EPA Microbiological Alternate Test 

Procedure (ATP) Protocol for 

Drinking Water, Ambient Water, and 

Wastewater Monitoring Methods 

Guidance 

April 2004 
U.S. Environmental Protection Agency


Office of Water


Engineering and Analysis Division


1200 Pennsylvania Avenue, NW (4303T)


Washington, DC 20460


EPA 821-B-03-004


July 2003

ACKNOWLEDGMENTS 

This protocol was prepared under the direction of the U.S. Environmental Protection Agency’s (EPA’s) 

Office of Water. This document was prepared by the DynCorp Biology Studies Group. 

DISCLAIMER 

The Engineering and Analysis Division, of the Office of Science and Technology, has reviewed and 

approved this report for publication. The Office of Science and Technology directed, managed, and 

reviewed the work of DynCorp in preparing this report. 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 company names, trade names, or commercial products in 

this protocol does not constitute endorsement or recommendation for use. 

Questions concerning this report should be addressed to: 

Robin K. Oshiro

Engineering and Analysis Division (4303T) 

U.S. EPA Office of Water, Office of Science and Technology

1200 Pennsylvania Avenue, NW

Washington, DC 20460

oshiro.robin@epa.gov

202-566-1075

202-566-1053 (facsimile)
FOREWORD 

This document describes a process for seeking EPA approval of microbiological alternate test procedures 

(ATPs) or new methods for use in monitoring drinking water, ambient water, and wastewater. This 

document serves as a supplement to the ATP guidelines at 40 CFR 136.4, 136.5, and 141.27. 

With this guidance document, EPA has revised and combined the three existing microbiological ATP 

protocols to make the ATP process more transparent to applicants while maintaining the same level of 

data quality. The guidelines in Quantitative Membrane Filter Methods (Reference 10.16), 

Presence/Absence Liquid Culture Methods for Finished Water (Reference 10.17), and Presence/Absence 

Membrane Filter Methods for Finished Waters (Reference 10.15) have been incorporated into this single 

protocol, which is applicable to a wider range of analytes and techniques. 

This revised ATP protocol describes a process for conducting side-by-side method comparisons and for 

conducting quality control (QC) acceptance criteria-based method studies for EPA-approved reference 

methods with QC acceptance criteria. Additionally, in some cases the revised protocol provides 

applicants an opportunity to demonstrate comparability by meeting QC acceptance criteria associated 

with the EPA-approved reference methods for different combinations of analyte and determinative 

technique. 

Under EPA’s ATP program, any person may apply for approval of the use of an ATP or new method to 

test for a regulated analyte. EPA anticipates that the standardized procedures described herein should 

generally expedite the approval of ATPs, encourage the development of innovative technologies, and 

enhance the overall utility of the EPA-approved methods for compliance monitoring under the National 

Pollution Discharge Elimination System (NPDES) permit program and national primary drinking water 

regulations (NPDWRs). 

This document is not a legal instrument and does not establish or affect legal obligations under Federal 

regulations. EPA reserves the right to change this protocol without prior notice. 

Questions or comments regarding this document should be directed to: 

Robin K. Oshiro

Engineering and Analysis Division (4303T) 

U.S. EPA Office of Water, Office of Science and Technology

1200 Pennsylvania Avenue, NW

Washington, DC 20460

oshiro.robin@epa.gov

202-566-1075

202-566-1053 (facsimile)
EPA Microbiological Alternate Test Procedure (ATP) Protocol 

TABLE OF CONTENTS 

Section 1.0 Introduction .......................................................... 1-1

1.1 Background and Objectives .............................................. 1-1

1.2 Types of Applications .................................................. 1-3

1.2.1 Limited Use .................................................... 1-3

1.2.2 Nationwide Use ................................................. 1-3

1.3 Types of Studies ....................................................... 1-4

1.4 Scope of Alternate Test Procedures ........................................ 1-4

1.4.1 EPA-Approved Reference Methods ................................. 1-4

1.4.2 Modifications to Sample Preparation Techniques ...................... 1-5

Section 2.0 Application ........................................................... 2-1

2.1 Submission Addresses and Approval Authority .............................. 2-1

2.2 Application Information ................................................. 2-1

2.3 Reason for ATP ....................................................... 2-2

2.4 Standard EPA Method Format ............................................ 2-3

2.5 Method Comparison Table .............................................. 2-3

2.6 Method Development Information ......................................... 2-3

2.7 Study Plan ........................................................... 2-3

2.8 Study Report ......................................................... 2-4

2.9 Proprietary Information in Applications .................................... 2-4

Section 3.0 Method Format ........................................................ 3-1

3.1 Scope and Application .................................................. 3-1

3.2 Summary of Method ................................................... 3-1

3.3 Method Definitions .................................................... 3-1

3.4 Interferences .......................................................... 3-1

3.5 Safety ............................................................... 3-1

3.6 Equipment and Supplies ................................................ 3-2

3.7 Reagents and Standards ................................................. 3-2

3.8 Sample Collection, Preservation, and Storage ................................ 3-2

3.9 Quality Control ....................................................... 3-2

3.10 Calibration and Standardization ........................................... 3-2

3.11 Procedure ............................................................ 3-2

3.12 Data Analysis and Calculations ........................................... 3-2

3.13 Method Performance ................................................... 3-3

3.14 Pollution Prevention ................................................... 3-3

3.15 Waste Management .................................................... 3-3

3.16 References ........................................................... 3-3

3.17 Tables, Diagrams, Flowcharts, and Validation Data ........................... 3-3

Section 4.0 Study Plan ........................................................... 4-1

4.1 Background .......................................................... 4-1

4.2 Objectives ........................................................... 4-1

4.3 Study Design ......................................................... 4-1

4.4 Coordination ......................................................... 4-2

4.4.1 Study Management .............................................. 4-2

4.4.2 Technical Approach ............................................. 4-2

4.5 Data Reporting ........................................................ 4-2

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

Section 5.0 Quality Assurance/Quality Control ........................................ 5-1

5.1 Quality Assurance ..................................................... 5-1

5.2 Quality Control ....................................................... 5-1

5.2.1 Analyst Counting Variability ...................................... 5-3

5.2.2 Autoclave Sterilization Verification ................................ 5-3

5.2.3 Dilution/Rinse Water Blanks ...................................... 5-3

5.2.4 Incubator/Waterbath Temperatures .................................. 5-3

5.2.5 Initial Demonstration of Capability ................................. 5-3

5.2.6 Initial Precision and Recovery ..................................... 5-4

5.2.7 Matrix Spike and Matrix Spike Duplicate Samples ..................... 5-4

5.2.8 Media Sterility Checks ........................................... 5-4

5.2.9 Method Blank .................................................. 5-4

5.2.10 Ongoing Demonstration of Capability (ODC) Samples .................. 5-5

5.2.11 Ongoing Precision and Recovery (OPR) Samples ...................... 5-5

5.2.12 Positive/Negative Controls ........................................ 5-5

5.2.13 Preparation Blanks (PB) .......................................... 5-6

5.2.14 Refrigerator/Freezer Temperatures .................................. 5-6

5.2.15 Sample Processing Equipment Sterility Checks ........................ 5-6

Section 6.0 Study Design ......................................................... 6-1

6.1 Side-by-Side Comparison Studies ......................................... 6-1

6.1.1 Number of Laboratories .......................................... 6-1

6.1.2 Number of Samples .............................................. 6-1

6.1.3 Verification of Results ........................................... 6-3

6.2 QC Acceptance Criteria-Based Comparison Studies ........................... 6-4

6.2.1 Number of laboratories ........................................... 6-4

6.2.2 Number of Matrices ............................................. 6-4

6.2.3 Number of Replicates per Matrix ................................... 6-4

Section 7.0 Sample Preparation and Analysis ......................................... 7-1

7.1 Collection of Samples for Analysis ........................................ 7-1

7.1.1 Source Water Characterization ..................................... 7-1

7.2 Sample Spiking and “Stressing” Procedures for Bacteriological Methods .......... 7-1

7.2.1 Drinking Water: Spiking and Chlorine-Stressing ....................... 7-2

7.2.2 Preparation of Enumerated Spiking Suspension ........................ 7-4

7.2.3 Log Phase Growth Curve ......................................... 7-5

7.2.4 Commercially Available Enumerated Spikes .......................... 7-5

7.3 Spiking Procedures for Virus Methods ..................................... 7-6

7.3.1 Cell Monolayer Propagation ....................................... 7-6

7.3.2 Propagation of Virus Stock Suspension .............................. 7-6

7.3.3 Titering of the Virus Stock Suspension .............................. 7-6

7.4 Spiking Procedures for Cryptosporidium and Giardia ......................... 7-6

7.5 Analysis of Samples .................................................... 7-7

7.5.1 Side-by-Side Comparison Studies .................................. 7-7

7.5.2 QC Acceptance Criteria-Based Comparison Studies .................... 7-7

7.6 Verification of Results .................................................. 7-7

7.6.1 Verification of Results from Bacteriological Methods ................... 7-8

7.6.2 Verification of Results from Virus Methods ........................... 7-8

7.6.3 Verification of Results from Cryptosporidium and Giardia Methods ....... 7-8

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

Section 8.0 Review of Study Results ................................................ 8-1

8.1 Assessment of Compliance with Approved Study Plan ......................... 8-1

8.2 Data Review .......................................................... 8-1

8.3 Data Validation ....................................................... 8-1

8.4 Development of Descriptive Statistics ...................................... 8-1

8.4.1 Mean Recovery ................................................. 8-1

8.4.2 Precision ...................................................... 8-2

8.4.3 False Positive Rates, False Negative Rates, Sensitivity, and Specificity ..... 8-2

8.5 Statistical Assessment of Method Comparability ............................. 8-3

8.5.1 Presence / Absence Methods ...................................... 8-3

8.5.2 Quantitative Methods ............................................ 8-5

8.5.3 QC Acceptance Criteria-Based Comparison Studies ................... 8-10

8.6 Method Recommendation and Approval ................................... 8-12

Section 9.0 Study Report ......................................................... 9-1

9.1 Background .......................................................... 9-1

9.2 Study Objectives and Design ............................................. 9-2

9.3 Study Implementation .................................................. 9-2

9.4 Data Reporting and Validation ........................................... 9-2

9.5 Results .............................................................. 9-2

9.6 Data Analysis and Discussion ............................................ 9-3

9.7 Conclusions .......................................................... 9-3

9.8 Appendix A - Method .................................................. 9-3

9.9 Appendix B - Study Plan ................................................ 9-3

9.10 Appendix C - Supporting Data ........................................... 9-3

9.10.1 Raw Data ...................................................... 9-3

9.10.2 Electronic Data Reporting ......................................... 9-4

9.10.3 Example Calculations ............................................ 9-4

9.11 Appendix D - Supporting References ...................................... 9-4

Section 10.0 References .......................................................... 10-1

LIST OF APPENDICES 

Appendix A .................................................................. Glossary 

Appendix B ....................................................... ATP Application Form 

Appendix C ................................................... Application Inventory Form 

Appendix D ....................................... Data Elements and Example Bench Sheets 

Appendix E .................................... EPA Headquarters and Regional ATP Contacts 

iii 

EPA Microbiological Alternate Test Procedure (ATP) Protocol 

SECTION 1.0 INTRODUCTION 

1.1 Background and Objectives 

In accordance with the Clean Water Act (CWA) and Safe Drinking Water Act (SDWA), the U.S. 

Environmental Protection Agency (EPA) promulgates guidelines establishing test procedures (analytical 

methods) for data gathering and compliance monitoring under National Pollution Discharge Elimination 

System (NPDES) permits and national primary drinking water regulations (NPDWRs). The approved test 

procedures can be found in the Code of Federal Regulations (CFR) at 40 CFR Part 136 for wastewater 

and ambient water and 40 CFR Part 141 for drinking water. In addition, EPA’s regulations at 40 CFR 

136.4, 136.5, and 40 CFR 141.27, allow entities to apply for Agency permission to use an alternate test 

procedure (ATP) in place of an EPA-approved reference method. Figure 1.1 below summarizes the ATP 

or new method review process within EPA. These regulations are the basis for the Agency’s alternate test 

procedure (ATP) program for water methods that is administered by the Office of Water, Office of 

Science and Technology, Director of Analytical Methods. 

An ATP is a modification of an EPA-approved reference method or a procedure that uses the same 

determinative technique (i.e., the physical and/or chemical process used to determine the identity and 

concentration of an analyte) and measures the same analyte(s) of interest as the EPA-approved reference 

method. The use of a different determinative technique to measure the same analyte(s) of interest as an 

EPA-approved reference method is considered a new method. 

Under the ATP program, an organization or individual may apply for approval of an ATP or new method 

to be used as an alternate to an EPA-approved reference method. The applicant is generally responsible 

for characterizing method performance of its proposed alternate test procedure prior to submission to the 

ATP program. EPA can provide assistance to applicants in the development of a study plan to 

demonstrate comparability with the EPA-approved reference method. Figure 1.1 summarizes the ATP or 

new method review process within EPA. The Agency reviews the ATP package, approves or disapproves 

the application, and, for nationwide applications, will generally propose to include successful ATPs in the 

CFR (unless the ATP is for limited use or constitutes a minor modification-- See Appendix A). 

Figure 1-1. Summary of the ATP or New Method Review Process LU W W & AW 

ATP 

application 

Apply through 

State Director 

RA provides 

letter of 

approval or 

disapproval 

NW 

DW , W W , & AW 

R egional 

review Forward Forward to OW for review 

OW 

review 

OW 

recom m ends 

approval 

or 

disapproval 

Acronym s: 

AW = Am bient W ater 

DW = Drinking W ater 

LU = Lim ited-use 

NW = Nationwide-use 

OW = Office of W ater 

RA = Regional 

Adm inistrator 

WW = Wastewater 

O W recommendation 

Optional 

EPA 

Adm inistrator 

approves 

through 

rulem aking 

1-1
EPA Microbiological Alternate Test Procedure (ATP) Protocol 

To make the ATP program more accessible to applicants while maintaining data quality, EPA has revised 

and combined the three microbiological ATP protocols into this comprehensive protocol. The guidelines 

in Quantitative Membrane Filter Methods (Reference 10.16), Presence/Absence Liquid Culture Methods 

for Finished Water (Reference 10.17), and Presence/Absence Membrane Filter Methods for Finished 

Waters (Reference 10.15) have been incorporated into a single protocol applicable to a wider range of 

analytes and techniques. Additionally, the revised protocol illustrates how applicants can demonstrate 

comparability by meeting quality control (QC) acceptance criteria associated with EPA-approved 

reference methods for which those criteria have been developed. 

The ATP program provides laboratories and regulated facilities with an opportunity to enhance 

compliance monitoring and encourages the use of innovative technologies. Approval for an ATP or new 

method may be sought when, for example, the alternate procedure reduces analytical costs, overcomes 

matrix interferences problems, improves laboratory productivity, or reduces the amount of hazardous 

materials used and/or produced in the laboratory. 

Any person or organization may apply to gain approval for the use of an ATP or new method for 

determination of a specific constituent that is regulated under the NPDES permit program or the 

NPDWRs. The ATP applicants generally may demonstrate comparability of its proposed ATP or new 

method with the EPA-approved reference method using the procedures described in this protocol. Other 

possible method comparison procedures include those provided by organizations such as ASTM 

(Reference 10.5), AOAC-International (Reference 10.1), and ISO (Reference 10.8). 

1.2 Types of Applications 

The types of applications submitted may depend on the intended use of the ATP. Methods intended for 

use in demonstrating compliance with the NPDES permit program (wastewater or ambient water ATPs) 

may be submitted for approval for limited-use (single laboratory) or for nationwide-use (all laboratories). 

Because only the Administrator has the authority to approve an alternate analytical technique for SWDA 

purposes, EPA will generally consider proposed methods intended for use in demonstrating compliance 

with NPDWRs (drinking water ATPs) that are submitted for approval for nationwide-use only. 

1.2.1 Limited Use 

The primary intent of the limited-use ATP is to allow use of an ATP or new method by a single 

laboratory. Limited-use ATPs can be applied to one or more matrix types, excluding drinking water 

matrices; limited-use applications generally will not apply to Office of Ground Water and Drinking Water 

(OGWDW) ATP applications (Reference 10.18). If a method developer intends to apply the method to 

more than one matrix type, method studies should be conducted on each matrix type. Generally, nine 

different wastewater types should be analyzed to demonstrate the ATP or new method will be applicable 

to most other matrix types. If method modifications are within the specified flexibility of the EPAapproved reference method and all QC acceptance criteria are met, it generally will not be necessary to 

submit the modification to the ATP program. 

1.2.2 Nationwide Use 

The primary intent of a nationwide-use ATP is to allow use of an ATP or new method by all regulated 

entities and laboratories for one or more matrix types including drinking water. Nationwide-use approval 

allows vendors to establish that new devices and reagents produce results that are acceptable for 

compliance monitoring purposes, and allows environmental laboratories across the United States to apply 

new technologies or modified techniques throughout their chain of laboratories to one or more matrix 

types. If a method developer intends to apply the method to more than one matrix type, method studies 

should be conducted on each matrix type. Generally, nine different wastewater types should be analyzed 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

to demonstrate the ATP or new method will be applicable to most other wastewaters. If method 

modifications are within the specified flexibility of the EPA-approved reference method and all QC 

acceptance criteria are met, it generally will not be necessary to submit the modification to the ATP 

program. 

1.3 Types of Studies 

The type of study most useful in seeking approval of an ATP or new method generally depends on 

whether or not the EPA-approved reference methods contain QC acceptance criteria. There are two basic 

types of studies described in this protocol: 

• Side-by-Side Method Comparison Study. A side-by-side method comparison study generally 

consists of parallel testing of an ATP or new method along side an EPA-approved reference method 

to determine whether the performance of the new or modified method is acceptable compared to the 

reference method. 

• QC Acceptance Criteria-Based Method Comparison Study. For EPA-approved reference 

methods that contain (or are supplemented with) QC acceptance criteria for most combinations of 

analyte(s) and determinative technique(s), the goal of the study is for the applicant to demonstrate that 

its ATP or new method is able to meet the QC acceptance criteria of the EPA-approved reference 

method (or other EPA-specified document) for the applicable combination of analyte and 

determinative technique through a QC acceptance criteria-based comparison study. 

Specific guidelines for the studies can be found in Section 5.0: Quality Control, Section 6.0: Study 

Design, Section 7.0: Sample Preparation and Analysis, and Section 9.0: Review of Study Results. 

1.4 Scope of Alternate Test Procedures 

This protocol for demonstration of comparability, submission, and approval of an ATP or new method 

offers flexibility to modify EPA-approved reference methods. Generally, an applicant should 

demonstrate and document that the modified method produces results better than or equal to those 

produced by an appropriate EPA-approved reference method for the applicable combination of analyte 

and determinative technique. 

1.4.1 EPA-Approved Reference Methods 

The ATP process is based on comparing the performance of an ATP or new method to an EPA-approved 

reference method through a side-by-side comparison study or a QC acceptance criteria-based comparison 

study. Method comparability is demonstrated when results produced by an ATP meet or exceed the 

performance criteria associated with the EPA-approved reference method. Table 1-1 below lists the EPAapproved reference methods for the analytes covered by this protocol. This table will be updated as 

necessary as additional pathogens are added to the list or advances in technology merit a change in the 

EPA-approved reference method. When performing a study, the applicant should use the reference 

method that uses the same determinative technique (e.g., MF, MPN) as the ATP for the analyte(s) of 

interest. If the applicant is validating a new method, which generally will use a determinative technique 

that is not currently approved for use with the analyte(s) of interest, then the applicant should consult 

EPA prior to commencing the study to determine which is the most appropriate reference method. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

Table 1-1. EPA-Approved Reference Methods 

Analyte Method Format1 EPA-Approved Reference 

Method2 40 CFR Citation

Total coliforms 

MPN SM 9221B 136.3, 141.21, 

141.74 MF SM 9222B 

Presence/Absence SM 9221D 141.21 

Fecal coliforms 

MPN SM 9221E 136.3, 141.21,

141.74, 503.8(b) MF SM 9222D 

E. coli 

MPN SM 9221F 136.3, 141.21, 

141.74 MF SM 9222G, SM 9213D 

HPC Pour Plate SM 9215B 141.74 

Fecal streptococcus 

MPN SM 9230B 

136.3 

MF SM 9230C 

Enterococcus 

MPN SM 9230B 

136.3 

MF SM 9230C 

Salmonella MPN Kenner and Clark 136.3, 503.8(b) 

Enteric virus Plaque Assay EPA Document 3 503.8(b) 

Helminth ova Microscopy EPA Document 4 503.8(b) 

Aeromonas MF USEPA Method 1605 141.40 

Coliphage 

Plaque Assay USEPA Method 1601 

141.403 

Two-Step Enrichment USEPA Method 1602 

Cryptosporidium Filtration/IMS/FA USEPA Method 1622/1623 

136.3, 141.74 

Giardia Filtration/IMS/FA USEPA Method 1623 

1

MPN = most probable number, MF = membrane filtration, IMS/FA = immunomagnetic separation/fluorescent

antibody

2

"SM” refers to Standard Methods for the Examination of Water and Wastewater. For the edition(s) approved for a

specific method, consult the CFR sections referenced in the column headed “40 CFR Citation.” (References 10.2,

10.3, and 10.4)

3

 See References 10.12 and 10.13

4

 See References 10.12 and 10.19

1.4.2 Modifications to Sample Preparation Techniques 

A sample preparation technique is any technique in the analytical process conducted at the laboratory that 

precedes the determinative technique (i.e., the physical and/or chemical process by which measurement of 

the identity and concentration of an analyte is made). Sample preparation techniques include the 

procedures, equipment, reagents, etc., that are used in the preparation and cleanup of a sample for 

analysis. Laboratories generally may modify sample preparation techniques, provided the modification is 

not explicitly prohibited in the EPA-approved reference method that is being modified and provided the 

modification can be demonstrated to produce results equal or superior to results produced by an EPAapproved reference method for each combination of analyte and determinative technique. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

SECTION 2.0 APPLICATION 

ATP applications should be submitted in triplicate to EPA to facilitate the review process. The 

application consists of a completed ATP application form (a sample application form is provided in 

Appendix D) with any attachments. Electronic submissions are also generally acceptable and often may 

accelerate the review process. 

2.1 Submission Addresses and Approval Authority 

A summary of ATP submission information and approval authorities is provided in Table 2-1. 

Table 2-1. Submission of Alternate Test Procedure Applications 

Level of Use Applicant Submit Application To1 Approval Authority 

EPA Regional laboratories 

EPA Regional 

Administrator (Regional 

ATP Coordinator)2 

Limited Use 

for Wastewater 

or Ambient Water 

States, commercial 

laboratories, individual 

dischargers, or permittees 

in States that do not have 

the authority to administer 

Clean Water Act and Safe 

Drinking Water Act 

monitoring programs 

States, commercial 

laboratories, individual 

dischargers, or permittees 

in States that have the 

authority to administer 

Clean Water Act and Safe 

Drinking Water Act 

monitoring programs 

EPA Regional 

Administrator (Regional 

ATP Coordinator)2 

Director of State Agency 

issuing the NPDES permit2 

EPA Regional 

Administrator 

Nationwide Use for 

Drinking Water, 

Wastewater, Ambient 

Water 

All applicants 

Director, Analytical 

Methods, Attn: ATP 

Program Coordinator, EPA 

Headquarters 

EPA Administrator 

1 See Appendix E for EPA addresses. 2 The Regional Administrator or the Director of State Agency issuing the NPDES permit may choose to forward 

limited-use applications to the Director of Analytical Methods, Attn: ATP Program Coordinator for an approval 

recommendation. Generally, the Regional Administrator or the Director of State Agency issuing the NPDES 

permit will forward a copy of the approval to the Director of Analytical Methods, Attn: ATP Program Coordinator. 

Generally, upon receipt, the application will be assigned an identification number, and a confirmation 

letter referencing this identification number will be sent to the applicant. The applicant should use the 

identification number in all future communications concerning the application. 

2.2 Application Information 

The following information should be provided on the ATP application form (Appendix B): 

• Name, mailing address, phone number, and email address of the applicant 

• Date of submission of the application 

• Method number, title, and revision date of the ATP or new method submitted for review 

• The analyte(s) included in the ATP or new method submitted for review 

2-1
EPA Microbiological Alternate Test Procedure (ATP) Protocol 

• The matrix or matrices to which the ATP applies 

• EPA-approved reference method used for demonstration of comparability 

• Type of application (i.e., wastewater, drinking water, ambient water, point source categories regulated 

at 40 CFR Parts 400-499) 

• The level of use desired (i.e., limited use or nationwide use) 

• Type of study (side-by-side comparison or QC acceptance criteria-based comparison study) 

• Applicant’s NPDES permit number, the issuing agency, the type of permit and the discharge serial 

number (if applicable) 

The following items should be submitted as attachments to the initial application: 

• Reason for proposing the ATP or new method 

• The proposed ATP or new method prepared in standardized format (Section 3.0) 

• A method comparison table that gives a side-by-side comparison of the steps of the proposed ATP or 

new method and the EPA-approved reference method (Section 2.5) 

• Method development information 

• Study plan for EPA review and comment (Section 4.0) 

A study plan is generally not needed with the application if an applicant is unsure whether or not a 

modification is allowed within the method-specified flexibility. In such cases, the applicant may request 

that EPA determine the usefulness of a full ATP comparability assessment based on the other information 

submitted with the application. From this information, EPA can determine whether a full ATP 

assessment will be helpful, whether the proposed modification is considered to be a minor modification 

(i.e., employs the same chemistry and/or biological principles as the EPA-approved reference method to 

determine the presence/absence or to quantify the amount of the target organism in a sample), or whether 

the proposed modification is considered to be within the specified flexibility of the EPA-approved 

reference method. 

The elements of a complete application are presented in Table 2-2. A list of the information discussed in 

detail in Sections 2.3 to 2.8 is provided in Appendix C. EPA will generally seek all application 

information and attachments before the application is considered complete. 

Table 2-2. Application Information 

Application Information 

• Completed application form 

• Reason for ATP 

• Method in EPA format 

• Method comparison table 

• Method development information 

• Study plan (to be approved by EPA before proceeding with study) 

• Study report (final report generally considered part of a complete application) 

Note: Although the application process generally begins with the initial submission of ATP materials, the 

application is not usually considered to be complete until the final study report has been submitted, and 

all EPA questions on the report have been resolved. 

2.3 Reason for ATP 

The entity that proposes an ATP should indicate why the ATP is being proposed. Examples include, but 

are not limited to, the following: 

• The ATP improves method performance 

• The ATP provides equivalent method components for a lower cost 

• The ATP enables laboratories to perform analyses more efficiently 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

• The ATP successfully overcomes some or all of the interferences associated with the EPA-approved 

reference method 

• The ATP significantly reduces the amount of hazardous wastes generated by the laboratory 

• The ATP provides another means for measuring a contaminant (i.e., provides more choices) 

2.4 Standard EPA Method Format 

In accordance with the standard EPA format advocated by EPA’s Environmental Monitoring 

Management Council (EMMC), methods should contain 17 specific topical sections in a designated order. 

The 17 sections are listed in Section 3.0 of this document. Additional numbered sections may be inserted 

starting with Section 11.0, Procedure, as appropriate for a particular method. For detailed information on 

the EPA format for proposed methods, see the Guidelines and Format document (Reference 10.14). 

2.5 Method Comparison Table 

As part of the application, the applicant should provide a two-column table comparing the proposed ATP 

or new method to the EPA-approved reference method. The two-column method comparison table 

should include the number and title of each method, the latest revision date of the proposed ATP, and a 

detailed discussion of each of the 17 topics specified by the standard EPA method format (as applicable). 

Each topic should be discussed on a separate row in the method comparison table. The applicant should 

highlight any differences between the proposed ATP and the EPA-approved reference method. 

2.6 Method Development Information 

Before EPA reviews the study plan and works with applicants on ATP studies, the applicant should 

provide data on performance of the modified or new method in the water matrix for which the ATP or 

new method is being applied. These data may have been generated during method development by the 

vendor, or through independent tests by third-party laboratories. Examples include, but are not limited to, 

replicate spiked reagent water or replicate spiked matrix water tests. It is the responsibility of the 

applicant to provide sufficient data to demonstrate that the ATP or new method performs sufficiently at a 

preliminary level in the matrix of interest to merit evaluation of an ATP. If sufficient data is not 

available, EPA may request additional studies be conducted prior to the review of the ATP study plan. 

In addition to data, the following descriptive method information will facilitate EPA’s evaluation of the 

ATP application: 

• The purpose and intended use of the method 

• The analytical basis for the method, noting any relationship of the method to other existing analytical 

methods and indicating whether the method is associated with a sampling method 

• Method limitations and an indication of any means of recognizing cases where the method may not be 

applicable to specific matrix types (e.g., turbidity greater than 50 NTU) 

• The basic steps involved in performing the test and data analysis 

• Options to the method, if applicable 

This information also will aid EPA in preparing the docket and the preamble for the proposed rule that 

will be published in the Federal Register if EPA proposes to approve the ATP for nationwide use. 

2.7 Study Plan 

Prior to conducting all studies, the applicant should submit a study design for EPA review and comment. 

A detailed procedure (Section 3.0) for the new method or the modification should be included as an 

attachment to the study plan. Generally, EPA will evaluate the study plan to ensure that the appropriate 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

data quality objectives identified in this protocol are defined and addressed. EPA comments will be 

incorporated into the study design and this process will be repeated until EPA has approved the study 

design. Generally, the study plan should contain the elements listed below: 

• Background 

• Objectives 

• Study Design 

• Coordination 

• Data Reporting 

These elements are further described in Section 4.0. 

2.8 Study Report 

The applicant should conduct a study and provide a comprehensive study report with the ATP or new 

method application. The study report should include the following elements: 

• Background 

• Study Objectives and Design 

• Study Implementation 

• Data Reporting and Validation 

• Results 

• Data Analysis and Discussion 

• Conclusions 

• Appendix A - Method 

• Appendix B - Study Plan 

• Appendix C - Supporting Data 

• Appendix D - Supporting References 

These elements are further described in Section 9.0. 

2.9 Proprietary Information in Applications 

All information provided to the Federal government is subject to the requirements of the Freedom of 

Information Act. Therefore, any proprietary information submitted with the proposed ATP application 

should be marked as confidential. However, EPA prefers that supporting documentation labeled as 

confidential business information not be submitted as part of the ATP application. If proprietary 

information is determined to be essential to the application, EPA staff will request the information and 

will handle such information according to the regulations in subparts A and B of 40 CFR Part 2. 

Specifically, in accordance with 40 CFR §2.203, a business that submits information to EPA may assert a 

business confidentiality claim covering the information by placing on (or attaching to) the information at 

the time it is submitted to EPA, a cover sheet, stamped or typed legend, or other suitable form of notice 

employing language such as trade secret, proprietary, or company confidential. Allegedly confidential 

portions of otherwise non-confidential documents should be clearly identified by the business, and may 

be submitted separately to facilitate identification and handling by EPA. If the business desires 

confidential treatment only until a certain date or until the occurrence of a certain event, the notice should 

so state. Please be advised, however, that any methods proposed in the Federal Register cannot be 

claimed as confidential business information. 

If a claim of business confidentiality is not made at the time of submission, EPA will make such efforts as 

are administratively practicable to associate a late claim with copies of previously submitted information 

in EPA files. However, EPA cannot ensure that such efforts will be effective due to the nature of 

application review that may already be in progress. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

SECTION 3.0 METHOD FORMAT 

Because alternate test procedures may be approved by EPA as comparable to the reference methods, and 

may be implemented by multiple laboratories, it is important that the written procedures include all of the 

information necessary to use the technique in the laboratory, including but not limited to: reagents and 

equipment, sample collection and preservation procedures, quality control, and a detailed description of 

the procedure. The information described below should be provided in the method. In addition, EPA 

recommends the Environmental Monitoring Methods Council (EMMC) format described below be used. 

Sections 3.1 through 3.17 provide a list of EMMC method sections and a general description of the type 

of information that should be included in each section. The date and revision number of the method 

should be included on the cover page. In addition, the date should be included as a footer on each page of 

the method. A detailed description of method format guidelines, as well as an example of a formatted 

method, is provided in Reference 10.14. The detailed information in Reference 10.14 is provided as 

guidance for the method write-up and as such, specific suggestions for font size, margins, etc. are 

optional. 

3.1 Scope and Application 

Include a list of target organisms (by common name), taxonomic group and their CAS registry numbers 

or other accepted numbering systems (if available), the matrices to which the method applies, a generic 

description of method sensitivity (the minimum number of organisms the method can detect per unit 

volume or mass, if known), and the data quality objectives that the method is designed to meet or 

monitoring programs for which the method was designed to support. 

3.2 Summary of Method 

Summarize the method in a few paragraphs. The purpose of the summary is to provide a succinct 

overview of the method procedure to aid the reviewer or data user in understanding the method and how 

the results are generated. Include a general description of the method procedure, sample volume, type of 

media used, preparation steps, incubation time and temperatures, and the techniques used for qualitative 

or quantitative determinations. 

3.3 Method Definitions 

Provide definitions of terms that are necessary to understand how the method is used or what the results 

represent. This should include a definition of the target organism or group of organisms, relative to the 

determinative step of the method. For extensive lists of definitions, this section may simply refer to a 

glossary attached at the end of the method document. 

3.4 Interferences 

This section should discuss any known method interferences such as toxic materials, particulates, nontarget organisms, etc. If known interferences in the reference method are not interferences in the alternate 

method, this also should be clearly stated. 

3.5 Safety 

This section should discuss only those safety issues specific to the method and beyond the scope of 

routine laboratory practices. Target analytes or reagents that pose specific health, toxicity, or safety 

issues should be addressed in this section. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

3.6 Equipment and Supplies 

For critical equipment that may affect the performance of the method, cite the manufacturer, model name, 

and catalog or product number of the equipment that was used to develop or validate the method; note 

that equivalent equipment can be used, if applicable. Use generic language for standard laboratory 

glassware and disposables. 

3.7 Reagents and Standards 

Provide sufficient details on the concentration and preparation of reagents and standards to allow the 

work to be duplicated, but avoid lengthy discussions of common procedures. If only pre-prepared 

proprietary reagents can be used, specify this. Include catalog and/or product numbers where appropriate. 

Indicate shelf life of packaged materials and special storage specifications. 

3.8 Sample Collection, Preservation, and Storage 

Provide information on sample collection, preservation, shipment, storage conditions, and holding times. 

If effects of holding time were specifically evaluated, provide reference to relevant data. 

3.9 Quality Control 

Describe specific quality control (QC) measures that enable one to establish the sensitivity, specificity, 

false positive rates, false negative rates, bias, and precision of measurements using the method, and that 

the measurements are free from contamination. Specific QC measures may include positive and negative 

controls, duplicate samples, method blanks, and media sterility checks. Indicate which QC measures are 

appropriate initially, before a laboratory uses the method, and which are appropriate on an ongoing basis. 

Indicate frequencies for each QC measure and list minimum specifications or acceptance ranges (see 

Section 5.0). Indicate corrective actions that should be taken when QC measures are not met. Define all 

terms in method definitions section. 

3.10 Calibration and Standardization 

Discuss initial calibration specifications for instruments used in the method (e.g., water baths, 

refrigerators, thermometers, balances, pH meters, microscopes, etc.). Indicate frequency of such 

calibrations; refer to performance specifications; and indicate corrective actions that should be taken 

when performance specifications are not met. This section may also include procedures for calibration, 

verification, or continuing calibration, or these steps may be included in the procedure section. 

3.11 Procedure 

Provide a detailed description of the sample processing and analysis steps. Avoid unnecessarily 

restrictive instructions, but provide sufficient detail for manual procedures so that analysts in other 

laboratories perform the method consistently. Ranges should be provided for temperature requirements, 

time requirements, etc. 

3.12 Data Analysis and Calculations 

Identify qualitative and quantitative aspects of the method. List criteria for the identification of target 

organism(s) and interpretation of results for all steps of the method, including criteria for presumptive and 

confirmed results. Provide equations used to derive final sample results. Provide discussion of 

estimating detection limits, recoveries, specificity, false positive/false negative rates, etc., if appropriate. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

3.13 Method Performance 

Provide detailed information on method performance, including data on precision, bias (for quantitative 

methods), specificity, detection limits (including the method by which they were determined and matrices 

to which they apply), statistical procedures used to develop performance specifications (i.e., recovery, 

precision, specificity, false positive/false negative rates, etc.). Where performance is tested relative to the 

reference method, provide a summary of the side-by-side comparison of performance versus reference 

method specifications. 

3.14 Pollution Prevention 

Describe aspects of this method that minimize or prevent pollution that may be attributable to the 

reference method. 

3.15 Waste Management 

Cite how waste is minimized and the proper disposal of samples and waste. 

3.16 References 

Include source documents, publications, etc. 

3.17 Tables, Diagrams, Flowcharts, and Validation Data 

Additional information may be presented at the end of the method. Lengthy tables may be included here 

and referred to elsewhere in the text by number. Diagrams should only include new or unusual 

equipment or aspects of the method. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

SECTION 4.0 STUDY PLAN 

Applicants should submit a study design for EPA review, comment, and approval prior to conducting the 

side-by-side comparison study or the QC acceptance criteria-based comparison study. This process 

protects the applicant by providing written approval of the study design before resources are spent to 

conduct the study. The ATP program is intended to be flexible, and thus EPA may modify the study 

design for a particular proposed method. Data from studies conducted without EPA review and approval 

may not meet EPA’s criteria, and may not adequately address the applicant’s study objectives. A detailed 

procedure (Section 3.0) for the ATP or new method should be included as an attachment to the study 

plan. EPA will generally evaluate the study plan to verify that the appropriate data quality objectives 

identified in this protocol are defined and addressed. EPA comments are incorporated into the study 

design. This review/revision process is repeated until EPA has approved the study design. 

Generally, the study design should include the information described in Sections 4.1 through 4.5. 

4.1 Background 

This section of the study plan should include the following information: 

• A statement identifying the ATP as a new method or a modification of a reference method 

• The EPA program(s) to which the ATP or new method applies (e.g., drinking water, wastewater, 

ambient water, point source categories regulated at 40 CFR Parts 400-499, etc.) 

• A short (one paragraph) summary of the ATP or new method 

• The organization and method number of the reference method if applicable 

• A description of the reasons for the extent of the modification, the logic behind the technical 

approach to the modification, and the result of the modification 

• The matrices (e.g., finished water, wastewater, ambient water, etc.), matrix types (e.g., turbidity 

greater than 10 NTU, etc.), and/or media to which the ATP or new method is believed to be 

applicable 

• A list of the analytes measured by the ATP or new method, including the corresponding CAS registry 

number (if available) or other identification number 

4.2 Objectives 

Include a description of the new method or modification, describe the goals of the study, and define data 

quality objectives. 

4.3 Study Design 

The following information should be included in the study design: 

• Laboratories that will participate in the study (Sections 5.0 and 6.0) 

• Number and type of samples to be analyzed (Section 6.0) 

• Description of the matrices that will be used (Sections 6.0 and 7.0) 

• Description of the spikes that will be used (Section 7.0) 

• Description of the spiking procedure (Section 7.0) 

• Positive and negative control organisms (Section 5.0) 

• Quality control procedures that will be followed (Section 5.0) 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

4.4 Coordination 

Describe how the study will be coordinated, how the spikes will be shipped to the laboratories, and who 

will compile the data for submission. Data compilation should not be performed by any of the analysts 

conducting the sample analyses. 

4.4.1 Study Management 

This section of the study plan should include the following information: 

• The organization responsible for managing the study 

• The laboratories, facilities, and other organizations that will participate in the study 

• A delineated study schedule including, but not limited to, sample collection, start of sample analysis, 

interpretation of sample results, completion of study, etc. 

4.4.2 Technical Approach 

This section of the study plan should include the following: 

• A description of how sample matrices and participating laboratories will be selected 

• A description of how samples will be collected and distributed 

• The numbers and types of analyses to be performed by the participating laboratories 

• A description of sample spiking procedures 

• A description of how analyses are to be performed 

4.5 Data Reporting 

List the data elements that will be collected and provide sample bench sheets (see Appendix D) that will 

be used to record raw data during the study. Raw data should be submitted as an attachment to the Study 

Report (Section 9.0). Address the statistical analysis of the study results that will be performed, if the 

statistical analyses will differ from those described in Section 8.0. Please note, however, that EPA’s 

evaluation of method performance will generally be based on the statistical analyses described in Section 

8.0. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

SECTION 5.0 QUALITY ASSURANCE/QUALITY CONTROL 

For side-by-side comparison studies in which only one laboratory is performing analyses, the 

comparability study should be conducted at an independent laboratory and the laboratory should be 

certified to perform microbiological analyses under EPA’s drinking water laboratory certification 

program. A laboratory with a vested interest in the method, instrumentation, apparatus, reagents, media, 

or associated kits may not participate in the side-by-side comparison study. 

For QC acceptance criteria-based comparison studies, at least three independent laboratories should 

participate. A laboratory with a vested interest in the ATP also may participate in the study, but in such 

instances there should be at least three independent laboratories participate and the majority of the 

laboratories participating are independent laboratories. All laboratories should be certified to perform 

microbiological analyses under EPA’s drinking water certification program, or a comparable certification 

program, if a laboratory located outside of the U.S. is included. At least three independent laboratories 

participating in the study should to be certified for microbiological analyses under EPA's drinking water 

certification program. If more than three laboratories participate, the majority of the laboratories should 

be certified for microbiological analyses under EPA's drinking water certification program. 

5.1 Quality Assurance 

The laboratory should have a comprehensive quality assurance (QA) program in place and operating at all 

times during the performance of the comparability study. General QA program guidance is provided at 

http://www.epa.gov/quality/qs-docs/r2-final.pdf (Reference 10.11). The laboratory should adhere closely 

to all QA and quality control (QC) measures in this protocol as well as the QC measures in the method(s). 

Laboratory QA/QC criteria for facilities, personnel, and laboratory equipment are included in Standard 

Methods 9020-Quality Assurance (Reference 10.4) and the U.S. EPA Manual for the Certification of 

Laboratories Analyzing Drinking Water, Fourth Edition (March 1997) (Reference 10.13). 

The laboratory should adhere to standard laboratory practices for cleanliness and environment, and to the 

methods for glassware and apparatus, reagents, solvents, and safety. Additional guidelines regarding 

general laboratory procedures should generally be followed, as specified in Sections 4 and 5 of the 

Handbook for Analytical Quality Control in Water and Wastewater Laboratories, EPA-600/4-79-019 

(Reference 10.19). 

5.2 Quality Control 

Laboratories participating in a comparability study should perform all QC procedures specified in the 

methods, except where explicitly stated in the approved study plan. The QC procedures listed in Table 5-

1 and described below should be performed, as appropriate, based on the technique used in the method. 

Other QC procedures may be necessary, based on the approved study plan. Laboratories participating in 

side-by-side comparison studies or QC acceptance criteria-based comparison studies should perform all 

QC procedures specified in the methods, except where explicitly stated in the approved study plan. The 

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

maintain a record of the date and results of all QC sample analyses. Laboratories should maintain reagent 

and material lot numbers along with samples analyzed using each of the lots. Laboratories should also 

maintain media preparation records. 

Table 5-1 lists quality control measures for each laboratory participating in side-by-side comparison 

studies and QC acceptance criteria-based comparison studies. Detailed descriptions of the QC measures 

are provided in Sections 5.2.1 to 5.2.15. If contamination is detected in any of the blanks or sterility 

checks described below, the source of contamination should be identified and corrected. The 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

blank/sterility check and all samples associated with that contaminated blank/sterility check should be 

reprepared and reanalyzed. Measures taken to eliminate contamination should be reported. 

Table 5-1. Quality control measures for each laboratory involved in the study 

Suggested for Study 

Quality Control Measure Frequency Side-by-Side 

Comparison Study 

QC acceptance 

criteria-based 

Comparison Study 

Analyst counting variability (Section 

5.2.1) 2 samples per study T T

Autoclave sterilization verification 

(Section 5.2.2) 

Within one week prior to 

the start of the study T

Dilution/rinse water blanks 

(Section 5.2.3) 

1 per every 20 samples or 

1 per day of study, 

whichever is greater 

T

Incubator/waterbath temperatures 

(Section 5.2.4) 

2 times per day when used 

in study T

Initial demonstration of capability 

(IDC) (Section 5.2.5) or Initial 

precision and recovery (IPR) 

(Section 5.2.6) 

Each laboratory 

participating in the study 

should generate 

acceptable IDC/IPR data. 

T

Matrix spike/matrix spike duplicate 

(Section 5.2.7) Per approved study plan T

Media sterility checks 

(Section 5.2.8) 

1 per each batch of media 

used in the study or per 

each test run, whichever is 

greater 

T

Method blank (Section 5.2.9) Per approved study plan T

Ongoing demonstration of 

capability (ODC) (Section 5.2.10) 

or Ongoing precision and recovery 

(OPR) (Section 5.2.11) 

Included as part of the 

study; not needed as 

separate QC. 

Optional 

Positive and negative controls 

(Section 5.2.12) 

1 positive control and 1 

negative control for each 

media/stain used in the 

study. See 5.2.12 below for 

more details. 

T

Preparation blanks 

(Section 5.2.13) 

Frequency depends on the 

type of method. See 5.2.13 

below for more details. 

T

Refrigerator/freezer temperatures 

(Section 5.2.14) 

Once per day when used in 

study T

Sample processing equipment 

sterility checks (Section 5.2.15) 

Prior to the analysis of 

samples. See 5.2.15 below 

for more details. 

T

T

T

T

T

T

T

T

T

T

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

5.2.1 Analyst Counting Variability 

If the laboratory has two or more analysts, each should count colonies, plaques, or positive wells on the 

same plate/tray from one positive field sample per month. Compare each analyst’s count of the colonies, 

plaques, or wells. Counts should fall within 10% between analysts. If counts fail to fall within 10% of 

each other, analysts should perform additional sets of counts, until the number of target colonies, plaques, 

or positive wells counted fall within 10% between analysts for at least three consecutive samples. If there 

is only one analyst replicate counts should be done and be within 5% of original counts. 

5.2.2 Autoclave Sterilization Verification 

Autoclave sterilization verification should be performed within one week of the start of the study by 

placing Bacillus stearothermophilus spore suspensions or strips inside glassware. Autoclave at 121°C for 

15 minutes. Place Bacillus stearothermophilus spore suspensions in trypticase soy broth tubes and 

incubate at 55°C for 48 hours. Check for growth to verify that sterilization was adequate. If sterilization 

was inadequate, determine appropriate time for autoclave sterilization. Repeat spore test. The laboratory 

should have historical data verifying that at a minimum, autoclave sterilization is performed on a monthly 

basis. 

5.2.3 Dilution/Rinse Water Blanks 

The laboratory should analyze dilution/rinse water blanks to demonstrate freedom from contamination. 

An aliquot of dilution/rinse water which is analyzed exactly like a field sample should be analyzed for 

every day of the study or for every 20 samples, whichever is more frequent, and observed for 

contamination with agent of interest. 

5.2.4 Incubator/Waterbath Temperatures 

Incubator or waterbath temperatures should be measured and recorded two times per day when in use. 

Temperatures should be taken at least 4 hours apart and should be within the range of the desired 

temperature as specified in each method. Thermometers used to measure “in-use” temperatures should be 

calibrated yearly against an NIST traceable thermometer. 

5.2.5 Initial Demonstration of Capability 

Laboratories participating in a QC acceptance criteria-based comparison study should have successfully 

performed an initial demonstration of capability (IDC) test for the new or modified method under 

evaluation in the study. An IDC test is performed when QC acceptance criteria are available for the 

evaluation of precision or recovery, but not both. The laboratory should perform an IDC as specified in 

the method to demonstrate acceptable performance. The laboratory should complete any additional 

analyses as specified in the study plan. 

For the IDC test, the laboratory spikes and analyzes reference matrix (e.g., reagent water, buffered water, 

etc.) samples to demonstrate acceptable performance with the method prior to the analysis of field 

samples. The number of samples involved in the IDC varies by method. If the results of the IDC test 

meets all IDC acceptance criteria (e.g., RSD, minimum number of samples positive, etc.), system 

performance will generally be acceptable. If any of the IDC test results fail to meet the acceptance 

criteria, system performance will generally be unacceptable. In this event, the laboratory should identify 

and correct the problem and repeat the test. IDC tests should be accompanied by a method blank (Section 

5.2.9). 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

5.2.6 Initial Precision and Recovery 

Laboratories participating in a QC acceptance criteria-based comparison study should have successfully 

performed an initial precision and recovery (IPR) test for the method using the modified version of the 

method under evaluation in the study. An IPR test is performed when QC acceptance criteria are 

available for the evaluation of both precision and recovery. The laboratory should perform an IPR as 

specified in the method to demonstrate acceptable performance. The laboratory should complete any 

additional analyses as specified in the study plan. 

For the IPR test, the laboratory spikes and analyzes reference matrix (e.g., reagent water, buffered water, 

etc.) samples to establish the laboratory’s ability to generate acceptable precision and recovery prior to 

the analysis of field samples. Using results of the analyses, the laboratory calculates mean percent 

recovery and relative standard deviation (RSD) of the recoveries for the analyte(s) and compares them 

with the corresponding limits for the IPR test criteria in the method. If the RSD and the mean percent 

recovery meet the acceptance criteria, system performance is acceptable and analysis of samples may 

begin. If the RSD or the mean percent recovery are unacceptable, system performance will generally be 

unacceptable. In this event, the laboratory should identify and correct the problem and repeat the test. 

IPR tests should be accompanied by method blank tests (Section 5.2.9). 

5.2.7 Matrix Spike and Matrix Spike Duplicate Samples 

Matrix spike (MS) and matrix spike duplicate (MSD) samples are spiked matrix water samples analyzed 

by the laboratory to verify acceptable method performance in the matrix being monitored. During routine 

performance of the method, MS/MSD samples are analyzed by the laboratory for the first sample of any 

new matrix that will be monitored, and on the 21st sample thereafter. During a QC acceptance criteriabased comparison study for some methods (e.g., Cryptosporidium and Giardia methods), the laboratory 

should analyze MS samples as part of routine laboratory QC, however these analyses may not be 

necessary during this study because MS/MSD samples using blinded spiking suspensions distributed by 

the study coordinator will be used for the study. 

5.2.8 Media Sterility Checks 

Before using newly prepared media, a representative portion of each media batch needs to be checked for 

sterility. The laboratory should test media sterility by incubating one unit (tube or plate) from each batch 

of medium specified in the method or per each test run, whichever is more frequent, at the appropriate 

temperature for the length of the method-specified incubation time and observing for growth. 

5.2.9 Method Blank 

Method blanks are reagent water blanks or other blanks including but not limited to buffered water, tap 

water, etc., depending on the method analyzed to demonstrate freedom from contamination. Method 

blanks should be analyzed at the frequency specified in the approved study plan. For QC acceptance 

criteria-based comparison studies, the laboratory needs to analyze a method blank with the IPR and IDC 

tests. During routine performance of the method, the laboratory should analyze at least one method blank 

per every 20 test samples or per every week. During a QC acceptance criteria-based comparison study for 

some methods (e.g., Cryptosporidium and Giardia methods), the laboratory should analyze method 

blanks as part of routine laboratory QC, but method blanks also should be shipped to the laboratory as 

double-blind samples by the study coordinator. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

5.2.10 Ongoing Demonstration of Capability (ODC) Samples 

Ongoing demonstration of capability samples are spiked reference matrix (e.g., reagent water, buffered 

water, etc.) samples that are analyzed to demonstrate that the analytical system is in control on an ongoing 

basis. 

5.2.11 Ongoing Precision and Recovery (OPR) Samples 

Ongoing precision and recovery samples are spiked reference matrix (e.g., reagent water, buffered water, 

etc.) samples that are analyzed by the laboratory to verify that method performance criteria are being met. 

During a performance based comparison study for some methods (e.g., Cryptosporidium and Giardia 

methods), the laboratory should analyze OPR samples as part of routine laboratory QC because generally 

OPR samples using blinded spiking suspensions distributed by the spiking coordinator will be used for 

the study. 

5.2.12 Positive/Negative Controls 

Positive and negative controls are target and non-target organisms processed to ensure the laboratories are 

familiar with the identification of the target organism and to ensure that confirmation test results are 

appropriate. 

5.2.12.1 Positive/Negative Culture Controls (Culture-Based Methods) 

Positive and negative culture controls refer to cultures that, when analyzed exactly like field samples, 

produce a known positive or a known negative result, respectively, for a given type of media. One 

positive culture control and one negative culture control should be prepared and analyzed for every media 

(including confirmation media) used in the method whenever a new batch of medium or reagents is used, 

every day of the study, or every 20 samples, or as specified in the method, whichever is more frequent. 

Each control should be carried through the entire procedure and should exhibit the expected positive or 

negative result. 

5.2.12.2 Positive/Negative Staining Controls (Cryptosporidium and Giardia Methods) 

A positive staining control for Cryptosporidium and Giardia methods is a slide containing positive 

antigen or intact Cryptosporidium oocysts and Giardia cysts, and that is stained using the same procedure 

as used for field samples or test samples. A negative staining control is a slide containing only phosphate 

buffered saline (PBS) that is stained using the same procedure as used for field samples or test samples. 

The laboratory should prepare and examine positive and negative staining controls with each batch of 

slides the laboratory prepares during the study. Positive staining controls should exhibit acceptable 

fluorescence and negative staining controls should not exhibit fluorescence. 

5.2.12.3 Positive/Negative Staining Controls (Other) 

At a minimum, the laboratory should prepare and examine a positive and negative control using the same 

procedure as used for field or test samples whenever a new batch of media or reagents is used every day 

of the study or every 20 samples, whichever is more frequent. Each control should be carried through the 

entire procedure and should exhibit the expected positive or negative result. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

5.2.13 Preparation Blanks (PB) 

5.2.13.1 Membrane Filter Preparation Blank (PB-MF) 

If membrane filtration is used, at the beginning and the end of each filtration series, a PB-MF is 

performed by filtering 20-30 mL of dilution water through the membrane filter and testing for growth. If 

the control indicates contamination with the target organism, all data from affected samples should be 

rejected. A filtration series ends when 30 minutes or more elapse between sample filtrations. 

5.2.13.2 Multiple-Tube Fermentation Test Preparation Blank (PB-MTF) 

If a multiple-tube fermentation test is used, a volume of sterile buffered water that is analyzed exactly like 

a field sample should be analyzed for every day of the study or every 20 samples, whichever is more 

frequent. The preparation blank should be incubated with the sample batch and observed for growth of 

the target organism. If the control indicates contamination with the target organism, all data from affected 

samples should be rejected. If buffered water is not used for dilutions, only the multiple-tube 

fermentation media should be included. 

5.2.13.3 Other Preparation Blank (PB-Other) 

A volume of sterilized water (e.g., reagent grade as defined in Specification D 1193, Annual Book of 

ASTM Standards) that is analyzed exactly like a field sample should be analyzed for every day of the 

study or every 20 samples, whichever is more frequent. The preparation blank should be incubated with 

the sample batch and observed for growth of the target organism. If the control indicates contamination 

with the target organism, all data from affected samples should be rejected. 

5.2.14 Refrigerator/Freezer Temperatures 

Refrigerator and freezer temperatures should be measured and recorded once per day when in use. 

Refrigerator temperature should be maintained at 1°C to 4°C. Freezer temperatures should be maintained 

at -15°C to -20°C. Special freezers capable of long-term storage of cultures or virus should be maintained 

at -70°C to -80°C. Thermometers used to measure “in-use” temperatures should be calibrated yearly 

against an NIST traceable thermometer. 

5.2.15 Sample Processing Equipment Sterility Checks 

A representative portion of non-disposable items such as sample containers, blender jars, etc., used to 

collect or process samples should be checked for sterility prior to use in analyses. To test for sterility add 

approximately 500 mL (or appropriate volume based on the size of the equipment being used) of a sterile 

non-selective broth (e.g., tryptic soy, trypticase soy, or tryptone broth) to the non-disposable item and 

incubate at 35°C ± 0.5°C for 24 hours and check for growth. Depending on the incubation times 

specified in the method, the length of the incubation time for the sample processing equipment sterility 

check may be increased. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

SECTION 6.0 STUDY DESIGN 

This section provides a description of the study design that is important for assessing comparability using 

either side-by-side method comparison studies (Section 6.1) or QC acceptance criteria-based studies 

(Section 6.2). The sections below address the number of laboratories, number of matrices, and the 

number of samples that should be analyzed in the studies. 

6.1 Side-by-Side Comparison Studies 

Methods are compared using the following parameters: 

• Recovery. Does the new method have similar, better or worse recoveries of the target organism as 

the reference method? 

• Precision. Are the recoveries by the new method significantly less or more variable than the 

reference method? 

• False positive rate/specificity. Is the new method significantly more likely or less likely to detect 

non-target organisms or other sample constituents that would be reported as the target organism by 

the analyst when compared to the reference method? 

• False negative rate/sensitivity. Is the new method significantly more likely or less likely to exhibit 

non-detects for samples with the target organism or to exhibit results that are biased low when 

compared to the reference method? 

To generate these parameters, samples are analyzed by a single laboratory (6.1.1). The number of 

samples (and matrix types) used in the study are determined using a historical EPA standard (6.1.2.1) 

6.1.1 Number of Laboratories 

A single laboratory should be used for a side-by-side comparability study. Since the study should be 

conducted in a single independent laboratory with no conflict of interest; the laboratory selected cannot 

be the method developer’s laboratory and cannot be affiliated with the method developer. 

6.1.2 Number of Samples 

The following standards generally provide the minimum number of samples that should be analyzed. 

Additional data are generally acceptable and may be very helpful when reviewing an ATP or new 

method. 

6.1.2.1 Method Comparison Study Design Summary 

Table 6.1 provides a summary of the method comparability requirements for nationwide microbiological 

ATPs and new methods. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

Number of Matrices 

Ten different water/wastewater matrices from geographically diverse locations should be included to 

obtain, as much as is practical, a good presentation of the wide range of water types with an even wider 

range of target organisms to which the method should appropriately respond. Each water or wastewater 

sample should be collected in sufficient volume to complete all replicate analyses of sample or dilution 

volumes by both the ATP or new method and the EPA-approved reference method. For ambient water 

studies, the turbidity of at least one matrix should be greater than 10 NTU. Generally, for matrices other 

than finished drinking water or ambient water, matrix composition will be addressed on a study-specific 

basis. 

Number of Samples and Replicates 

Twenty replicate analyses should be performed by each method for each of the 10 matrices for a total of 

200 replicate analyses per method. The replicate analyses should be performed on the same day for both 

the proposed and reference methods. 

Verification of Results 

For quantitative reference methods, 10 typical colonies must be verified from 4 randomly chosen 

replicates (of the 20 replicates) of each of the 10 samples for a total of 400 colony verifications. For 

presence/absence tests, all positive samples should be verified for up to 200 samples. See Section 6.1.3 

for additional information pertaining to the verification of results. Calculations for false positive rates 

and false negative rates are described in Section 8.4.3. 

6.1.3 Verification of Results 

6.1.3.1 False Positives 

To assess whether the false positive rates are significantly different between methods, replicates known to 

contain non-target organisms that could be falsely identified as the target organism should be analyzed by 

both the ATP or new method. The determination that the samples do not contain the target organism 

should be based on a third independent standard method (see Section 7.6) rather than by the EPAapproved reference method being used in the comparison. This is because it should not be assumed that 

the accepted method has a false positive or negative rate of zero. In side-by-side comparison studies 

using the EPA standard for determining the number of matrices and the number of samples, at least 200 

positive results should be verified for the ATP or new method in order to adequately compare false 

positive rates. 

6.1.3.2 False Negatives 

To assess whether the false negative rates are significantly different between methods, replicates known 

to contain target organisms should be analyzed by both the ATP or new method. The determination that 

the samples do not contain the target organism should be based on a third independent standard method 

(see Section 7.6) rather than by the EPA-approved reference method being used in the comparison. This 

is because it should not be assumed that the accepted method has a false positive or negative rate of zero. 

In side-by-side comparison studies using the EPA standard for determining the number of matrices and 

the number of samples, at least 200 negative results should be verified for the ATP or new method in 

order to adequately compare false negative rates. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

6.2 QC Acceptance Criteria-Based Comparison Studies 

QC acceptance criteria-based comparison studies are generally conducted to demonstrate that the ATP or 

new method is able to meet the QC acceptance criteria of the EPA-approved reference method. In some 

instances, the quality control (QC) acceptance criteria specified in a method may not be sufficient to 

demonstrate comparability between the ATP and the EPA-approved reference method and a side-by-side 

comparison study should be conducted. Generally, EPA will make this decision based on review of the 

application materials. 

6.2.1 Number of laboratories 

A minimum of three laboratories should be used for a QC acceptance criteria-based comparison study. 

All three laboratories should meet all QC acceptance criteria in the EPA-approved reference method. If 

more than three laboratories participate, at least 75% of the participating laboratories should meet all QC 

acceptance criteria in the EPA-approved reference method. 

6.2.2 Number of Matrices 

For all QC acceptance criteria-based comparison studies, there should be one matrix per laboratory. For 

ambient water studies, the turbidity of at least one matrix should be greater than 10 NTU. Generally, for 

matrices other than finished drinking water or ambient water, matrix composition should be addressed on 

a study-specific basis. 

6.2.3 Number of Replicates per Matrix 

The number of replicates per matrix and laboratory should be specific to the QC criteria to which the 

results will be compared. Generally, the number of reagent results should be 4 and the number of source 

water results per matrix should be 2. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

SECTION 7.0 SAMPLE PREPARATION AND ANALYSIS 

7.1 Collection of Samples for Analysis 

Each sample should be collected in sufficient volume to complete all replicate analyses by both the ATP 

or new method and the EPA-approved reference method. Samples should be spiked (if necessary) and 

analyzed as soon as possible after collection. 

7.1.1 Source Water Characterization 

Source water characterization information should be collected at the time of sample collection. This 

information will be useful in characterizing the matrix to identify potential interferences and generally 

includes, but is not limited to, the following: 

• Sample collection location 

• Source of water (e.g., ground water, stream, river, lake, etc.) 

• Plant treatment processes (if sample is collected from a water treatment facility) 

• Temperature 

• pH 

• Turbidity 

• Total organic carbon (TOC) 

• Free and total disinfectant residual at time of sample collection 

• Heterotrophic plate count (HPC) unless heterotrophic bacteria are the target analytes, in which case 

total coliforms should be measured 

In addition to this information, data on the concentration of potential interferences (e.g., competing 

bacteria, interfering chemicals, etc.) in the water collected for analysis may be necessary. Known 

interferences should be discussed in the method and addressed in the study plan. The final results, as well 

as bench sheets, log sheets, instrument printouts, and any associated quality control analyses should be 

submitted as part of Appendix C of the study report (Section 9.10). 

7.2 Sample Spiking and “Stressing” Procedures for Bacteriological Methods 

Depending on the matrix and the analyte of interest, it may not always be necessary to spike samples prior 

to analysis. Rangefinding analyses should be performed to assess the ambient concentration of target 

organism(s) in the matrix of interest to determine whether sample spiking will be useful. Samples are 

collected and dilutions are prepared in order to enumerate the number of organisms in the sample without 

qualifiers (i.e. less than, greater than, or too numerous to count). In addition, rangefinding can also be 

used to obtain environmental isolates for use in sample spiking or to determine the concentration of target 

organism(s) in a spiking suspension or spiked sample. 

If samples are spiked, environmental isolates should be used, as pure strains may exhibit different 

recovery and precision characteristics than natural flora. NELAC 

(http://www.epa.gov/ttn/nelac/standard/5qs-15-0-jhrev-corr.pdf, Reference 10.9) and ATCC 

(http://www.atcc.org/SearchCatalogs/faqBacteriology.cfm#Q15, Reference 10.6) recommend that 

bacterial cultures be transferred monthly and passed no more than five times before returning to the 

original culture. 

Sections 7.2.1 through 7.2.4 below, detail several different procedures for sample spiking and stressing. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

7.2.1 Drinking Water: Spiking and Chlorine-Stressing 

Drinking water samples should be spiked and stressed with chlorine as described in Sections 7.2.1.1 and 

7.2.1.2 below. 

7.2.1.1 Drinking Water Spiking 

Because finished drinking water does not typically contain the target analyte(s) of interest, finished 

drinking water should be spiked. For the evaluation of drinking water samples, a single finished drinking 

water matrix is spiked with target organism(s) from other matrices, such as non-chlorinated secondary 

sewage effluents or polluted surface waters. Sewage effluent generally has the advantage of providing a 

wide range of strains, whereas surface waters typically have the advantage of providing organisms more 

variable in quality. Depending on the study design (Section 6.0), the source of spiking suspensions (i.e., 

wastewaters) should come from geographically dispersed sites. 

To prepare spiked drinking water samples: 

(1) Collect at least five liters of each non-chlorinated secondary sewage effluents or polluted surface 

water to be used as spiking suspensions. 

(2) Perform rangefinding analyses as described above on each spiking suspension to determine target 

organism density as soon as possible after receipt of the sample to ensure that target organism 

density and diversity are not reduced. 

(3) For each spiking suspension, spike a sufficient volume of drinking water with a sufficient volume 

of spiking suspension (based on rangefinding) to obtain 103

-105 target organisms/100 mL. Please 

note: A single drinking water, with negligible concentrations of oxidants and reductants (e.g., 

chlorine and sodium thiosulfate, respectively) should be used for the entire study. Refrigerate all 

spiked drinking waters at 1°C - 4°C for use in preliminary chlorination study and comparability 

study. High oxidant or reductant levels in the drinking water could interfere with organism 

stressing. 

7.2.1.2 Preliminary Chlorination Study to Determine Appropriate Exposure Time 

Microorganisms in the spiked drinking water samples (from Section 7.2.1.1) should be stressed by 

chlorination at ambient temperatures under conditions similar to those in drinking water treatment 

facilities. The goal of chlorinating the spiked samples is to simulate drinking water treatment by reducing 

the number of organisms in the spiked drinking water samples from 103

-105 target organisms/100 mL to 

1-10 chlorine-stressed target organisms/100 mL for most probable number methods or to 20 - 100 

chlorine-stressed target organisms/100 mL for membrane filtration methods (i.e., a 2-4 log removal). 

After chlorination, no two samples are expected to produce the same levels of injured (stressed) target 

organisms because the disinfection process is impacted by physical and biological factors. These include: 

the type of spiked drinking water sample to be disinfected (e.g., spiked with sewage effluent or polluted 

source water), the initial concentration of the target organism(s), the chlorine demand of the spiked 

sample, the type and concentration of chlorinating agent, the exposure time, the sample mixing , pH, and 

temperature. As a result, a preliminary chlorination study should be performed to establish the exposure 

time necessary to reduce the number organisms in the spiked drinking water samples from 103

-105 target 

organisms/100 mL to 1-10 chlorine-stressed target organisms/100mL (or a 2-4 log removal). During this 

preliminary chlorination exposure time study, the physical and biological parameters in Section 7.1.1 

should be carefully monitored and recorded for each sample. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

The exposure time is directly dependent upon the initial concentration of the target organism present, the 

matrices’ chlorine demand, and the form of chlorine present. For testing, the spiked sample is generally 

exposed to 2.0-2.5 mg total chlorine/L for over a range of times such as 10, 20, and 30 minutes to reduce 

the density of target organisms from 103

-105 CFU/100mL to 1-10 CFU/100 mL sample. However, the 

period of exposure of a sample with a low chlorine demand may be significantly shorter than 20-30 

minutes. 

Suggested Preliminary Chlorination Study 

(1) Determine and record the total residual and free residual chlorine concentrations using an EPAapproved N, N diethyl-p-phenylenediamine (DPD) colorimetric method (e.g., Standard Method 

4500-Cl- G) initially, at midpoint, and at the end of the exposure time just prior to dechlorination. 

(2) For each exposure time, place 2 L of each spiked drinking water sample (from Section 7.2.1.1) in 

a glass container. 

(3) Add an appropriate volume of a diluted solution of reagent grade sodium hypochlorite (e.g., a 

1:20 dilution of 5% (w/v) stock solution), to achieve the desired level of chlorinating agent and 

stir the sample continuously during exposure to chlorination. If the spiked sample has an 

appreciable chlorine demand (e.g., spiked with a primary effluent or a sewage sample), add dilute 

sodium hypochlorite solution until a total residual chlorine level between 2.0 and 2.5 mg/L is 

maintained in the absence of free chlorine. If a sample has a low chlorine demand, avoid overstressing or killing the organisms by prolonged exposure to free residual chlorine. The free 

residual chlorine concentration should not exceed 0.5-1.0 mg/L. 

(4) Stop the chlorine oxidation (dechlorinate) at the end of the exposure period by adding 0.8 mL of a 

10% (w/v) sodium thiosulfate solution/L sample. 

(5) Enumerate the target organism density in an aliquot of the spiked, chlorine-stressed, 

dechlorinated drinking water sample using the appropriate EPA-approved reference method from 

Table 1-1. 

(6) For each exposure time, repeat Steps 3 - 5, above. 

7.2.1.3 Suggested Procedure for Chlorination and Dilution of Samples for Comparability Study 

(1) Determine and record the total residual and free residual chlorine concentrations initially, at 

midpoint, and at the end of the exposure time just prior to dechlorination using an EPAapproved N, N diethyl-p-phenylenediamine (DPD) colorimetric method (e.g., Standard 

Method 4500-Cl- G). 

(2) Place a sufficient volume of each spiked drinking water in a glass container to perform 

sufficient repeat analyses at multiple dilutions. 

(3) Immediately prior to chlorination, perform enumeration to determine target organism density 

as described in 7.2.1.1(2). This value should be used to determine the log reduction due to 

chlorination. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

(4) Add reagent-grade sodium hypochlorite to achieve the same concentration, as in the 

preliminary chlorination exposure study. 

(5) To reduce the density of target organisms from 103

-105 CFU/100mL to 1-10 CFU/100 mL, 

chlorinate each spiked drinking water sample for the appropriate time, based upon the 

preliminary chlorination exposure study. Stir the sample continuously during the 

chlorination. 

(6) Stop the chlorine oxidation (dechlorinate) at the end of the exposure period by adding 0.8 mL 

of a 10% (w/v) sodium thiosulfate solution/L sample. 

(7) Enumerate target organism density in an aliquot of the spiked, chlorine-stressed, 

dechlorinated drinking water sample using the appropriate EPA-approved reference method 

from Table 1-1. 

(8) Refrigerate the spiked, chlorine-stressed, dechlorinated drinking water samples at 1°C - 4°C 

for use in comparability testing. 

(9) Read the plates to determine the approximate density of the target organisms. Use these 

results to estimate the appropriate dilution necessary to reach the target organism density of 

1-10 CFU/100 mL. 

(10) Evaluate three dilutions of each spiked drinking water sample. Samples should be diluted 

with the same, original, oxidant-free and reductant-free drinking water, as necessary to reach 

a target organism density of 1-10 target organisms/100 mL for most probable number 

methods or 20-100 target organisms/100 mL for membrane filtration methods. Make the 

dilution and at least two others that bracket the target density, for example, half and double 

that dilution. One of these dilutions should contain the desired 1-10 target organisms per 100 

mL. Immediately conduct the comparability analyses with each sample using these three 

dilutions. 

Please note: For the evaluation of presence/absence methods, the data used for comparison should be 

from the dilution(s) which produces results closest to an equal number of positive and negative results for 

the reference method. A 25% to 75% split in responses (in either direction) should be sought. For 

comparability, the evaluated results for the ATP or new method should be from the same dilution as the 

reference method. If one of the dilutions does not produce an acceptable split in positive and negative 

results for the reference method, the applicant should return to the original, spiked sample. 

7.2.2 Preparation of Enumerated Spiking Suspension 

This dilution scheme is adapted from Standard Methods for the Examination of Water and Wastewater, 

19th Edition, Section 9020 B (Reference 10.3). This entire process should be performed quickly to avoid 

loss of viable organisms. There should be approximately 1010 organisms per slant. Therefore, dilution 

bottles “A” through “E,” below contain approximately 1010, 108

, 106, 104, and 103 organisms per dilution 

bottle, respectively. Depending on the growing conditions, these numbers may vary. As a result, until 

experience has been gained, more dilutions should be filtered to determine the appropriate dilution. 

Inoculate bacterial culture onto the entire surface of several nutrient agar slants with a slope 

approximately 6.3 cm long in a 125 × 16 mm screw-cap tube. Incubate for 24 ± 2 hours at 35°C ± 0.5°C. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

From the slant that has the best growth, prepare serial dilutions using four dilution bottles with 99 mL of 

sterile buffered dilution water (bottles A, B, C, and D) and one dilution bottle containing 90-mL of sterile 

buffered dilution water (bottle E). 

Pipette 1 mL of buffered dilution water from bottle “A” to one of the slants. Emulsify the growth on the 

slant by gently rubbing the bacterial film with the pipette, being careful not to tear the agar. Pipette the 

suspension back into dilution bottle “A.” Repeat this procedure a second time to remove any remaining 

growth on the agar slant, without disturbing the agar. 

Make serial dilutions as follows: 

(1) Shake bottle “A” vigorously and pipette 1 mL to bottle “B” containing 99 mL buffer 

(2) Shake bottle “B” vigorously and pipette 1 mL to bottle “C” containing 99 mL buffer 

(3) Shake bottle “C” vigorously and pipette 1 mL to bottle “D” containing 99 mL buffer 

(4) Shake bottle “D” vigorously and pipette 10 mL to bottle “E” containing 90 mL buffer; this should 

result in a final dilution of approximately 10 organisms / mL. If it is more convenient for your 

laboratory, an acceptable alternative to the dilution scheme presented for this step, is to pipette 

11 mL of dilution D into dilution bottle E, which contains 99 mL of dilution water. 

Filter 1- to 5-mL portions in triplicate from bottles “D” and “E" according to standard membrane 

filtration methods to determine the number of CFU in the dilutions. The recommended target dilution and 

spike volume depends on the method and the target analyte(s). Typically, dilutions should be stored at 

1°C to 5°C and may be used throughout the day they are prepared, however, storage conditions should be 

adjusted as necessary since both storage conditions and viability may vary from organism to organism. 

7.2.3 Log Phase Growth Curve 

Inoculate 100 mL of broth media with a single isolated colony and incubate organisms at optimum 

temperature. If possible, shake at 200 RPM during incubation. 

Take optical density (OD) measurements at 550-600 nm at 30 minute intervals for the first 8 hours and 

record readings. In addition to OD readings at 30 minute intervals, using aseptic technique, remove a 

0.1-mL portion of the culture and make a series of 1:10 dilutions in sterile buffer. Initially plate 0.1 mL 

of the 1.0, 10-1, and 10-2 dilutions in duplicate and incubate overnight at optimum temperature. Count 

colonies and record data. As the optical density increases, evaluate serial dilutions to accommodate the 

increased numbers of bacteria (i.e., when the optical density exceeds 1.0 then plate 0.1 mL from dilutions 

10-1, 10-2, and 10-3). 

Based on OD and the CFU/mL results, the OD of fresh cultures (in log-phase growth) can be used to 

determine the concentration of bacteria in the tubes by a simple graphic representation of the combined 

OD and CFU/mL results. 

Commercially available McFarland standards may be used to determine the bacterial density instead of 

actually doing a growth curve within the lab. In order to determine bacterial densities using McFarland 

standards, the OD of the standards are compared to the OD of the log phase culture. 

7.2.4 Commercially Available Enumerated Spikes 

One time use, commercially available spiking suspensions may be obtained from a variety of vendors. 

Prepare spiking suspensions according to manufacturer’s instructions. It should be noted that a different 

spiking volume than that recommended by the manufacturer may be necessary to achieve the target 

density. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

7.3 Spiking Procedures for Virus Methods 

7.3.1 Cell Monolayer Propagation 

In order to propagate virus stock suspensions cell monolayers should be propagated. The type of assay 

cytopathic effect (CPE) or plaque assay (PA), being performed determines the size of the flask and the 

number of days the monolayer incubates prior to use for the assay. Inoculate T75 or T25 flasks with 1 

x105

 cells containing cell specific growth medium. Flasks for CPE should incubate for 5-7 days at 37°C 

± 0.5°C with 95% relative humidity (and 5% CO2 concentration, if necessary). One CPE flask should be 

stained with crystal violet and microscopically checked to ensure a 95% confluent cell monolayer prior to 

use in the assay. Flasks used for PA incubate for 7-10 days under the same conditions as the monolayers 

used for CPE. PA flasks should also be checked to ensure 95% confluency before use for assay. 

7.3.2 Propagation of Virus Stock Suspension 

For (CPE) analysis, utilizing the appropriate cell line (e.g., Buffalo green monkey kidney cells; BGMK), 

inoculate the cell monolayer with virus and incubate culture flasks at 37°C ± 0.5°C until the entire 

monolayer has been destroyed by virus replication (approximately 72 - 96 hours). Freeze (to 

approximately -80°C) and thaw the flasks three times, then pool contents of the flasks and spin at 

10,000 x g for 30 minutes. Filter supernatant using a 0.2μm pore nylon filter to remove any remaining 

cell debris. The filtrate is the virus stock suspension. 

7.3.3 Titering of the Virus Stock Suspension 

Titer the virus stock suspension (from Section 7.3.2) by performing a plaque assay. Inoculate monolayers 

with 0.2 mL of serial diluted viral stock suspension. After rocking and rinsing the monolayers, add the 

agar overlay to the monolayer. Incubate the culture flasks for seven days while reading the number of 

plaques each day for the entire seven days. After the viral stock suspension has been titered, appropriate 

volumes of the suspension can be used to spike test matrices to obtain plaque forming units (PFU)/mL. 

7.4 Spiking Procedures for Cryptosporidium and Giardia 

Enumerated spiking suspensions are needed for initial and ongoing precision and recovery (IPR and 

OPR) samples (often referred to as positive controls) and matrix spike (MS) samples. Flow 

cytometer–sorted organisms are necessary for these spiking suspensions, rather than manual techniques. 

Flow cytometer–sorted spikes generally are characterized by lower variability than manually enumerated 

spikes. 

Spiking suspensions should be prepared using unstained organisms that have not been irradiated, heatfixed or formalin-fixed. Immediately before sorting spiking suspensions, initial calibration of the flow 

cytometer should be performed by conducting a series of sequential sorts directly onto membranes or well 

slides. These initial sorts should be stained and counted microscopically to verify the accuracy of the 

system. When sorting the spiking suspensions, ongoing calibration samples should also be prepared and 

counted at regular intervals. The mean of the ongoing calibration counts should be used as the estimated 

spike dose. Flow-cytometer-sorted spiking suspensions should be used by the expiration date noted on 

the suspension. Flow-cytometer-sorted spiking suspensions containing live organisms should be used 

within two weeks of the preparation date. General procedures for preparing flow-cytometer-counted 

spikes for Cryptosporidium and Giardia can be found in EPA Method 1622 and 1623 (April 2001). 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

A potential commercial source of flow-sorted Cryptosporidium and Giardia spiking suspensions is: 

Wisconsin State Laboratory of Hygiene

Flow Cytometry Unit

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. 

7.5 Analysis of Samples 

Samples should be analyzed in accordance with the EPA-approved study plan. Any deviations from the 

study plan may suggest the need for additional analyses and potentially, rejection of data generated from 

the study. If any deviations from the approved study plan are necessary prior to or during the study, the 

applicant should consult EPA and receive approval for the modification to the study plan. Deviations 

from the approved study plan should be documented in the study report (Section 9.0). 

Example analysis schemes are provided in Sections 7.5.1 and 7.5.2 below. Generally, the analysis 

schemes will differ depending on the type of organism and the type of method. The study plan (Section 

4.0) should detail the order in which samples will be analyzed. 

7.5.1 Side-by-Side Comparison Studies 

Method Blank 1

Sample 1 up to Sample 20

Method Blank 2

Positive Control

Negative Control

Method Blank 3

Media Sterility Checks

7.5.2 QC Acceptance Criteria-Based Comparison Studies 

Replicate 1 for IPR

Replicate 2 for IPR

Replicate 3 for IPR

Replicate 4 for IPR

Method Blank

Matrix Spike

Matrix Spike Duplicate

Unspiked Matrix Sample

7.6 Verification of Results 

The number of positive and negative results to be verified in a side-by-side comparison study is discussed 

in Section 6.1.3. Sections 7.6.1 to 7.6.3 below discuss the types of independent standards that may be 

used for the verification of results from bacteriological methods, virus methods, and Cryptosporidium and 

Giardia methods, respectively. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

7.6.1 Verification of Results from Bacteriological Methods 

7.6.1.1 Biochemical Tests that May be Used for Verifications 

Oxidase Citrate

Catalase Lysine decarboxylase

ONPG Methyl Red and Voges Proskauer (MRVP)

Indole Triple sugar iron (TSI)

Coagulase Lysine iron agar (LIA)

Esculin Urease

Sugars (e.g., Trehalose, lactose, mannitol, and sorbitol)

It is recommended that multiple biochemical tests be utilized to verify colony identification. The above 

list of biochemical tests is not exhaustive. The choice of which biochemical tests to use is based on type 

of organism (i.e., gram stain results) being identified/verified. Biochemical tests used for verification 

should be discussed in the study plan (Section 4.0). It may be appropriate to perform a gram stain prior to 

biochemical identification. A description of the biochemical tests listed above, as well as additional tests 

is provided in Standard Methods for the Examination of Water and Wastewater, Method 9225. 

7.6.1.2 Commercially Available Biochemical Testing Products 

Commercial biochemical test systems incorporate multiple biochemical tests to allow for identification to 

the genus and/or species level, which may be difficult when using individual biochemical tests prepared 

in house. 

Commercial biochemical test systems are available in two formats: systems that depend on the analyst to 

manually interpret the results (e.g., API strips, BBL crystal, and enterotube) and systems that automate 

the interpretation of results (e.g., Vitek and Biolog). 

7.6.2 Verification of Results from Virus Methods 

Viral protocol testing confirmations tend to be method-specific, therefore a detailed description of the 

confirmation/verification procedure should be included in the study plan and reviewed on a study-specific 

basis. 

7.6.3 Verification of Results from Cryptosporidium and Giardia Methods 

Verification of results may not be necessary for Cryptosporidium and Giardia IFA methods beyond the 

analyst’s microscopic examination of the organism. If verification is necessary, a detailed description of 

the confirmation/verification procedure should be included in the study plan and reviewed on a studyspecific basis. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

SECTION 8.0 REVIEW OF STUDY RESULTS 

Generally, upon receipt of the applicant’s data, EPA will perform the following reviews discussed in 

Sections 8.1 to 8.5. 

1. Assessment of compliance with the approved study plan 

2. Data review 

3. Data validation 

4. Development of descriptive statistics 

5. Statistical assessment of method comparability 

Methods that are deemed acceptable will generally be recommended for approval (Section 8.6). 

8.1 Assessment of Compliance with Approved Study Plan 

Generally, EPA will review the study report and associated data to ensure the study was conducted 

according to the approved study plan. The applicant should explain and justify (possibly with additional 

studies) any deviations from the study plan. Deviations from the approved study plan that occur without 

prior approval from EPA may result in the rejection of some or all study data. 

8.2 Data Review 

Upon receipt of the applicant’s data, EPA will generally verify that all raw data described in Section 9.10 

are present and complete. Generally, all calculations used in the method will be verified. This may 

include calculations used for spiking enumeration, preliminary or presumptive stages of the method, and 

the determination of the final result. 

8.3 Data Validation 

After verifying data completeness and reviewing of all calculations, EPA will generally verify that all 

measurements were performed in accordance with the method. This may include, but is not limited to, 

the following: 

• Temperature logs for incubator/waterbath/refrigerator 

• Media preparation records 

• Sample incubation times 

• Associated QC samples (as described in Section 5.0) 

• Method blanks 

• Preparation blanks 

• Sterility checks 

• Positive and negative controls 

8.4 Development of Descriptive Statistics 

8.4.1 Mean Recovery 

To determine if matrix characteristics effect method performance, mean recoveries should be calculated 

separately for each matrix and method. Mean recoveries should also be calculated for each method over 

all matrices. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

8.4.2 Precision 

Precision can be expressed both on an absolute scale (i.e., standard deviation) and on a relative scale (i.e., 

relative standard deviation). The RSD (sometimes referred to as coefficient of variation) is calculated as 

the standard deviation divided by the mean, expressed as a percent. For the purpose of summarizing the 

data, both standard deviations and RSDs should be calculated, and the one which is most appropriate for 

assessing comparability will be used in analyses. Generally, RSDs are most appropriate for summarizing 

precision when variability increases as concentration increases. 

To give an indication of the effect of multiple matrices on precision, standard deviations should be 

calculated separately for each matrix and method. Standard deviations also should be calculated for each 

method over all matrices. 

8.4.3 False Positive Rates, False Negative Rates, Sensitivity, and Specificity 

False positive (FP) and false negative (FN) rates of approved and reference methods should be evaluated 

when assessing comparability. An independent standard (described in Section 7.6) may sometimes be 

necessary to confirm positives and negatives of both the ATP or new method and the EPA reference 

method. The confirmation methods used should be discussed in the validation study plan. 

Generally, performance of the ATP or new method and EPA-approved reference methods will be defined 

in terms of false positive rates and false negative rates. For the purposes of the ATP protocol, false 

positive rates and false negative rates are equivalent to (1- Specificity) and (1-Sensitivity), respectively. 

Specificity is defined as the percent of negative samples correctly identified as negative, and sensitivity is 

defined as the percent of positive samples correctly identified as positive (see equations on the next page). 

In order to calculate estimates of false positives, false negatives, sensitivity, and specificity for each 

method, 2-by-2 tables for each matrix, and over all matrices, should be set up as follows in Table 8-1. 

Table 8-1. Standard Format for 2-by-2 Tables 

Independent Standard 

+ - Total 

ATP or New 

Method 

+ TP1 FP1 TP1 + FP1 

- FN1 TN1 FN1 + TN1 

Total TP1 + FN1 FP1 + TN1 TP1 + FP1 + TN1 + FN1 

Independent Standard 

+ - Total 

EPAApproved 

Reference 

Method 

+ TP2 FP2 TP2 + FP2 

- FN2 TN2 FN2 + TN2 

Total TP2 + FN2 FP2 + TN2 TP2 + FP2 + TN2 + FN2 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

Estimates of sensitivity, specificity, false positive rates, and false negative rates as percentages for the two 

methods should be calculated as follows: 

TPi

Sensitivityi = * 100 % 

TPi

 + FNi 

TNi 

* 100 % 

TNi

 + FPi 

Specificityi = 

FPi TNi

* 100 % = ( 1 - ) * 100 % = 1 - Specificityi

TNi + FPi TNi + FPi 

False positive ratei = 

FNi TPi

* 100 % = ( 1 - ) * 100 % = 1 - Sensitivityi

TPi + FNi TPi + FNi 

False negative ratei = 

Where F = False, N = Negative, T = True and “ i” refers to the specified method (i=1 for new method, 

i=2 for reference method) 

8.5 Statistical Assessment of Method Comparability 

8.5.1 Presence / Absence Methods 

For presence/absence methods, the chi-square test and Breslow-Day test will generally be used to 

compare the percent of false positives and false negatives in the proposed method analyses with the 

percent of false positives and false negatives in the reference method analyses. 

Generally, tests for significant differences in false positive and false negative rates between methods will 

be run using the results summarized in Table 8-1 in Section 8.4. To perform the chi-square and BreslowDay tests, it may help to first rearrange the data as follows in Tables 8-2 and 8-3: 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

Table 8-2. False Negative Rate Comparison 

Method 

New Reference Total 

Result 

True + TP1 TP2 TP1 + TP2 

False - FN1 FN2 FN1 + FN2 

Total TP1+FN1 TP2 + FN2 TP1 + TP2 + FN1 + FN2 

Table 8-3. False Positive Rate Comparison 

Method 

New Reference Total 

Result 

False + FP1 FP2 FP1 + FP2 

True - TN1 TN2 TN1 + TN2 

Total FP1 + TN1 FP2 + TN2 FP1 + FP2 + TN1 + TN2 

Before running the chi-square tests, positives and negatives should be confirmed using the independent 

standard method. The chi-square test will be used to determine whether the false positive rate and false 

negative rate have a statistically significant difference between the ATP or new method and the EPAapproved reference method. 

8.5.1.1 Assessing Method Differences (Chi-Square Test) 

In order to assess whether the false positive and false negative rates differ between methods, chi-square 

tests should be run over all matrices; additional tests in each matrix also may be necessary depending on 

the presence of matrix interactions (see Section 8.5.1.2). For false negative rates, the chi-square test(s) 

indicate whether the proportions of negative samples correctly identified as negative by the two methods 

are significantly different, and for false positive rates, the chi-square test(s) indicate whether the 

proportions of true positive results correctly identified as positive for the two methods are significantly 

different. 

8.5.1.2 Assessing Method/Matrix Interactions (Breslow-Day Test) 

In order to assess whether there are false positive and false negative rate differences between methods, it 

is also necessary to establish if there is a matrix effect on these parameters. The effect of matrices on 

false positive and false negative rate differences between methods is assessed using the Breslow-Day test. 

(Reference 10.7). The Breslow-Day test is used to test whether there is an interaction between matrix and 

method in terms of the likelihood of a false positive result (for specificity) and in terms of the likelihood 

of a false negative result (for sensitivity). If a significant interaction is found between method and matrix, 

then the chi-square tests for a difference between methods for that attribute should be done separately for 

each matrix. Otherwise, separate chi-square tests for each matrix are not necessary, and a single chisquare test can be run using the data from all matrices. 

8.5.1.3 Method Comparability Conclusions 

A decision on whether the ATP or new method is comparable to the EPA-approved reference method will 

generally be made based on the results of the false positive and false negative rate comparisons. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

Generally, the decision on acceptability of the new method should be made based on the chi-square test 

using data from all matrices. However, if the results of the Breslow-Day test indicate that there is a 

significant interaction between method and matrix for false positive and/or false negative rates, then 

further review should be made for those matrices which yielded a higher false positive and/or false 

negative rate for the proposed method. 

If the results of the chi-square tests indicate that the false positive and false negative rates of the ATP or 

new method are not significantly different from the EPA reference method, this generally will be 

interpreted as not having enough evidence to conclude that the performance of the ATP or new method is 

worse than the EPA-approved reference method. However, if the results of the chi-square test indicate 

that the false positive and/or false negative rates of the new method are significantly less than that of the 

EPA-approved reference method, this will generally be interpreted as worse performance and would lead 

to rejection of the ATP or new method. 

8.5.2 Quantitative Methods 

8.5.2.1 Testing for Normality 

Many of the statistical analyses used to assess differences in recovery and precision between methods 

require certain assumptions about the data be met. Because the validity of these assumptions affect which 

statistical tests will be used to assess recovery and precision differences, testing these assumptions is a 

prerequisite as a first step. There are two assumptions that should be evaluated prior to comparing the 

two methods statistically. One assumption is that the variability of the replicates is constant for each 

matrix and method. This assumption is discussed in Section 8.5.2.2. The other main assumption that 

should be met is that the data follow a normal distribution. 

An assessment of normality can be done either graphically or with statistical tests, or both. A normal 

distribution looks like a bell curve (i.e., symmetric around the mean). A graphical assessment can be 

done using a histogram, stem-and-leaf or Normal probability plot. Appropriate statistical tests include the 

Shapiro-Wilk test, D’Agostino test, and Kolmogorov-Smirnov test. These tests are computation 

intensive, and it may be necessary to use software (e.g., SAS) to run them. The Shapiro-Wilk test is 

generally inappropriate for testing the normality assumption of a data set with greater than 50 results. 

For environmental data (especially field data, though sometimes spiked data as well), the distribution of 

results will often be positively skewed (i.e., with a few unusually high results). A common corrective 

action of this that can be utilized is the logarithmic transformation, base e (natural log). If the data are 

positively skewed, this transformation should be attempted, assuming that none of the results are negative 

or equal to zero. When zero-valued results are present in a skewed data set, a common approach is to add 

a small constant to every result prior to transformation. However, the arbitrary choice of this constant 

will have a large effect on the transformed data (the log of 1 and the log of 0.1 are very different, for 

example), and this approach is not recommended. The use of non-parametric tests, discussed later in this 

guidance, are considered to be the appropriate alternatives. 

If no zero-valued results are present in a positively skewed data set, then the log-transformed data should 

be tested for normality. If the log-transformed data do follow a Normal distribution, then all analyses 

should be done using the log-transformed results. 

8.5.2.2 Evaluating Precision 

The decision of what statistical test to use to evaluate precision will generally be made on the results of 

the normality test described in Section 8.5.2.1. If the assumption of normality is met, then the F-test for 

differences in variance should be used. If the assumption of normality is not met, a non-parametric test 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

such as the Conover Squared-Rank test should be used. The F-test is based on comparisons of variances 

(i.e., the squared standard deviations). The Conover Squared-Rank test is based on ranking absolute 

deviations from the mean. Figure 8-1 provides a summary of the procedure for evaluating precision. 

Prior to testing whether there is a precision difference between methods, the precision of the different 

matrices should be compared for each method. 

If the results of the F-test indicate that variances differ significantly by matrix, or if the results of the 

Conover Squared-Rank test indicate that absolute deviations from the mean differ significantly by matrix, 

then comparisons of precision between methods should be done separately for each matrix. Otherwise, a 

single comparison of method precision can be done using the data from all matrices. If the results of the 

F-test indicate that there is not enough evidence to conclude that the variances differ significantly by 

matrix, then the pooled within-matrix variance (i.e., the average of the matrix variances for the given 

method) should be calculated for each method. The F-test should then be used to compare the pooled 

within-matrix variance for the two methods. If the Conover Squared-Rank test was used and there was 

not enough evidence to conclude that the mean absolute deviation differed significantly between matrices, 

then the Conover Squared-Rank test should be used to compare the mean absolute deviations from the 

overall method mean for the two methods. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

Figure 8-1. Evaluating Precision 

Compare absolute deviations from 

method mean for two methods using 

Conover squared rank test. 

Run test for 

normality 

Data follow Normal 

distribution 

Data do not follow 

Normal distribution 

Compare variances between matrices for 

each method using F-test. 

Are all 

matrices 

equal? 

Yes No 

Pool variances over 

matrices for each 

method. Compare 

pooled variances for 

two methods using the 

F-test. 

Compare 

variances between 

methods 

separately for 

each matrix using 

the F-test. 

Is the new 

method at least 

as precise for at 

least 80% of 

matrices? 

Evaluate 

Recovery 

Yes 

Failure 

No 

Is the new 

method 

significantly less 

precise? 

Yes No

Evaluate 

Recovery Failure 

Is the new 

method 

significantly less 

precise? 

No 

Failure 

Yes 

Evaluate 

Recovery 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

8.5.2.3 Evaluating Recovery 

Comparisons of mean recovery of the different methods should be done using either an analysis of 

variance (ANOVA) model or non-parametric test such as the Wilcoxon-Mann-Whitney (WMW) test. If 

the assumption of normality is not met, or if the precision differs between methods or matrices based on 

the precision evaluation described in the previous section, the WMW test should be used. If all 

assumptions are met, the ANOVA model should be used. Figure 8-2 provides a summary of the 

procedure for evaluating recovery. 

If an ANOVA model is used, method-by-matrix interactions should be tested for significance first using 

the corresponding ANOVA F-test. If the method-by-matrix interaction is significant, it would mean that 

the difference between methods is not the same for each matrix. In this case, no conclusion regarding an 

overall difference between the recovery of methods can be made. If the interaction is not significant, then 

the test for a significant difference between methods can be run in the ANOVA model, using the F-test 

for a significant method main effect. 

If the WMW test is used, between-matrix and between-laboratory variability cannot be separated from 

replicate variability. Therefore, significant interactions between method and laboratory or matrix cannot 

be tested when the WMW test is used. Instead the WMW model only tests for an overall difference 

between methods. The model can be run separately for each matrix, but the diminished power will limit 

the value of this approach. It is therefore recommended that a single WMW test be run comparing 

method recoveries, without stratifying by or controlling for matrix type. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

Figure 8.2 Evaluating Recovery 

Evaluate false 

positive and 

false negative 

rates. 

Is recovery 

significantly lower for 

the new method? 

Run test for 

normality 

Data do not follow 

Normal distribution 

Failure 

Run ANOVA model 

testing for method 

difference, controlling 

for matrix. 

No interaction Interaction 

Yes No

Run single WMW test 

to compare method 

recovery, disregarding 

matrices. 

Is recovery 

significantly lower for 

the new method? 

Failure 

Yes No

Evaluate false 

positive and 

false negative 

rates. 

Evaluate false 

positive and 

false negative 

rates. 

Failure 

Yes No

Run ANOVA model 

testing for method 

difference separately 

for each matrix. Count 

number of matrices 

where the new method 

is equal to or 

significantly better. 

Is recovery 

significantly 

lower for the new 

method for at least 20% 

of all matricies? 

Data follow Normal 

distribution 

Using analysis of 

variance 

(ANOVA), test for 

method/matrix 

interaction. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

8.5.2.4 False Positive Rates, False Negative Rates, Sensitivity, and Specificity 

Comparisons of false positive rates, false negative rates, sensitivity, and specificity should be conducted 

according to the methods described in Sections 8.5.1.1 and 8.5.1.2. 

8.5.2.5 Method Comparability Conclusions 

If there is not enough evidence to conclude that there is a significant interaction between method and 

matrix for recovery, or if there is not enough evidence to conclude that there is a statistically significant 

difference in precision between matrices for either method, then it can be concluded that any such 

differences of the method will be consistent. Therefore, lack of sufficient evidence to conclude a 

significant difference between methods will generally be interpreted as equal or better performance, and 

might lead to acceptance of the ATP or new method, pending review of false positive and false negative 

rates. However, a statistically significant test (worse) statistic will be interpreted as worse performance of 

the ATP or new method, and would generally lead to rejection of the new method. 

In cases where significant interactions between matrix and method are found when assessing recovery, or 

in cases where differences in precision between matrices for at least one method (and therefore a 

comparison of method precision could not be done over all matrices), some judgment will be necessary in 

deciding whether the proposed method should be deemed acceptable. The decision should be based on 

the attribute (i.e., recovery or precision) for each matrix. As a general rule, if there was not enough 

evidence to conclude that the new method was similar or better than the EPA-approved reference method 

for that attribute for at least 80% of the matrices used in the study, then the new method can generally be 

recommended for approval, pending review of false positive and false negative rates. 

If there is not enough evidence to conclude that the new method is worse than the EPA-approved 

reference method for at least 80% of the matrices for both precision and recovery, the false negative rate 

and false positive rate should next be compared, based on the methods described in sections 8.5.1.1 and 

8.5.1.2. If the results of the chi-square test indicate that there is not enough evidence to conclude that the 

false negative rate or false positive rate of the ATP or new method is worse than that of the EPAapproved reference method, this will be interpreted as equal or better performance and might lead to 

acceptance of the new methods. However, if the results of the chi-square test indicate that the false 

negative rate or false positive rate of the new method is significantly greater than that of the EPAapproved reference method, this will generally be interpreted as worse performance for 100% of the 

matrices used in the study, and would lead to rejection of the ATP or new method. 

For example, suppose side-by-side testing is done in one laboratory for ten different matrices. For 

recovery, there is not enough evidence to conclude that the new method is significantly worse than the 

EPA-approved reference method for eight of the ten matrices. For precision, there is not enough evidence 

to conclude a significant matrix effect, and there is not enough evidence to conclude a significant 

difference between methods. There is not enough evidence to conclude the false negative rate or false 

positive rate of the ATP or new method is worse than the EPA-approved reference method. Under these 

circumstances, the ATP or new method may be recommended for approval. However, if there is not 

enough evidence to conclude that the new method is not worse than the approved method for only seven 

of ten matrices for recovery, then the ATP or new method will not, generally, be recommended for 

approval. 

8.5.3 QC Acceptance Criteria-Based Comparison Studies 

For methods with QC acceptance criteria, necessary calculations and QC criteria are typically provided in 

the method. Generally, QC criteria will include both recovery and precision specifications. Recovery 

criteria take the form of a recovery interval for either a single result or mean of multiple results. Precision 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

criteria will generally be either a maximum standard deviation, RSD, or RPD. Details on calculations that 

may be necessary for the comparisons are described below. 

8.5.3.1 Recovery 

Recovery results will generally be necessary for data spiked into both reagent water (IPR, IDC, etc.) and 

source water (MS, MSD, etc.). For reagent water, percent recovery should be calculated as follows: 

Result 

% Recovery = * 100 % 

Spike 

where Result is the amount recovered from the sample after spiking, and 

Spike is the estimated amount spiked into the sample. 

For source water, percent recovery should be calculated as follows: 

Result - Background

% Recovery = * 100 % 

Spike 

where Result is the amount recovered from the sample after spiking, 

Spike is the estimated amount spiked into the sample, and 

Background is the estimated background amount measured in the sample prior to spiking.

For some QC criteria, it may be necessary to calculate the mean of the sample recoveries prior to 

comparing the results to the criteria. Generally, this will be specified with that given method. 

8.5.3.2 Precision 

Calculation of an RSD, RPD, or standard deviation is generally necessary for precision criteria. For 

source water data, the calculation should be based on the recovered amount, rather than the percent 

recovery. This is because the calculated percentage will be inflated if the background amount is large 

compared to the total amount recovered. For reagent water-spiked data, where there the background 

count is zero, the calculation can be made either using the recovered amount or the percent recovery. 

Where the precision criteria is to be based on two results, the precision criterion will generally be an 

RPD. The equation for an RPD is given below: 

*Amount1 - Amount2 *

RPD = * 100 %

(Amount1 + Amount2) / 2

Where Amount1 and Amount2 are the two recovered amounts. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

Where the precision criterion is to be based on more than two results, the precision criterion will 

generally be either a standard deviation or RSD. An RSD is calculated based on the equation below: 

SD

RSD = * 100 % 

Mean 

Where SD is the standard deviation of all recovered amounts, and 

Mean is the mean of all recovered amounts. 

8.5.3.3 Presence/Absence Criteria 

QC criteria for presence/absence methods may be defined as false positive and/or false negative rates, 

sensitivity and/or specificity, or as a specified proportion of positive and/or negative results out of a given 

number of samples. All calculations should be specific to the given situation and should be defined 

explicitly therein. 

8.6 Method Recommendation and Approval 

Generally, after completion of the technical and statistical reviews for nationwide-use applications, the 

Director of Analytical Methods, Attn: ATP Program Coordinator (see Table 2-1 and Appendix E) will 

prepare a recommendation for approval/disapproval of the ATP or new method and notify the applicant of 

the recommendation. 

If the data evaluation demonstrates that the applicant’s method performs at least as well as the EPAapproved reference method, the Director of Analytical Methods, Attn: ATP Program Coordinator will 

generally recommend approval to the Office of Ground Water and Drinking Water (OGWDW) or 

appropriate office, which begins the regulation development process. Regulation development includes a 

Federal Register notice proposing to approve an ATP, public comment on the proposed method, and 

(depending on public comment) a final rule published in the Federal Register that approves the method. 

Generally, the regulation development process may take one year or more. 

For limited-use ambient water or wastewater applications the Regional Administrator (see Table 2-1 and 

Appendix E) will generally prepare a recommendation of approval/disapproval of the ATP or new method 

and notify the applicant of the recommendation after completion of the technical and statistical reviews. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

SECTION 9.0 STUDY REPORT 

Laboratories or other organizations responsible for developing ATPs or new methods should document 

the results of the side-by-side comparison study or QC acceptance criteria-based comparison study in a 

formal study report that contains the elements described in this section. 

The information and supporting data included in the study report should be sufficient to enable EPA to 

evaluate the performance of the ATP or new method and make a decision on whether it is comparable to 

the reference method. The applicant is responsible for ensuring that all method-specified criteria are met 

by the laboratory(ies) involved in the study and that the study report contains all data from the 

laboratory(ies). A copy of all comparison study data should be maintained at the participant 

laboratory(ies) or other organization responsible for developing the ATP or new method. 

Like the study plan developed and approved by EPA before the study was performed, the study report 

contains background information and describes the study design. However, the study report also details 

the process and results of the study, provides an analysis and discussion of the results, and presents study 

conclusions. The approved study report should also identify and discuss any deviations from the study 

plan that were made in implementing the study, and the study plan should be appended to and referenced 

in the study report. Significant deviations from the study plan without prior EPA approval could result in 

the rejection of the study data. 

The study report should be organized into the following sections: 

• Background 

• Study Objectives and Design 

• Study Implementation 

• Data Reporting and Validation 

• Results 

• Data Analysis and Discussion 

• Conclusions 

• Appendix A - Method 

• Appendix B - Study Plan 

• Appendix C - Supporting Data 

• Appendix D - Supporting References 

Details on the information that should be included in each of these sections are provided below, in 

Sections 9.1 through 9.11. 

9.1 Background 

This section of the study report should describe the ATP or new method that was tested, and identify the 

organization responsible for developing the ATP or new method. The background section of the study 

report should include the following information: 

• A method summary 

• The organization, method number, and title for the ATP or new method 

• The method number or title and publication number (given in 40 CFR parts 136, 141, and 405 - 503) 

for the EPA-approved reference method that is being used for demonstrating method comparability 

(i.e., the reference method) 

• A description of the nature of the ATP (e.g., alternate media, alternate concentration technique, etc.) 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

• The matrices, matrix types, and/or media to which the ATP or new method is believed to be 

applicable 

• The analyte(s) measured by the ATP or new method, including corresponding CAS Registry or other 

identification numbers, when available 

9.2 Study Objectives and Design 

This section of the study report should identify the overall objectives and data quality objectives of the 

study and briefly describe the study design. This information should be consistent with the study 

objectives and design specified in the approved study plan. Any study limitations should be identified. 

The approved study plan should be appended to the study report. 

9.3 Study Implementation 

This section of the study report should describe the methodology and approach undertaken in the study. 

This section should include the following information: 

• The organization that was responsible for managing the study 

• The laboratories, facilities, and other organizations that participated in the study; describe how 

participating laboratories were selected; and explain the role of each organization involved in the 

study 

• The type of study performed (i.e., side-by-side or QC acceptance criteria-based comparison study) 

• The study schedule that was followed 

• A brief description of how sample matrices were chosen, including, for QC acceptance criteria-based 

comparison studies, a statement of compliance with recommendations for matrix type selection 

• A description of any preliminary testing conducted prior to the side-by-side or QC acceptance 

criteria-based comparison study (e.g., method validation, physical and chemical assessment of the 

matrices, preliminary range-finding analyses) 

• The numbers and types of analyses performed by the participating laboratories 

• A description of how samples were collected, distributed, and stored 

• The source and strain of the organism used for sample spiking 

• If spikes were quantified, a description of how estimated true spike values were determined and 

provide all supporting data 

• The type of water used in the preparation of sample dilutions if not specified by the method (e.g., 

reagent water, phosphate buffered water, phosphate buffered saline, etc.) 

• Any problems encountered with samples, spiking organisms, equipment, etc. and their subsequent 

resolution 

• Any communications with EPA relevant to the study, such as clarification of the study design or 

approved changes to the study plan 

• Any deviations from the study plan and their impact on study performance and/or results 

9.4 Data Reporting and Validation 

This section of the study report should describe the procedures that were used to report and validate study 

data. While EPA generally does not use a standard format for analytical data submission, a list of 

necessary data elements and an example bench sheet may be found in Appendix D of this document. 

9.5 Results 

This section of the study report presents the study results. Results may be presented in a summary table 

that lists the recovery or concentration of each sample, by test method, laboratory, and matrix. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

For QC acceptance criteria-based comparison studies, results should indicate the QC test associated with 

each sample (e.g. IPR, method blank, MS/MSD, unspiked matrix sample). Raw data and example 

calculations should be submitted, and should be included in an appendix to the study report (see Section 

9.10.1). 

9.6 Data Analysis and Discussion 

This section of the study report provides a statistical analysis and discussion of the study results. 

Recovery, precision, false positive rates, false negative rates, specificity, and sensitivity, as appropriate, 

should be calculated by test method, laboratory, and matrix, and summarized in a tabular format that 

includes the mean, standard deviation, and relative standard deviation. The discussion should address any 

discrepancies between the results and comparability guidelines, or, for QC acceptance criteria-based 

comparison studies, any discrepancies between the results and the QC acceptance criteria of the EPAapproved reference method. 

9.7 Conclusions 

This section of the study report should describe the conclusions drawn from the study based on the data 

analysis discussion. The section should contain a statement(s) regarding achievement of the study 

objective(s). 

9.8 Appendix A - Method 

The ATP or new method, prepared in accordance with EPA's Guidelines and Format document (Section 

3.0 and Reference 10.14), should be appended to the study report. 

9.9 Appendix B - Study Plan 

The study plan approved by EPA (Section 4.0) should be appended to the study report. 

9.10 Appendix C - Supporting Data 

The study report should be accompanied by raw data, quality control information, and example 

calculations that support the summary results presented in the report. 

9.10.1 Raw Data 

Appendix C of the study report should include sufficient raw data so that an independent reviewer can 

verify each determination and calculation performed by the laboratory or the study coordinator. This 

verification consists of tracing all steps of the method to the final result reported. The raw data are, 

generally, method-specific and may include but are not limited to the following: 

• Sample numbers or other identifiers used by the laboratory 

• Sample collection dates and times 

• Verification that method-specified QC procedures were met for test samples and all associated QC 

samples 

• Analysis dates and times for all steps in the method 

• Sample volume 

• Any measurements of ancillary parameters (i.e., temperature, pH, turbidity, percent solids, etc.) 

• Results for all intermediate steps in the method 

• Preliminary data steps to determine the final result 

• Final result 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

• If appropriate, quantitation reports, data system outputs, and other data to link the raw data to the 

results reported 

• Laboratory bench sheets and copies of all pertinent logbook pages for all sample preparation and 

cleanup steps, and for all other parts of the determination 

• Temperature logs for waterbaths, incubators, refrigerators, etc. 

• Media preparation information 

• If appropriate, direct instrument readouts and other data to support the final results 

Raw data are generally needed for all samples, positive and negative controls, sterility checks, 

verifications, blanks, matrix spikes and duplicates, and other QC analyses specified in the EPA-approved 

reference method. Data should be organized so that a microbiologist can clearly understand how the 

analyses were performed. The names, titles, addresses, and telephone numbers of the analysts who 

performed the analyses and of the quality assurance officer who verified the analyses should be provided. 

For instruments involving data systems, raw data on magnetic tape or disk should be made available on 

request. 

9.10.2 Electronic Data Reporting 

In addition to the hard copy raw data, applicants should also submit data in electronic format (Excel 

spreadsheet, or equivalent) so that EPA can to create a database of study results. EPA anticipates that this 

database will facilitate automated review and statistical analysis of study results. The information 

included in electronic format may include: laboratory, analyst, method, sample type, sample number, date 

and time of analysis, volume analyzed, replicate number, raw data, and calculated results. The applicant 

should discuss an appropriate electronic format with EPA prior to data submission. 

9.10.3 Example Calculations 

Generally, the study report should provide example calculations that will allow the data reviewer to 

determine how the laboratory used the raw data to arrive at the final results. Useful examples include 

both detected analytes and undetected analytes. If the laboratory or the method employs a standardized 

reporting level for undetected analytes, this should be made clear in the example, as should adjustments 

for sample volume, etc. 

9.11 Appendix D - Supporting References 

Hard copies of all references and supporting documentation for the ATP or new method should be 

attached to the study report as an appendix. The list of references may contain links to web sites, or 

documents available on-line. However, a hard copy should be submitted with the final study report. 

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EPA Microbiological Alternate Test Procedure (ATP) Protocol 

SECTION 10.0 REFERENCES 

10.1 AOAC. 1999. Qualitative and Quantitative Microbiology Guidelines for Methods Validation, 

Journal of AOAC International, Vol. 82, No. 2. 

10.2 APHA. 1998. Standard Methods for the Examination of Water and Wastewater. 20th Edition. 

American Public Health Association. 1015 15th Street, NW, Washington, DC 20005. 

10.3 APHA. 1995. Standard Methods for the Examination of Water and Wastewater. 19th Edition. 

American Public Health Association. 1015 15th Street, NW, Washington, DC 20005. 

10.4 APHA. 1992. Standard Methods for the Examination of Water and Wastewater. 18th Edition. 

American Public Health Association. 1015 15th Street, NW, Washington, DC 20005. 

10.5 ASTM. 1999. D4855-91: Standard Practice for Comparing Test Methods, ASTM Standards on 

Precision and Bias for Various Applications. 1999 Annual Book of ASTM Standards: Water and 

Environmental Technology, Volume 7.02. 100 Barr Harbor Drive, West Conshohocken, PA 

19428. 

10.6 ATCC. http://www.atcc.org 

10.7 Fleiss, J. F. Statistical Methods for Rates and Proportions, 2nd ed., John Wiley & Sons, New 

York, NY. 

10.8 ISO. 2001. CD17994. Water Quality - Criteria for the Establishment of Equivalence Between 

Microbiological Methods, Final Version, June 15, 2001. 

10.9 National Environmental Laboratory Accreditation Conference. 2001. National Environmental 

Laboratory Accreditation Conference: Quality Systems. Approved May 25, 2001, effective July 

1, 2003 unless otherwise noted. Appendix D - Essential Quality Control Requirements, section 

D.3, pp D-15 to D-16. 

10.10 Title 40, Code of Federal Regulations, Sections 136.4, 136.5, and 141.27. 

10.11 USEPA. 2001. EPA Requirements for Quality Management Plans. USEPA Office of 

Environmental Information. EPA/240/B-01/002. 

10.12 USEPA. 1999. Environmental Regulations and Technology Control of Pathogens in Vector 

Attraction in Sewage Sludge. USEPA Office of Research and Development. EPA/625/R-92/013, 

revised October 1999. 

10.13 USEPA. 1997. Manual for the Certification of Laboratories Analyzing Drinking Water: Criteria 

and Procedures Quality Assurance, Fourth Edition. USEPA Office of Ground Water and 

Drinking Water. EPA-815-B-97-001. 

10.14 USEPA. 1996. Guidelines and Format for Methods to be Proposed at 40 CFR Part 136 or Part 

141. USEPA Office of Science and Technology. EPA-821-B-96-003. 

10-1
EPA Microbiological Alternate Test Procedure (ATP) Protocol 

10.15 USEPA. 1995a. Presence/Absence Membrane Filter Methods for Finished Waters, USEPA 

Protocol for Alternate Test Procedures for Coliform Bacteria in Compliance with Drinking Water 

Regulations, Version 1.2, December 1995. USEPA Office of Research and Development, 

Cincinnati, OH. 

10.16 USEPA. 1995b. Quantitative Membrane Filter Methods, USEPA Protocol for Alternate Test 

Procedures for Coliform Bacteria in Compliance with Water and Wastewater Regulations, 

Version 1.0, December 1995. USEPA Office of Research and Development. Cincinnati, OH. 

10.17 USEPA. 1995c. Presence/Absence Liquid Culture Methods for Finished Waters, USEPA 

Protocol for Alternate Test Procedures for Coliform Bacteria in Compliance with Drinking Water 

Regulations, Version 1.2, December 1995. USEPA Office of Research and Development. 

Cincinnati, OH. 

10.18 USEPA 1989. Memorandum: Analytical Methods for Compliance and Limited Alternate Test 

Procedures Approvals. December 27, 1989. 

10.19 USEPA. 1979. Handbook for Analytical Quality Control in Water and Wastewater Laboratories. 

EPA-600/4-79-019. Environmental Monitoring and Support Laboratory, Cincinnati, OH. March 

1979. 

10-2
APPENDIX A

GLOSSARY
APPENDIX A: GLOSSARY 

40 CFR part 136—Title 40, part 136 of the Code of Federal Regulations. This part specifies 

approved test procedures for the analysis of pollutants regulated under the Clean Water Act. 

40 CFR part 141—Title 40, part 141 of the Code of Federal Regulations. This part specifies 

EPA’s National Primary Drinking Water Regulations pursuant to the Safe Drinking Water Act; 

Subpart C of 40 CFR part 141 lists analytical methods required for monitoring under the Act. 

95% confidence interval— A statistical level indicating a 95% probability that the parameter 

variable is enclosed within the given data interval. 

A Acceptable version— An acceptable version is a method that is either identical to the approved 

B 

C 

method or exercises the flexibility explicitly allowed in the method. See “minor modification.” 

Accuracy— The degree of agreement between an observed value and an accepted reference 

value. Accuracy includes random error (precision) and systematic error (recovery) that are 

caused by sampling and analysis. 

Aliquot— A representative portion of a sample. 

Ambient water— Ambient water refers to any fresh, marine, or estuarine surface water used for 

recreation; propagation of fish, shellfish, or wildlife; agriculture; industry; navigation; or as 

source water for drinking water facilities. 

Analysis of variance (ANOVA)— A study of the effect of a set of qualitative variables on a 

quantitative response variable, based on a decomposition of the variance of the response variable. 

Analyte— The target organism or class of organisms that are measured by the method. 

Analyte of concern— An analyte designated by EPA to adversely affect or have the potential to 

adversely affect human health, the environment, aesthetics, or the senses. Analytes of concern 

are listed in approved methods. 

Approved method— A testing procedure (analytical method) promulgated at 40 CFR parts 136, 

141, 405-500, and other parts of the CFR that support EPA's water programs. 

Average percent recovery— The average of the recovery, expressed as percent. See recovery. 

Bias— A systematic or persistent distortion of a measurement process that deprives the result of 

representativeness; i.e., the expected sample measurement is different than the sample’s true 

value. A data quality indicator. (QAMS) 

Blank— See "method blank." 

Bulk sample— A large sample that is aliquoted into smaller volumes prior to analyses. 

Calibration— The process of establishing the relationship between the concentration or amount 

of material introduced into an instrument or measurement process and the output signal. 

Calibration verification— Means of establishing that the instrument performance remains 

within pre-established limits. 
D 

Code of Federal Regulations— A codification of the general and permanent rules published in 

the Federal Register by the Executive departments and agencies of the Federal Government. 

Comparability test— See side-by-side comparison. 

Confidence interval— The numerical interval constructed around a point estimated of a 

population parameter, combined with a probability statement (the confidence coefficient) linking 

it to the population’s true parameter value. If the same confidence interval construction technique 

and assumptions are used to calculate future intervals, they will include the unknown population 

parameter with the same specified probability. (EMMC) 

Confirmed counts— Organism counts that have been verified to ensure proper identification. 

Conover Squared-Rank test— A nonparametric test for equality of variability, based on 

the joint squared ranks of deviations from the means. (SPRENT) 

Contract laboratory— Private, academic, or commercial laboratory under contract to EPA or 

other organization to perform testing. 

D’Agostino test— A statistical test for determining whether a given set of results follow a normal 

or log-normal distribution. Best used for datasets with at least 50 results. (GILBERT) 

Data quality objective— Qualitative and/or quantitative statement of the overall level of 

uncertainty that a decision-maker is willing to accept in results or decisions derived from 

environmental data. Data quality objectives provide the statistical framework for planning and 

managing environmental data operations consistent with the data user's needs. (EMMC) 

Determinative technique— The physical and/or chemical process by which measurement of the 

identity and concentration of an analyte is made. 

Differential medium— A solid culture medium that makes it easier to distinguish colonies of the 

target organism. 

Dilution/rinse water blank— An aliquot of dilution/rinse water that is treated exactly like a 

sample and carried through all portions of the procedure until determined to be negative or 

positive. The dilution/rinse water blank is used to determine if the sample has become 

contaminated by the introduction of a foreign microorganism through poor technique. 

Discharge—Generally, any spilling, leaking, pumping, pouring, emitting, emptying or dumping 

(40 CFR 109.2; 110.1; 116.3); also, see "discharge of a pollutant" (40 CFR 122.2); the medium 

that is spilled, leaked, pumped, poured, emitted, emptied, or dumped. 

Discharge of pollutant— Any addition of any pollutant or combination of pollutants to (1) 

waters of the U.S. from any point source or (2) to the waters of the contiguous zone or the ocean 

from any point source other than a vessel or other floating craft which is being used as a means of 

transportation (40 CFR 122.2; 401.11) 

Duplicate— A second sample collected from the same sampling point at the same time the 

original sample is collected and analyzed exactly like the original sample. Duplicate samples can 

be used as a measure of sample variability. 

Effluent— A medium that flows out of a point source, e.g., the discharge from a sewage 

treatment plant. 

E 
F 

G 

H 

I 

Enrichment— Using a culture medium to enhance growth of the target organism prior to 

isolation of that organism. 

Explicit flexibility— Modifications that are explicitly allowed in an approved method. 

F distribution— A type of sampling distribution for a random variable. The ratio of 2 chi-square 

distributions, each divided by their respective degrees of freedom. 

F-test— In an Analysis of Variance, a test for the equality of factor level means (such as for 

different methods) or for the presence of an interaction between two factors (such as method and 

matrix). (RICE) (ASTM) 

Facility— A plant or group of plants within a single location that is regulated under the CWA 

and/or SDWA. A single facility may have multiple water supplies, discharges, waste streams, or 

other environmental media that are subject to compliance monitoring. For example, a single 

facility within the Pulp, Paper, and Paperboard industrial category may have a direct discharge, 

an indirect discharge, and an in-process waste stream, all of which are subject to compliance 

monitoring. 

False negative— A target organism incorrectly identified as a non-target organism or not 

identified at all using the method of interest. 

False positive— A non-target organism incorrectly identified as the target organism using the 

method of interest. 

False negative error rate— The proportion of target organisms incorrectly identified as a nontarget organism or not identified at all using the method of interest, equal to (1 - Sensitivity). In 

statistical testing, the rate at which one falsely accepts a statistical hypothesis (such as that a 

difference between methods does not exist) based on a statistical test, when the hypothesis is 

actually false. Abbreviated as $, and also referred to as the Type II error rate. (ASTM) 

False positive error rate— The proportion of non-target organisms incorrectly identified as the 

target organism using the method of interest, equal to (1 - Specificity). In statistical testing, the 

rate at which one falsely rejects a statistical hypothesis (such as that a difference between 

methods does not exist) based on a statistical test, when the hypothesis is actually true. 

Abbreviated as ", and also referred to as the Type I error rate. (ASTM) 

Federal Register— A daily publication that provides a uniform system for publishing 

Presidential and Federal agency documents. Documents published in the Federal Register make 

changes to the CFR to keep the CFR current. (OFR) 

Guidelines and Format— The document titled Guidelines and Format for Methods to be 

Proposed at 40 CFR Parts 136 and 141; available from the National Technical Information 

Service (NTIS), U.S. Department of Commerce, Springfield, Virginia, 22161 (703-487-4600) as 

NTIS publication PB96-210448. 

Histogram— A bar diagram of the distribution of a set of analytical results. The range of values 

is categorized into sets of subintervals, or bins, and the number of results within each bin are 

displayed as the height of the bars. (BERRY) 

Industrial category— A category listed in 40 CFR parts 405-503. 

Industrial subcategory— A subcategory defined at 40 CFR parts 405-503. 

Initial demonstration of capability— A test performed to establish the ability to demonstrate 

control over the analytical system and to demonstrate acceptable performance. 
J 

K 

L 

M 

Initial precision and recovery— The analysis of a minimum of four spiked reagent water 

samples under the same conditions as will be used for analysis of environmental samples. The 

IPR is used to demonstrate that a laboratory is able to produce reliable results with the method 

prior to analysis of environmental samples. 

Interaction— The situation where the effect of one variable (such as method type) on a 

dependent variable (such as recovery) is affected by the value of a third variable (such as matrix). 

Interference— A positive or negative effect on a measurement caused by a substance other than 

the one being investigated. (QAD) 

Interlaboratory— Occurring in multiple laboratories. 

Intralaboratory— Occurring within a single laboratory. 

Kolmogorov-Smirnov test— A statistical test for determining whether a given set of 

results follow a normal distribution, or any other specified distribution. (GILBERT) 

Limited use— Use of a method by a single regulated entity or laboratory for analysis of one or 

more matrix types. 

Log-normal— A distribution of a random variable X such that the natural logarithm of X is 

normally distributed. 

Log-phase— Bacterial growth phase in which the logarithm of the bacterial biomass increases 

linearly with time. 

Main effect— Situation where a variable (such as method type) has a consistent effect on a 

dependent variable (such as recovery). 

Matrix— The component or substrate that contains the analytes of interest. 

Matrix effect— Variability in the analytical performance of a method that can be attributed to 

the type of sample analyzed. 

Matrix spike— A sample prepared by adding a known mass of target analyte to a specified 

amount of a sample matrix for which an independent estimate of target analyte concentration is 

available. A matrix spike is used, for example, to determine the effect of the matrix on a 

method’s recovery efficiency. (QAMS) 

Matrix spike duplicate— A replicate of the matrix spike to test precision. The MS/MSD are 

used in combination to test the precision of an analysis. (QAD) 

Matrix type— A sample medium with common characteristics across a given industrial category 

or subcategory. For example, C-stage effluents from chlorine bleach mills, effluent from the 

continuous casting subcategory of the iron and steel industrial category, POTW sludge, and inprocess streams in the Atlantic and Gulf Coast Hand-shucked Oyster Processing subcategory are 

each a matrix type. For the purposes of this initiative all drinking waters constitute a single 

matrix type. 

May— This action, activity, or procedural step is neither required nor prohibited. 

May not— This action, activity, or procedural step is prohibited. 
N 

Measurement quality— Critical level which, if exceeded, is considered to append objective 

additional, and possibly unacceptable, measurement uncertainty to the corresponding data. 

Method— A body of procedures and techniques for performing a task (e.g. sampling, 

characterization, quantitation) systematically presented in the order in which they are to be 

executed. (QAD) 

Method blank— An aliquot of reagent water or designated matrix 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. 

Method-defined analyte— An analyte without a specific, known composition where the 

analytical result depends totally on the measurement procedure. 

Method modification— A change made to an approved method. 

Method validation— A process by which a laboratory or vendor establishes the performance of 

a new method or substantiates the performance of a method modification. 

Methods and Criteria— The document titled: Analysis of Pollutants in Municipal Water and 

Industrial Wastewater: Test Procedures and Quality Control Acceptance Criteria; available from 

the National Technical Information Service (NTIS), U.S. Department of Commerce, Springfield, 

Virginia, 22161 (703-487-4600) as NTIS publication PB96-210463, and incorporated by 

reference into this part. 

Mid-point response factor— The response factor at the concentration at which calibration is 

verified. 

Minor modification— A modified method that has been reviewed by EPA and has been 

determined to be technically equivalent to a method approved for use in compliance monitoring. 

A minor modification employs the same chemistry and/or biological principles as the approved 

method to determine the presence/absence or to quantify the amount of the target organism in a 

sample. Supporting data may be necessary to demonstrate that a minor modification will yield 

results equivalent to those obtained using the approved method but does not require approval as 

an alternate test procedure through proposal and promulgation in the Federal Register. 

Modified method— An approved method that has been modified to change a front-end 

technique. EPA will judge a modified method to be: 1) an acceptable version or minor 

modification of a previously promulgated method, which does not require approval as an ATP or 

2) a significantly different method which requires an application for an ATP approval. 

Nationwide use— Use of a method by all regulated entities and laboratories for analysis of one 

or more matrix types. 

Navigable waters— All waters of the United States, including the territorial seas. (40 CFR 

110.1) 

Negative control— A non-target organism processed to ensure the laboratories are familiar with 

the identification of the target organism and to ensure that confirmation test results are 

appropriate. 
O 

P 

New method— A method that employs a determinative technique for an analyte of concern that 

differs from determinative techniques employed for that analyte in methods previously approved 

at 40 CFR part 136 or 141. 

Nonparametric— A type of statistical analysis for which no assumptions about the underlying 

distribution of the data are necessary. 

Non-selective media— An enrichment media that allows most bacteria to grow. 

Normal probability plot— A graphical depiction of the distribution of a set of analytical results. 

A normal probability plot is a scatterplot depicting the observed results compared to the expected 

results based on a normal distribution. If the observed data follow a normal distribution, the 

graph will display a line at a 45 degree angle from the x-axis. Also known as a Q-Q plot. 

Ongoing demonstration of capability— The laboratory needs to demonstrate that the analytical 

system is in control on an ongoing basis through the analysis of ODC samples (positive 

control/positive control duplicate). 

Ongoing precision and recovery— A reagent water sample 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 within the method for 

precision and recovery. 

Other approved methods— Promulgated methods that are not designated as a reference method, 

but continue to carry the same regulatory status. 

Physical phase— The physical phase of a sample matrix (e.g., air, water, soil). 

Positive control— A target organism that is analyzed to ensure that the laboratory is performing 

the method acceptably and that the media is providing appropriate results. 

Power— The probability that a statistical test will conclude that a difference (for example, 

between methods) exists, when a difference truly does exist. Equal to 1 - $. 

Precision— The degree to which a set of observations or measurements of the same property, 

usually obtained under similar conditions, conform to themselves; a data quality indicator. 

Precision is usually expressed as standard deviation, variance, or range, in either absolute or 

relative terms. 

The precision obtainable from an environmental measurement method may be estimated from 

replicate analyses of subsamples taken from the same (homogenous) sample. Generally speaking, 

the more carefully one executes the various steps of a method and controls the variables affecting 

the method’s capability, the more precise will be the results. The use of a nonhomogeneous 

sample will compound the precision estimate with the sample variability. 

Preparation— Processing performed on a sample prior to analysis, e.g. extraction, concentration, 

cleanup, etc. 

Presumptive counts— Numbers of organisms based on results that have not been confirmed or 

verified. 

Procedures— A set of systematic instructions for performing an activity. (QAD) 
Q 

R 

Promulgated method— A method that has been published or incorporated by reference into 40 

CFR parts 136, 141, 405-500, or other parts that support EPA's water programs (i.e., an approved 

method). 

Promulgation— Publication of a final rule in the FR. 

Public water system (PWS)— A system for the provision to the public of piped water for human 

consumption, if such system has at least fifteen service connections or regularly serves an average 

of at least twenty-five individuals daily at least 60 days out of the year. Such term includes (1) 

any collection, treatment, storage, and distribution facilities under control of the operator of such 

system and used primarily in connection with such system, and (2) any collection or pretreatment 

storage facilities not under such control which are used primarily in connection with such system. 

A public water system is either a “community water system” or a “noncommunity water system.” 

Quality assurance — An integrated system of activities involving planning, quality control, 

quality assessment, reporting, and quality improvement to ensure that a product or service meets 

defined standards of quality with a stated level of confidence. (QAD) 

Quality control — The overall system of technical activities whose purpose is to measure and 

control the quality of a product or service so that it meets the needs of users. The aim is to 

provide quality that is satisfactory, adequate, dependable, and economical. (QAD) 

QC acceptance criteria— Performance specifications developed from validation data and used 

to control the limits within which an analytical method is operated. 

QC acceptance criteria-based comparison study— A study performed to evaluate the 

performance of a modified method against the quality control acceptance criteria of a reference 

method. 

Range finding— Preliminary analyses conducted to assess the ambient concentration of the 

target organism in a matrix to be used in a study or preliminary analyses of spiked samples 

involving few, if any, replicates, to assess method performance to identify the spike dose to be 

used in a study. 

Raw data— Data that have not been processed. 

Reagent water— Water conforming to Specification D 1193, Annual Book of ASTM Standards, 

or specifications in Standard Methods 9020B.4.d. 

Recovery— The total amount of the analyte found in the sample divided by the amount of the 

analyte added into the sample as a spike. 

Reference method— A method that serves as a standard against which method modifications can 

be compared. 

Regulated entity— Permittees, PWSs, POTWs, and other entities responsible for compliance 

with provisions of the CWA or SDWA. 
S 

Relative percent difference (RPD)— An estimate of the variability of two numbers expressed in 

relative terms. Calculated as the absolute value of the difference of the two numbers, divided by 

their mean: 

A B − RPD = 1 *100% 

2 *( A B + )

Equal to the relative standard deviation of the two numbers multiplied by the square root of 2. 

Relative standard deviation (RSD)— The standard deviation expressed as a percentage of the 

mean (100F/X); i.e., the coefficient of variation. 

Replicate— Multiple samples collected from the same sampling point at the same time and 

analyzed exactly the same way. Replicate samples can be used as a measure of sample variability. 

Sample matrix— See "matrix." 

Sample preparation technique— Any technique in the analytical process that precedes the 

determinative technique, including all procedures, equipment, solvents, etc. that are used in the 

preparation and cleanup of a sample for analysis. Sample preparation techniques do not include 

conditions and/or procedures for the collection, preservation, shipment, and storage of the sample. 

Sensitivity— In presence/absence testing, sensitivity is the proportion of target organisms in the 

sample that were correctly detected by the method of interest. 

Shapiro-Wilk test— A statistical test for determining whether a given set of results follow a 

normal or log-normal distribution. Best used for datasets with at most 50 results. (GILBERT) 

Side-by-side comparison— Parallel testing of a new or modified method and a reference method 

to determine whether the performance of the new or modified method is acceptable compared to 

the reference method. 

Specificity— In presence/absence testing, the proportion of non-target organisms in the sample 

that were correctly identified as not being the target organism by the method of interest. 

Spike— The process of adding a known amount of target analyte to a sample; used to determine 

the recovery efficiency of the method. (QAD) 

Spiking suspension— Diluted stock suspension containing the organism(s) of interest at a 

concentration appropriate for spiking samples. 

Standard deviation (F)— The measure of the dispersion of observed values expressed as the 

positive square root of the sum of the squares of the difference between the individual values of a 

set and the arithmetic mean of the set, divided by one less than the number of values in the set. 

For a total of n numbers: 

SD = n 

X ean i 

i 

n 

− − =

∑ 1 

1 

2 

1 

( M )

Statistical power— See power. 
T 

U 

V 

Stem-and-leaf plot— A graphical depiction of a set of analytical results, that conveys 

information about the shape of the distribution while retaining the numerical information. The 

stem-and-leaf plot separates the digits of the values as leaves (the last digit of the values) and 

stems (the remaining digit of the values). The individual results are grouped according to the 

stems, and the leaves are listed separately in a format similar to a histogram. (RICE) 

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 

organisms. 

Study plan— A study design submitted for EPA review, comment, and approval prior to 

conducting the side-by-side or QC acceptance criteria-based method comparability study. This 

process protects the applicant by providing written approval of the study design before resources 

are spent to conduct the study. Data from studies conducted without EPA review and approval 

may not adequately address the applicant’s study objectives. A detailed procedure for the ATP or 

new method should be included as an attachment to the study plan. EPA will evaluate the study 

plan to verify that the appropriate data quality objectives identified in this protocol are defined 

and addressed. EPA comments are incorporated into the study design and this review process is 

repeated until EPA has approved the study design. 

Study report— A formal report developed by laboratories or other organizations responsible for 

developing ATPs or new methods documenting the results of the side-by-side or QC acceptance 

criteria-based method comparability study. The information and supporting data needed in the 

study report should be sufficient to enable EPA to evaluate the performance of the ATP or new 

method and make a decision on whether it is comparable or superior to the reference method. 

The approved study report should also identify and discuss any deviations from the study plan 

that were made in implementing the study, and the approved study plan should be appended to 

and referenced in the study report. 

Summary results— Overall study statistics (not sample-specific results). 

Target organism— The organism the method is designed to detect. 

Validate— Reliably assess the performance (bias and precision) of a method in a reference 

matrix (such as reagent water) and the matrix in which the validated method will be used (such as 

drinking water, surface water, or municipal wastewater effluent). 

Validation, single-laboratory— Assessment of method performance (see “validate”) in one 

laboratory. 

Validation, interlaboratory— Assessment of method performance (see “validate”) at multiple 

laboratories. 

Variance— A measure of the dispersion of a set of values. The sum of the squares of the 

difference between the individual values of a set and the arithmetic mean of the set, divided by 

one less than the number of values in the set. (The square of the sample standard deviation) 

(QAD) 
Wilcoxon-Mann-Whitney (WMW) test— A nonparametric analysis used to determine whether 

the medians from two levels of a given factor (such as method) differ from each other, based on 

the ranks of all results. (SPRENT) 

W 

X 

Y 

Z 

The above definitions are referenced to the following organizations: 

ASTM ASTM D 4855-91. Standard Practice for Comparing Test Methods. 

BERRY Berry, D. A. and B. Lindgren. Statistics Theory and Methods. Wardsworth, 

Belmonst, CA, 1990. 

EMMC Environmental Monitoring Management Council 

GILBERT Gilbert, R. O. Statistical Methods for Environmental Pollution Monitoring. Van 

Nostrand Reinhold, New York, 1987. 

NELAC QS National Environmental Laboratory Accreditation Conference, Quality Systems 

OFR Office of Federal Register 

QAD Quality Assurance Division, National Center for Environmental Research and 

Quality Assurance, Office of Research and Development, USEPA 

RICE Rice, J. A. Mathematical Statistics and Data Analysis, Second Edition. 

Wadsworth, Belmont, CA, 1995. 

SPRENT Sprent, P. Applied Nonparametric Statistical Methods, Second Edition. Chapman 

& Hall, London, 1993. 
APPENDIX B

ATP APPLICATION FORM
EPA Office of Water 

Alternate Test Procedure or New Method Preliminary Application Form 

for Microbiological Analytes 

Applicant Name and 

Address: 

EPA Use Only 

ATP Case Number: 

Date Application Submitted: 

Type of Application:

Circle appropriate application 

type 

Alternate Test Procedure New Method 

Method Number 

Title of Method 

Revision Date 

EPA-Approved Reference 

Method: 

Analyte(s): 

Applicable Matrices:

Circle all that apply Ambient Water Drinking Water Wastewater 

Level of Use: Limited Use Nationwide 

Study Design:

Circle appropriate study design Side-by-Side Comparison Study QC Acceptance Criteria-Based 

Comparison Study 

FOR LIMITED USE APPLICATIONS ONLY 

ID Number of Existing or 

Pending Permit: 

Issuing Agency: 

Type of Permit: 

Discharge Serial Number: 

Attachments 

Check all that apply 

G Justification for ATP 

G Alternate Test Procedure or New Method (in standard EPA format) 

G Method Comparison Table 

G Study Plan 

G Other: 

Submit Application and Attachments in Triplicate 

Biosolids 
EPA Office of Water 

Alternate Test Procedure or New Method Final Application Form 

for Microbiological Analytes 

Applicant Name and Address: 

EPA Use Only 

ATP Case Number: 

Date Application Submitted: 

Type of Application:

Circle appropriate application 

type 

Alternate Test Procedure 

Method Number 

Title of Method 

Revision Date 

EPA-Approved Reference 

Method: 

Analyte(s): 

Applicable Matrices:

Circle all that apply Ambient Water Drinking Water Wastewater 

Level of Use: Limited Use Nationwide 

Study Design:

Circle appropriate study design Side-by-Side Comparison Study QC Acceptance Criteria-Based 

Comparison Study 

FOR LIMITED USE APPLICATIONS ONLY 

ID Number of Existing or 

Pending Permit: 

Issuing Agency: 

Type of Permit: 

Discharge Serial Number: 

Attachments 

Check all that apply 

G Justification for ATP 

New Method 

Biosolids 

G Alternate Test Procedure or New Method (in standard EPA format)

G Method Comparison Table

G Study Plan

G Study Report

G Other: 

Submit Application and Attachments in Triplicate 
APPENDIX C

APPLICATION INVENTORY FORM
1. Completed application form. 

Includes the name and address of the applicant; the date of submission of the application; 

the method number, title, and revision date; the EPA-approved reference method; the 

analyte(s) for which the ATP or new method is proposed; the type of application; applicable 

matrices; study design; level of use; NPDES permit information, if applicable; and the 

attachments submitted with the application. 

Section 2.2 

Appendix C G

2. Justification for ATP or new method. 

Brief justification for why the ATP or new method is being proposed. Section 2.3 G

3. Method in EPA format. 

Scope and application Section 3.1 G

Summary of method Section 3.2 G

Definitions of method Section 3.3 G

Interferences Section 3.4 G

Safety Section 3.5 G

Equipment and supplies Section 3.6 G

Reagents and standards Section 3.7 G

Sample collection, preservation, and storage Section 3.8 G

Quality control Section 3.9 G

Calibration and standardization Section 3.10 G

Procedure Section 3.11 G

Data analysis and calculations Section 3.12 G

Method performance Section 3.13 G

Pollution prevention Section 3.14 G

Waste management Section 3.15 G

References Section 3.16 G

Tables, diagrams, flowcharts, and validation data Section 3.17 G

4. Method comparison table. 

A two-column table comparing the proposed ATP or new method with the EPA-approved 

reference method. This table should include the number and title of each method, the 

latest revision date of the ATP or new method, and a detailed discussion of each of the 

method sections listed in Section 3.0. Each topic should be discussed in a separate row of 

the table and the applicant should highlight any differences between the ATP or new 

method and the EPA-approved reference method. 

Section 2.5 G
5. Study plan. 

Background Section 4.1 G

Objectives Section 4.2 G

Study design Section 4.3 G

Coordination Section 4.4 G

Data reporting Section 4.5 G

6. Study report. 

Background Section 9.1 G

Study objectives and design Section 9.2 G

Study implementation Section 9.3 G

Data reporting and validation Section 9.4 G

Results Section 9.5 G

Data analysis and discussion Section 9.6 G

Conclusions Section 9.7 G

Appendix A: Method Section 9.8 G

Appendix B: Approved study plan Section 9.9 G

Appendix C: Supporting data Section 9.10 G

Appendix D: Supporting references Section 9.11 G
APPENDIX D

DATA ELEMENTS AND EXAMPLE BENCH SHEETS
Data Elements 

The data elements listed below should be reported on the bench sheets or in the lab notebook for each 

method, as applicable. EPA will review the information during the data validation process to ensure the 

method-specific QC measures are met, as agreed to in the approved study plan. 

• Laboratory name 

• Method number 

• Media 

• Procedure 

• Matrix 

• Sample collection date/time 

• Dates and times for all method steps associated with holding times or incubation times 

• Analyst initials for each processing step in the method 

• Presumptive results for all applicable media 

• Confirmed/completed results for all applicable media 

• All measured volumes 

• Dilution information 

• Final result per units of measurement 

Example Benchsheets 

Example bench sheets for the following EPA-approved reference methods are included in this appendix: 

• Aeromonas (USEPA Method 1605) 

• Cryptosporidium (USEPA Methods 1622 and 1623) 

• E. coli (SM 9221F, SM 9222G) 

• Enterococci (SM 9230C) 

• Fecal coliforms (SM 9221E, 9222D) 

• Fecal streptococcus (SM 9230B, SM 9230C) 

• Giardia (USEPA Method 1623) 

• Total coliforms (SM 9221B, SM 9222B) 

Note: Additional example bench sheets or electronic copies of the attached bench sheets are available 

upon request. 
Multiple-Tube Fermentation: Total Coliform, Fecal Coliform, E. coli 

(SM 9221B, SM 9221E, SM 9221F) 

Laboratory: Sample Collection Time: Sample Collection Date: Sampling Point: 

Analyst 

Initials Read Temp 

10 

mL 

1.0 

mL 

0.1 

mL 

0.01 

mL 

0.001 

mL 

Analyst 

Initials Read Temp 

10 

mL 

1.0 

mL 

0.1 

mL 

0.01 

mL 

0.001 

mL 

Analyst 

Initials 

24 hr fecal read 

(From 24 hr LTB) 

24 hr fecal read 

(From 48 hr LTB) 24 hr E. coli read 

(From 24 hr LTB) 

LTB start 

24 hr E. coli read 

(From 48 hr LTB) 

LTB 24 hr read 

Final tube combination: Total coliforms/100 mL: 

Fecal tube combination: E. coli tube combination: 

Analyst 

Initials Read Temp 

10 

mL 

1.0 

mL 

0.1 

mL 

0.01 

mL 

0.001 

mL 

Analyst 

Initials 

BGB 48 hr read (from 48 hr LTB) 

24 hr fecal read 

(From 24 hr LTB) 

24 hr fecal read 

(From 48 hr LTB) 24 hr E. coli read 

(From 24 hr LTB) 

Analyst 

Initials Read Temp 

10 

mL 

1.0 

mL 

0.1 

mL 

0.01 

mL 

0.001 

mL 

24 hr E. coli read 

(From 48 hr LTB) 

Final tube combination: Total coliforms/100 mL: Fecal tube combination: 

E. coli tube combination: 

Analyst 

Initials Read Temp 

10 

mL 

1.0 

mL 

0.1 

mL 

0.01 

mL 

0.001 

mL 

Analyst 

Initials Read Temp 

10 

mL 

1.0 

mL 

0.1 

mL 

0.01 

mL 

0.001 

mL 

Analyst 

Initials 

24 hr fecal read 

(From 24 hr LTB) 

24 hr fecal read 

(From 48 hr LTB) 24 hr E. coli read 

(From 24 hr LTB) 

24 hr E. coli read 

(From 48 hr LTB) 

Final tube combination: Total coliforms/100 mL: Fecal tube combination: 

E. coli tube combination: 

Comments: 24 hr / 48 hr 

BGB read 

(From 48 hr LTB) 

LTB: Replicate 3 BGB: Replicate 3 LTB: Replicate 2 

24 hr / 48 hr 

LTB read 

BGB: Replicate 2 

24 hr / 48 hr 

BGB read 

(From 24 hr LTB) 

24 hr / 48 hr 

BGB read 

(From 24 hr LTB) 

LTB: Replicate 1 BGB: Replicate 1 E

24 hr / 48 hr 

LTB read 

24 hr / 48 hr 

BGB read 

(From 48 hr LTB) 

Please enter date and time for the following: 

LTB 48 hr read, BGB 24 hr read (from 24 hr LTB), ECMUG 24 hr read (from 24 hr LTB) 

E

24 hr / 48 hr 

BGB read 

(From 24 hr LTB) 

E

BGB 48 hr read (from 24 hr LTB), BGB 24 hr read (from 48 hr LTB), ECMUG 24 hr read (from 48 hr LTB) 

24 hr / 48 hr 

BGB read 

(From 48 hr LTB) 

24 hr / 48 hr 

LTB read 
Membrane Filtration: mEndo/NA-MUG 

(SM 9222B/SM 9222G) 

Laboratory: Sample collection time: 

Sample collection date: Sampling point: 

mEndo incubation start temperature (o

C): mEndo incubation start date/time: 

mEndo incubation end temperature (o

C): mEndo incubation end date/time: 

NA-MUG incubation start temperature (o

C): NA-MUG incubation start date/time: 

NA-MUG incubation end temperature (o

C): NA-MUG incubation end date/time: 

Replicate number 1 

Analyst initials Sample volume 

filtered 

No. colonies 

per filter 

Total Coliforms 

per 100 mL 

E. coli 

per 100 mL 

100 mL 

10.0 mL 

1.0 mL 

0.1 mL 

Replicate number 2 

Analyst initials Sample volume 

filtered 

No. colonies 

per filter 

Total Coliforms 

per 100 mL 

E. coli 

per 100 mL 

100 mL 

10.0 mL 

1.0 mL 

0.1 mL 

Replicate number 3 

Analyst initials Sample volume 

filtered 

No. colonies 

per filter 

Total Coliforms 

per 100 mL 

E. coli 

per 100 mL 

100 mL 

10.0 mL 

1.0 mL 

0.1 mL 

Total coliforms E. coli

E. coli 

No. colonies 

per filter 

Total coliforms 

E. coli 

No. colonies 

per filter 

No. colonies 

per filter 

Total coliforms 
Membrane Filtration: mFC/NA-MUG 

(SM 9222D/SM 9222G) 

Laboratory: Sample collection time: 

Sample collection date: Sampling point: 

mFC incubation start temperature (o

C): mFC incubation start date/time: 

mFC incubation end temperature (o

C): mFC incubation end date/time: 

NA-MUG incubation start temperature (o

C): NA-MUG incubation start date/time: 

NA-MUG incubation end temperature (o

C): NA-MUG incubation end date/time: 

Replicate number 1 

Analyst initials Sample volume 

filtered 

No. colonies 

per filter 

Fecal coliforms 

per 100 mL 

E. coli 

per 100 mL 

100 mL 

10.0 mL 

1.0 mL 

0.1 mL 

Replicate number 2 

Analyst initials Sample volume 

filtered 

No. colonies 

per filter 

Fecal coliforms 

per 100 mL 

E. coli 

per 100 mL 

100 mL 

10.0 mL 

1.0 mL 

0.1 mL 

Replicate number 3 

Analyst initials Sample volume 

filtered 

No. colonies 

per filter 

Fecal coliforms 

per 100 mL 

E. coli 

per 100 mL 

100 mL 

10.0 mL 

1.0 mL 

0.1 mL 

E. coli 

No. colonies 

per filter 

No. colonies 

per filter 

Fecal coliforms 

E. coli 

No. colonies 

per filter 

Fecal coliforms 

Fecal coliforms E. coli
E. coli Membrane Filtration: mTEC 

(EPA 1103.1, SM 9213D) 

Laboratory: Sample collection time: 

Sample collection date: Sampling point: 

mTEC incubation start temperature (o

C): mTEC incubation start date/time: 

mTEC incubation end temperature (o

C): mTEC incubation end date/time: 

Urease substrate incubation start date/time: Urease substrate incubation end date/time: 

Replicate number 1 

Analyst initials 

Replicate number 2 

Analyst initials 

Replicate number 3 

Analyst initials 

E. coli 

E. coli 

Sample volume filtered 

100 mL 

10.0 mL 

1.0 mL 

10.0 mL 

1.0 mL 

100 mL 

10.0 mL 

1.0 mL 

0.1 mL 

E. coli 

per 100 mL 

Sample volume filtered No. colonies 

per filter 

E. coli 

per 100 mL 

E. coli 

Sample volume filtered No. colonies 

per filter 

E. coli 

per 100 mL 

100 mL 

0.1 mL 

0.1 mL 

No. colonies 

per filter 
E. coli Membrane Filtration: Modified mTEC 

(EPA 1603) 

Laboratory: Sample collection time: 

Sample collection date: Sampling point: 

modified mTEC incubation start temperature (o

C): modified mTEC incubation start date/time: 

modified mTEC incubation end temperature (o

C): modified mTEC incubation end date/time: 

Replicate number 1 

Analyst initials 

Replicate number 2 

Analyst initials 

Replicate number 3 

Analyst initials 

100 mL 

E. coli 

E. coli 

0.1 mL 

100 mL 

10.0 mL 

1.0 mL 

10.0 mL 

1.0 mL 

E.coli 

per 100 mL 

E.coli 

per 100 mL 

No. colonies 

per filter 

1.0 mL 

0.1 mL 

10.0 mL 

Sample Volume filtered No. colonies 

per filter 

E.coli 

per 100 mL 

E. coli 

100 mL 

No. colonies 

per filter 

0.1 mL 

Sample Volume filtered 

Sample Volume filtered 
 Multiple-Tube Fermentation: Fecal streptococcus 

(SM 9230B) 

Laboratory: Sample Collection time: Sample Collection Date: Sampling point: 

10 

mL 

1.0 

mL 

0.1 

mL 

0.01 

mL 

0.001 

mL 

10 

mL 

1.0 

mL 

0.1 

mL 

0.01 

mL 

0.001 

mL 

24 hr ADB read 

24 hr BEA read 

from 24 hr ADB 

48 hr ADB read 

24 hr BEA read 

from 48 hr ADB 

Final tube combination: Fecal streptococcus/100 mL: 

10 

mL 

1.0 

mL 

0.1 

mL 

0.01 

mL 

0.001 

mL 

10 

mL 

1.0 

mL 

0.1 

mL 

0.01 

mL 

0.001 

mL 

24 hr ADB read 

24 hr BEA read 

from 24 hr ADB 

48 hr ADB read 

24 hr BEA read 

from 48 hr ADB 

Final tube combination: Fecal streptococcus/100 mL: 

Replicate Number 2 

BEA Plates 

Analyst 

Initials 

Replicate Number 1 

Comments: 

Azide Dextrose Broth (ADB) 

Analyst 

Initials 

Analyst 

Initials 

Comments: 

Azide Dextrose Broth (ADB) 

Analyst 

Initials 

BEA Plates 

10 

mL 

1.0 

mL 

0.1 

mL 

0.01 

mL 

0.001 

mL 

10 

mL 

1.0 

mL 

0.1 

mL 

0.01 

mL 

0.001 

mL 

24 hr ADB read 

24 hr BEA read 

from 24 hr ADB 

48 hr ADB read 

24 hr BEA read 

from 48 hr ADB 

Final tube combination: Fecal streptococcus/100 mL: 

BEA Plates 

Comments: 

Azide Dextrose Broth (ADB) 

Analyst 

Initials 

Analyst 

Initials 

Replicate Number 3 

ADB incubation start dateStart temp: ADB incubation end date/Start temp: End temp: Start temp: End temp: BEA incubation end date/(From 48 hr ADB) End temp: BEA incubation start date(From 24 hr ADB) BEA incubation end date/(From 24 hr ADB) BEA incubation start date(From 48 hr ADB) 
 Membrane Filtration: Fecal Streptococcus 

(SM 9230C) 

Laboratory: Sample collection time: 

Sample collection date: Sampling point: 

mEnterococcus incubation start temperature ( oC): mEnterococcus incubation start date/time: 

mEnterococcus incubation end temperature ( o

C): mEnterococcus incubation end date/time: 

Replicate number 1 

Analyst initials 

Replicate number 2 

Analyst initials 

Replicate number 3 

Analyst initials 

Fecal streptococcus 

Fecal streptococcus 

Fecal streptococcus 

1.0 mL 

0.1 mL 

Sample volume filtered 

100 mL 

10.0 mL 

100 mL 

10.0 mL 

1.0 mL 

0.1 mL 

Sample volume filtered 

0.1 mL 

Sample volume filtered 

100 mL 

10.0 mL 

1.0 mL 

No. colonies per filter Fecal streptococcus 

per 100 mL 

Fecal streptococcus 

per 100 mL 

Fecal streptococcus 

per 100 mL 

No. colonies per filter 

No. colonies per filter 
 Membrane Filtration: Enterococcus (mE-EIA) 

(EPA 1106.1, SM 9230C) 

Laboratory: Sample collection time: 

Sample collection date: Sampling point: 

mE incubation start temperature ( o

C): mE incubation start date/time: 

mE incubation end temperature (o

C): mE incubation end date/time: 

EIA incubation start temperature ( o

C): EIA incubation start date/time: 

EIA incubation end temperature (o

C): EIA incubation end date/time: 

Replicate number 1 

Analyst initials 

Replicate number 2 

Analyst initials 

Replicate number 3 

Analyst initials 

Enterococcus 

Enterococcus 

Enterococcus 

0.1 mL 

0.1 mL 

Volume filtered 

100 mL 

10.0 mL 

0.1 mL 

Volume filtered 

1.0 mL 

Enterococci 

per 100 mL 

100 mL 

10.0 mL 

1.0 mL 

No. colonies 

per filter 

Enterococci 

per 100 mL 

Enterococci 

per 100 mL 

No. colonies 

per filter Volume filtered 

100 mL 

No. colonies 

per filter 

10.0 mL 

1.0 mL 
Membrane Filtration: Enterococcus (mEI) 

(EPA 1600) 

Laboratory: Sample collection time: 

Sample collection date: Sampling point: 

mEI incubation start temperature (o

C): mEI incubation start date/time: 

mEI incubation end temperature (o

C): mEI incubation end date/time: 

Replicate number 1 

Analyst initials 

Replicate number 2 

Analyst initials 

Replicate number 3 

Analyst initials 

Enterococcus 

0.1 mL 

100 mL 

10.0 mL 

1.0 mL 

0.1 mL 

Enterococcus 

Volume filtered No. colonies 

per filter 

Enterococcus 

per 100 mL 

100 mL 

10.0 mL 

Volume filtered 

100 mL 

10.0 mL 

1.0 mL 

No. colonies 

per filter 

Enterococcus 

per 100 mL 

Enterococcus 

per 100 mL 

No. colonies 

per filter 

Enterococcus 

Volume filtered 

1.0 mL 

0.1 mL 
Batch-specific Cover Sheet:

Method 1605 (Aeromonas) - ADA with Vancomycin

Note: Please complete one sheet per week of analysis. 

Laboratory name: 

Section 1. Media Preparation Information 

1 Kovac's expiration date 

2 Oxidase dry slides expiration date 

3 Date of nutrient agar plate preparation 

4 Date of nutrient agar slant preparation 

5 

Date nutrient agar slants for positive 

controls and matrix spikes were inoculated 

6 

Time nutrient agar slants for positive 

controls and matrix spikes were inoculated 

Section 2: Sample Processing Information 

7 Dilution preparation date (for IDC, ODC, and MS/MSD samples) 

8 Dilution preparation time (for IDC, ODC, and MS/MSD samples) 

9 Analyst preparing dilutions for IDC, ODC, and MS/MSD samples) 

10 Sample spiking date 

11 Sample spiking time 

12 Analyst spiking samples 

13 Analyst performing filtration 

14 Funnel decontamination method 

Section 3: Sample Analysis Information 

15 Incubator temperature at start date oC 

16 Incubator temperature at read date oC 

16 ADA-V incubation start date / time 

17 ADA-V read date / time 

18 ADA-V read analyst 

19 Nutrient agar plate incubation start date / time 

20 Nutrient agar plate read date / time 

21 Nutrient agar plate read analyst 

22 Oxidase confirmation date / time 

23 Oxidase read analyst 

24 Trehalose incubation start date / time 

25 Trehalose read date / time 

26 Trehalose read analyst 

27 Tryptone (indole) incubation start date / time 

28 Tryptone (indole) read date / time 

29 Tryptone (indole) read analyst 
QC Checklist:

Method 1605 (Aeromonas) - ADA with Vanomycin

Note: Please complete one sheet per week of analysis. Please circle the appropriate response and provide supporting information as 

necessary. 

Laboratory name: 

Batch identification: 

1 

Did all method blanks (dilution/rinse water) exhibit the appropriate response? Yes No

If no, please list contaminated method blank(s) and associated samples. 

2 

Did ADA-V media sterility check exhibit the appropriate response? Yes No

If no, please explain. 

3 

Did nutrient agar plate media sterility check exhibit the appropriate response? Yes No 

If no, please explain. 

4 

Did nutrient agar slant media sterility check exhibit the appropriate response? Yes No 

If no, please explain. 

5 

Did trehalose media sterility check exhibit the appropriate response? Yes No 

If no, please explain. 

6 

Did tryptone media sterility check exhibit the appropriate response? Yes No

If no, please explain. 

7 

Did the unspiked reagent water sample exhibit the appropriate response? Yes No 

If no, please explain. 

8 

Did negative controls for oxidase, trehalose, and indoleexhibit the appropriate responses? Yes No 

If no, please explain. 
1

23456789

10

Sample-specific Data Report Form: 

Method 1605 (Aeromonas) - ADA With Vanomycin 

Laboratory name: 

Section 1: Sample information 

1 Sample number: 5 Volume (mL) of spike (QC samples only): 2 Utility: 6 Volume filtered (mL): 3 Sampling point: 7 Dilution bottle (for laboratory-prepared QC samples only, plea D 

sterility check, direct streak, streak with filter reagent, finished, dilution/rinse 

4 QC Analysis or Matrix filtered (please circle one): 8 If this report form is for a method blank, please indicate samples that are associated with this method blank: 

Please note: It is important to record the number of colonies for each presumptively positive morphological type so that the final density of Aeromonas per sample can be reported based on percent co

Section 2: Sample results 

ADA-V colony description 

(this section is optional) 

No. of presumptive 

positive colonies for 

this colony type 

No. of presumptive colonies 

submitted to confirmation 

for this colony type 

Nutrient agar colony description 

(this section is optional) 

No. oxidase 

positive 

per colony type 

Color Morphology* size (mm) Color Morphology* size (mm) 

pale yellow 1358 2-4 mm 60 2 off-white 1358 2-4 mm 2 of 2 

dark yellow 1358 1-3 mm 5 1 off-white 1358 1-2 mm 1 of 1 

*Morphology choices (list all that apply) : (1) Round, (2) oval, (3) symmetric, (4) asymmetric, (5) shiny, (6) dull, (7) translucent, (8) opaque, (9) grainy, (10) fuzzy, (11) other 

Section 3: Calculations (Use one row for each presumptive positive colony color and morphology. If more than five colony types, please attach another sheet.) 

A 

No. presumptive positive colonies 

for each colony type 

B 

No. of each colony type 

submitted to confirmation 

C 

How many submitted colonies per 

colony type confirmed (oxidase 

positive, ferments trehalose, and 

produces indole)? 

A * (C/B) = D 

Calculated no. of 

confirmed Aeromonas per colony type 

D1 + D2 + 

Total confirmed Ae

60 2 1 D1= 30 

30 + 

5 1 1 D2= 5 

D3= D4= D5= 

Section 5: Comments 
1

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10

Sample-specific Data Report Form: 

Method 1605 (Aeromonas) - ADA With Vanomycin

Laboratory name: 

Section 1: Sample information 

1 Sample number: 5 Volume (mL) of spike (QC samples only): 2 Utility: 6 Volume filtered (mL): 3 Sampling point: 7 Dilution bottle (for laboratory-prepared QC samples only, please circle one): D 

sterility check, direct streak, streak with filter reagent, finished, dilution/rinse 

4 QC Analysis or Matrix filtered (please circle one): 8 If this report form is for a method blank, please indicate samples that are associated with this method blank: 

Please note: It is important to record the number of colonies for each presumptively positive morphological type so that the final density ofAeromonas per sample can be reported based on percent confirmatio

Section 2: Sample results 

ADA-V colony description 

(this section is optional) No. of presumptive positive colonies for 

this colony type 

No. of presumptive colonies 

submitted to confirmation 

for this colony type 

Nutrient agar colony description 

(this section is optional) 

No. oxidase 

positive 

per colony ty

Color Morphology* size (mm) Color Morphology* size (mm) 

*Morphology choices(list all that apply): (1) Round, (2) oval, (3) symmetric, (4) asymmetric, (5) shiny, (6) dull, (7) translucent, (8) opaque, (9) grainy, (10) fuzzy, (11) other 

Section 3: Calculations (Use one row for each presumptive positive colony color and morphology. If more than five colony types, please attach another sheet.) 

A 

No. presumptive positive colonies 

for each colony type 

B 

No. of each colony type 

submitted to 

confirmation 

C 

How many submitted colonies per 

colony type confirmed (oxidase 

positive, ferments trehalose, and 

produces indole)? 

A * (C/B) = D 

Calculated no. of 

confirmed Aeromonas per colony type 

D1 + D2 + D

Total confirmed Aer

D1= D2= D3= D4= D5= 

Section 5: Comments 
Method 1622/23 Bench Sheet 

Laboratory name: Laboratory ID: 

1. Client sample number 

2. Internal laboratory sample ID (if applicable) 

3. Date and time of sample receipt 

4. Received by 

5. Temperature of sample and condition of sample upon arrival 

6. Storage location and storage temperature 

7. Sample turbidity, in NTU 

8. Sample type (IPR, method blank, field sample, OPR, MS, PT sample) 

9. Spiking suspension number (for IPR, OPR, MS, and PT samples only) 

10. Estimated number of oocysts/cysts spiked (for IPR, OPR, MS, and PT samples only) Crypto Giardia 

11. Spiking date and time 

12. Sample volume spiked, in L 

13. Sample filtration start date and time 

14. Type of filter used (Envirochek, Envirochek HV, FiltaMax, CrypTest, other [specify]) and lot number: 

15. Name of analyst performing filtration 

16. Sample volume filtered, to nearest 1⁄4 L (do not include rinse volume) 

17. Did filter clog? 

18. Elution date and time (must be performed within 96 hours of sample collection/filtration) 

These steps must be completed in one working day 

19. Elution procedure: 9 wrist shaker 9 FiltaMax wash station 9 stomacher 9 backflush/sonication 

20. Name of analyst performing elution 

21. Elution buffer:_________________________ Elution buffer lot number and expiration date: 

22. Concentration procedure (centrifugation, FiltaMax concentrator, other [specify]) 

23. Name of analyst performing concentration 

24. Pellet volume after concentration, in mL 

25. (a) Total volume of resuspended concentrate; (b) volume transferred to IMS (in mL) (a) (b) 

26. Number of subsamples processed independently through the remainder of the method 

27. IMS system used (Dynal anti-Cryptosporidium, Dynal GC-Combo, other [specify]) and lot number 

27. Name of analyst performing IMS procedure 

28. Slide(s) used (Meridian, Dynal, other [specify]) and lot number 

29. Date and time sample applied to slide(s) to dry (must be completed same working day as Row 18) 

30. Detection kit used (Merifluor, AquaGlo, Crypt-a-Glo, Giardi-a-Glo, other [specify]) and lot number 

31. Analyst performing staining procedure 

32. Staining completion date and time (must be complete within 72 hours of Row 29) 

33. Total number of oocysts and cysts counted in sample (sum of counts in subsamples, if applicable) Crypto Giardia 

Comments: 

HV=high volume IPR=initial precision and recovery 

June 15, 2001, Revision - Draft NTU=nephelometric turbidity unit OPR=ongoing precision and recover PT=performance test 

MS=matrix spike 
1

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Method 1622/1623 Cryptosporidium Report Form 

Laboratory name: Laboratory ID: Client sample number: Internal laboratory sample ID (if applicable): 10-mL subsample ID (if packed pellet > 0.5 mL): Volume examined (in L) on this slide: Analyst: Positive staining control acceptable 9 YES 

Negative staining control acceptable 9 YES 

Object 

located 

by FA 

No. 

Shape 

(oval 

or 

round) 

Size 

L x W 

(Fm) 

DAPI - DAPI + D.I

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) 

Total FA number from this slide: 

Examination completion date: Examination completion time (must be complete within 7 

DAPI -: Total number (A): D.I.C. - Total number of empty oocysts (D): 

DAPI +: Total number (B): D.I.C. - Total number of oocysts with amorphous structure 

DAPI +: Total number (C): D.I.C. - Total number of oocysts with internal structure (F)

Total count DAPI + (C) that show structure by D.I.C. (F): 

June 15, 2001, Revision - Draft 
1

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Method 1623 Giardia Report Form 

Laboratory name: Laboratory ID (if applicable): Client sample number: Internal laboratory sample ID (if applicable): 

10-mL subsample ID (if packed pellet > 0.5 mL): Volume examined (in L) on this slide: Analyst: Pos. staining control acceptable 9 YES 

Neg. staining control acceptable 9 YES 

Object 

located 

by FA 

No. 

Shape 

(oval 

or 

round) 

Size 

L x W 

(Fm) 

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 cysts 

(D) 

Cysts with 

amorphous 

structure 

(E) 

Cysts wi

Number of 

nuclei 

Total FA number from this slide: 

Examination completion date: Examination completion time (must be complete within 7 days of s

DAPI-: Total number (A): D.I.C.: Total number of empty cysts (D): DAPI+: Total number (B): D.I.C.: Total number of cysts with amorphous structure (E): 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): D.I.C.: Total number of cysts with >one internal structure (F): 

June 15, 2001, Revision - Draft 
APPENDIX E

EPA HEADQUARTERS AND REGIONAL ATP CONTACTS
Headquarters

William Telliard

Director, Analytical Methods 

Attn: ATP Program Coordinator

Mail Code 4303T 

U.S. EPA Office of Water, Office of Science

and Technology

1200 Pennsylvania Avenue, NW

Washington, DC 20460

Region 1

Arthur Clark

QA Chemist

USEPA Region 1

EQA

60 Westview Street

Lexington, MA 02173

Region 2

Carol Lynes

ATP Program Coordinator

USEPA Region 2

Division of Science and Monitoring

2890 Woodbridge Avenue (MS-220)

Building 10

Edison, NJ 08837-3679

Region 3

Charles Jones

Regional QA Officer

USEPA Region 3

Environmental Assessment and Protection

Division

1650 Arch Street, 3ES-10

Philadelphia, PA 19103-2029

Region 4

Wayne Turnbull 

Chemist/ATP Program Coordinator

USEPA Region 4

Room: SESD

960 College Station Road

Athens, GA 30605-2720

Region 5

Kenneth Gunter

ATP Program Coordinator

USEPA Region 5

77 W. Jackson Blvd., WT-15J

Chicago, IL 60604

Region 6

David Stockton

USEPA Region 6 Laboratory

Houston Branch

10625 Fallstone Road (6MD-HI)

Houston, TX 77099

Region 7

Doug Brune

ATP Program Coordinator

USEPA Region 7

726 Minnesota Avenue, ENSV/QA

Kansas City, KS 66101

Region 8

Rick Edmonds

Regional Quality Assurance Officer

USEPA Region 8

999 18th Street - Suite 500 (8TMS-L)

Denver, CO 80202-2466

Region 9

Roseanne Sakamoto

ATP Program Coordinator

USEPA Region 9

75 Hawthorne Street, PMD-3

San Francisco, CA 94105

Region 10

Bruce Woods

QAO

USEPA Region 10

200 Sixth Avenue, OEA-095

Seattle, WA 98101