1 Determining Volume Analyzed when Less than 100% of Sample Was 

Examined 

When <100% of the sample filtered is processed through the remainder of the method and examined 

(such as when the volume filtered yields >2 mL of packed pellet volume after centrifugation), then the 

volume analyzed should be determined using the following equations to determine the percentage of the 

sample that was examined. 

39 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

percent examined = total volume of resuspended concentrate transferred to IMS (mL) (see Section 3.6.1.2 

(expressed as a of Method 1622/1623) 

decimal) total volume of resuspended concentrate (mL) 

volume analyzed (L) = percent examined × sample volume filtered (L) 

3.6.1.2 Determining the Volume of Resuspended Concentrate to Use for Packed 

Pellets > 0.5 mL 

Packed pellets with a volume >0.5 mL must be divided into subsamples. You should use the formula 

below to determine the total volume of resuspension required in the centrifuge tube before separating the 

concentrate into two or more subsamples and transferring to IMS. 

pellet volume (mL) after centrifugation 

total volume of resuspended concentrate (mL) required = x 5 mL

0.5 mL 

3.6.1.3 Example Calculation 

Example. A 10 L field sample was filtered and processed, producing a packed pellet volume of 2.7 

mL. The laboratory transferred 20 mL of the total resuspended concentrate (27 mL) to 

IMS and examination (because a minimum of 2 mL of pellet is required for analysis if 10 

L cannot be examined or 2 filters did not clog [see Section 3.1.3 of this manual]). The 

laboratory detected 20 oocysts during examination. The following calculations were 

performed to determine the volume analyzed and final concentration. 

2.7 mL 

total volume of resuspended concentrate (mL) required = × 5 mL = 27 mL 

0.5 mL 

20 mL 

percent examined = = 0.74 (74%) 

27 mL 

volume analyzed (L) = 0.74 × 10 L = 7.4 L 

20 oocysts 

final concentration (oocysts/L) = = 2.7 oocysts/L 

7.4 L 

3.6.2 Matrix Spike Recovery Calculations 

To determine the percent recovery for a matrix spike (MS) sample the following information is needed: 

• The number of oocysts counted in the MS sample 

• The estimated number of oocysts spiked into the MS sample 

• The number of oocysts counted in the unspiked field sample (to correct for background 

concentration) 

40 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

oocysts counted in MS sample - oocysts counted in unspiked field sample

percent recovery = × 100%

oocysts spiked into MS sample

This calculation assumes that the same sample volume was examined for both the field and MS samples. 

If the sample volumes examined are different, you should calculate the number of oocysts per L for both 

the field and MS samples before calculating percent recovery. If both a matrix spike (MS) and a matrix 

spike duplicate (MSD) are analyzed, then the mean recovery and relative percent difference should be 

calculated and compared to the acceptance criteria in Tables 3 and 4 of the December 2005 version of 

EPA Method 1623. The percent recovery for each sample should be calculated as described above to 

determine the mean recovery. 

To calculate the mean percent recovery, you should calculate the percent recovery for each sample, as 

described above, and then use the following formula: 

percent recovery of MS sample + percent recovery of MSD sample 

mean percent recovery = 

2 

To calculate the relative percent difference (RPD), the absolute value (without sign) of the difference 

between the number of oocysts counted in the MS and MSD should be divided by the mean of the oocysts 

counted in both samples to yield a percentage of the difference. This calculation assumes that the same 

volume is analyzed for both the MS and MSD. You should calculate the number counted per L before 

calculating the RPD if volumes analyzed are different. 

*oocysts counted in MS - oocysts counted in MSD*

RPD = × 100% 

((oocysts counted in MS + oocysts counted in MSD)/2) 

Example. The laboratory prepared both the MS and MSD by spiking two 10 L samples with 100 

oocysts each. The laboratory detected 45 oocysts in the MS sample and 50 oocysts in the 

MSD. In the 10 L unspiked field sample only 2 oocysts were detected. To determine the 

percent recovery for each sample and the mean recovery and relative percent difference 

of the MS and MSD, the following calculations were performed. For both the MS and 

MSD, as well as the unspiked field sample, the entire 10 L sample was filtered and 100% 

of the sample was examined. 

45 oocysts - 2 oocysts 

MS percent recovery = × 100% = 43% 

100 oocysts 

50 oocysts - 2 oocysts 

MSD percent recovery = × 100% = 48% 

100 oocysts 

41 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

43% + 48% 

mean recovery = = 45.5% 

2 

*45 oocysts - 50 oocysts*

RPD = × 100% = 10.5% 

((45 oocysts + 50 oocysts) / 2) 

3.6.3 OPR Sample Calculations 

The percent recovery of an OPR sample should be calculated using the following formula: 

oocysts detected 

percent recovery = × 100% 

oocysts spiked 

Example: The laboratory prepared the OPR sample by spiking 50 L with 150 oocysts. The entire 

sample was filtered and examined. The laboratory detected 76 oocysts. 

76 oocysts 

percent recovery = × 100% = 50.7% 

150 oocysts 

OPR recoveries are compared to the limits for ongoing recovery in Tables 3 and 4 of the December 2005 

version of EPA Method 1623. These recoveries are tracked over time using control charts to assess 

precision, as discussed in Section 3.3.5, above. 

3.7 Electronic Data Reporting 

During the LT2 Rule, laboratories will report Cryptosporidium data to their PWS clients electronically 

through EPA’s LT2/Stage2 Data Collection and Tracking System (DCTS). The DCTS is a web-based 

application that allows laboratory users to enter or upload data, then electronically “release” the data to 

the PWS for review, approval, and submission to EPA and the State. Although ownership of the data 

resides with the PWS throughout this process, the DCTS increases the ease and efficiency of the data 

entry and transfer process from one party to another by transferring the ability to access the data from the 

laboratory to the PWS to EPA and the State, and ensuring that data cannot be viewed or changed by 

unauthorized parties. A summary of the data entry, review, and transfer process through the DCTS is 

provided in Table 3-8, below. 

The data reporting process is described in more detail below, in Sections 3.7.1 through 3.7.3, and is also 

described in detail in the Users’ Manual for the LT2/Stage 2 Data Collection and Tracking System 

(DCTS). The DCTS users’ manual also provides detailed information on the PWS user registration 

process. Information on the DCTS and a downloadable users’ manual are available at 

http://www.epa.gov/safewater/disinfection/lt2/. 

42 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

Table 3-8. LT2/Stage 2 Data Collection and Tracking System Data Entry, Review, and Transfer 

Laboratory actions 

• Laboratory posts analytical results to the DCTS 

• DCTS reduces data and checks data for completeness and compliance with LT2 Rule requirements 

• Laboratory Principal Analyst confirms that data meets quality control requirements 

• Laboratory “releases” results electronically to the PWS for review 

• Laboratory user cannot edit data after it is released to the PWS 

EPA does not have access to data 

PWS actions 

• PWS reviews electronic data through the DCTS 

• PWS cannot edit data - only review data and either return to laboratory to resolve errors or submit to 

EPA 

• PWS “releases” data back to the laboratory if it has questions 

• If no questions, PWS submits data to EPA as “approved” or “contested” (indicating that samples have 

been correctly analyzed, but that the PWS contends that they are not valid for use in LT2 binning) 

• If the PWS does not review the sample result by the deadline for submitting it to EPA (no later than 10 

days after the end of the first month following the month when the sample was collected) the sample 

result status in the DCTS is automatically changed to “approved” to prevent a monitoring violation report 

from generating. 

EPA and State actions 

• EPA and State users cannot edit data - only review data 

• EPA and State review data through the DCTS and approve results where appropriate 

• Contested results 

- If EPA/the State rejects the PWS explanation for the contested sample, the sample is marked 

“EPA approved” in the DCTS 

- If EPA/the State accepts the PWS explanation for the contested sample, the sample is invalidated 

and the PWS must resample 

3.7.1 Data Entry/Upload 

The analyst or another laboratory staff member enters a subset of the data recorded at the bench (Section 

3.5 of this manual) into the DCTS, either by entering the data using web forms or by uploading data in 

XML format (see the DCTS users’ manual). In accordance with 40 CFR § 141.706(e)(1), this information 

includes the following: 

• PWS ID 

• Facility ID 

• Sample collection date 

• Sample type (field or MS) 

• Sample volume filtered (L), to nearest 1⁄4 L 

43 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

• Was 100% of filtered volume examined? 

• Number of oocysts counted 

• For samples in which less than 10 L is filtered or less than 100% of the sample volume is 

examined, the laboratory also must enter or upload the number of filters used and the packed 

pellet volume 

• For samples in which less than 100% of sample volume is examined, the laboratory also must 

report the volume of resuspended concentrate and volume of this resuspension processed through 

immunomagnetic separation 

• For matrix spike samples, the laboratory also must report the sample volume spiked and 

estimated number of oocysts spiked; these data are not required for field samples 

By entering Cryptosporidium data into the system, the laboratory acknowledges that the following QC 

requirements were met including: all holding times, sample condition on receipt, results of associated 

method blank, OPR, and positive and negative staining controls. The DCTS allows for replacement 

samples to be entered and marked as replacement samples. See the DCTS users’ manual for guidance. 

After the information has been entered or uploaded into the data system, the system will reduce the data 

to yield final sample results, in oocysts/L, verify that LT2 Rule Cryptosporidium sample volume analysis 

requirements were met for samples in which less than 10 L were analyzed, and calculate MS recoveries. 

The laboratory’s Principal Analyst under the Lab QA Program is generally responsible for verifying the 

quality and accuracy of all sample results in the laboratory. If inaccuracies or other problems are 

identified, the Principal Analyst discusses the sample information with the analyst or data entry staff and 

resolves the issues before the data are released for PWS review. 

If no inaccuracies or other issues are identified, the laboratory approves the reported data for “release” to 

the PWS for review (EPA does not receive the data at this point). When the data are approved, the rights 

to the data are transferred electronically by the system to the PWS, and the data can no longer be changed 

by the laboratory. 

3.7.2 PWS Data Review 

After the laboratory has released Cryptosporidium data electronically to the PWS using the DCTS, the 

PWS will review the results. The PWS user cannot edit the data, but if the PWS has an issue with the 

sample result, such as if the PWS believes that the sample collection point ID or collection date is 

incorrect, the PWS can release the results back to the laboratory for issue resolution. In addition to noting 

the reason in the DCTS for the return of the data to the laboratory, the PWS may also contact the 

laboratory verbally to discuss the issue. 

If the PWS determines that the data are accurate, the PWS releases the results to EPA (and the State, if 

applicable) as “approved” results. If the PWS determines that the data are accurate, but believes that the 

data are not valid for LT2 binning purposes, the PWS can release the results to EPA and the State as 

“contested.” Contested samples are those that have been correctly analyzed, but that the PWS contends 

are not valid for use in LT2 binning, and have been submitted to EPA for evaluation. 

44 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

3.7.3 EPA/State Review 

After the PWS has released the results as approved or contested, they are available to EPA and State users 

to review through the DCTS. EPA and State users cannot edit the data. EPA or State users approve 

results where appropriate. Pursuant to 141.702(a)(2), resampling must occur whenever EPA or the State 

rejects results or indicates agreement with a PWS action to contest a result. 

3.8 Data Archiving 

The PWS is required to keep all original, hardcopy monitoring results associated with LT2 sample 

analyses (both initial and second round of monitoring) for 3 years after bin classification for filtered 

systems or determination of mean Cryptosporidium level for unfiltered systems [40 CFR § 141.722(a)]. 

Although it is the PWS’s responsibility to meet LT2 Rule data storage requirements for compliance 

monitoring samples, including MS samples, the PWS may contract this work to the laboratory. 

3.8.1 Hardcopy Data 

The following data should be archived: 

• Bench sheets and slide examination forms for all LT2 monitoring samples, including both field 

samples and MS samples 

• Bench sheets and slide examination forms for all OPR samples and method blank samples, and 

records of the compliance monitoring samples associated with each OPR sample and blank 

sample 

• Spike enumeration information received from Cryptosporidium spiking suspension vendors 

• Bench sheets and slide examination forms for all OPT samples 

As part of the Lab QA Program, the laboratory also should maintain the same documentation for their IPR 

and IPT data for each method variation used for LT2 samples. 

3.8.2 Slides 

Although not required, laboratories also may want to archive slides and/or take photographs of slides to 

maintain for clients. Slides should be stored in the dark between 1E and 10EC and not frozen, and in the 

appropriate type of chamber for the mounting medium used. 

As an alternate to the DABCO/glycerol mounting medium, currently specified in EPA Method 1622 and 

1623, laboratories may wish to evaluate the use of the elvanol mounting medium, which hardens, and 

may be useful for archiving slides. EPA recommends quality control assessment before changing the 

mounting medium currently used by the laboratory including initial precision and recovery, method 

blank, matrix spike/matrix spike duplicate, and unspiked field samples. Reagents for the mounting 

medium include the following: 

• 8.0 g elvanol (polyvinyl alcohol (PVA))—MP Biomedical cat. no. 151937, Solon, Ohio, or 

equivalent 

• 48.0 g (40 mL) glycerol 

• 10% NaN3 (sodium azide) 

• DABCO—Sigma-Aldrich, cat no. D-2522, or equivalent 

45 February 2006 
Section 3: Guidance for Cryptosporidium Laboratories 

• Tris buffer—Dissolve 1.2 g Tris (Fisher cat. no. BP152) in 95 mL reagent water, adjust pH to 8.5 

with 1 N HCl 

To prepare the medium, you should use the following procedure: 

• Add 48.0 g (40 mL) glycerol to 8.0 g elvanol and stir. 

• Add 49.0 mL of reagent water and 1.0 mL 10% NaN3 and stir. Let stand 4 hours at room

temperature. 

• Add DABCO in Tris buffer (4.75 g of DABCO in 100 mL Tris buffer, adjusted to pH 8.5 with 

conc. HCl) and stir. 

• Place mixture in a boiling water bath until the mixture becomes homogenous. 

• Centrifuge mixture at 2000XG for 10 minutes. Centrifugation of entire mixture in one tube is 

preferable. 

• Dispense 3- to 5 mL aliquots of the mixture into tubes and store at 0EC to 10EC. 

If the mounting medium sets up in the tube during storage, re-heat in boiling water bath or microwave for 

a short time to restore mixture to liquid state. 

Make sure to test any new reagent first on QC samples to verify that the mounting medium performs 

properly before using the medium on any monitoring samples. 

Commercially prepared mounting media for archiving slides are also available. For example, Waterborne 

Inc. produces an archiving mounting medium (product no. M102) that is fade-retardant and self-sealing. 

Vector Laboratories produces both an anti-fade mounting medium and an archiving anti-fade mounting 

medium (Vectashield® Mounting Medium and HardSetTM Mounting Medium, product no. H-1000 and 

H-1400). 

3.9 Equipment, Supplies, Reagents, and Standards 

See Sections 6 and 7 of EPA Method 1622/1623 for details on the materials needed to perform the 

Cryptosporidium analyses specified in the methods. 

46 February 2006 
SECTION 4: GUIDANCE FOR E. COLI LABORATORIES

The Long-Term 2 Enhanced Surface Water Treatment (LT2) rule requires that large filtered PWSs (those 

serving ≥10,000) perform E. coli and turbidity analyses on source water samples [40 CFR § 141.701(a)]. 

The Cryptosporidium, E. coli, and turbidity data will be analyzed to confirm or refine the relationship 

between Cryptosporidium and E. coli levels in source waters and refine the previously established trigger 

levels for E. coli. To reduce costs, small filtered PWSs (those serving <10,000) will use E. coli 

monitoring to determine the need for implementing more expensive Cryptosporidium monitoring or 

improved treatment. 

Turbidity measurement requirements and recommendations are addressed in the Source Water 

Monitoring Guidance Manual for Public Water Systems for the Long Term 2 Enhanced Surface Water 

Treatment Rule (LT2 Rule) (http://www.epa.gov/safewater/disinfection/lt2/). Laboratories performing E. 

coli analyses under LT2 must be certified under the Drinking Water Laboratory Certification program 

(described below) to analyze drinking water compliance samples. The procedures required of a 

laboratory to become certified are described in the Manual for the Certification of Laboratories Analyzing 

Drinking Water, 5th Edition (Reference 5.6). This manual can also be found online at 

http://www.epa.gov/safewater/labcert/labindex.html. 

LT2 compliance monitoring differs from monitoring currently being conducted under the Surface Water 

Treatment Rule (SWTR) and Total Coliform Rule (TCR). It is important for both PWSs and laboratories 

to note the differences in monitoring requirements so that there is no confusion surrounding the samples 

that are taken and analyzed for these three regulations. 

• LT2 requires samples to be analyzed for E.coli while the SWTR requires that samples be 

analyzed for fecal coliforms. 

• LT2 requires all E. coli analyses to be quantitative [40 CFR 141.704(b)], while the TCR requires 

only presence/absence analyses. 

• LT2 allows the use of mTEC medium (Standard Methods 9213 or EPA Method 1103.1) and mColiBlue 24® to be used for the detection of E. coli in source water samples. These methods are 

not approved for use in analysis of samples under the SWTR. 

• The holding time for LT2 samples is 30 hours, with a possible extension to 48 hours for samples 

analyzed by the Colilert reagent version of Standard Method 9223B if approved by the State [40 

CFR 141.704(b)]. This extended holding time is for use only with LT2 samples; SWTR samples 

are still required to be analyzed within the 8 hour holding time. 

Compliance monitoring for LT2 is in addition to, and does not replace, the monitoring mandated under 

SWTR and TCR 

47 February 2006 
Section 4: Guidance for E. coli Laboratories 

4.1 Laboratory Certification Program 

Since 1978, the U.S. Environmental Protection Agency has implemented a certification program for 

laboratories performing drinking water analyses for compliance with regulations issued pursuant to the 

Safe Drinking Water Act and subsequent National Primary Drinking Water Regulations (NPDWR). All 

laboratories analyzing drinking water compliance samples must be certified for the analyses they perform 

[40 CFR 141.28]. The laboratory certification process and detailed specifications for certification are 

described in the Manual for the Certification of Laboratories Analyzing Drinking Water, referred to as 

“laboratory certification manual”, 5th Edition (Reference 5.6). This manual can be found online at: 

http://www.epa.gov/safewater/labcert/labindex.html. Laboratories performing E. coli analyses for LT2 

monitoring must be certified by EPA, the National Environmental Laboratory Accreditation Conference 

(NELAC), or the State to conduct total or fecal coliform analysis in drinking water under 40 CFR 141.74 

using the same E.coli technique that they are certified to use for coliform analysis in drinking water [40 

CFR § 141.705(b)]. Approved E. coli methods and references are provided in Table 4-1 [40 CFR § 

141.704(b)]. 

EPA notes that this approach deviates from the approach typically used in its Laboratory Certification 

program in that the latter program is based on certification for the specific method (not simply the same 

technique) being used in compliance monitoring. EPA strongly encourages all laboratories using these E. 

coli methods to seek certification for those methods as soon as is practical. 

Table 4-1. Approved E. coli Methods for LT2 Rule 

E. coli Methods Approved for 

LT2 Rule Method Format Method Citation 

Standard Methods 

9221B.1/9221F (LTB-EC-MUG) multiple tube fermentation/ 

most probable number 

Standard Methods for the 

Examination of Water and 

Wastewater (Reference 5.8)2 

Standard Methods for the 

Standard Methods 9223B 

(Colilert®/Colilert-18®) multiple tube/multiple well 

Examination of Water and 

Wastewater (Reference 5.8)2

; 

IDEXX Laboratories, Inc. 

(Reference 5.9) 

Standard Methods 9222B/9222G1 

(mEndo/LES-Endo-NA-MUG) membrane filtration, two step 

Standard Methods for the 

Examination of Water and 

Wastewater (Reference 5.8)2 

Standard Methods 9222D/9222G 

(mFC-NA-MUG) membrane filtration, two step 

Standard Methods for the 

Examination of Water and 

Wastewater (Reference 5.8)2 

Standard Methods 9213D/ 

EPA Method 1103.1 (mTEC) membrane filtration, one step 

EPA Method 1103.1 (Reference 

5.10); Standard Methods for the 

Examination of Water and 

Wastewater (Reference 5.8)2 

EPA Method 1603 Modified 

mTEC membrane filtration, one step EPA Method 1603 (Reference 

5.11) 

EPA Method 1604 MI medium1 membrane filtration, one step EPA Method 1604 (Reference 

5.12) 

m-ColiBlue24® Broth1 membrane filtration, one step Hach Company (Reference 5.13) 1

 If high levels of non-E. coli total coliforms interfere with the ability to accurately enumerate E. coli despite additional 

dilutions, an alternate method should be used (i.e., SM 9222D/9222G, SM 9213D/EPA Method 1103.1, EPA Method 

1603, SM 9221B.1/9221F, and SM 9223B) 

2 18th, 19th, or 20th Editions of Standard Methods for the Examination of Water and Wastewater may be used. 

48 February 2006 
Section 4: Guidance for E. coli Laboratories 

4.2 Summary of LT2 Rule E. coli Methods 

The methods approved under the LT2 Rule were developed by EPA, voluntary consensus standards 

bodies (VCSB) (i.e., American Public Health Association (APHA), American Water Works Association 

(AWWA), and Water Environment Foundation (WEF) who jointly publish Standard Methods for the 

Examination of Water and Wastewater, referred to as “Standard Methods”), and commercial vendors with 

methods submitted to the EPA Office of Water Alternate Test Procedure (ATP) process. For several 

procedures, an EPA Method, VCSB method, and/or a commercially available method (submitted to the 

ATP program) are approved. 

Laboratories should obtain a copy of the methods approved for LT2 monitoring prior to seeking 

certification for these methods. The method summaries provided below only offer a brief overview of the 

methods and are not meant to provide details of the methods. Copies of analytical methods may be 

obtained from the citations listed in Table 4-1. Copies of analytical methods published by EPA are 

available for a nominal cost through the National Technical Information Service (NTIS); U.S. Department 

of Commerce; 5285 Port Royal Road; Springfield, VA 22161. Copies of the EPA methods may also be 

downloaded from the EPA Office of Research and Development; National Exposure Research Laboratory 

(NERL)-Cincinnati Microbiology home page at www.epa.gov/microbes/. All other methods must be 

obtained from the publisher. Publishers for all methods are included in Table 4-1; addresses for 

publishers can be found in Section 5. 

E. coli sample analyses performed under the LT2 Rule must be quantitative; presence/absence E. coli 

results are unacceptable. The analytical methods approved under LT2 are for enumeration of E. coli in 

ambient water, and the results are reported as number of E. coli per 100 mL water. Public Water Systems 

monitoring for E. coli under the LT2 Rule should collect and analyze at least 100 mL of sample to ensure 

sufficient volume for sample analysis. In order to obtain quantitative results for the source water samples 

for LT2 monitoring, the laboratory will need to select the appropriate configuration of tubes/filtrations 

and dilutions/volumes to account for the quality, character, consistency, and anticipated E. coli density of 

the water sample. See Section 4.5 below for guidance on sample volume and dilutions for LT2 sample 

analyses. 

To assess the comparability of results obtained with individual methods, it is suggested that side-by-side 

tests be conducted across seasons of the year with water samples routinely tested in accordance with the 

most current Standard Methods for the Examination of Water and Wastewater or EPA alternate test 

procedure (ATP) guidelines. The methods summarized below are approved for the analysis of E. coli 

samples under the LT2 Rule [40 CFR § 141.704(b)]. 

4.2.1 Most Probable Number (MPN) Methods 

4.2.1.1 Standard Methods 9223B: Colilert® and Colilert-18® 

Colilert® and Colilert-18® tests are chromogenic/fluorogenic enzyme substrate tests for the simultaneous 

determination of total coliforms and E. coli in water. All tests must be conducted in a format that 

provides quantitative results, such as the multiple tube or multiple well formats, e.g., Quanti-tray® (51 

well analysis) and Quanti-tray® 2000 (97 well analysis). Using multiple-tube procedures, laboratories 

will need to employ an appropriate tube and dilution configuration of the sample as needed. After the 

appropriate sample dilutions/volumes are added, the tubes or trays are incubated for 18 hours when using 

Colilert-18® or 24 hours when using Colilert®. Each tube or well is then compared to the reference color 

“comparator” available from the manufacturer. A yellow color greater or equal to the comparator 

indicates the presence of total coliforms in the sample. The tube or well is then checked for fluorescence 

under long-wavelength UV light. A yellow well with fluorescence greater than or equal to the 

49 February 2006 
Section 4: Guidance for E. coli Laboratories 

comparator is positive for E. coli. The most probable number (MPN) value is determined by the number 

of positive tubes or wells using MPN tables provided by the manufacturer. E. coli densities are then 

calculated and reported as MPN/100 mL (discussed in Section 4.6.1 of this manual below). 

4.2.1.2 Standard Methods 9221B.1/9221F: LTB-EC-MUG 

The multiple-tube fermentation (MTF) method uses multiple tubes and serial dilutions/volumes in a twostep procedure to determine E. coli densities in water. In the first step a series of tubes containing lauryl 

tryptose broth (LTB) are inoculated with undiluted sample and/or dilutions/volumes of the sample and 

mixed. After incubation, tubes are examined for growth (turbidity) and gas, which constitute a positive 

presumptive test for coliforms, which include E. coli. Note: Lactose broth may be used in lieu of LTB, if 

at least 25 parallel tests are conducted between this broth and LTB using the water samples normally 

tested, and this comparison demonstrates that the false-positive rate and false-negative rate for total 

coliform using lactose broth is less than 10 percent. 

Growth from all presumptive tubes (showing any amount of gas, growth or activity within 48+3 hours of 

incubation) is transferred to tubes containing EC-MUG broth for E. coli enumeration. After incubation, 

tubes that exhibit turbidity and fluorescence are considered to be positive for E. coli. The density of E. 

coli is determined from the number of positive tubes using the MPN table provided in SM 9221C. E. coli 

densities are reported as MPN/100 mL (See Section 4.6.3 of this manual for guidance on determining the 

MPN). Note: there is no requirement to run the completed phase on 10 percent of all total coliformpositive tubes on a seasonal basis. 

4.2.2 Membrane Filtration (MF) Methods 

Note: When the MF method has not been used previously to test ambient water with high turbidity, large 

number of non-coliform bacteria, or samples that may contain organisms stressed by chlorine, a parallel 

test should be conducted with a multiple-tube technique to demonstrate applicability and comparability of 

results. 

4.2.2.1 Standard Methods 9222B/9222G: mEndo/LES-Endo—NA-MUG and 

Standard Methods 9222D/9222G: mFC — NA-MUG 

These membrane filter methods are two-step MF procedures that provide a direct count of E. coli in 

water. First, a sample is filtered through a 0.45 μm, white gridded membrane filter. The filter is then 

placed on a pad saturated with mEndo broth or a plate containing mEndo or LES-Endo agar and 

incubated. Pink to red colonies with a metallic (golden-green) sheen on mEndo (or LES-Endo) are total 

coliforms. If initial determination of fecal coliforms is desired, mFC media can be substituted for 

mEndo/LES-Endo. Blue colonies on the filter are fecal coliforms. 

In the second step of these methods, the filter is transferred to nutrient agar containing MUG (NA-MUG) 

medium and incubated. Sheen colonies from mEndo/LES-Endo or blue colonies from mFC that fluoresce 

under a long-wavelength UV light after incubation on NA-MUG are positive for E. coli. E. coli densities 

are reported as CFU/100 mL. Guidance on determining the number of E.coli/100 mL is discussed below 

in Section 4.6.2. 

If high levels of total coliforms interfere with the ability to accurately enumerate E. coli despite additional 

dilutions, an alternate method (e.g., SM 9213D, EPA Method 1603 or 9223B) should be used. 

50 February 2006 
Section 4: Guidance for E. coli Laboratories 

4.2.2.2 Standard Methods 9213D/EPA Method 1103.1: mTEC 

The mTEC agar method is a two-step MF procedure that provides a direct count of E. coli in water. In this 

method, a water sample is filtered through a 0.45 μm white gridded membrane filter, the filter is placed 

on mTEC agar (a selective primary isolation medium), and the plate is incubated at 35EC ± 0.5EC for 2 

hours to resuscitate injured or stressed bacteria and then at 44.5EC ± 0.2EC for 22-24 hours in a water 

bath. Following incubation, the filter is transferred to a pad saturated with urea substrate medium. After 

15 minutes, all yellow, yellow-brown, or yellow-green colonies are counted as E. coli. E. coli densities 

are reported as CFU/100 mL. 

4.2.2.3 EPA Method 1603: Modified mTEC 

The modified mTEC agar method is a single-step MF procedure that provides a direct count of E. coli in 

water. This is a modification of the standard mTEC media (SM 9213D). In this method, a water sample is 

filtered through a 0.45 μm white gridded membrane filter, the filter is placed on modified mTEC agar, 

incubated at 35EC ± 0.5EC for 2 hours to resuscitate injured or stressed bacteria, and then incubated for 

22-24 hours in a 44.5EC ± 0.2EC water bath. Following incubation, all red or magenta colonies are 

counted as E. coli. E. coli concentrations are reported as CFU/100 mL. 

4.2.2.4 EPA Method 1604: MI Medium 

The MI medium method is a single-step MF procedure used to simultaneously enumerate total coliforms 

and E. coli in water. In this method, a water sample is filtered through a 0.45 μm white gridded membrane 

filter, the filter is placed on an MI medium and incubated. Following incubation, all blue colonies under 

ambient light are counted as E. coli. These colonies can be fluorescent or non-fluorescent under longwavelength ultraviolet light. E. coli concentrations are reported as CFU/100 mL. 

If high levels of total coliforms interfere with the ability to accurately enumerate E. coli despite additional 

dilutions, an alternate method (e.g., SM 9213D, EPA Method 1603 or 9223B) should be used. 

4.2.2.5 m-ColiBlue24® Broth 

This broth method is a single-step MF test for enumerating total coliforms and E. coli in water. In this 

method, a water sample is filtered through a 0.45 μm white gridded membrane filter. The filter is placed 

on a plate containing an absorbent pad saturated with m-ColiBlue24® broth and incubated. Following 

incubation, all blue colonies are counted as E. coli. E. coli concentrations are reported as CFU/100 mL. 

If high levels of total coliforms interfere with the ability to accurately enumerate E. coli despite additional 

dilutions, an alternate method (e.g., SM 9213D, EPA Method 1603) should be used. 

4.3 Quality Assurance/Quality Control for E. coli Analyses 

The Manual for the Certification of Laboratories Analyzing Drinking Water (Reference 5.6) describes 

criteria and procedures that should be considered when a laboratory undergoes the certification process. 

This manual contains elements that are required by federal regulations as well as criteria that are 

recommended by EPA for laboratory certification. Additional QA/QC specifications beyond those found 

in the laboratory certification manual may be found in Standard Methods, Section 9020 (Reference 5.8). 

51 February 2006 
Section 4: Guidance for E. coli Laboratories 

Each laboratory should have a Quality Assurance plan describing the QA program and QC activities 

necessary to meet the laboratory’s specific needs. The QA Plan for LT2 monitoring must meet the 

specifications of the laboratory’s certifying authority and the method used. The QA plan should address 

the following issues: personnel policies, equipment and instrument specifications, specifications for 

supplies, analytical methods and QC measures, standard operating procedures (SOPs), documentation 

specifications, performance evaluation samples, internal and external lab audits, and corrective actions. 

For more suggestions on what items should be included in a QA plan, see the Laboratory Certification 

Manual, Chapter 3. 

Chapter V, Critical Elements of Microbiology, of the Laboratory Certification Manual details both 

required and recommended criteria for a laboratory that is analyzing water samples for microbes. E. coli 

sample results reported under the LT2 Rule should meet the quality control (QC) specifications set forth 

in the Laboratory Certification Manual and the methods listed in Table 4-1 above. 

4.3.1 Quality Control Specifications Applicable to LT2 Samples 

4.3.1.1 Holding Time and Temperature Requirements for Field Samples 

Source water samples are dynamic environments and, depending on sample constituents and 

environmental conditions, E. coli present in a sample can grow or die off, biasing analytical results. 

Samples that are not analyzed immediately after sample collection during LT2 Rule monitoring must be 

chilled to reduce biological activity, and preserve the state of source water samples between collection 

and analysis. Samples for E. coli analyses must be maintained between 0°C and 10°C if they are shipped 

[40 CFR 141.704(b)(3)]. Samples should not be allowed to freeze. 

For best results, samples should be analyzed as soon as possible after collection. Due to the need by some 

utilities to ship samples overnight to an off-site laboratory for analysis, the holding time for LT2 E. coli 

samples is 30 hours. If the State determines on a case-by-case basis that analyzing an E. coli sample 

within 30 hours is not feasible, the State may authorize the holding of an E. coli sample for up to 48 hours 

between collection and initiation of analysis. E. coli samples held between 30 to 48 hours must be 

analyzed by the Colilert® reagent version of Standard Method 9223B [40 CFR § 141.704(b)(2)]. The E. 

coli holding time established for source water monitoring under the LT2 Rule does not apply to E.coli 

sample holding time requirements that have been established under other programs and regulations. 

Given the importance of maintaining sample temperatures for E. coli, laboratories should establish 

acceptance criteria for receipt of E. coli samples transported to their laboratory. Several options are 

available to measure sample temperature upon receipt at the laboratory and, in some cases, during 

shipment; these are provided in Section 2.2 of this manual. 

4.3.2 Quality Control Specifications for E. coli Methods 

In addition to the overall QC specifications set forth in the Laboratory Certification Manual and in the 

analytical methods, laboratories analyzing samples for LT2 Rule E. coli analyses must meet methodspecific incubation time and temperature requirements. 

4.3.2.1 Incubation Time/Temperature Specifications for MPN Methods 

The required incubation times and temperatures for MPN methods are provided in Table 4-2. 

52 February 2006 
Section 4: Guidance for E. coli Laboratories 

Table 4-2. Incubation Time and Temperature Specifications for MPN Methods 

Method Media Incubation Time/Temperature 

Standard Methods 9223B Colilert® 24 - 28 hours at 35EC ± 0.5EC 

Coliert-18® 18 - 22 hours at 35EC ± 0.5EC 

Standard Methods 9221B/9221F LTB 24 ± 2 hours at 35EC ± 0.5EC and 

48 ± 3 hours at 35EC ± 0.5EC 

EC-MUG 24 ± 2 hours at 44.5EC ± 0.2EC 

4.3.2.2 Incubation Time and Temperature Specifications for MF Methods 

The required incubation times and temperatures for MF methods are provided in Table 4-3. 

Table 4-3. Incubation Time and Temperature Specifications for Membrane Filter Methods 

Method Media Incubation Time/Temperature 

Standard Methods 9222B/9222G 

mENDO6NA-MUG 22 - 24 hours at 35EC ± 0.5EC6

4 hours at 35EC ± 0.5EC 

Les-ENDO6NA-MUG 22 - 24 hours at 35EC ± 0.5EC6

4 hours at 35EC ± 0.5EC 

Standard Methods 9222D/9222G mFC6NA-MUG 22 - 26 hours at 44.5EC ± 0.2EC6

4 hours at 35EC ± 0.5EC 

Standard Methods 9213D/EPA 

Method 1103.1 mTEC agar 2 hours at 35EC ± 0.5EC 6

22 - 24 hours at 44.5EC ± 0.2EC 

EPA 1603 Modified mTEC 2 hours at 35EC ± 0.5EC 6

22 - 24 hours at 44.5EC ± 0.2EC 

EPA 1604 MI agar 24+2 hours at 35EC ± 0.5EC 

m-ColiBlue24® Broth m-ColiBlue24® Broth 24 hours at 35EC ± 0.5EC 

4.4 Sample Collection Procedures 

PWSs are required to collect source water samples for the LT2 Rule from the plant intake prior to 

chemical treatment, unless approved by the State to collect the source water sample after chemical 

treatment [40 CFR § 141.703(b)(2)]. Systems that recycle filter backwash water must collect source water 

samples prior to the point of filter backwash water addition [40 CFR § 141.703(c)]. All Cryptosporidium, 

E. coli, and turbidity source water samples collected under LT2 Rule requirements must be collected from 

the same appropriate sampling location. Detailed guidance on sample collection procedures for E. coli 

samples for on-site analysis and for shipment to an off-site laboratory for analysis are provided in the 

Source Water Monitoring Guidance Manual for Public Water Systems for the Long Term 2 Enhanced 

Surface Water Treatment Rule (LT2 Rule). This manual is available for download from 

http://www.epa.gov/safewater/disinfection/lt2/. 

53 February 2006 
Section 4: Guidance for E. coli Laboratories 

4.5 Sample Volume and Dilution Guidance 

Because E. coli analyses will be performed on source waters, rather than finished drinking waters, 

multiple sample volumes/dilutions may be necessary to assess high E. coli levels. Initially all laboratories 

should consider analyzing four sample volumes (100, 10, 1.0, and 0.1 mL) for all methods except MTF. 

For MTF methods, the PWS should initially consider analyzing five sample volumes of 10, 1.0, 0.1, 0.01, 

and 0.001 mL. Sample volumes may need to be adjusted based on confounding conditions (e.g., high 

turbidity, heavy rainfall, etc.). 

If the PWS has historical data demonstrating that E. coli levels are consistently low, they may drop the 

smaller sample volumes. If there has been a substantial rainfall in the 24 hours prior to sample collection 

causing runoff, sample volumes may need to be adjusted in order to obtain valid results and avoid data 

that are above the analytical range of the method. This could also be a concern if the PWS eliminates the 

100 mL sample volume due to historical data indicating that values tend to be very high and the E. coli 

levels are significantly lower than anticipated, requiring the PWS to report data that are below the 

analytical range of the method. EPA recommends bracketing the target sample volume (i.e., analyzing a 

sample volume above and below the sample volume expected to yield useable data) to account for 

potential variability. 

4.5.1 Sample Volume and Dilution Guidance for Multiple-Well Methods 

The analysis of water samples under LT2 using multiple-well methods generally requires the use of four 

aliquots of samples (100, 10, 1.0, and 0.1 mL). As mentioned above, sample volumes may need to be 

adjusted based on confounding conditions (e.g., high turbidity, heavy rainfall, etc.). Because a 100 mL 

volume is necessary to fill all of the wells, it will be necessary to add the aliquots of the sample to sterile 

reagent water blanks. The total volume after the sample aliquot is added to the reagent water blank should 

be approximately 100 mL (i.e., a 90 mL blank should be used for the 10 mL sample volume, a 99- mL 

reagent water blank should be used for the 1.0 mL aliquot). Colilert® or Colilert18® reagent should be 

added to the sterile reagent water blanks prior to adding sample. If the sample is added to the reagent 

water before the Colilert® or Colilert18® reagent, bacterial cells may lyse. (Reference 5.14) 

4.5.2 Sample Volume and Dilution Guidance for Multiple-Tube Methods 

For the analysis of water samples by a multiple-tube technique (e.g., Colilert® in a multiple-tube format, 

SM 9222B LTB/EC-MUG) under LT2, laboratories should consider the use of a 20-tube most probable 

number format (i.e., 5 tubes at each of the four dilutions, 10.0, 1.0, 0.1, and 0.01 mL). Initially, it is 

recommended that five sample volumes (i.e., 5 tubes for each sample volume, 10.0, 1.0, 0.1, 0.01, and 

0.001 mL) are analyzed. Additional guidance on selection of dilutions for multiple-tube methods is 

available in Standard Methods 9221 (Reference 5.8). 

4.5.3 Sample Volume and Dilution Guidance for Membrane Filtration 

For the analysis of water samples under LT2, it is recommended that four different aliquots of sample 

(100, 10, 1.0, and 0.1 mL) be analyzed when using membrane filtration methods. Alternate sample 

volumes may be used if necessary. To ensure adequate distribution of bacteria during filtration, dispense 

aliquots of the sample, with the exception of the 100 mL volume, into sterile buffered water blanks (at 

least 30 mL, depending on the sample volume analyzed). Alternately, 10 mL of sterile buffer may be 

added to the filter apparatus prior to adding the sample. The buffered water minimizes clumping of the 

bacteria on the filter surface. 

54 February 2006 
Section 4: Guidance for E. coli Laboratories 

4.6 E. coli Data Recording and Calculations 

Laboratories performing E. coli analyses during the LT2 Rule must follow any data recording practices 

required by the certification authority granting the certification to the laboratory, and should follow any 

recommended data recording practices outlined in the Laboratory Certification Manual, Chapter V, 

Section 8. 

Some of the recommended information to be recorded for each E. coli sample is as follows. Data for each 

sample should be recorded on a sample collection form used in the field and maintained in the same file 

as the E. coli laboratory data or on the laboratory data form. Note that some of these data will be entered 

into the LT2/Stage 2 Data Collection and Tracking System (DCTS): 

• PWS ID 

• Facility ID 

• Sample Identification (if any) 

• Sample collection point ID 

• Sample collection date and time 

• Sample type 

• Analytical method number 

• Method type 

• Name of sampler 

• Turbidity 

• Source water type 

The laboratory should record all of the primary measurements associated with each analysis, as they are 

needed to calculate the final concentration of E. coli per 100 mL. Primary measurements for membrane 

filtration methods will include the volumes filtered and the plate counts for each volume filtered. The 

multiple-well and multiple-tube formats will include the volumes or dilutions of samples analyzed and the 

number of positive wells or tubes per each volume analyzed. Method-specific data to record for each of 

the individual method types are discussed in Sections 4.6.1 ― 4.6.3 of this manual. 

The suggested data elements shown below are important as they allow the laboratory to ensure that final 

sample results can be verified using primary data and to demonstrate that sample analyses were 

performed within method-specified holding times. Please note that not all of the data that may be 

recorded will be reported in the DCTS. E. coli data reporting under the LT2 Rule is discussed in Section 

4.7 of this manual. 

The final E. coli concentration for field samples will be reported as CFU/100 mL or MPN/100 mL 

depending on the method used for analysis. If no E. coli are detected in the sample, the detection limit 

based on the volume of sample analyzed may be reported (e.g., <1 CFU /100 mL or <1.8 MPN/100 mL) 

or a zero for purposes of the DCTS. 

In addition, this section also provides standardized procedures for determining E. coli concentration for 

LT2 samples for the various analytical techniques that are approved for use under the rule. Because these 

analyses will be performed on source waters rather than finished drinking waters, and multiple dilutions 

may be necessary to assess higher E. coli levels, it is recommended that laboratories consistently use the 

same analytical procedures for determining E. coli sample concentrations to reduce variability. 

55 February 2006 
Section 4: Guidance for E. coli Laboratories 

4.6.1 Multiple-Well Data 

In addition to the data elements contained in the bulleted list in Section 4.6 of this manual, laboratories 

using Colilert® and Colilert-18® methods for E. coli sample analyses in support of the LT2 Rule should 

record the data elements specific to multiple-well techniques. Data elements to be recorded for the 97­

well format (Quanti-Tray 2000®) are noted in Table 4-4; data to be recorded for the 51-well format 

(Quanti-Tray®) are noted in Table 4-5. These elements include the primary measurements needed to 

calculate the E. coli concentration in the sample as well as all method-required incubation and read times 

needed to verify that the sample analyses were conducted under analytical control. 

Calculations for determining the E. coli concentration using the Quanti-Tray 2000® (97-well) and 

Quanti-Tray® (51-well) formats are provided after each table. 

4.6.1.1 Data Elements for Quanti-Tray 2000® (97-well) Analyses 

The recommended data elements to record for Quanti-Tray 2000® (97-well) analyses are provided in 

Table 4-4. 

Table 4-4. Recommended Minimum Record for Quanti-tray 2000® Colilert® and Colilert-18® 

Analyses 

Primary Measurements 

mL of sample added to tray (does not include reagent water volume) 

Large wells positive: Total coliform positive and UV fluorescence 

Small wells positive: Total coliform positive and UV fluorescence 

Holding Time / Incubation Time Information 

Incubation start: date/time 

Incubation end: date/time 

Additional incubation start: date/time 

Additional incubation end: date/time 

4.6.1.2 Determining E. coli Concentration Using Colilert® and Colilert-18® QuantiTray 2000® Data 

A. Select appropriate dilution to yield countable results. If multiple dilutions are used, the tray 

exhibiting positive wells in the 40 percent and 80 percent range (39 to 78 total positive large and 

small wells) should be used to determine MPN value. 

Note: The analytical result can be automatically calculated using the LT2/Stage 2 Data 

Collection and Tracking System. See Section 4.7 of this manual for additional 

information. 

56 February 2006 
Section 4: Guidance for E. coli Laboratories 

B. Determine MPN. Use the MPN tables provided by the vendor. To determine the MPN using 

these tables, locate the number at the intersection of large positive wells and small positive wells 

from the appropriate dilution, identify the corresponding MPN/100 mL in the table provided by 

the vendor. Large well values are located in the left column; small well values are located in the 

top row. For example, if a 100 mL sample was analyzed, and there were 29 large positive wells 

and 5 small positive wells, the corresponding MPN would be 49.6 MPN/100 mL. 

C. Adjust for dilution factor. Because the MPN/100 mL values in the table are based on 100 mL 

samples, the MPN value should be adjusted if less than 100 mL of sample volume was analyzed. 

Use the following calculation to adjust the MPN to account for the dilution: 

100

Analytical result = MPN value × 

mL of sample analyzed

Example: 

Volume analyzed = 10 mL of sample (in 90 mL of dilution water) 

Large wells positive = 39 

Small wells positive = 5 

The MPN value calculated based on the intersection of 39 and 5 in the table. 

 MPN = 81.3 

100

Analytical result = 81.3 × = 813 E. coli MPN/100 mL

10

4.6.1.3 Data Elements for Quanti-Tray® (51-well) Analyses 

The recommended data elements to record for Quanti-Tray® (51-well) analyses are provided in Table 4­

5. 

Table 4-5. Recommended Data to Record for Quanti-Tray® Colilert® and Colilert-18® 

Analyses 

Primary Measurements 

mL of sample added to tray (does not include reagent water volume) 

Number of wells positive: Total coliform positive and UV fluorescence 

Holding Time / Incubation Time Information 

Incubation start: date/time 

Incubation end: date/time 

Additional incubation start: date/time 

Additional incubation end: date/time 

57 February 2006 
Section 4: Guidance for E. coli Laboratories 

4.6.1.4 Determining E. coli Concentration Using Colilert® and Colilert-18® (51-well) 

Data 

A. Select appropriate dilution. If multiple dilutions are used, the tray exhibiting 80 percent positive 

wells (41 positive wells) should be used to determine MPN value. 

Note: The analytical result can be automatically calculated using the LT2/Stage 2 Data 

Collection and Tracking System (DCTS). See Section 4.6 of this manual for additional 

information. 

B. Determine MPN. Using the number of positive wells from the appropriate dilution, identify the 

corresponding MPN/100 mL using the MPN table provided by the vendor. For example, if a 100 

mL sample was analyzed, and there were 41 positive wells, the corresponding MPN would be 

83.1 MPN/100 mL. 

C. Adjust for dilution factor. Because the MPN/100 mL values in the table are based on 100 mL 

samples, the MPN value should be adjusted if less than 100 mL of sample volume was analyzed. 

Use the following calculation to adjust the MPN to account for the dilution: 

100 

MPN value x = E. coli MPN/100 mL 

mL of sample analyzed 

Example: 

Volume analyzed (mL) = 10 mL (in 90 mL of dilution water) 

Number of positive wells = 41 

 MPN = 83.1 

The analytical result is calculated as follows: 

100

Analytical result = 83.1 × = 831 E. coli MPN/100 mL

10

4.6.2 Membrane Filtration Data 

In addition to the general sample data contained in the bulleted list in Section 4.6 of this manual, 

laboratories using membrane filtration methods for E. coli sample analyses in support of the LT2 Rule 

should record the data elements specific to this technique. These are noted in Table 4-6, and include the 

primary measurements needed to calculate the E. coli concentration in the sample, as well as all methodrequired incubation and read times needed to verify that the sample analyses were conducted under 

analytical control. 

4.6.2.1 Data Elements for Membrane Filtration Analyses 

The recommended data elements to record for membrane filtration analyses are provided in Table 4-6. 

58 February 2006 
Section 4: Guidance for E. coli Laboratories 

Table 4-6. Recommended Data Elements for Record for Membrane Filtration Analyses 

Primary Measurements 

Filter 1 volume (mL) (e.g., 100 mL)

CFU on Filter 1

Filter 2 volume (mL) (e.g., 10 mL)

CFU on Filter 2

Filter 3 volume (mL) (e.g., 1.0 mL)

CFU on Filter 3

Filter 4 volume (mL) (e.g., 0.1 mL)

CFU on Filter 4

Holding Time / Incubation Time Information 

Primary isolation medium (e.g., mENDO, mFC) incubation start: date/time 

Primary isolation medium (e.g., mENDO, mFC) incubation end: date/time 

Secondary isolation medium (e.g, NA-MUG) incubation start: date/time 

Secondary isolation medium (e.g, NA-MUG) incubation end: date/time 

4.6.2.2 Determining E. coli Concentrations Using Membrane Filter Data 

A. E. coli counts should be determined from the volume(s) filtered that yielded 20 to 80 

E. coli colonies (20-60 for mFC-NA-MUG), and not more than 200 total colonies per plate. 

(Guidance for samples that do not yield countable plates is provided in Sections E and F below) 

(References 5.8 and 5.10-5.12) 

Note: The analytical result can be automatically calculated using the LT2/Stage 2 Data 

Collection and Tracking System (DCTS). See Section 4.7 of this manual for additional 

information. 

B. If there are greater than 200 colonies per membrane, even for the lowest dilution, the result is 

recorded as “too numerous to count” (TNTC). These results should not be reported for LT2 

monitoring, as they cannot be used for the required data analyses. During the next sampling event, 

analyze an additional, lower dilution volume (the highest dilution volume may be omitted) unless 

conditions were unusual (e.g., heavy rains, flooding, etc.) during the sampling event yielding TNTC 

for all dilutions. 

C. If colonies are not sufficiently distinct for accurate counting, the result is recorded as “confluent 

growth” (CNFG). To prevent CNFG from occurring, smaller sample aliquots should be filtered. For 

example, if sample volumes of 100, 10, 1 and 0.1 mL are analyzed and even the 0.1 mL plate results 

in CNFG, then potentially 0.01 mL should be analyzed during the next sampling event. For sample 

volumes less than 1 mL, serial dilutions should be used, and 1 mL volumes of the dilutions should be 

filtered. The 100 mL volume can be eliminated. Note: If growth is due to high levels of total coliforms 

but low E. coli then another method should be chosen for analyses that does not rely on total coliform 

determination prior to or simultaneously with E. coli determination. 

59 February 2006 
Section 4: Guidance for E. coli Laboratories 

Note: Results that are TNTC or CNFG are not appropriate for LT2 microbial data 

analysis, and cannot be entered into the LT2/Stage 2 Data Collection and 

Tracking System (DCTS). These results should not be reported. 

D. Using the E. coli counts from the appropriate dilution, E. coli CFU/100 mL should be calculated 

based on the following equation: 

100 

E. coli CFU × = E. coli CFU/100 mL

mL sample filtered

Example 1: 

Filter 1 volume = 100 mL CFU = TNTC 

Filter 2 volume = 10 mL CFU = 40 

Filter 3 volume = 1.0 mL CFU = 9 

Filter 4 volume = 0.1 mL CFU = 0 

Using the guidance on countable colonies in Step A, the count from the 10 mL plate will be used to 

calculate the E. coli concentration for the sample: 

100

40 E. coli CFU × = 400 E. coli CFU/100 mL

10 mL

E. If no E. coli colonies are present, the detection limit (i.e., 1 CFU per volume filtered) is 

calculated and reported per 100 mL (see example below). 

Example 2: 

Filter 1 volume (mL) = 100 mL CFU = 0 

Filter 2 volume (mL) = 10 mL CFU = 0 

Filter 3 volume (mL) = 1.0 mL CFU = 0 

Detection 100 mL

= E. coli CFU/100 mL

limit = Largest volume filtered

100 mL

= <1 E. coli /100 mL

100 mL

60 February 2006 
Section 4: Guidance for E. coli Laboratories 

Example 3: 

Filter 1 volume (mL) = 100 mL

Filter 2 volume (mL) = 10 mL

Filter 3 volume (mL) = 1.0 mL

CFU = Lab accident, no data available 

CFU = 0 

CFU = 0 

Calculation of E. coli/100 mL: 

100 mL

= <10 E. coli CFU /100 mL

10 mL

F. If there are no filters with E. coli counts in the 20-80 colony range (20-60 for mFC-NA-MUG), 

sum the E. coli counts on all filters, divide by the volume filtered and report as number per 100 

mL. 

Example 4: 

Filter 1 volume (mL) = 50 mL CFU = 15 

Filter 2 volume (mL) = 25 mL CFU = 6 

Filter 3 volume (mL) = 10 mL CFU = 0 

The analytical result is calculated as: 

100

(15 + 6 + 0) × = 25 E. coli CFU/100 mL

 (50+25+10)

 Example 5: 

Filter 1 volume (mL) = 50 mL CFU = 105 

Filter 2 volume (mL) = 25 mL CFU = 92 

Filter 3 volume (mL) = 10 mL CFU = 85 

The analytical result is calculated as: 

100

(105 + 92 + 85) × = 332 E. coli CFU/100 mL

(50 + 25 + 10)

 Example 6: 

Filter 1 volume (mL) = 100 mL CFU = 82 

Filter 2 volume (mL) = 10 mL CFU = 18 

Filter 3 volume (mL) = 1.0 mL CFU = 0 

The analytical result is calculated as: 

100

 (82 + 18 + 0) × = 90 E. coli CFU/100 mL

(100 + 10 + 1)

61 February 2006 
Section 4: Guidance for E. coli Laboratories 

 Example 7: 

Filter 1 volume (mL) = 50 mL CFU = TNTC 

Filter 2 volume (mL) = 25 mL CFU = TNTC 

Filter 3 volume (mL) = 10 mL CFU = 83 

The analytical result is calculated as: 

100

 83 ×

 10 

= 830 E. coli CFU/100 mL 

4.6.3 Multiple-Tube Data 

In addition to the general sample data contained in the bulleted list in Section 4.6 of this manual, 

laboratories using multiple-tube methods for E. coli sample analyses in support of the LT2 Rule should 

record the data elements specific to these techniques. These data elements are noted in Table 4-7 for 15­

tube most probable number methods and Table 4-8 for 15-tube multiple-tube fermentation methods. The 

data elements include the primary measurements needed to calculate the E. coli concentration in the 

sample, as well as all method-required incubation and read times needed to verify that the sample 

analyses were conducted under analytical control. 

Calculations for determining the E. coli concentration using multiple tube formats are provided in Section 

4.6.3.2. 

4.6.3.1 Data Elements for Multiple-Tube Analyses 

The recommended data elements to record for 15-tube most probable number methods are provided in 

Table 4-7 and Table 4-8 for 15-tube multiple-tube fermentation methods. 

Table 4-7. Recommended Data Elements to Record for 15-Tube MPN Methods (Colilert®) 

Primary Measurements (Note: not all dilutions listed below may be used) 

Number of positive 10.0 mL tubes: Total coliform positive and UV fluorescence 

Number of positive 1.0 mL tubes: Total coliform positive and UV fluorescence 

Number of positive 0.1 mL tubes: Total coliform positive and UV fluorescence 

Number of positive 0.01 mL tubes: Total coliform positive and UV fluorescence 

Number of positive 0.001 mL tubes: Total coliform positive and UV fluorescence 

Holding Time / Incubation Time Information 

Incubation start: date/time 

Incubation end: date/time 

Additional incubation start: date/time 

Additional incubation end: date/time 

62 February 2006 
Section 4: Guidance for E. coli Laboratories 

Table 4-8. 

) 

Recommended Data Elements to Record for 15-Tube Fermentation Methods 

Primary Measurements (Note: not all dilutions listed below may be used

Number of positive tubes 10.0 mL 

Number of positive tubes 1.0 mL 

Number of positive tubes 0.1 mL 

Number of positive tubes 0.01 mL 

Number of positive tubes 0.001 mL 

Number of positive tubes 0.0001 mL 

Holding Time / Incubation Time Information 

LTB incubation start date/time 

LTB 24-hour incubation end date/time 

LTB 48-hour incubation read date/time 

EC-MUG incubation 24-hour read date/time (from 24-hour LTB) 

EC-MUG incubation 24-hour read date/time (from 48-hour LTB) 

4.6.3.2 Determination of E. coli Concentrations Using Multiple-Tube Methods1

: 

The guidance and examples for determining E. coli concentrations using multiple-tube methods are based 

on the revision of Standard Methods 9221C included in the 2001 Supplement to the 20th Edition of 

Standard Methods, approved by the Standard Methods Committee in 1999. 

Note: The analytical result can be automatically calculated using the LT2/Stage 2 Data 

Collectionand Tracking System (DCTS). See Section 4.7 of this manual for 

additional information. 

A. For each sample volume (e.g., 10, 1, 0.1, and 0.01 mL or additional sample volumes as 

necessary), determine the number of positive tubes out of five. 

B. A dilution refers to the volume of original sample that was inoculated into each series of tubes. 

Only three of the dilution series will be used to estimate the MPN. The three selected dilutions are 

called significant dilutions and are selected according to the following criteria. Examples of 

significant dilution selections are provided in Table 4-9, below. 

• Choose the highest dilution (the most dilute, with the least amount of sample) giving positive 

results in all five tubes inoculated and the two succeeding higher (more dilute) dilutions. 

(Table 4-9, Example A). 

• When the lowest dilution (least dilute) tested has less than five tubes with positive results, 

select it and the two next succeeding higher dilutions (Table 4-9, Examples B and C). 

1

Adapted from 2001 Supplement to the 20th Edition of Standard Methods 9221 C: Explanation of Bacterial 

Density. This supplement is available for download at http://www.techstreet.com/cgi-bin/detail?product_id=923645. 

63 February 2006 
Section 4: Guidance for E. coli Laboratories 

• When a positive result occurs in a dilution higher (more dilute) than the three significant 

dilutions selected according to the rules above, change the selection to the lowest dilution 

(least dilute) that has less than five positive results and the next two higher dilutions (more 

dilute) (Table 4-9, Example D). 

• When the selection rules above have left unselected any higher dilutions (more dilute) with 

positive results, add those higher-dilution positive results to the results for the highest 

selected dilution (Table 4-9, Example E). 

• When there are not enough higher dilutions tested to select three dilutions, then select the 

next lower dilution (Table 4-9, Example F). 

C. MPN values must be adjusted based on the significant dilutions series selected above. Because 

the MPN/100 mL values in the table are based on the analysis of 10, 1, and 0.1 mL dilutions, per 

method requirements, the MPN value must be adjusted if these are not the significant dilutions 

selected. Use the following calculation to adjust the MPN when the 10, 1, and 0.1 mL dilutions 

are not the significant dilutions selected: 

MPN value

Analytical result = = E. coli MPN/100 mL

# of mL in middle dilution

Table 4-9. Examples of Different Combinations of Positive Tubes (Significant Dilution 

Results Are in Bold and Underlined) 

Example 

Least dilute 

Most dilute 

(Lowest) 

(Highest) Combination 

of positives 

MPN Index from 

Standard Methods 

E. coli/100 mL 

(after adjustment) 

10 

mL 1 mL 0.1 mL 

0.01 

mL 

0.001 

mL 

A 5 5 1 0 0 5-1-0 33 330 

B 4 5 1 0 0 4-5-1 48 48 

C 0 0 1 0 0 0-0-1 1.8 1.8 

D 5 4 4 1 0 4-4-1 40 400 

E 5 4 4 0 1 4-4-1 40 400 

F 5 5 5 5 2 5-5-2 540 54,000 

Example A: The significant dilution series for the 5-1-0 combination of positives includes the 

1, 0.1, and 0.01 mL dilutions. Because the 10, 1, and 0.1 mL dilutions were not 

selected, an adjustment is necessary to account for the dilutions selected: 

33 

Analytical result = = 330 E. coli / 100 mL 

0.1 

64 February 2006 
Section 4: Guidance for E. coli Laboratories 

Example B: Because the 10, 1, and 0.1 mL dilutions are the significant dilutions, no 

adjustment is necessary and the result is 48 E. coli/100 mL. 

Example C: Because the 10, 1, and 0.1 mL dilutions are the significant dilutions, no 

adjustment is necessary and the result is 1.8 E. coli/100 mL. 

Examples D and E: The significant dilution series for the 4-4-1 combination of positives includes the 

1, 0.1, and 0.01 mL dilutions. Because the 10, 1, and 0.1 mL dilutions were not 

selected, an adjustment is necessary to account for the dilutions selected: 

40 

Analytical result = = 400 E. coli / 100 mL 

0.1 

Example F: The significant dilution series for the 5-5-2 combination of positives includes the 

0.1, 0.01 and 0.001 mL dilutions. Because the 10, 1, and 0.1 mL dilutions were 

not selected, an adjustment is necessary to account for the dilutions selected: 

540 

Analytical result = = 54,000 E. coli / 100 mL 

0.01 

4.7 Electronic Data Reporting 

During the LT2 Rule, laboratories will report E. coli data electronically through EPA’s LT2/Stage 2 Data 

Collection and Tracking System (DCTS) to the PWS staff responsible for approving and submitting 

monitoring results to EPA. The DCTS is a web-based application that allows laboratory users to enter or 

upload data, then electronically “release” the data to the appropriate PWS staff for review, approval, and 

submission to EPA and the State. Although ownership of the data resides with the PWS throughout this 

process, the DCTS increases the ease and efficiency of the data entry and transfer process from one party 

to another by transferring the ability to access the data from the laboratory to the PWS to EPA and the 

State, and ensuring that data cannot be viewed or changed by unauthorized parties. A summary of the data 

entry, review, and transfer process through the DCTS for both Cryptosporidium and E. coli samples is 

provided in Table 3-8, in Section 3.7. 

The data reporting process is summarized below, in Sections 4.7.1 through 4.7.3, and discussed in detail 

in the Users’ Manual for the LT2/Stage 2 Data Collection and Tracking System (DCTS). The DCTS 

users’ manual also provides detailed information on the laboratory registration process. Information on 

the DCTS and a downloadable users’ manual are available at 

http://www.epa.gov/safewater/lt2/index.html. 

4.7.1 Data Entry/Upload 

The analyst or another laboratory staff member enters a subset of the data recorded at the bench (Section 

4.6 of this manual) into the DCTS either by entering the data using web forms or by uploading data in 

XML format. This information includes the following: 

• Sample ID (optional) 

• PWS ID 

65 February 2006 
Section 4: Guidance for E. coli Laboratories 

• Facility ID 

• Sample collection date 

• Analytical method number 

• Method type 

• Source water type (provided by PWS on sample collection form) 

• E. coli/100 mL (see note below) 

• Turbidity result (provided by PWS on sample collection form) 

Note: The laboratory may enter the final result for the E. coli sample or may enter the primary 

measurements recorded at the bench, and have the DCTS automatically calculate the final E. coli 

concentration. Because this information is specific to method type (membrane filtration, multiple-tube, 

51-well, and 97-well), the system provides different entry screens for each method type. By entering E. 

coli data into the system, the laboratory acknowledges that the following QC requirements were met: all 

holding and incubation times and temperatures, sample condition on receipt, all method-specific QC 

requirements, and all QA/QC criteria and procedures specified in the Lab Certification Manual. 

The laboratory should establish a contact person that is responsible for verifying the quality and accuracy 

of all sample results in the laboratory, and should review and approve the results before they are 

submitted to the PWS for review. If inaccuracies or other problems are identified, the official contact 

discusses the sample information with the analyst or data entry staff and resolves the issues before the 

data are released to the PWS for review. 

If no inaccuracies or other issues are identified, the laboratory’s official contact approves the data for 

“release” to the PWS for review (EPA does not receive the data at this point). When the data are approved 

by the laboratory, the rights to the data are transferred electronically by the system to the PWS, and the 

data can no longer be changed by the laboratory. 

4.7.2 PWS Data Review 

After the laboratory has released E. coli data electronically to the PWS using the DCTS, the PWS will 

review the results. The PWS user cannot edit the data, but if the PWS has an issue with the sample result, 

such as if the PWS believes that the sample collection point or collection date is incorrect, the PWS can 

release the results back to the laboratory for issue resolution. In addition to noting the reason in the DCTS 

for the return of the data to the laboratory, the PWS should contact the laboratory verbally to discuss the 

issue. 

If the PWS determines that the data are accurate, the PWS releases the results to EPA (and the State, if 

applicable) as “approved” results. If the PWS determines that the data are accurate, but believes that the 

data are not valid for other reasons, the PWS can release the results as “contested.” 

4.7.3 EPA/State Review 

After the PWS has released the results as approved or contested, they are available to EPA and State users 

to review through the DCTS. EPA and State users cannot edit the data. EPA or State users approve 

results where appropriate. Pursuant to 141.702(b)(2), resampling must occur whenever EPA or the State 

rejects results or indicates agreement with a PWS action to contest a result. 

66 February 2006 
Section 4: Guidance for E. coli Laboratories 

4.8 Data Archiving 

The PWS is required to keep all original, hardcopy monitoring results associated with LT2 sample 

analyses (both initial and second round of monitoring) for 3 years after bin classification for filtered 

systems or determination of the mean Cryptosporidium level for unfiltered systems [40 CFR § 

141.722(a)]. Although it is the PWS’s responsibility to meet LT2 Rule data storage requirements for 

compliance monitoring samples, including MS samples, the PWS may contract this work to the 

laboratory. 

67 February 2006 
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68 February 2006 
SECTION 5: REFERENCES 

5.1 USEPA. 2006. National Primary Drinking Water Regulations: Long Term 2 Enhanced Surface 

Water Treatment Rule. 40 CFR §s 9, 141, and 142. 

5.2 USEPA. 2005. Method 1622: Cryptosporidium in Water by Filtration/IMS/FA. U.S. 

Environmental Protection Agency, Office of Water, Washington, D.C. EPA-815-R-05-001. 

5.3 USEPA. 2005. Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA. U.S. 

Environmental Protection Agency, Office of Water, Washington, D.C. EPA-815-R-05-002. 

5.4 USEPA. 1998. National Primary and Secondary Drinking Water Regulations: Analytical 

Methods for Regulated Drinking Water Contaminants, Final Rule, Federal Register: September 

3, 1998 63(171), FR 47097-47114.. 

5.5 USEPA. 2002. Laboratory Quality Assurance Evaluation Program for Analysis of 

Cryptosporidium Under the Safe Drinking Water Act; Agency Information Collection: Proposed 

Collection; Comment Request. Federal Register: March 4, 2002. 67 (42) FR 9731 - 9734. 

5.6 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 Martin Luther King Drive, 

Cincinnati, OH 45268. 

5.7 USEPA. 2004. EPA Microbiological Alternate Test Procedure (ATP) Protocol for Drinking 

Water, Ambient Water, and Wastewater Monitoring Methods Guidance. Washington, DC. EPA 

821-B-03-004. 

5.8 American Public Health Association. 1998. Standard Methods for the Examination of Water and 

Wastewater; 20th Edition. American Public Health Association, Washington, D.C. Standard 

Methods may be ordered from: American Water Works Association Bookstore, 6666 West 

Quincy Avenue, Denver, CO 80235. 

5.9 IDEXX Laboratories, Inc., Description of Colilert®, Colilert-18®, Quanti-Tray®, QuantiTray®/2000, and ColisureTM methods may be obtained from: IDEXX Laboratories, Inc., One 

IDEXX Drive, Westbrook, Maine 04092. 

5.10 USEPA. 2002. Method 1103.1: Escherichia coli in Water by Membrane Filtration Using 

membrane-Thermotolerant Escherichia coli Agar (mTEC). U.S. Environmental Protection 

Agency, Office of Water, Washington, D.C. EPA-821-R-02-020. 

5.11 USEPA. 2002. Method 1603: Escherichia coli (E.coli) in Water by Membrane Filtration Using 

Modified membrane-Thermotolerant Escherichia coli agar (Modified mTEC). U.S. 

Environmental Protection Agency, Office of Water, Washington, D.C. EPA-821-R-02-023. 

5.12 USEPA. 2002. Method 1604: Total coliforms and Escherichia coli (E. coli) in Water by 

Membrane Filtration Using a Simultaneous Detection Technique (MI Medium). U.S. 

Environmental Protection Agency, Office of Water, Washington, D.C. EPA-821-R-02-024. 

69 February 2006 
Section 5: References 

5.13 Hach Company, Inc. m-ColiBlue24 Method is available from: Hach Company, P.O. Box 389, 

Loveland, CO 80539 

5.14 Noble, Rachel T., Dorsey, J., Leecaster, M., Mazur, M., McGee, C., Moore, D., Victoria, O., 

Reid, D., Schiff, K., Vainik P., Weisberg, S. 1999. Southern California Bight 1998 Regional 

Monitoring Program, Vol I: Summer shoreline microbiology. Southern California Coastal Water 

Research Project, Westminster, CA. 

5.15 USEPA. 2005. Source Water Monitoring Guidance Manual for Public Water Systems for the Long Term 2 

Enhanced Surface Water Treatment Rule (LT2 Rule). This manual is available for download from 

http://www.epa.gov/safewater/disinfection/lt2/pwsguide.html 

70 February 2006 
Appendix A 

Biosafety Guidelines for Laboratories Analyzing Environmental 

Samples for Cryptosporidium 
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BIOSAFETY GUIDELINES FOR LABORATORIES 

ANALYZING ENVIRONMENTAL SAMPLES FOR CRYPTOSPORIDIUM

1.0 Introduction 

Laboratory-related infections with Cryptosporidium can occur if personnel are not properly trained in 

biosafety techniques. In addition to waterborne, fecal-oral, person to person transmission, and animal to 

person, circumstantial evidence suggests that airborne transmission of oocysts may occur1

. Adoption of a 

biosafety policy by laboratory management that includes commitment to technician safety, training and 

supervision, as well as, rigid adherence to biosafety guidelines will prevent the occurrence of 

Cryptosporidium infection (cryptosporidiosis) in laboratory personnel. 

Symptoms associated with cryptosporidiosis may include: watery diarrhea, abdominal cramps, nausea, 

low-grade fever, dehydration, weight loss, and loss of appetite. Symptoms may develop within 2 to 10 

days after infection. There are no antibiotics or drug treatments that will cure cryptosporidiosis. For 

additional information : www.cdc.gov/ncidod/dpd/parasites/cryptosporidiosis/default.htm 

Biosafety Level 2 (BSL 2) practices and facilities are recommended for activities with infective stages of 

Cryptosporidium. A BSL 2 facility is appropriate for agents known to cause disease in humans. 

Biosafety Level 2 practices and facilities include the following requirements: 

• Laboratory personnel have specific training in handling pathogenic agents and are directed by 

competent scientists 

• Access to the laboratory is limited when work is being conducted 

• Certain procedures in which infectious aerosols or splashes may be created are conducted in 

biological safety II cabinets 

2.0 Scope and Application 

The biosafety guidelines described in this document are adapted from Laboratory Safety: Principles and 

Practices, Second Edition (Reference 8.1) and Biosafety in Microbiological and Biomedical Laboratories

 (BMBL) Fourth Edition (Reference 8.2). A readily available laboratory-specific biosafety manual may 

be developed, and maintained, to address the safety, handling, and laboratory practices described below. 

The manual can be distributed to all employees and available at all times. It can be reviewed annually, or 

as recommended, by the laboratory safety officer. Personnel may read and sign off on the document on a 

regular basis, as determined by the laboratory safety officer. 

3.0 General Safety Practices 

3.1 Basic Laboratory Safety Recommendations 

3.1.1 Closed-toed shoes worn in the laboratory. 

3.1.2 All work surfaces and floors cleaned regularly and free of clutter. 

3.1.3 All emergency numbers posted in the laboratory. 

1

Hojlyng, N., Holten-Andersen, W., and S. Jepsen. 1987. Cryptosporidiosis: a case of airborne 

transmission. Lancet. 2:271-272. 

Blagburn, B.L., and W.L. Current. 1983. Accidental infection of a researcher with human 

Cryptosporidium. J. Infect. Dis. 142:772-773. 

A-1 February 2006 
LT2 Biosafety Guidance 

3.1.4 All employees trained in the use and location of all safety/emergency equipment in each 

work area. 

3.1.5 Biological safety II cabinets tested and certified annually. 

3.1.6 All laboratory personnel trained in the proper procedures to clean up biological spills. 

4.0 Recommended Microbiological Practices 

4.1 Access to the laboratory is limited or restricted at the discretion of the laboratory director or 

laboratory safety officer when experiments are in progress. 

4.2 Persons wash their hands after they handle viable materials and animals, after removing gloves, 

and before leaving the laboratory. 

4.3 Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in 

the work areas. Persons who wear contact lenses in laboratories should also wear goggles or a 

face shield. Food is stored outside the work area in cabinets or refrigerators designated for this 

purpose only. 

4.4 Mouth pipetting recognized as poor practice. 

4.5 All procedures are performed carefully to minimize the creation of splashes or aerosols. Ensure 

that lids are used during all centrifugation and vortexing. Any procedures with open containers 

are performed inside a BSL 2 hood. 

4.6 Work surfaces are decontaminated before and after each use and after any spill of viable material. 

4.7 All cultures, stocks, and other regulated wastes are decontaminated before disposal by an 

approved decontamination method such as autoclaving. 

4.7.1 Materials to be decontaminated outside of the immediate laboratory are to be placed in a 

durable, leakproof container and closed for transport from the laboratory. 

4.7.2 Materials to be decontaminated off-site from the laboratory are packaged in accordance 

with applicable local, state, and federal regulations before removal from the facility. 

5.0 Recommended Special Practices when Processing Cryptosporidium 

Samples 

5.1 In general, persons who are at increased risk of acquiring infection or for whom infection may be 

unusually hazardous are not allowed in the laboratory or animal rooms. For example, persons 

who are immunocompromised may be at risk of acquiring infections. 

5.2 The laboratory safety officer has the final responsibility for assessing each circumstance and 

determining who may enter or work in the laboratory. The laboratory safety officer will report to 

the laboratory director on a regular basis on the status of safety in the laboratory, conduct training 

and maintain outside professional contacts to exchange safety information relevant to laboratory 

operations. 

5.3 The laboratory director or laboratory safety officer, establishes policies and procedures, whereby 

only persons who have been advised of the potential hazard and meet specific entry requirements 

are allowed to enter the laboratory or animal rooms. 

5.4 When the infectious agent(s) in use in the laboratory require special provisions or special training 

for entry, a hazard warning sign incorporating the universal biohazard symbol is posted on the 

access door to the laboratory work area. The hazard warning sign identifies the infectious agent, 

A-2 February 2006 
LT2 Biosafety Guidance 

lists the name and telephone number of the laboratory director or other responsible person(s), and 

indicates the special requirement(s) for entering the laboratory. 

5.5 Laboratory personnel receive appropriate training on the potential hazards associated with the 

work involved, the necessary precautions to prevent exposures, and the exposure evaluation 

procedures. Personnel receive annual updates, or additional training as necessary for procedural 

or policy changes. 

5.6 Materials containing Cryptosporidium are placed in a container that prevents leakage during 

collection, handling, processing, storage, transport, or shipping. 

5.7 Laboratory equipment is decontaminated with an appropriate disinfectant before and after the 

equipment is used, and especially after overt spills, splashes, or other contamination by infectious 

materials. Contaminated equipment is decontaminated according to any local, state, or federal 

regulations before it is sent for repair or maintenance or packaged for transport in accordance 

with applicable local, state, or federal regulations before removal from the facility. 

5.8 Spills and accidents which result in overt exposures to infectious materials are immediately 

reported to the laboratory director or laboratory safety officer. Medical evaluation, surveillance, 

and treatment are provided as appropriate and written records are maintained. 

6.0 Recommended Safety Equipment (Primary Barriers) 

6.1 Properly maintained biological safety II cabinets, and other appropriate personal protective 

equipment or physical containment devices are used whenever: 

6.1.1 Procedures with potential for creating infectious aerosols or splashes are conducted. 

These may include centrifuging, vortexing, grinding, blending, vigorous shaking or 

mixing, sonic disruption, or opening containers of infectious materials whose internal 

pressures may be different from ambient pressures. 

6.1.2 High concentrations or large volumes of infectious agents are used. Such materials may 

be centrifuged in the open laboratory if sealed rotor heads or centrifuge safety cups are 

used, and if these rotors or safety cups are opened only in a biological safety cabinet. 

6.2 Face protection (goggles, mask, faceshield, or other splatter guards) is used for anticipated 

splashes or sprays of infectious or other hazardous materials to the face, when the 

microorganisms are manipulated outside the biological safety cabinet. 

6.3 Protective laboratory coats, gowns, smocks, or uniforms designated for lab use are worn while in 

the laboratory. This protective clothing is removed and left in the laboratory before leaving for 

non-laboratory areas (e.g., cafeteria, library, administrative offices). All protective clothing is 

either disposed of in the laboratory or laundered by the institution; and is not taken home by 

personnel. 

6.4 Gloves are worn when handling infected animals and when hands may contact infectious 

materials, contaminated surfaces, or equipment. 

6.4.1 Wearing two pairs of gloves may be appropriate; if a spill or splatter occurs, the hand will 

be protected after the contaminated glove is removed. 

6.4.2 Gloves are disposed of when contaminated, removed when work with infectious 

materials is complete, and are not worn outside the laboratory. 

6.4.3 Disposable gloves are not washed or reused. 

A-3 February 2006 
LT2 Biosafety Guidance 

7.0 Recommendations for Laboratory Facilities (Secondary Barriers) 

7.1 Each laboratory contains a sink for handwashing. 

7.2 The laboratory is designed so that it can be easily cleaned. Rugs in laboratories are not 

appropriate, because proper decontamination following a spill is extremely difficult to achieve. 

7.3 Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate 

heat. 

7.4 Laboratory furniture is sturdy, and spaces between benches, cabinets, and equipment are 

accessible for cleaning. 

7.5 If the laboratory has windows that open, they are fitted with fly screens. 

7.6 A method for decontamination of infectious or regulated laboratory wastes is available (e.g., 

autoclave, chemical disinfection, incinerator, or other approved decontamination system). 

7.7 An eyewash facility is readily available. 

7.8 The laboratory facilities are clean, temperature and humidity controlled, and have adequate 

lighting at bench tops. 

8.0 References 

8.1 D. O. Fleming, J. H. Richardson, J. J. Tulis, and D. Vesley. Laboratory Safety: Principles and 

Practices. 1995. Second Edition. American Society for Microbiology, Washington, D.C., 

publisher. 

8.2 US Department of Health and Human Services. Biosafety in Microbiological and Biomedical 

Laboratories. 1999. Fourth Edition. U.S. Department of Health and Human Services, Centers for 

Disease Control and Prevention, and National Institutes of Health, US Government Printing 

Office, Washington, D.C., publisher. 

8.3 Fayer, Ronald, Ed. 1997. Cryptosporidium and Cryptosporidiosis. CRC Press, Inc., Boca Raton. 

A-4 February 2006 
 

Appendix B

Method 1622: Cryptosporidium in Water by Filtration/IMS/FA 
This page intentionally left blank 
Method 1622: Cryptosporidium in Water 

by Filtration/IMS/FA 

December 2005

Office of Water (4607) 

EPA 815-R-05-001 

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 

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. To implement 

these requirements, EPA must assess Cryptosporidium occurrence in raw surface waters used as source 

waters for drinking water treatment plants. EPA Method 1622 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. 

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 Version of the Method 

The method was 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 revision of the method (EPA-821-R-01-025) 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 

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 

iii 
Changes in the June 2003 Version of the Method 

The method was 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 version 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 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 Version of the Method 

The method was revised again in 2005 to support promulgation of EPA’s Long Term 2 Enhanced Surface 

Water Treatment Rule. Changes incorporated into the June 2003 version 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. 

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

Methods 1622. 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 1622 is a performance-based method applicable to the determination of Cryptosporidium in 

aqueous matrices. EPA Method 1622 requires filtration, immunomagnetic separation of the oocysts from 

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

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

interference contrast microscopy. 

The interlaboratory validation of EPA Method 1622 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. 

iv 
Since the interlaboratory validation of EPA Method 1622, 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 for use in routine QC samples 

The validation studies for the 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 ......................................................... 3


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 Suspension Enumeration and Sample Spiking ................................. 28


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


13.0 Sample Concentration and Separation (Purification) .................................. 45


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


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


16.0 Analysis of Complex Samples ................................................... 53


17.0 Method Performance ........................................................... 54


18.0 Pollution Prevention ........................................................... 54


19.0 Waste Management ............................................................ 54


20.0 References ................................................................... 54


21.0 Tables and Figures ............................................................ 56


22.0 Glossary of Definitions and Purposes .............................................. 65


vi 
Method 1622: Cryptosporidium 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 in 

water by concentration, immunomagnetic separation (IMS), and immunofluorescence assay (FA) 

microscopy. Cryptosporidium 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, but not the species. The method cannot 

determine the host species of origin, nor can it determine the viability or infectivity of detected 

oocysts. 

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

the supernatant fluid is aspirated. 

2.2.2	 The oocysts are magnetized by attachment of magnetic beads conjugated to antiCryptosporidium antibodies. The magnetized oocysts are separated from the extraneous 

materials using a magnet, and the extraneous materials are discarded. The magnetic bead 

complex is then detached from the oocysts. 

2.3	 Enumeration 

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

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

fluorescence and differential interference contrast (DIC) microscopy. 

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

size, shape, and fluorescence characteristics of Cryptosporidium oocysts. 

1	 December 2005 
Method 1622 - Cryptosporidium 

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

confirmed as oocysts. 

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

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

provided in Section 9.0. 

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	 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. 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 contribute to false positives by immunofluorescence assay (FA) 

(Reference 20.3). 

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

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

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

should be used wherever possible. 

December 2005	 2 
Method 1622 - Cryptosporidium 

5.0	 Safety 

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

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

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

safety and health practices prior to use of this method. In particular, 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 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. 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 or other infectious agents (e.g., during an outbreak), samples should be shipped 

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

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

CDC or state regulations, the sample should be shipped in accordance with these regulations. 

6.0	 Equipment and Supplies 

NOTE: Brand names, suppliers, and part numbers are for illustrative purposes only. No 

endorsement is implied. Equivalent performance may be achieved using apparatus and 

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

that meets the requirements of this method is the responsibility of the laboratory. 

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

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

one use. 

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

with Method 1622. Other options may be used if their acceptability is demonstrated according to 

the procedures outlined in Section 9.1.2. 

3	 December 2005 
Method 1622 - Cryptosporidium 

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). 

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, 

December 2005	 4 
Method 1622 - Cryptosporidium 

provided the laboratory demonstrates acceptable performance on initial and ongoing 

spiked reagent water and matrix samples (Section 9.1.2). 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 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 

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 

5	 December 2005 
Method 1622 - Cryptosporidium 

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. 

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 

December 2005	 6 
Method 1622 - Cryptosporidium 

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 

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 

7	 December 2005 
Method 1622 - Cryptosporidium 

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 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 

December 2005	 8 
Method 1622 - Cryptosporidium 

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

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

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

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

7.4.1.4). Dilute to 1000 mL with reagent water. 

7.4.2	 Reagents for eluting 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® anti-Cryptosporidium beads, 

Dynal cat. nos. 730.01/730.11, or equivalent 

7.6	 Direct antibody labeling reagents for detection of oocysts. 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, Waterborne cat. no. A400FLR, 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 1622 - Cryptosporidium 

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 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 —Wisconsin State Laboratory of 

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

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

oocysts —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	 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 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 

December 2005	 10 
Method 1622 - Cryptosporidium 

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 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 determination, laboratories performing 

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

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

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

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

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

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

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

receipt to estimate the temperature of the filter. Temperature sample 

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

potential effect that the difference in sample volume may have in 

temperature equilibration in the sample cooler. Example product: Cole 

Parmer cat. no. U-06252-20. 

11	 December 2005 
Method 1622 - Cryptosporidium 

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. 

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. 

December 2005	 12 
Method 1622 - Cryptosporidium 

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 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 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). 

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). 

13 December 2005 
Method 1622 - Cryptosporidium 

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: 

9.1.2.2.1	 The names, titles, addresses, and telephone numbers of 

the analyst(s) who performed the analyses and 

December 2005	 14 
Method 1622 - Cryptosporidium 

9.1.2.2.2 

9.1.2.2.3 

9.1.2.2.4 

9.1.2.2.5 

modification, and of the quality control officer who 

witnessed and will verify the analyses and modification.

A listing of the analyte(s) measured (Cryptosporidium). 

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. 

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. 

15	 December 2005 
Method 1622 - Cryptosporidium 

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, 

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

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 

December 2005	 16 
Method 1622 - Cryptosporidium 

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	 Calculate the percent recovery (R) using the following equation: 

N 

R = 100 x 

T 

where: 

R = the percent recovery 

N = the number of oocysts counted 

T = the number of oocysts 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. 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 Table 3 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 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 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 

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) 

17	 December 2005 
Method 1622 - Cryptosporidium 

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 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 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 counted in the spiked sample 

Ns is the number of oocysts counted in the unspiked sample 

T is the true value of the oocysts spiked 

9.5.1.3 Compare the recovery with the acceptance criteria in Table 3 in Section 

21.0. 

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

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 Calculate the percent recovery (R) using the equation in Section 9.5.1.2. 

December 2005	 18 
Method 1622 - Cryptosporidium 

9.5.2.2	 Calculate the mean of the number of oocysts in the MS and MSD (Xmean) 

(= [MS+MSD]/2). 

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

following equation: 

| NMS - NMSD |

RPD = 100 x 

XMEAN 

X

N

N

where 

RPD is the relative percent difference 

MS is the number of oocysts counted in the MS 

MSD is the number of oocysts counted in the MSD 

mean is the mean number of oocysts counted in the MS and MSD 

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

criteria in Table 3 in Section 21.0. 

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 or potentially interfering organisms or 

materials that may be misidentified as oocysts 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 (as defined in Section 3) or any potentially 

interfering organism or materials that may be misidentified as oocysts 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 each week to verify all 

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

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

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

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

performance criteria are met should samples be analyzed. 

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

19	 December 2005 
Method 1622 - Cryptosporidium 

9.7.1.1	 Using 200X to 400X magnification, more than 50% of the oocysts 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 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 report form, as well as any 

additional comments on organism appearance, if notable. 

9.7.2	 Calculate the percent recovery (R) using the following equation: 

N 

R = 100 x 

T 

where:


R = the percent recovery


N = the number of oocysts detected


T = the number of oocysts spiked


9.7.3	 Compare the recovery with the acceptance criteria for ongoing precision and recovery in 

Table 3 in Section 21.0. 

9.7.4	 Actions 

9.7.4.1	 If the recoveries for Cryptosporidium meet the acceptance criteria, system 

performance is acceptable and analysis of samples may proceed. 

9.7.4.2	 If the recovery for Cryptosporidium 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 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. 

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), 

December 2005	 20 
Method 1622 - Cryptosporidium 

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 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 by this method. 

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

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

the precision of the analysis. 

10.0 Microscope Calibration and Analyst Verification 

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

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

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

placement of slides and ancillary materials. 

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

operation of the microscope. 

10.3 Microscope adjustment and calibration (adapted from Reference 20.10) 

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 will not be 

21 December 2005 
Method 1622 - Cryptosporidium 

possible. Consequently, it is imperative that all portions of the 

microscope from the light sources to the oculars are properly adjusted. 

10.3.1.2	 While microscopes from various vendors are configured somewhat 

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

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

procedures below work for a particular instrument. 

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

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

and that the condenser is adjusted to produce Köhler illumination. 

10.3.1.4	 Persons with astigmatism should always wear contact lenses or glasses 

when using the microscope. 

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

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

premature failure of the bulb. 

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

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

eye damage. 

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

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

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

illumination are experienced. 

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

out of the transmitted light path. 

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

oculars is sharply defined. 

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

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

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

location of the center of the field of view. 

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

diffuser lens in place and turn on the mercury bulb. 

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

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

appear on the card after focusing the image with the appropriate 

adjustment. 

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

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

the transmitted light dot in between them. 

10.3.2.8	 Reattach the objective to the nosepiece. 

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

house and the microscope. 

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

fluorescent material. Check the field for even fluorescent illumination. 

Adjustment of the diffuser lens probably will be required. Additional 

slight adjustments as in Section 10.3.2.7 above may be required. 

December 2005	 22 
Method 1622 - Cryptosporidium 

10.3.2.11	 Maintain a log of the number of hours the UV bulb has been used. Never 

use the bulb for longer than it has been rated. Fifty-watt bulbs should not 

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

than 200 hours. 

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

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

changed. 

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

out of the transmitted light path. 

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

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

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

filament image at the bottom of the tube. 

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

lamp house. 

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

appropriate adjustment(s) on the lamp house. 

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

house and the microscope. 

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

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

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

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

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

10.3.4.1	 Interpupillary distance 

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

the transmitted light, and focus the specimen image using 

the coarse and fine adjustment knobs. 

10.3.4.1.2	 Using both hands, move the oculars closer together or 

farther apart until a single circle of light is observed 

while looking through the oculars with both eyes. Note 

interpupillary distance. 

10.3.4.2 Ocular adjustment for microscopes capable of viewing a photographic 

frame through the viewing binoculars: This procedure assumes both 

oculars are adjustable. 

10.3.4.2.1	 Place a card between the right ocular and eye keeping 

both eyes open. Adjust the correction (focusing) collar on 

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

same as the interpupillary distance. Bring an image 

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

focus as possible. 

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

Again keeping both eyes open, bring the same image into 

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

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

right ocular. 

23	 December 2005 
Method 1622 - Cryptosporidium 

10.3.4.3 Ocular adjustment for microscopes without binocular capability: This 

procedure assumes a single focusing ocular. The following procedure 

assumes that only the right ocular is capable of adjustment. 

10.3.4.3.1 Place a card between the right ocular and eye keeping 

both eyes open. Using the fine adjustment, focus the 

image for the left eye to its sharpest point. 

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

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

into sharp focus by adjusting the ocular collar at the top 

of the ocular without touching the coarse or fine 

adjustment. 

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

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

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

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

for it to become contaminated with dust particles. This calibration should be done for 

each objective in use on the microscope. If there is a top lens on the microscope, the 

calibration procedure must be done for the respective objective at each top lens setting. 

The procedure must be followed when the microscope is first used and each time the 

objective is changed. 

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

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

fine adjustment knobs for the objective to be calibrated. Continue 

adjusting the focus on the stage micrometer so you can distinguish 

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

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

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

micrometer. 

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

from the two 0 lines where two other lines are exactly superimposed. 

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

of millimeters on the stage micrometer between the two points of 

superimposition. For example: Suppose 48 ocular micrometer spaces 

equal 0.6 mm. 

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

0.6 mm 0.0125 mm 

= 

48 ocular micrometer spaces ocular micrometer space 

10.3.5.6 Because most measurements of microorganisms are given in μm rather 

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

multiplying it by 1000 μm/mm. For example: 

0.0125 mm 1,000 μm 12.5 μm 

x = 

ocular micrometer space mm ocular micrometer space 

December 2005 24 
Method 1622 - Cryptosporidium 

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. 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 

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. 

25 December 2005 
Method 1622 - Cryptosporidium 

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


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 by FA, 4',6-diamidino-2-


phenylindole (DAPI), and DIC that the analysts (Section 22.2) determine are accurate 

(Section 15.2). 

December 2005	 26 
Method 1622 - Cryptosporidium 

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. Quantify the size, 

shape, microscope settings, and other characteristics that can be used to differentiate 

oocysts 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, this method shall rely upon the ability of the analyst 

for identification and enumeration of oocysts. 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. More than 50% of the oocysts must be 

DAPI positive and undamaged under DIC. 

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

slide meeting the criteria in 10.6.1. For the same 10 oocysts, 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 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 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 

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 in QC samples and field samples be identified and enumerated. 

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

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

photographs of oocysts and 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. 

27	 December 2005 
Method 1622 - Cryptosporidium 

11.0 Oocyst 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 1622 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. 

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 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 

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 (Section 7.10.2) received from suppliers can be used to 

December 2005 28 
Method 1622 - Cryptosporidium 

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 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 

before proceeding (these criteria are based on the pooled RSDs of 105 manual 

Cryptosporidium enumerations 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 

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). 

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. 

29	 December 2005 
Method 1622 - Cryptosporidium 

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, 

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. 

December 2005	 30 
Method 1622 - Cryptosporidium 

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 before proceeding. If the RSD is unacceptable, or the 

mean number is outside the expected range, add additional oocysts from 

stock suspension or dilute the contents of the beaker appropriately with 

reagent water. Repeat the process to confirm counts. Refer to Section 

11.3.3.14 for factors that may introduce errors. 

31	 December 2005 
Method 1622 - Cryptosporidium 

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 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 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. 

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. 

December 2005	 32 
Method 1622 - Cryptosporidium 

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

filter support. Repeat for the other two filters. 

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

allow to stand. 

33	 December 2005 
Method 1622 - Cryptosporidium 

11.3.6.9	 Vortex the tube containing the spiking suspension (diluted stock 

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

the tube three times. 

11.3.6.10	 Using a micropipettor, sequentially remove two, 10-μL aliquots from the 

spiking suspension and pipet into the 5 mL of 0.01% Tween® 20 

standing in the unit. Rinse the pipet tip twice after each addition. Apply 

10 μL of 0.01% Tween® 20 to the third unit to serve as the negative 

control. Apply vacuum at 2" Hg and allow liquid to drain to miniscus, 

then close off vacuum. Pipet 10 mL of reagent water into each funnel and 

drain to miniscus, closing off the vacuum. Repeat the rinse and drain all 

fluid, close off the vacuum. 

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

mm petri dish for each sample. 

11.3.6.12	 Unclamp the top funnel and transfer each cellulose acetate support 

membrane/ polycarbonate filter combination onto the drop of stain using 

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

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

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

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

minutes at room temperature. 

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

time with 20 mL of reagent water. 

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

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

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

aliquots must be prepared and counted, and the counts averaged, to 

sufficiently enumerate the spike dose. Include a filter blank sample at a 

frequency of every five samples; rotate the position of filter blank to 

eventually include all three filter placements. 

11.3.6.15	 Repeat Sections 11.3.6.4 through 11.3.6.10 until the 10-μL spiking 

suspensions have been filtered. The last batch should include a 10-μL 

0.01 Tween® 20 blank control and 20 μL of positive control antigen as a 

positive staining control. 

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

cellulose acetate filter support and polycarbonate filter from drop of stain 

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

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

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

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

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

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

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

all slides.) 

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 

December 2005	 34 
Method 1622 - Cryptosporidium 

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

#16% for Cryptosporidium 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 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 

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 

35	 December 2005 
Method 1622 - Cryptosporidium 

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 

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. 

December 2005	 36 
Method 1622 - Cryptosporidium 

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 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. 

12.2.6.1.2 Prepare sufficient quantity of elution buffer to elute all

12.2.6.1.3

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. 

Designate at least one 250-mL conical centrifuge tube for

each sample and label with the sample number. 

37 December 2005 
Method 1622 - Cryptosporidium 

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 

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. 

December 2005	 38 
Method 1622 - Cryptosporidium 

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 

12.3.1.5.3 

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.

Allow the carboy discharge tube and filter housing to fill


with sample water. Turn on the pump to start water


flowing through the filter. Verify that the flow rate is


between 1 and 4 L per min.


39	 December 2005 
Method 1622 - Cryptosporidium 

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

off the pump. Allow the pressure to decrease until flow 

12.3.1.6 Disassembly 

stops.

12.3.1.6.1 Disconnect the inlet end of the filter housing assembly 

while maintaining the level of the inlet fitting above the 

level of the outlet fitting to prevent backwashing and the 

loss of oocysts from the filter. Restart the pump and 

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

12.3.1.6.2 

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 

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 

December 2005	 40 
Method 1622 - Cryptosporidium 

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. 

(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 

41	 December 2005 
Method 1622 - Cryptosporidium 

concentrated using centrifugation, as described in 

Section 12.3.3.3. 

12.3.2.3	 Stomacher elution procedure 

12.3.2.3.1 

12.3.2.3.2 

12.3.3 Concentration 

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.

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). 

12.3.3.1	 The eluate can be concentrated using the Filta-Max® concentrator 

apparatus, which pulls most of the eluate through a membrane filter 

leaving the oocysts concentrated in a small volume of the remaining 

eluting solution (Section 12.3..2), or by directly centrifuging all of the 

eluting solution used to wash the filter (Section 12.3.2.3). 

12.3.3.2	 The Filta-Max® concentrator procedure 

12.3.3.2.1 Concentration of first wash 

(a) If the stomacher was used to elute the sample 

(Section 12.3.2.3), transfer 600 mL of eluate from the 

pooling beaker to the concentrator tube. Otherwise 

proceed to Step (b). 

(b) Stand the concentrator tube on a magnetic stirring 

plate and attach the lid (with magnetic stirrer bar). 

Connect the waste bottle trap and hand or electric 

vacuum pump to the valve on the concentrator base. 

Begin stirring and open the tap. Increase the vacuum 

using the hand pump. 

NOTE: The force of the vacuum should not exceed 30 cmHg. 

(c) Allow the liquid to drain until it is approximately 

level with the middle of the stirrer bar then close the 

valve. Remove the magnetic stirrer, and rinse it with 

December 2005	 42 
Method 1622 - Cryptosporidium 

12.3.3.2.2


12.3.3.2.3


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: 

Perform a fourth wash using another 5 mL of PBST, 

by hand-kneading an additional minute or placing the 

bag on a flat-headed vortexer and vortexing for one 

minute. Transfer the eluate to the 50-mL tube.) 

12.3.3.2.4	 If the membrane filter clogs before concentration is 

complete, there are two possible options for completion 

43	 December 2005 
Method 1622 - Cryptosporidium 

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 during this step. 

(d) Vortex each 250-mL tube vigorously until pellet is 

completely resuspended. Swirl the centrifuge tube 

gently to reduce any foaming after vortexing. 

Combine the contents of each 250-mL centrifuge 

tube into a 50-mL centrifuge tube. Rinse each of the 

December 2005	 44 
Method 1622 - Cryptosporidium 

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). 

13.0 Sample Concentration and Separation (Purification) 

13.1 During concentration and separation, the filter eluate is concentrated through centrifugation, and 

the oocysts in the sample are separated from other particulates through 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. 

45 December 2005 
Method 1622 - Cryptosporidium 

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 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 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 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 

subsamples would be 12 mL / 5 mL = 2.4, rounded = 3 

subsamples). 

13.2.4.1.2	 Determine volume of resuspended concentrate per 

subsample. Divide the total volume in the centrifuge 

tube by the calculated number of subsamples (for 

example, if the resuspended volume in Section 13.2.4 is 

12 mL, then the volume to use for each subsample = 12 

mL / 3 subsamples = 4 mL). 

13.2.4.1.3	 Process subsamples through IMS. Vortex the tube 

vigorously for 10 to 15 seconds to completely resuspend 

the pellet. Record the resuspended pellet volume on the 

December 2005	 46 
Method 1622 - Cryptosporidium 

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. 

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 

47	 December 2005 
Method 1622 - Cryptosporidium 

13.3.2.2 

13.3.2.3 

13.3.2.4 

13.3.2.5 

13.3.2.6 

13.3.2.7 

13.3.2.8 

13.3.2.9 

13.3.2.10

13.3.2.11 

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).

Vortex the Dynabeads® anti-Cryptosporidium beads vial from the IMS


kit for approximately 10 seconds to suspend the beads. Ensure that the


beads are fully resuspended by inverting the sample tube and making sure


that there is no residual pellet at the bottom. 


Add 100 μL of the resuspended Dynabeads® anti-Cryptosporidium beads


(Section 13.3.2.2) to the sample tube(s) containing the water sample


concentrate and SL-buffers.


Affix the sample tube(s) to a rotating mixer and rotate at approximately


18 rpm for 1 hour at room temperature.

After rotating for 1 hour, remove each sample tube from the mixer and


place the tube in the magnetic particle concentrator (MPC®-1 or MPC®-


6) with flat side of the tube toward the magnet.


Without removing the sample tube from the MPC®-1, place the magnet


side of the MPC®-1 downwards, so the tube is horizontal and the flat side


of the tube is facing down.

Gently rock the sample tube by hand end-to-end through approximately


90°, tilting the cap-end and base-end of the tube up and down in turn.


Continue the tilting action for 2 minutes with approximately one tilt per


second.


Ensure that the tilting action is continued throughout this period to


prevent binding of low-mass, magnetic or magnetizable material. If the


sample in the MPC®-1 is allowed to stand motionless for more than 10


seconds, remove the flat-sided tube from the MPC®-1, shake the tube to


resuspend all material, replace the sample tube in the MPC®-1 and repeat


Section 13.3.2.9 before continuing to Section 13.3.2.11.

Return the MPC®-1 to the upright position, sample tube vertical, with


cap at top. Immediately remove the cap and, keeping the flat side of the


tube on top, pour off all of the supernatant from the tube held in the


MPC®-1 into a suitable container. Do not shake the tube and do not


remove the tube from MPC®-1 during this step. Allow more supernatant


to settle; aspirate additional supernatant with pipette.

Remove the sample tube from the MPC®-1 and resuspend the sample in 


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.

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


December 2005 48 
Method 1622 - Cryptosporidium 

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. 

13.3.2.12	 Place the microcentrifuge tube into the second magnetic particle 

concentrator (MPC®-M or MPC®-S), with its magnetic strip in place. 

13.3.2.13	 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. 

13.3.2.14	 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 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. 

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. 

49	 December 2005 
Method 1622 - Cryptosporidium 

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. 

•	 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 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. 

December 2005	 50 
Method 1622 - Cryptosporidium 

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 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. 

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. 

51	 December 2005 
Method 1622 - Cryptosporidium 

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. 

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 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 must 

be recorded by the analyst on a microscope log. The analyst also must 

indicate on each sample examination form whether the positive staining 

control was acceptable. 

15.2.1.2	 Examine the negative staining control to confirm that it does not contain 

any oocysts (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 within the expected range 

and at the appropriate fluorescence for both FA and DAPI, and the 

negative staining control does not contain any oocysts (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 

December 2005	 52 
Method 1622 - Cryptosporidium 

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 Record the date and time that sample examination was completed on the examination 

form. 

15.2.4 Report Cryptosporidium concentrations as oocysts/L. 

15.2.5 Record analyst name. 

16.0 Analysis of Complex Samples 

16.1 Some samples may contain high levels (>1000/L) of oocysts and/or interfering organisms, 

substances, or materials. Some samples may clog the filter (Section 12.0); others will not allow 

separation of the oocysts from the retentate or eluate; and others may contain materials that 

preclude or confuse microscopic examination. 

16.2 If the sample holding time has not been exceeded and a full-volume sample cannot be filtered, 

dilute an aliquot of sample with reagent water and filter this smaller aliquot (Section 12.0). This 

dilution must be recorded and reported with the results. 

53 December 2005 
Method 1622 - Cryptosporidium 

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 1622 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 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	 Rodgers, Mark R., Flanigan, Debbie J., and Jakubowski, Walter, 1995. Applied and 

Environmental Microbiology 61 (10), 3759-3763. 

20.3	 Fleming, Diane O., et al.(eds.), Laboratory Safety: Principles and Practices, 2nd edition.1995. 

ASM Press, Washington, DC 

December 2005	 54 
Method 1622 - Cryptosporidium 

20.4	 "Working with Carcinogens," DHEW, PHS, CDC, NIOSH, Publication 77-206, (1977). 

20.5	 "OSHA Safety and Health Standards, General Industry," OSHA 2206, 29 CFR 1910 (1976). 

20.6	 "Safety in Academic Chemistry Laboratories," ACS Committee on Chemical Safety (1979). 

20.7	 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. 

20.8	 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. 

20.9	 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). 

20.10	 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. 

20.11	 "EnvirochekTM Sampling Capsule," PN 32915, Gelman Sciences, 600 South Wagner Road, Ann 

Arbor, MI 48103-9019 (1996). 

20.12	 "Dynabeads® GC-Combo,” Dynal Microbiology R&D, P.O. Box 8146 Dep., 0212 Oslo, Norway 

(September 1998, Revision no. 01). 

20.13	 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). 

20.14	 USEPA. Implementation and Results of the Information Collection Rule Supplemental Surveys. 

EPA-815-R-01-003. Office of Water, Office of Ground Water and Drinking Water, Standards and 

Risk Management Division, Washington, DC (2001). 

20.15	 Connell, K., J. Scheller, K. Miller, and C.C. Rodgers, 2000. Performance of Methods 1622 and 

1623 in the ICR Supplemental Surveys. Proceedings, American Water Works Association Water 

Quality Technology Conference, November 5 - 9, 2000, Salt Lake City, UT. 

55	 December 2005 
Method 1622 - Cryptosporidium 

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 

December 2005 56 
Method 1622 - Cryptosporidium 

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 Table 3 are based on results from 293 Cryptosporidium 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). 

57	 December 2005 
Method 1622 - Cryptosporidium 

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 - 143 

Precision (as maximum relative percent difference) 9.5.2.3 67 

(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 

4. 

December 2005 58 
Method 1622 - Cryptosporidium 

Table 4. 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 

<10% 6.7% 

>10% - 20% 6.3% 

>20% - 30% 14.9% 

>30% - 40% 14.2% 

>40% - 50% 18.4% 

>50% - 60% 17.4% 

>60% - 70% 11.2% 

>70% - 80% 8.4% 

>80% - 90% 2.3% 

>90% 0.2% 

59 December 2005 
Method 1622 - Cryptosporidium 

Figure 1. Hemacytometer Platform Ruling. Squares 1, 2, 3, and 4 

are used to count stock suspensions of Cryptosporidium 

oocysts (after Miale, 1967) 

December 2005 60 
Method 1622 - Cryptosporidium 

Figure 2.	Manner of Counting Oocysts in 1 Square mm. 

Dark organisms are counted and light 

organisms are omitted (after Miale, 1967). 

61	 December 2005 
Method 1622 - Cryptosporidium 

Figure 3a. Filtration Systems for EnvirochekTM or 

EnvirochekTMHV Capsule (unpressurized source - top, 

pressurized source - bottom) 

62 December 2005 
Method 1622 - Cryptosporidium 

Figure 3b. Filtration Systems for Filta-Max® filters (unpressurized 

source - top, pressurized source - bottom)

December 2005 63 
Method 1622 - Cryptosporidium 

Figure 4. Methods for Scanning a Well Slide 

December 2005 64 
Method 1622 - Cryptosporidium 

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 analyte in this method is 

Cryptosporidium. 

Cyst—A phase or a form of an organism produced either in response to environmental conditions 

or as a normal part of the life cycle of the organism. It is characterized by a thick and 

environmentally resistant cell wall. 

Flow cytometer—A particle-sorting instrument capable of counting protozoa. 

65 December 2005 
Method 1622 - Cryptosporidium 

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. 

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. In Cryptosporidium oocysts, there is one 

nucleus per sporozoite. 

Oocyst—The encysted zygote of some sporozoa; e.g., Cryptosporidium. The oocyst is a phase or 

form of the organism produced as a normal part of the life cycle of the organism. It is 

characterized by a thick and environmentally resistant outer wall. 

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 

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 

December 2005 66 
Method 1622 - Cryptosporidium 

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. 

67 December 2005 
 

Appendix C

Method 1623: Cryptosporidium and Giardia in Water by 

Filtration/IMS/FA 
This page intentionally left blank 
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 
Appendix D 

Abbreviated Checklist for Cryptosporidium Sample Results 
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Abbreviated Checklist for Cryptosporidium Sample Results 

Sample analyzed by EPA Method 1622 or 1623 U 

Laboratory has current approval status through Lab QA Evaluation Program U 

Quality Control (QC) Requirements Before Sample Processing 

Frequency 

The ongoing precision and recovery (OPR) and method blank samples associated 

with the sample were performed within the same week and associated with no 

more than 19 other samples 

Method blank results No Cryptosporidium oocysts or potentially interfering materials were found in the 

method blank 

OPR sample results Recovery for the OPR sample was at least 11%) 

Spike for OPR The associated OPR was spiked with no more than 500 oocysts as counted by 

flow-cytometry 

Analyst Performance Analysts’ examination criteria was verified monthly 

Key Sample Requirements

Location/Schedule Description of sampling location and schedule submitted to the State 

Temperature The sample temperature upon receipt was #20°C, and not frozen 

Volume analyzed The volume analyzed for the sample was at least 10 L OR 2 mL of packed pellet 

volume OR as much volume as can be filtered by 2 EPA-approved filters 

Staining Controls 

Positive staining control for this sample had appropriate fluorescence and 

characterization of FITC, size, shape, DIC and DAPI characteristics for 3 oocysts 

was recorded. The negative staining control for this sample did not contain any 

oocysts or potentially interfering materials 

Holding Time Requirements 

Sample collection The elution step for the sample was initiated within 96 hours of sample collection (if 

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

Sample processing The sample was processed (eluted, concentrated, purified, and applied to the 

slide) in 1 working day 

Sample drying The slide(s) for the sample were stained within 72 hours of application of the 

sample to the slide 

Sample examination The slide(s) for the sample were examined within 168 hours (7 days) of staining 

Matrix Spike (MS) Sample Requirements

Sample volume The same sample volume (within 10%) was analyzed for the MS sample and the 

associated, unspiked field sample 

MS Frequency MS sample analyzed with first field sample and at least every 20 samples 

thereafter 

Method version The same method version (type of filter, IMS kit, and staining kit) was used for the 

MS sample and the associated, unspiked field sample 

Spike for MS The MS sample was spiked with no more than 500 oocysts as counted by flowcytometry 

D-1 February 2006 
Appendix E 

Cryptosporidium Laboratory Quality Assurance Program 

Application 
This page intentionally left blank 
OMB Control Number: 2040-0246 

United States Environmental Protection Agency 

Office of Ground Water and Drinking Water 

Standards and Risk Management Division 

February 21, 2006 

Dear Laboratory Manager: 

Thank you for your interest in the U.S. EPA’s Laboratory Quality Assurance Evaluation Program 

for Analysis of Cryptosporidium under the Safe Drinking Water Act (Lab QA Program). This is a 

voluntary program open to laboratories analyzing Cryptosporidium in water using EPA Method 1622 and 

EPA Method 1623. To increase the likelihood that laboratories analyzing water samples for 

Cryptosporidium generate reliable data, EPA has established the following process for evaluating 

laboratory performance and quality assurance practices: 

Step 1. Application. Laboratories must first submit the Lab QA Program application. The application 

forms are enclosed with this letter, and the application process is described in detail below. EPA 

will evaluate laboratory applications to confirm the following: (1) the laboratory has the 

equipment required in EPA Method 1622 and/or EPA Method 1623 (December 2005 version), (2) 

laboratory personnel have the recommended experience to analyze samples, and (3) the 

laboratory has successfully completed the initial precision and recovery and matrix spike/matrix 

spike duplicate tests specified in the method. Laboratories will be requested to correct any 

deficiencies in these areas before proceeding to the next step in the evaluation process. 

Step 2. Proficiency testing. After an application has been accepted, the laboratory will be sent a set of 

eight initial proficiency testing (IPT) samples consisting of a suspension of oocysts in a 

concentrated matrix. Laboratories will resuspend these spikes in reagent water to produce 

simulated source water samples, and analyze the samples using the 2005 version of Method 

1622/1623 that the laboratory plans to use for routine Cryptosporidium analyses. If a laboratory 

wishes to be evaluated for more than one variation of the method, the laboratory will receive a set 

of eight proficiency test (PT) samples for each variation. Laboratory IPT data will be evaluated 

against the mean recovery and precision (as relative standard deviation) for the IPT samples from 

other laboratories. 

Laboratories already participating in the EPA Cryptosporidium PT Program, may use the initial 

round of samples from the PT program to meet the IPT sample requirement. 

Step 3. On-site evaluation. After a laboratory completes the IPT, an on-site evaluation of the laboratory 

will be scheduled. The on-site evaluation will include two separate but concurrent assessments: 

(1) assessment of the laboratory’s sample processing and analysis procedures, including 

microscopic examination, and (2) evaluation of the laboratory’s personnel qualifications, quality 

control program, equipment, and record keeping procedures. 

Each laboratory will receive an audit report, which will document deficiencies, if any, that should be 

corrected by the laboratory. After a laboratory has corrected any deficiencies noted in the audit report, 

EPA will confirm that the laboratory is approved under the Lab QA Program. 

E-1 February 2006 
Laboratories that meet the program performance criteria will also receive a set of three ongoing 

proficiency testing (OPT) samples approximately every four months that must be analyzed in the same 

manner as the IPT samples. EPA will evaluate the precision and recovery data for OPT samples to 

determine if the laboratory continues to meet the performance criteria of the Laboratory QA Program. 

Application Requirements 

The first step in the laboratory evaluation process is submission of a laboratory application 

package. The following materials should be submitted for each laboratory application package: 

1. Signed, completed application form (attached). 

2. Completed self-audit checklist (attached). This checklist is similar to the checklist that will be used to 

audit your laboratory during the on-site evaluation. 

3. Resumes detailing qualifications of your laboratory’s proposed principal analyst/supervisor and each 

analyst and technician listed on the application form and documentation of the training, including the 

list of samples analyzed by each and the time period during which the samples were performed (the 

list for each analyst and technician should include at a minimum the number of samples specified 

below for personnel prerequisites). 

The recommended personnel prerequisites for the laboratory evaluation program are as follows: 

Principal Analyst/Supervisor (one required per laboratory) 

• BS/BA in microbiology or closely related field 

• A minimum of 1 year of continuous bench experience with Cryptosporidium and IFA 

microscopy 

• A minimum of 6 months experience using EPA Method 1622 and/or EPA Method 1623 

• A minimum of 100 samples analyzed using EPA Method 1622 and/or EPA Method 1623 

(minimum 50 samples if the person was an approved analyst for Cryptosporidium under the 

Information Collection Rule(ICR)) 

Other Analysts (no minimum requirement per laboratory)

$ Two years of college in microbiology or equivalent or closely related field 

$ A minimum of 6 months of continuous bench experience with Cryptosporidium and IFA 

microscopy 

$ A minimum of 3 months experience using EPA Method 1622 and/or EPA Method 1623 

$ A minimum of 50 samples analyzed using EPA Method 1622 and/or EPA Method 1623 

(minimum 25 samples if the person was an ICR-approved analyst) 

Technician (no minimum requirement per laboratory)

$ Three months experience with the specific parts of the procedure he/she will be performing 

$ A minimum of 50 samples analyzed using EPA Method 1622 and/or EPA Method 1623 

(minimum 25 samples if the person was an ICR-approved technician) for the specific 

analytical procedures they will be using. 

E-2 February 2006 
4. Detailed laboratory standard operating procedures (SOP) for each variation of the method your 

laboratory plans on using for routine Cryptosporidium analyses. SOP’s for the following should be 

included: 

$ Performance of each method step including, sample spiking, filtration, elution, concentration, 

purification, slide preparation, sample staining and examination 

$ Dividing pellets greater than 0.5mL 

$ Preparation of reagents 

$ Dishwashing 

$ Staff training 

$ Corrective action procedures for failing to meet OPR, method blank, staining controls, sample 

acceptance, and performance verification criteria 

$ Sampling procedures to be followed by field or utility personnel 

$ Procedures for data recording, checking manual calculations, and checking accuracy of all data 

transcriptions 

5. EPA Method 1622/1623 initial demonstration of capability (IDC) data which include initial precision 

and recovery (IPR) test results and matrix spike and matrix spike duplicate (MS/MSD) test results for 

Cryptosporidium. The IPR test consists of four reagent water samples spiked with between 100 - 500 

oocysts and one method blank. The MS/MSD test consists of one unspiked and two spiked source 

water samples. These tests are described in Section 9 of EPA Method 1622/1623 and the results 

should meet the criteria in the method (December 2005 version). The following data should be 

submitted: 

• Completed EPA Method 1622/1623 bench sheets and report forms for each of the eight samples 

(attached) 

• Initial demonstration of capability summary form (attached) 

• Spiking suspension preparation data. This should include completed flow-cytometer calibration 

forms. 

Laboratories wishing to be evaluated for more than one variation of the method (different volumes, 

filters, elution and concentration procedures, and immunomagnetic separation kits) should submit a 

complete set of IDC data for each variation. 

If your laboratory currently participates in the EPA PT sample program and the required IDC data 

have already been submitted, the data do not need to be resubmitted. Please indicate this is the case 

on the initial demonstration of capability summary form. 

6. Table of contents from your laboratory’s quality assurance plan. The quality assurance plan should 

specifically address the requirements of Cryptosporidium analysis under the Lab QA Program. 

7. An example of the data reporting form used to submit Cryptosporidium results to your clients. 

8. A statistical summary of percent recoveries for all OPR and MS samples analyzed at your laboratory 

for the past six months. 

E-3 February 2006 
Application materials should be submitted to the following address: 

Cryptosporidium Laboratory QA Program Coordinator 

 CSC Water Programs 

 6101 Stevenson Avenue 

 Alexandria, VA 22304 

Send comments on the Agency's need for this information, the accuracy of the provided burden 

estimates, and any suggested methods for minimizing respondent burden, including through the use of 

automated collection techniques to the Director, Collection Strategies Division, U.S. Environmental 

Protection Agency (2822T), 1200 Pennsylvania Ave., NW, Washington, D.C. 20460. Include the OMB 

control number in any correspondence. Do not send the completed form to this address. 

When your application package has been received and reviewed, you will be notified whether it is 

complete or has any deficiencies. After your application has been accepted, you will be notified of when 

you should expect your initial set of PT samples. If you have any questions about the laboratory 

application materials or evaluation process, please feel free to contact either me at 

moulton.carrie@epamail.epa.gov or Jennifer Scheller at jscheller@csc.com. 

Sincerely, 

Carrie Moulton 

Manager, Cryptosporidium Laboratory Approval Program 

Technical Support Center 

26 West Martin Luther King Drive 

Cincinnati, OH 45268 

Attachments 

E-4 February 2006 
Burden Statement: The public reporting and recordkeeping burden for this collection of 

information is estimated to average 18 hours per response or 72 hours per respondent annually. 

Burden means the total time, effort, or financial resources expended by persons to generate, 

maintain, retain, or disclose or provide information to or for a Federal agency. This includes the 

time needed to review instructions; develop, acquire, install, and utilize technology and systems for 

the purposes of collecting, validating, and verifying information, processing and maintaining 

information, and disclosing and providing information; adjust the existing ways to comply with any 

previously applicable instructions and requirements; train personnel to be able to respond to a 

collection of information; search data sources; complete and review the collection of information; 

and transmit or otherwise disclose the information. An agency may not conduct or sponsor, and a 

person is not required to respond to, a collection of information unless it displays a currently valid 

OMB control number. 

E-5 February 2006 
Appendix F 

Checklist for the Laboratory Quality Assurance Evaluation 

Program for Analysis of Cryptosporidium under the Safe 

Drinking Water Act 
This page intentionally left blank 
Checklist for the Laboratory Quality Assurance Evaluation Program for An

Cryptosporidium under the Safe Drinking Water Act

Part A: Personnel, Facilities, Equipment, and Quality Assurance 

Laboratory Name Date of Evaluation Name and Affiliation of Eva

Part A: Facilities, Equipment, and Quality Assurance 

Item to be Evaluated Classification Yes, No, U

1 Laboratory Equipment and Supplies 1.1 Reagent-grade water testing 

1.1.1 Is reagent water tested monthly for these minimum parameters: conductivity, total 

chlorine residual; and annually for metals-Pb, Cd, Cr, Cu, Ni, Zn? Critical 

1.1.2 Were the results for the above parameters acceptable, total chlorine residual not greater 

than 0.1 mg/L, conductivity not greater than 2 μmhos/cm, and each metal not greater Critical than 0.05 mg/L and collectively not greater than 0.1 mg/L? 

1.1.3 Is reagent water tested monthly for heterotrophic plate count? Critical 1.1.4 Are the results for the heterotrophic plate count acceptable, < 500 CFU/mL? Critical 

1.2 Laboratory pH meter: 1.2.1 Accuracy ± 0.1 units, scale graduations, 0.1 units? Critical 1.2.2 Is a record maintained for pH measurements and calibrations used? Critical 1.2.3 Is pH meter standardized each use period with pH 7, 4 or 10 standard buffers (selection 

 dependant upon desired pH)? Critical

 1.2.4 All pH buffers are dated when received and opened and are discarded before expiration 

date? Critical 

1.3 Balances (top loader or pan balance): 1.3.1 Are balances calibrated monthly using Class S/S-1 weights, or weights traceable to 

 Class S/S-1 weights? Critical

 1.3.2 Is correction data available with S/S-1 weights? Critical 1.3.3 Is preventative maintenance conducted yearly at a minimum? Recommendation 

Note: All section references in [ ] refer to Method 1623 December 2005 

F-1 
Item to be Evaluated Classification Yes, No, U

1.4 Autoclave: 1.4.1 Is unit equipped with a temperature gauge/operational safety valve? Critical 1.4.2 Are date, contents, sterilization time and temperature recorded for each cycle? Critical 1.4.3 Is a maximum registering thermometer or continuous monitoring device used during  each autoclave cycle? Critical 1.4.4 Is automatic timing mechanism checked with stopwatch quarterly? Critical 1.4.5 Are spore strips or ampules used monthly to confirm sterilization? Critical 1.5 Refrigerator/Freezer: 

1.5.1 Is refrigerator able to maintain temperature of 1EC to 5EC? Critical

 1.5.2 Is temperature recorded once daily for days in use? Critical 1.6 Temperature recording device: 

1.6.1 Are calibration of glass/mercury thermometers checked annually (dial thermometers 

quarterly) at the temperature used against a reference NIST thermometer or equivalent? Requirement [Section 8.1.4] 

1.7 Micropipetters: 1.7.1 Have micropipetters been calibrated within the past year? [Section 9.2.1] Requirement 1.8 Centrifuge 

1.8.1 Is a maintenance contract in place, or internal maintenance protocol available? [Section 

9.1] Critical

 1.8.2 Are RPM and RCF calibrated yearly? Critical 1.9 General 1.9.1 Are calibration and maintenance records complete and well organized? [Section 9.1] Recommendation 2 Quality Assurance 2.1 Does the laboratory have a formal QA laboratory plan prepared and ready for examination? 

[Section 9.1] Requirement 

2.2 Are employee resumes present and complete? [Section 9.1] Requirement 2.3 Is a training protocol for new employees present? [Section 9.1] Requirement 2.4 Is the laboratory performing analyst verification of examination monthly and does the lab have 

corrective action procedures in place if criteria are not met? [Section 10.6] Requirement 

2.5 Are employee training records available and up to date? [Section 9.1] Requirement 

Note: All section references in [ ] refer to Method 1623 December 2005 

F-2 
Item to be Evaluated Classification Yes, No, U

 2.5.1 Have technicians/analysts analyzed the required number of samples using Method 

 1622/1623? [Section 22.2] Critical 

2.6 Are all relevant SOPs present and current? Critical 2.7 Are sampling instructions present for clients collecting and/or filtering samples in the field? Critical 2.8 Does the laboratory have criteria for sample acceptance and corrective action procedures? 

[Section 8.1.4] Requirement 

2.9 Are data recording procedures present? Critical 2.9.1 Does the laboratory have an SOP for checking all manual calculations? Critical 2.10 Are corrective action contingencies present? Requirement 

2.10.1 For OPR failures? [Section 9.7.4] Requirement 

2.10.2 For method blank contamination? [9.6.2.2] Requirement 

2.10.3 For positive/negative staining control failures? Critical 

2.11 Does the quality assurance plan specifically address requirements for protozoa analysis under 

the programs for which the laboratory intends to analyze samples? Critical 

2.12 Is a laboratory organization chart or other information available listing staff organization and 

responsibilities? Does it identify the QA manager? Recommendation 

2.12.1 Is the QA manager separate from the lab manager? Recommendation 

2.13 Does the laboratory have a list of preventative maintenance procedures and schedules? [Section 

9.1] Requirement 

2.14 Date range covered for quality control (QC) sample audit? 2.15 When did the laboratory begin processing samples with the Envirochek filter? / / 2.16 When did the laboratory begin processing samples with the Filta-Max filter (if applicable)? / / 2.17 When did the laboratory begin processing samples with the CrypTest filter (if applicable)? / / 2.18 Approximately how many field samples were analyzed using methods 1622/1623 since the lab 

started using Method 1622/1623? Field samples ___ MS_____

2.19 Have acceptable initial precision and recovery analyses been performed for each version of the 

method the laboratory is using? [Section 9.1.2.1.1] Requirement 

2.20 Were method blanks run once per week or per 20 samples during this period? [Section 9.6.1] Requirement 

2.20.1 If the answer to 2.20 is no, then at what frequency where method blanks performed? 

2.20.2 What percentage of method blanks evaluated were without contamination? 

Note: All section references in [ ] refer to Method 1623 December 2005 

F-3 
Item to be Evaluated Classification Yes, No, U

2.20.3 Was an acceptable method blank associated with each field sample examined? [Section 

9.6.2.2] Requirement 

2.20.4 How many method blanks were evaluated? 

2.21 Were ongoing precision and recovery (OPR) samples run once per week or per 20 samples 

during this period? [Section 9.7] Requirement 

2.21.1 If the answer to 2.21 is no, then at what frequency where OPR samples performed? 

2.21.2 What percentage of OPR samples evaluated met the recovery criteria? [Table 3; 

 Section 9.7.3] 

2.21.3 Does the laboratory maintain control charts of OPR results? [Section 9.7.6] Recommendation 

2.21.4 Was an acceptable OPR associated with each field sample examined? [Section 9.7.4.2] Requirement 

2.21.5 How many OPR samples were evaluated? 2.21.6 How many OPR samples were analyzed during the past six months? 2.21.7 What is the mean and relative standard deviation (RSD) of the recoveries of the OPR 

samples analyzed during the past six months? 

Mean_______ RSD________ 

2.22 Were matrix spike (MS) samples analyzed at the method -specified frequency? [Section 9.1.8] Requirement 

2.22.1 If the answer to 2.22 is no, then at what frequency were MS samples analyzed? 2.22.2 How many MS samples were evaluated? 2.22.3 How many MS samples were analyzed during the past six months? 2.22.4 What is the mean and relative standard deviation of the MS samples analyzed during 

 the past six months? 

Mean_______ RSD________ 

2.23 Were OPR and MS samples spiked with 100 - 500 organisms? [Section 9.7] Requirement 

2.23.1 If the answer to 2.23 is no, then at what level were samples spiked? 

2.24 Are the laboratory personnel performing the QC analyses representative of the personnel 

seeking approval under this program? Critical 

2.25 Does the laboratory have records of all QC checks available for inspection? [Section 9.1] Requirement 2.26 Does the laboratory have an adequate record system for tracking samples from collection 

through log-in, analysis, and data reporting? Critical 

2.27 Are results from each sample maintained electronically? 2.28 If data are stored electronically, are files backed up on more than one disk to ensure data are 

not lost in the eventuality of some hardware failure? Critical 

2.29 If data is stored electronically, does the laboratory have an SOP for checking the accuracy of  data entry into an electronic system? Critical 

Note: All section references in [ ] refer to Method 1623 December 2005 

F-4 
Item to be Evaluated Classification Yes, No, U

2.30 Is the laboratory using the December 2005 version of Method 1622/1623 for LT2 samples? [CFR 

40 Part 141.704] Requirement 

3 Data Recording Procedures 3.1 Is shipping information complete, including the time and date of sample receipt, sample condition, 

and noting any discrepancies between samples on the traffic report and samples received? 

 [Section 8.1.3] 

Requirement 

3.2 Do sample numbers on the shipping forms match the sample numbers on the report forms? Requirement 3.3 Are current Method 1622/1623 bench sheets used to record sample processing data? Recommendation 3.4 Are all primary measurements during each step recorded, including all raw data used in 

calculations? [Section 11.0, 12.0, 13.0] Requirement 

3.5 Name of analyst or technician performing the elution is recorded? Critical 3.6 Date and time of elution is recorded? [Section 12.2.6.2.1] Requirement 3.7 Name of analyst or technician performing the concentration is recorded? Critical 3.8 Date and time of concentration is recorded? [Section12.3.3.3.2] Requirement 3.9 Are batch and lot numbers of reagents used in the analysis of the sample recorded? Critical 3.10 Lot number for the IMS kit is recorded? Critical 3.11 Are Method 1622/1623 Cryptosporidium report forms used to record sample examination results? 

[Section 15.2] Requirement 

3.12 Name of examining analyst is recorded? [Section 15.2.6] Requirement 3.13 Date and time of sample examination is recorded? [Section 15.2.4] Requirement 3.14 Are calculations of final concentrations and recoveries complete and correct? Requirement 3.15 Do values recorded on the data sheets match the reported values? Requirement 3.16 Are mistakes on all forms crossed out with a single line, initialed, and dated? Critical 3.17 Are data always recorded in pen? Critical 3.18 Are hardcopy records well organized, complete, and easily accessible? Critical 3.19 Does the laboratory include a disclaimer on the report to the client if method QC requirements 

were not met? Recommendation 

3.20 Is the manually recorded data legible? Critical 3.21 Do records demonstrate each analyst's characterization of 3 oocysts and 3 cysts from positive 

control for each microscopy session? [Section 15.2.1.1] Requirement 

3.22 Data shows that no more than 0.5 mL of pellet was used per IMS? [Section 13.2.4] Requirement 

Note: All section references in [ ] refer to Method 1623 December 2005 

F-5 
Item to be Evaluated Classification Yes, No, U

4 Holding Times 4.1 Samples analyzed according to December 1999 version of Method 1622/1623

 4.1.1 Is time from initiation of sample collection to completion of concentration 72 hours or

 less? Requirement

 4.1.2 Concentrate is held no longer than 24 hours between IMS and staining? Requirement 4.1.3 Are stained slides read and confirmed within 72 hours of staining? Requirement 

4.2 Samples analyzed according to the April 2001, 2003, or December 2005 version of Method 1622/1623

 4.2.1 Is sample elution initiated within 96 hours of sample collection or field filtration? [Section 

8.2.1] Requirement

 4.2.2 Are sample elution, concentration, and purification steps completed in one work day?

 [Section 8.2.2] Requirement

 4.2.3 Are slides stained within 72 hours of application of the purified sample to the slide? 

 [Section 8.2.3] Requirement

 4.2.4 Are stained slides read and confirmed within 7 days of staining? [Section 8.2.4] Requirement 

5 Spike enumeration procedures 5.1 What method does the laboratory currently use to estimate spike doses:(A) flow-sorted spikes, (B) 

well-slide-counted spikes, (C) hemacytometer-counted spikes, or (D) membrane-filter-counted Circle one: A B C D

 spikes 

5.1.1 If flow-sorted spikes are used, on what date did the laboratory begin using flow-sorted 

 spikes? 

 5.1.2 If counted manually, does the laboratory follow Method 1622/1623 procedures for  establishing spike level? [Section 11.3] Requirement 

5.1.3 What were the relative standard deviations of the last four spike enumerations? 

1. 2. 3.

4. 

5.2 Source of oocysts for spikes 5.3 If 50-L samples are analyzed, what positive control procedure does the laboratory follow for OPR 

and MS samples: (A) spike entire 50 L, (B) spike and filter 10 L before filtering 40 L, or (C) filter 40 L before spiking and filtering 10 L. 

Note: All section references in [ ] refer to Method 1623 December 2005 

F-6 
Part B: Sample Processing and Examination 

Date of 

Evaluation 

Laboratory Name Name and Affiliation of Ev

Item to be evaluated Classification Yes, No, NA 

6 Laboratory Facilities and Laboratory Safety 6.1 Are laboratory coats and gloves worn in the laboratory? [Section 5.3] Critical 6.2 No other safety or facility issues were observed? 7 Sample Spiking Technician: 7.1 What method does laboratory currently use to estimate spike doses:(A) flow-sorted spikes, (B) 

well-slide-counted spikes, (C) hemacytometer-counted spikes, or (D) membrane-filter-counted 

spikes 

Circle one: A B C D 

7.2 With what filter type did the laboratory demonstrate their spiking procedure? 7.3 Is the carboy used for negative control randomly selected from carboy stock to check efficacy of 

 cleaning system? Critical 

7.4 If flow-sorted spikes are used, was suspension vial vortexed for 30 seconds or per manufacturer’s 

instructions? [Section 11.4.3] Method Procedure 

7.5 Was the suspension vial adequately rinsed? [Section 11.4.3.1] Method Procedure 7.6 Does the laboratory have an acceptable SOP for sample spiking? Critical 7.7 Other than the issues noted for items 7.2 through 7.6 (if any) was sample spiking demonstrated  successfully? 8 Envirochek (Complete Sections that apply) 8.1 Envirochek Filtration Technician: 8.1.1 Are all components required for sample filtration present and in good condition?  [Section 6.2] Requirement 8.1.2 Is the filter assembly set up correctly? [Figure 3a, pg 63] Method Procedure 8.1.3 Is the pump adequate for needs? [Section 6.3.3] Requirement 

Note: All section references in [ ] refer to Method 1623 December 2005 

F-7 
Item to be evaluated Classification Yes, No, NA 

 8.1.4 Is the appropriate flow rate maintained (approximately 2 L/min)? [Section 12.2.1.2] Method Procedure 8.1.5 Is the volume filtered measured using a flow totalizer or calibrated carboy? [Section 

12.2.4.2] Requirement

 8.1.6 Is the system well maintained and cleaned appropriately following use? Critical

 8.1.7 Is the system able to maintain seal during use with no leaks? Requirement

 8.1.8 Does the laboratory have an acceptable SOP for Envirochek filtration? Critical 8.1.9 Other than the issues noted in items 8.1.1 through 8.1.8, was Envirochek filtration  demonstrated successfully? 8.2 Envirochek capsule filter elution Technician: 8.2.1 Is the elution buffer prepared as per Method 1622/1623? [Section 7.4.1] Method Procedure 8.2.2 Is the wrist-shaker assembly set up correctly? [Section 12.2.6.1.1] Method Procedure 8.2.3 Does the eluting solution cover the membrane? [Section 12.2.6.2.2] Method Procedure 8.2.4 Are the samples shaken at an appropriate speed? [Section 12.2.6.2.3] Method Procedure 8.2.5 Are the samples shaken three times for 5 minutes each time, and each in a different 

orientation? [Section 12.2.6.2] Method Procedure

 8.2.6 Does the laboratory have an acceptable SOP for Envirochek capsule filter elution? Critical 8.2.7 Other than the issues noted for items 8.2.1 through 8.2.7 (if any) was Envirochek filter 

elution demonstrated successfully? 

9 This section is no longer applicable and has been deleted. 10 Filta-Max 10.1 Filta-Max filtration Technician: 

10.1.1 Are all components required for sample filtration present and in good condition? 

 [Section 6.2.3] Requirement 

10.1.2 Is the filter assembly set up correctly? [Fig. 3b, pg. 64] Method Procedure 10.1.3 Is appropriate flow rate maintained of <4 L per minute? [Section 12.3.1.1.3} Method Procedure 10.1.4 Is the volume filtered measured correctly using a flow meter or calibrated carboy? 

 [Section 12.3.1.5.2] Requirement 

10.1.5 Is system well maintained and cleaned appropriately following use? [Section 12.3.4] Requirement 

10.1.6 Is system able to maintain seal during use with no leaks? Requirement 

Note: All section references in [ ] refer to Method 1623 December 2005 

F-8 
Item to be evaluated Classification Yes, No, NA 

10.1.7 Does the laboratory have an acceptable SOP for Filta-Max filtration? Critical 

10.1.8 Does the laboratory indicate on the filter housing the correct direction of flow? [Section 

12.3.1.3] Critical 

10.1.9 Other than the issues noted in items 10.1.1 through 10.1.8 (if any) was Filta-Max 

filtration demonstrated successfully? 

10.2 Filta-Max filter wash station elution Technician: 

10.2.1 Is an automatic or manual wash station used? 10.2.2 Is the filter wash station set up correctly? [Section 12.3.2.1] Requirement 10.2.3 Is PBST used to elute the filter? [Section 7.4.2.4] Method Procedure 10.2.4 Is an appropriate amount of PBST used for each wash? (approx. 600 mL) [Section 

12.3.2.2.1] Method Procedure 

10.2.5 During the first wash, is the plunger moved up and down 20 times? [Section 12.3.2.2.1] Method Procedure 10.2.6 Is the plunger moved up and down gently to avoid generating excess foam? Method Procedure 10.2.7 During the second wash, is the plunger moved up and down 10 times? [Section 

12.3.2.2.2] Method Procedure 

10.2.8 If the automatic washer is used, is the machine operating properly? [Section 12.3.2.1] Requirement

 10.2.9 Is the wash station cleaned adequately between samples? [Section 12.3.4.2] Requirement 

10.2.10 Does the laboratory have an acceptable SOP for Filta-Max elution with the wash 

station? Critical 

10.2.11 Other than the issues noted for items 10.2.2 through 10.2.10 (if any) was elution of the 

Filta-max filter using the wash station demonstrated successfully? 

10.3 Filta-Max filter stomacher elution Technician: 

10.3.1 Is PBST used to elute the filter? [Section 7.4.3.4] Method Procedure 10.3.2 Is an appropriate amount of PBST used for each wash? (approx. 600 mL) [Section 

12.3.2.3] Method Procedure 

10.3.3 Are two washes performed for 5 minutes each? [ Section 12.3.2.3] Method Procedure 10.3.4 Is the stomacher in good condition and operating properly? Requirement 10.3.5 Does the laboratory have an acceptable SOP for Filta-Max elution using a stomacher? Critical 10.3.6 Other than the issues noted for items 10.3.1 through 10.3.5 (if any) was elution of the 

Filta-Max filter using the stomacher demonstrated successfully? 

10.4 Filta-Max filter sample concentration (as an alternative to Section 11) Technician: 

Note: All section references in [ ] refer to Method 1623 December 2005 

F-9 
Item to be evaluated Classification Yes, No, NA 

10.4.1 Is concentrator set up correctly? [Section 12.3.3.2.1 b.] Requirement 10.4.2 Is the force of the vacuum maintained below 30 cm Hg? [note, pg. 43] Method Procedure 10.4.3 Is concentration performed after each of the washes? Method Procedure 10.4.4 Is the concentrate from the first wash added to the 600 mL of eluate from the second

 wash? Method Procedure

 10.4.5 Is the sample concentrated so that some liquid remains above the filter (enough to 

cover the stirbar about half-way)? [Section 12.3.3.2.1] Method Procedure 

10.4.6 Are the stir bar and concentration tube rinsed after each concentration and the liquid 

added to the concentrate? [Section 12.3.3.2.1 c.] Requirement

 10.4.7 Was the filter membrane washed twice? [Section 12.3.3.2.3] Method Procedure 10.4.8 Was 5 mL of PBST used each time? [Section 12.3.3.2.3] Method Procedure 

10.4.9 Is the membrane adequately washed to remove oocysts from filter? Method Procedure 10.4.10 Is the pellet volume determined? [Section 12.3.3.3] Requirement

 10.4.11 Is there a set of standards for comparison of pellet size? Recommendation 10.4.12 Does the laboratory have an acceptable SOP for concentration using the Filta-Max 

concentrator? Critical 

10.4.13 Other than the issues noted in items 10.4.1 through 10.4.12 (if any) was sample 

concentration using the Filta-Max concentrator demonstrated successfully? 

11 Concentration 11.1 Envirochek, CrypTest, and Filta-Max filter sample centrifugation Technician: 

11.1.1 Is the sample centrifuged at 1500 x G using a swinging bucket rotor? [Section 13.2.1] Method Procedure 11.1.2 Are the centrifuge tubes properly balanced prior to centrifugation? Critical 11.1.3 Is the sample centrifuged for 15 minutes? [Section 13.2.1] Method Procedure 11.1.4 Is the centrifuge slowly decelerated at the end without the brake? [Section 13.2.1] Method Procedure 11.1.5 Is the pellet volume determined? [Section 13.2.1] Requirement 11.1.6 Is there a set of standards for comparison of pellet size? Recommendation 11.1.7 Does the laboratory have an acceptable SOP for sample concentration? Critical 

11.1.8 Is residual suspension rinsed from all containers and gloves? Critical 

Note: All section references in [ ] refer to Method 1623 December 2005 

F-10 
Item to be evaluated Classification Yes, No, NA 

11.1.9 Other than the issues noted in items 11.1.1 through 11.1.8 (if any) was sample 

concentration demonstrated successfully? 

12 Reagents, equipment and clean-up 12.1 Source for reagent-grade water: 

12.1.1 Is still or DI unit maintained according to manufacturer's instructions? Critical 12.1.2 Is reagent grade water used to prepare all media and reagents? [Section 7.3] Requirement 

12.2 Centrifuge: 

12.2.1 Does centrifuge have a swinging bucket rotor? [Section 6.8.1] Requirement 12.2.2 Does lab have easily accessible method for determining relative centrifugal force of

 centrifuges? Critical 

12.3 SOP’s for Reagents

 12.3.1 Are SOP’s available for the preparation of all essential chemicals and reagents? Critical 

12.3.2 Are SOP’s posted or easily accessible at the bench? Recommendation 

12.3.3 Are all reagents clearly labeled with date of preparation, technician initials, and 

 expiration date? Critical 

12.4 Clean-up

 12.4.1 Is all glassware and plasticware washed well and stored appropriately between uses? Critical 12.4.2 Is distilled or deionized water used for final rinse? Critical

 12.4.3 Is an SOP available for glassware washing? Critical 

13 Purification and Slide Preparation Technician: 13.1 What IMS kit/manufacturer is used? 13.2 Is the supernatant from the centrifuged sample aspirated no lower than 5 mL above the 

pellet? [Section 13.2.2] Requirement 

13.3 Is the pellet vortexed a sufficient time for resuspension? [Section 13.2.3] Method Procedure 13.4 Does the lab have an appropriate SOP for dividing pellets greater than 0.5 mL into 

subsamples and analyzing? Critical 

13.5 Is no more than 0.5 mL of pellet used per IMS? [Section 13.2.4] Method Procedure 13.6 Is the resuspended pellet volume quantitatively transferred to the Leighton tube (2 rinses)? 

 [Section 13.3.2.1] Method Procedure 

Note: All section references in [ ] refer to Method 1623 December 2005 

F-11 
Item to be evaluated Classification Yes, No, NA 

13.7 Are the IMS beads thoroughly resuspended prior to addition to the Leighton tube? 

 [Section 13.3.2.2] Method Procedure 

13.8 Is the leighton tube rotated at 18 rpm for 1 hour at room temperature? [Section 13.3.2.6] Method Procedure 13.9 Is Leighton tube correctly placed in magnet and rocked through 90 degrees about once per 

 second? [Section 13.3.2.9] Method Procedure 

13.10 Is all the liquid removed when decanting is performed with the magnet up? [Section 

13.3.2.11] Method Procedure 

13.11 Is the sample quantitatively transferred from the Leighton tube to the microcentrifuge tube (2 

rinses)? [Section 13.3.2.13] Method Procedure 

13.12 Are extra rinses to minimize debris performed appropriately when needed? Does the 

laboratory rinse A) IMS beads in the Leighton tube prior to transfer, B) Leighton tube, not IMS beads, prior to transfer, C) IMS beads in microcentrifuge tube prior to dissociation? 

Method Procedure 

Circle one: A B 

13.13 Is standard NaOH (5 FL, 1N) and standard HCl (50 FL, 0.1N) used? [See note on pg 49] Requirement 13.14 Is sample vortexed vigorously for 50 seconds immediately after the addition of acid and 30 

seconds after the sample has set for 10 minutes at room temperature? [Section 13.3.3] Method Procedure 

13.15 Is a second dissociation performed? [Section 13.3.3.10] Method Procedure 13.16 When the second dissociation is performed, does the laboratory: (A) use a second slide, or 

(B) add the additional volume to the original slide? Circle one: A B 

13.17 Are the slides clearly labeled so they can be associated with the correct sample? [Section 

13.3.3.7] Requirement 

13.18 What type of slides are used? 13.19 Is slide dried at a) room temperature or b) 35 to 42 C? [Section 13.3.3.12] Circle one: A B 13.20 If the slide is warmed, is incubator or slide tray calibrated and labeled? Critical 13.21 Does the laboratory have an acceptable SOP for sample purification? Critical 13.22 Other than the issues noted in items 13.1 through 13.21 (if any) were sample purification and 

slide preparation performed successfully? 

14 Sample staining Technician: 14.1 What staining kit/manufacturer is used? [Section 14.2] 14.2 Is FITC stain applied according to manufacturer’s directions? [Section 14.2] Method Procedure 14.3 Are positive and negative staining controls performed? [Section 14.1] Requirement 

Note: All section references in [ ] refer to Method 1623 December 2005 

F-12 
Item to be evaluated Classification Yes, No, NA 

14.4 Are the slides incubated in a humid chamber in the dark at room temperature for 

approximately 30 minutes or per manufacturer’s directions? [Section 14.3] Method Procedure 

14.5 Are the labeling reagents rinsed away properly after incubation, without disturbing the 

sample? [Section 14.5] 

Method Procedure 

14.6 Was the working DAPI stain prepared the day it was used? [Section 7.7.2] Method Procedure 14.7 Is stock DAPI stored at 1 to 10oC in the dark? [Section 7.7.2] Method Procedure 14.8 Is the DAPI stain applied properly and allowed to stand for a minimum of 1 minute? 

[Section 14.6] 

Method Procedure 

14.9 Is the DAPI stain rinsed away properly without disturbing the sample? [Section 14.7] Method Procedure 14.10 Is the mounting media applied properly? Method Procedure 

14.10.1 What type of mounting media is used? 

14.10.2 Are all the edges of the cover slip sealed well with clear fingernail polish, unless 

Elvenol is used? [Section 14.9] Method Procedure 

14.11 Are the finished slides stored in a humid chamber in the dark at 1 to 10oC (humid chamber 

not required for Evenol)? [Section 14.10] Method Procedure 

14.12 Does the laboratory have an acceptable SOP for sample staining? Critical 14.13 Other than the issues noted in items 14.2 through 14.13 (if any) was sample staining

 demonstrated successfully? 

15 Microscope and Examination 15.1 Is microscope equipped with appropriate excitation and band pass filters for examining FITC 

labeled specimens? (Exciter filter - 450-490 nm, dichroic beam-splitting mirror - 510 nm, 

barrier or suppression filter: 515-520 nm)? [Section 6.9.2] 

Requirement 

15.2 Is microscope is equipped with appropriate excitation and band pass filters for examining 

DAPI labeled specimens? (Exciter filter - 340-380 nm, dichroic beam-splitting mirror - 400 nm, barrier or suppression filter - 420 nm) [Section 6.9.3] 

Requirement 

15.3 Does the microscope have HMO or DIC, objectives? [Section 6.9.1] Requirement 15.4 Is microscope operation easily changed from epifluorescence to DIC/HMO? Recommendation 15.5 Does the microscope have a 20 X scanning objective? [Section 6.9.1] Requirement 15.6 Does the microscope have a 100 X oil immersion objective? [Section 6.9.1] Requirement 15.7 Is the microscope equipped with an ocular micrometer? [Section 6.9.1] Requirement 15.8 Is a stage micrometer available to laboratory? [Section 10.3.5] Requirement 

Note: All section references in [ ] refer to Method 1623 December 2005 

F-13 
Item to be evaluated Classification Yes, No, NA 

15.9 Is a calibration table for each objective located close to the microscope(s)? [Section 10.3.5] Requirement 15.10 Has the mercury bulb been used less than the maximum hours recommended by the 

manufacturer? [Section 10.3.2.11] Recommendation 

15.11 Does the positive control contain Cryptosporidium oocysts at the appropriate fluorescence 

intensity for both FITC and DAPI? [Section 15.2.1.3] Requirement 

15.12 Does the laboratory have an acceptable SOP for sample examination? Requirement 15.13 Other than the issues noted for items 15.1 through 15.13 (if any) were other microscope or 

examination issues acceptable? 

Note: All section references in [ ] refer to Method 1623 December 2005 

F-14 
16 Evaluation of sample processing

 Method Step Name Position Demonstrated Recorded data as 

(filtration, elution, concentration, purification Technique sample was 

or staining) Successfully analyzed 

yes/no yes/no 

Note: All section references in [ ] refer to Method 1623 December 2005 February 2006 

F-15 
17 Was analyst microscope examination acceptable? (yes/no) 

Classification Requirement Requirement [Section 10.3.4] Requirement [Section 10.3.4.2] Requirement [Section 10.3.6] Requirement [Section 15.2.1.1] Requirement [Section 10.6] Requirement [Section 15.2.1.1] 

Name Position Adjust Interpupillary Distance Focus both eyepieces Establish Kohler Illumination Examine and Record Characteristics of Three Oocysts Crypto Count Measurement with 1000X objective 

Comments: Note: All section references in [ ] refer to Method 1623 December 2005 

F-16 
Appendix G 

Method 1622/1623 Bench Sheet 
This page intentionally left blank 
Laboratory Name: Laboratory ID: 

Method 1622/1623 Bench Sheet 

Sample Identification Information 

* Lab Sample ID: Person Receiving Sample: 

* PWS ID: *Sample collection date and time: 

* Facility ID: Turbidity (NTU): 

* Sample Collection Point ID: Temperature, date and time @ sample receipt: 

* Initial precision and recovery (IPR) Method blank Field (monitoring) sample Sample type (circle one): 

Ongoing precision and recovery (OPR) Matrix spike (MS) Proficiency testing (PT)

Sample Spiking Information (for IPR, OPR, MS, and PT samples only) 

Crypto Giardia * Estimated number spiked: Spiking time: 

* Sample volume spiked (L): Spiking date:

Spiking suspension ID: Spiking analyst: 

Sample Filtration 

Filter type (circle one): Envirochek Envirochek HV Filta-Max CrypTest Other (specify)

Did filter clog? (circle one): Yes No Filtration time: Filter lot number: 

* Number of filter(s) used?: Filtration date: 

* Volume filtered (L) to nearest 1/4L: Filtration analyst: 

Sample Elution (must be initiated within 96 hours of sample collection/filtration) 

Elution procedure (circle one): Wrist shaker Filta-Max wash station Stomacher Backflush/sonication

Type of Elution buffer: Elution time:

Elution buffer lot number: Elution date:

Elution buffer expiration date: Elution analyst: 

Concentration, IMS, and Slide Preparation 

(must be completed on same working day that samples are eluted)

Procedure (circle one): Centrifugation Filta-Max concentrator Other (specify)

* Pellet volume after concentration (mL) to nearest 0.1mL: Concentration analyst: 

* Total volume of resuspended concentrate (mL): IMS analyst: 

* Volume of resuspended concentrate transferred to IMS (mL): Slide preparation time:

Number of subsamples processed through entire method: Slide preparation date:

IMS lot number: Slide preparation analyst:

IMS system (circle one): Dynal GC-Combo Dynal anti-Crypto Other (specify)

Slides (circle one): Meridian Dynal Waterborne Other (specify)

Slide Staining (must be completed within 72 hours of application of sample to the slide) 

Detection kit (circle one): BTF EasyStain Merifluor Crypt-a-glo Giardi-a-glo Aqua-glo Other (specify)

Detection kit lot number: Staining date and time:

Number of slides for this sample: Staining analyst: 

* Examination Results as Total FA number from all slides for sample Cryptosporidium: Giardia: 

Comments: 

* = Data entered into LT2/Stage2 Data Collection and Tracking System G-1 February 2006 
Appendix H 

Method 1622/1623 Slide Examination Form 
This page intentionally left blank 
Laboratory name: Laboratory ID: 

Method 1622/1623 Slide Examination Form 

Sample ID: Analyst: 

Examination/verification completion date and time : Slide number: 

(must be completed within 168 hours (7 days) of staining) Total number of slides for this sample: 

Positive staining control acceptable 9 YES 9 NO Negative staining control acceptable 9 YES 9 NO 

FITC, Size, Shape, DIC and DAPI Characteristics of 3 Oocysts Recorded 9 YES 9 NO 

Cryptosporidium Results 

Object 

located 

by FA 

No. 

Shape 

(oval 

or 

round) 

Size 

L x W 

(μm) 

DAPI ­ DAPI + D.I.C. 

Light blue internal 

staining, no distinct 

nuclei, green rim 

(A) 

Intense 

blue 

internal 

staining 

(B) 

Number of 

nuclei stained 

sky blue 

(C) 

Empty 

oocysts 

(D) 

Oocysts with 

amorphous 

structure 

(E) 

Oocysts with internal 

structure (F) 

Number of sporozoites 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

Total FA number from this slide: D.I.C. - Total number of empty oocysts (D): 

DAPI -: Total number (A): D.I.C. - Total number of oocysts with amorphous structure (E): 

DAPI +: Total number (B): D.I.C. - Total number of oocysts with internal structure (F): 

DAPI +: Total number (C): Total count DAPI + (C) that show structure by D.I.C. (F): 

Giardia Results 

DAPI ­ DAPI + D.I.C. 

Object 

located 

by FA 

No. 

Shape 

(oval 

or 

round) 

Size 

L x W 

(μm) 

Light blue internal 

staining, no distinct 

nuclei, green rim 

(A) 

Intense 

blue 

internal 

staining 

(B) 

Number of 

nuclei 

stained sky 

blue 

(C) 

Empty 

cysts 

(D) 

Cysts with 

amorphous 

structure 

(E) 

Cysts with internal structure (F) 

Number 

of nuclei 

Median 

body Axonemes 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

Total FA number from this slide: D.I.C.: Total number of empty cysts (D): 

DAPI-: Total number (A): D.I.C.: Total number of cysts with amorphous structure (E): 

DAPI+: Total number (B): D.I.C.: Total number of cysts with one internal structure (F): 

DAPI+: Total number (C): D.I.C.: Total number of cysts with >one internal structure (F): 

Total number DAPI + (C) that show structure by D.I.C. (F): 

H-1 February 2006