United States

Environmental Protection

Agency

_________________________________________________________________________

Manual for the 

Certification of 

Laboratories 

Analyzing

Drinking Water

Criteria and

Procedures

Quality Assurance

Fifth Edition

EPA 815-R-05-004

January 2005

Manual for the 

Certification of 

Laboratories 

Analyzing

Drinking Water

Criteria and

Procedures

Quality Assurance

Fifth Edition

Supersedes EPA/570/9-90/008, April 1990

EPA 814-B-92-002, September 1992

EPA 570/9-90-008A, October 1991

EPA 815-B-97-001, March 1997

US Environmental Protection Agency

Office of Water

Office of Ground Water and Drinking Water

Technical Support Center

Cincinnati, Ohio 45268 tape on recycle logo
ii

DISCLAIMER

The U.S. Environmental Protection Agency’s (EPA’s) Office of Ground Water and Drinking Water, in the Office of

Water, and the Office of Research and Development of the U.S. Environmental Protection Agency prepared this manual.

Those Offices as well as EPA’s ten Regional Offices have reviewed this manual. EPA intends to use this manual for its

own use in certifying laboratories for analysis of drinking water contaminants. In order to assume primary enforcement

responsibility for the drinking water regulations, a State must either have available laboratory facilities, certified by the

Administrator, capable of conducting analytical measurements of drinking water contaminants, or establish and maintain

its own program for certification of laboratories. States wishing to adapt the procedures and criteria of this manual for

their own certification program should revise it to accurately reflect accurately their State certification program.

This is a guidance manual and not a regulation. It does not change or substitute for any legal requirement. While EPA

has made every effort to ensure the accuracy of the manual’s discussion, the obligations of the regulated community are

determined by the relevant statutes, regulations or other legally binding requirements. The manual obviously can only

reflect the regulations in place at this time of its preparation. Consequently, for any definitive description of current legal

obligations, the public should not rely only on the discussion in the manual. This manual is not a rule, is not legally

enforceable, and does not confer legal rights or impose legal requirements upon any member of the public, States or any

other Federal agency. In the event of a conflict between the discussions in this manual and any statute or regulation, this

document is not controlling. The word “should” in this manual does not connote a requirement but does indicate EPA’s

strongly preferred approach to ensure the quality of laboratory results. EPA may decide to revise this manual without

public notice to reflect changes to its approach or to clarify and update the text.

The mention of commercial products in this manual does not constitute an endorsement of the use of that product by

EPA.
iii

Acknowledgments

This edition of the manual was prepared through the efforts of many individuals, including representatives from the U.S.

Environmental Protection Agency’s Office of Ground Water and Drinking Water (OGWDW), Office of Research and

Development (ORD), Regional Offices and the States. It has as its foundation previous editions of the manual.

Contributors to the previous editions of the manual are listed in EPA documents EPA 815-B-97-001 March 1997,

EPA/570/9-90/008 April 1990, EPA-570/9-82-002 October 1982 and EPA 600/8-78-008 May 1978. Contributors to

this edition are listed below.

Manual Revision Committee:

Paul Berger, OGWDW

Edward Glick, OGWDW

Patricia Hurr, OGWDW

Caroline Madding, OGWDW

Microbiology Subcommittee:

Paul S. Berger, Chair

Kristen Brenner, ORD

Terry C. Covert, ORD

Mary Ann Feige, OGWDW

Nancy H Hall, UI HL

Stephanie Harris, Region 10

Margo Hunt, OEI

Kimberly K Laurie, IN DOH

Ted Pass II, KY DEP

Gene Rice, ORD

Mark Rodgers, ORD

Peggy Ryker, Consultant

Lois Shadix, OGWDW

Jim Sinclair, OGWDW

John Standridge, WI SLH

Phillip R Zillinger, IN DOH

Radiochemistry Subcommittee:

John Dehart, Dyncorp

John Griggs, OAR

Richard Sheibley, PA DEP

Other Manual Reviewers:

Ann Marie Allen, MA DEP

Dale Alley, RI DOH

Cheryl Baker, ME DHS

Rusty Baldwin, AR DOH

Brenda Barnett, WV DOH

Aimee Bennett, WA

Eve Berry AR DOH

Dave Bingham, WA

George Bowman, WI LOH

Ellen Braun-Howland, NYSDOH

David Brierley, MA DEP

Douglas Brune, Region 7

John Bourbon, Region 2

Donald Buckley, MA DPH

Joan Burkart, LA DOHH

Dwayne Burkholder, PA DEP

Isa Chamberlain, Region 10

Fred Choske, CA ELAP

Art Clark, Region 1

Terry Covert, ORD

Chris Dean, NM DOH

Veronica DeBoer, AK

Mary Dillon, NJDHSS

Ron Donaly, ID

Charles Dyer, NH DES

Stan Falk, AR DOH

Charles Feldmann, OGWDW

Ray Flores, Region 6

Paul Gray, NM DOH

Stephanie Harris, Region 10

Garry Haworth, NH DES

Paul Hickey, NYSDOH

Myron Honda, HI DOH

Jane Jensen, CA ELAP

Charles Jones, Jr., Region 3

Cecylia Karch, VT DOH

Ewa King, RI DOH

Michael Kitto, NYSDOH

RJ Kiyokane, HI DOH

Sharon Kluender, WDNR

Margaret Knight, Region 10

George Kulasingam, CA ELAP

Don LaFara, NV BLC

Mary Beth Lawhorn, IL EPA

Andrew Lincoff, Region 9

Alan Love, AK

Nicholas Macelletii, CT DHS

DeLois Manor, AR DOH

Ted Martin, ORD

Gerald McKenna, Region 2

Joseph Mierzwicki, NJDHSS

William George Mills, VT DOH

Cathy Moore, AR DOH

Jean Munch, ORD

David Munch, OGWDW

Thomas O’Connell, MA DPH
iv

Jim O’Dell, ORD

Alicia P. Ordona, VA DCLS

Pavin Parekh, NYSDOH

Virgina Palomo, OR

Oscar Pancorbo, MA DEP

Bahman Parsa, NJDHSS

Gary Perryman, Region 8

Barry Pharoah, ID

Max Phillips, TCEQ

Sandra Radwin, ID

Sara Rairick, AK

Robert Rieck, Region 10

J.Jane Roll, AR DOH

Irene Ronning, OR PHL

Jack Ruckman, NV BLC

David Russell, Region 3

Eileen Sanders, VA DCLS

Tim Sanders, Region 6

Philip Schlossberg, CT DPH

Richard Sheibley, PA DEP

Thomas Semkow, NYSDOH

Jody Shoemaker,ORD

Sandra Spence, Region 8

Mary E. T. Stancavage, MD HMH

David Stockton, Region 6

Miguel R. Suarez, Region 5

Marilyn Thornton, Region 4

Lisa Touet, MA DEP

Laura Traas, WDOTCP

Timothy Troup, AR DOH

Wayne Turnbull, Region 4

Debra Waller, NJDEP

Ted Witten, OK DEQ

Bruce Woods, Region 10

Kenneth Wunschel, RI DOH

Yue Zhang, TDH

Editing

Patricia R. Louis, FSMD, ORD
v

Preface

Since 1978, the U. S. Environmental Protection Agency (EPA) has implemented a certification program for laboratories

performing drinking water analyses for compliance with regulations issued pursuant to the Safe Drinking Water Act.

These laboratories include EPA Regional laboratories, certain Federal laboratories, Tribal Nation laboratories, principal

State laboratories in primacy States, and drinking water laboratories in non-primacy States. This manual describes

criteria and procedures that EPA uses in evaluating laboratories for certification. EPA has concluded that laboratories

that adopt the approaches discussed in this manual will generate reliable analytical data. Consequently, EPA

recommends that States follow these procedures and criteria in their certification decisions. 

This document is the fifth edition of the manual, describing the program's implementation procedures and technical

criteria. It supersedes the Manual for the Certification of Laboratories Analyzing Drinking Water, EPA-815-B-97-001

(March 1997). 

The manual was revised to address: 1)the recently promulgated drinking water regulations and methods; and 2) Agency

policy (Office of Ground Water and Drinking water memo, October 1, 2002)(see Appendix F) that, at the discretion of

each state’s Certification Authority (CA), allows National Environmental Laboratory Accreditation Program (NELAP)

accreditation to be accepted in lieu of drinking water certification in terms of producing data for compliance monitoring

purposes.

A committee chaired by the EPA's Office of Ground Water and Drinking Water (OGWDW) with participation of the

National Exposure Research Laboratories in Cincinnati (NERL-Ci) and representatives from the EPA Regions and the

States prepared this document. Its goal is to improve implementation of the SDWA in light of newly approved

methodology and additional experience with the program.

Like previous editions, this edition is in loose-leaf format which will allow the EPA to update it more easily. Holders

of this manual should check with the EPA Region or the State Certification Officers to make sure their manual is current.

Additional copies of this manual may be obtained from the EPA, OGWDW, 26 W M.L. King Dr., Cincinnati, OH,

45268, fax number 513 569-7191, or by calling the Safe Drinking Water Hotline at 800 426-4791. The manual is also

posted on the Internet at www.epa.gov/safewater/certlab/labindex.html.

To ensure uniformity in its program in all the Regions, EPA uses the certification criteria in this manual for evaluating

all drinking water laboratories that it certifies. The Agency also uses this manual to determine the adequacy of State

certification programs for drinking water laboratories. States are encouraged to use the criteria in this manual to evaluate

all laboratories that they certify.

Generally the term "must" in this manual refers to elements that are required by the National Primary Drinking Water

Regulations or the approved drinking water methods. This manual uses the term "should” to describe criteria and

procedures that in OW’s judgement are necessary for laboratories to produce data that are scientifically valid and

defensible, and are of known and acceptable precision and accuracy. 
vi
vii

Table of Contents

Page 

CHAPTER I: INTRODUCTION I-1

CHAPTER II: RESPONSIBILITIES II-1

CHAPTER III: IMPLEMENTATION III-1

 Evaluation of Certification Programs III-1

 Requirements for Certification of Laboratories III-1

 Individual(s) Responsible for the Certification Program III-1

 On-Site Laboratory Audit Team III-2

 Plans for Certification of Laboratories III-2

 Principal State Laboratories III-2

 Certification Process III-2

 Types of Certification III-3

 Drinking Water Laboratories III-3

 Other Considerations for Laboratory Certification III-4

 Laboratory Quality Assurance Plan III-4

 Chain of Custody Procedures III-6

 Requirements for Maintaining Certification Status III-6

 Criteria and Procedures for Downgrading/Revoking Certification Status III-7

 Record Keeping III-9

 Reciprocity III-9

 Training III-9

 Alternate Test Procedures (ATPs) III-9

CHAPTER IV: CRITICAL ELEMENTS FOR CHEMISTRY IV-1

 Personnel IV-1

 Laboratory Facilities IV-1

 Laboratory Equipment and Instrumentation IV-2

 General Laboratory Practices IV-2

 Analytical Methods IV-3

 Sample Collection, Handling and Preservation IV-3

 Quality Control IV-4

 Records and Data Reporting IV-9

 Action in Response to Noncompliant Laboratory Results IV-10

 Tables IV-11-37

 Forms for On-site Evaluation of Laboratories IV-39-56

CHAPTER V: CRITICAL ELEMENTS FOR MICROBIOLOGY V-1

 Personnel V-1

 Laboratory Facilities V-1

 Laboratory Equipment and Supplies V-2

 General Laboratory Practices V-6

 Analytical Methodology V-8
Page 

viii

 Sample Collection, Handling, and Preservation V-29

 Quality Assurance V-30

 Records and Data Reporting V-31

 Action Response to Laboratory Results V-32

 Forms for On-site Evaluation of Laboratories V-34-77

CHAPTER VI: CRITICAL ELEMENTS FOR RADIOCHEMISTRY VI-1

 Personnel VI-1

 Laboratory Facilities VI-2

 Laboratory Equipment and Instrumentation VI-2

 General Laboratory Practices VI-6

 Analytical Methods VI-6

 Sample Collection, Handling, and Preservation VI-7

 Quality Assurance VI-7

 Records and Data Reporting VI-11

 Action Response to Laboratory Results VI-13

 Tables VI-14-16

 Sample Forms for On-site Evaluation of Laboratories VI-17-29

APPENDICES

 Appendix A: Chain of Custody Evaluations A-1

 Appendix B: Recommended Protocol for On-Site Laboratory Evaluations B-1

 Appendix C: Definitions and Abbreviations C-1

 Appendix D: Memorandum Regarding Third Party Auditors D-1

 Appendix E: Required Analytical Capability for Principal State Laboratory Systems E-1

 Appendix F: Memorandum Regarding National Environmental Laboratory Accreditation F-1

 Appendix G: Analytical Methods for Microbiology G-1

TABLES

 Chemistry

 Table IV-1 Glassware Cleaning Procedures IV-11

 Table IV-2 Approved Methods for Primary Inorganic Chemicals IV-13

 Table IV-3 Approved Methods for Primary Organic Chemicals IV-17

 Table IV-4 Approved Methods for Disinfectant Residuals IV-20

 Table IV-5 Recommended Methods for Secondary Drinking Water Contaminants IV-21

 Table IV-6 Sample Containers,Preservation and Holding Times for Regulated Parameters IV-23

 Table IV-7 MCLc and Detection Limit Requirements to Composite IV-27

 Table IV-8 VOC MCLs and MDLs IV-28

 Table IV-9 SOC MCLs and Detection Requirements to Reduce Monitoring (mg/L) IV-29

 Table IV-10 MCL and Proficiency Performance Evaluation Sample Acceptance Criteria IV-30

 Table IV-11 Promulgated Organic Drinking Water Methods IV-35

 Radiochemistry

 Table VI-1 Methods for Radionuclide Analysis CFR 141.25 VI-14

 Table VI-2 Sample Handling, Preservation, and Instrumentation VI-16
I-1

Chapter I

Introduction

Public water systems serving at least 25 persons or having at least 15 service connections must comply with the Safe

Drinking Water Act (SDWA) and the requirements of the National Primary Drinking Water Regulations (NPDWR) (40

CFR 141). Section 1401(1)(D) of the Act defines a National Primary Drinking Water Regulation to include "criteria

and procedures . . . [for] quality control and testing procedures to insure compliance . . . " EPA regulations require

community water systems to conduct monitoring for compliance with the drinking water standards. In addition,

regulations provide that analyses must be conducted by laboratories certified by EPA or the States. The regulations also

established requirements for laboratory certification..

The regulations governing primacy at 40 CFR 142.10(b)(4) require, as a condition of primary enforcement responsibility

(primacy), that a state have laboratory facilities available (the Principal State Laboratory) certified by the regional

administrator. In addition, the regulations governing certification (40 CFR 141.28) require that all testing for compliance

purposes be performed by certified laboratories except that turbidity, free chlorine residual, temperature, pH, alkalinity,

calcium, conductivity, orthophosphate, TOC, SUVA, daily chlorite, and silica may be performed by anyone acceptable

to the State. This manual is intended to assist EPA in implementing 40 CFR 142.10(b)(4) by specifying criteria and

procedures EPA uses in evaluating principal State laboratories for certification. States with primacy may also choose

to use equivalent criteria and procedures similar to those in this manual for their own certification programs.

To obtain and maintain primacy, a State must comply with 40 CFR 142.10, which includes the following provisions:

The establishment and maintenance of a State program for the certification of laboratories conducting

analytical measurements of drinking water contaminants pursuant to the requirements of the State

primary drinking water regulations including the designation by the State of a laboratory officer, or

officers, certified by the Administrator, as the official(s) responsible for the State's certification

program. The requirements of this paragraph may be waived by the Administrator for any State where

all analytical measurements required by the State's primary drinking water regulations are conducted

at laboratories operated by the State and certified by the Agency. (40 CFR 142.10(b)(3)(i))

Assurance of the availability to the State of laboratory facilities certified by the Administrator and

capable of performing analytical measurements of all contaminants specified in the State primary

drinking water regulations . . . (40 CFR 142.10(b)(4)). 

NOTE: Reference to the Administrator of EPA also refers to his or her designee.

The requirement for a laboratory certification program extends to EPA Regional laboratories, Tribal Nation laboratories,

principal State laboratories in primacy States, and laboratories that perform analyses under the Safe Drinking Water Act

in States without primacy. If all required analyses are not performed in principal State laboratories, primacy States must

have a certification program for certifying other drinking water laboratories (40 CFR 142.10(b)(3)(i)). 

EPA's Technical Support Center (TSC) in Cincinnati, Ohio, with the assistance of the National Exposure Research

Laboratory in Cincinnati, Ohio (NERL-Ci), is responsible for determining the certification status for EPA’s Regional

laboratories in microbiology, chemistry, and radiochemistry. Regional certification officers are responsible for the

certification of the principal State laboratory in each primacy State, and are also responsible for certifying all Tribal

Nation laboratories and laboratories in non-primacy States. Primacy States with certification programs are responsible

for certifying the other drinking water laboratories in their State, (i.e., laboratories other than the principal State

Laboratory). 

Regional Laboratories must successfully analyze a set of proficiency testing samples (PTs) at least annually for all

regulated contaminants for which they wish to be certified, by each method for which they wish to be certified (40 CFR
I-2

141.23 and 141.24), and pass an on-site evaluation at least every three years. An on-site evaluation determines

conformance with the criteria specified in this manual. Principal State laboratories must (40 CFR 141.23 and 141.24)

successfully analyze a complete set of proficiency testing (PT) samples from a source acceptable to the Region at least

annually for the contaminants and methods included in the regulations which the State has adopted, and pass an on-site

evaluation every three years. EPA auditors use the criteria in this manual for the on-site audits of the Regional and

principal State laboratories.

Chapter II describes the responsibilities of each of the parties involved in the certification program. Chapter III describes

how the program operates. Chapters IV, V, and VI cover the technical criteria to be used during the on-site evaluation

of a laboratory for chemistry, microbiology, and radiochemistry, respectively. Optional audit forms are also included

in Chapters IV, V, and VI. The appendices include the following: a recommended protocol and format for conducting

on-site laboratory evaluations which may be used by the laboratory auditors; frequently used abbreviations and

definitions; EPA's policy on third-party auditors; EPA’s policy on National Environmental Laboratory Accreditation

Program (NELAP) accreditation as an alternate for drinking water certification; a list of contaminants a principal State

laboratory must (primacy citation 142.10(b)(4)) have the capability to analyze, analytical methods for microbiology

analyses; and recommended chain-of-custody procedures to be used when necessary.
II-1

Chapter II

Responsibilities

The success of the laboratory certification program depends upon cooperation among the organizations responsible for

its implementation. Within the Agency, responsibilities for laboratory certification are shared by the Office of Ground

Water and Drinking Water (OGWDW), the Office of Research and Development (ORD), and the Regional Offices.

Office of Ground Water and Drinking Water (OGWDW) and Office of Research and

Development (ORD)

OGWDW, with assistance from ORD, has the responsibility for developing and implementing the national certification

program for laboratories that analyze drinking water samples and for implementing the Safe Drinking Water Act. These

responsibilities include the following:

• Propose and promulgate regulations;

• Assess national laboratory capacity and capability;

• Review the EPA Regional certification programs annually and evaluate the resources and personnel available

in each EPA Region to carry out the certification program;

• Develop guidance and respond to questions and comments; 

• Develop technical and administrative certification criteria to support future regulations;

• Revise this manual when necessary;

• Conduct triennial on-site audits of each Regional laboratory for chemistry, microbiology, and radiochemistry

• Maintain a database of laboratory ID Codes

• Develop and participate in training courses to support the certification program;

• Provide technical assistance to EPA and the States; 

• Develop and evaluate methods for the analysis of drinking water contaminants.

EPA Regions

The Regions oversee the certification programs in the States and Tribal Nations. The Regions' responsibilities are to:

• Determine the certification status for the principal State/Tribal laboratory system in each primacy State/Tribe;

• Perform an annual review of State/Tribal certification programs and proficiency testing results and monitor the

adequacy of State/Tribal programs for certifying laboratories, as described in Chapter III;

• Sponsor annual meetings for the state Certification Officers (COs);

• Provide technical assistance to the States' EPA-certified drinking water laboratories, as needed; 

• Manage the certification program for drinking water laboratories in non-primacy States using the criteria in this

manual.

• Manage the certification program for drinking water laboratories on Tribal Nation lands using the criteria in

this manual.

This last duty may be performed by the State, but the Region retains responsibility for the on-site evaluation of the

designated Tribal Nation principal laboratory. Drinking water laboratories may be evaluated by the Region, or under

a Region-approved program carried out by a designated State program. In either case, this manual is the basis for the

on-site audits, conducted by EPA, of principal State laboratories, laboratories on Tribal Nation lands, and drinking water

laboratories in non-primacy States.

The Regional laboratory should maintain certification for as many regulated contaminants as its resources permit. This

enhances both EPA's technical assistance capabilities and credibility with those it certifies. It also ensures the laboratory

capability to analyze samples for possible enforcement actions and for States which do not have primacy. Reciprocal

agreements with other regions to share scarce resources are recommended.
II-2

Primacy States

Primacy States, in which all drinking water compliance samples are not analyzed at State operated laboratories, are

required to establish and maintain a State program for the certification of laboratories conducting analyses of drinking

water compliance samples [see 40 CFR 142.10(b)(3)(i)]. EPA encourages the States to base certification of drinking

water laboratories either upon criteria contained in this manual or upon state-developed equivalents that are at least as

stringent as this manual. Primacy States must establish and maintain a state program for the certification of laboratories

conducting analytical measurements of drinking water contaminants pursuant to the requirements of the State primary

drinking water regulations. The States must designate a “laboratory officer or officers, certified by the Administrator

or designee as the official(s) responsible for the certification program.” (40 CFR §142.10 (a)(3)(i))

States are responsible for the certification of the public and private laboratories in their State. This includes auditing the

laboratories and reviewing the PT data. States should also provide technical assistance to laboratories. They may also

choose to certify laboratories outside their State either by an on-site evaluation or reciprocity.
III-1

Chapter III

Implementation

1. Evaluation of Certification Programs

OGWDW and the Regions monitor the certification programs under their purview annually. These offices assess the

adequacy of programs for certifying laboratories by evaluating each program's scope, staffing, resources, policies,

procedures, and effectiveness. This should be done in person during an on-site audit when possible, and at least by

means of a questionnaire in the other years. The adequacy of these essential program elements is evaluated by:

• Reviewing the program plan, responsibilities, organizational structure, staff (including educational background

and experience), scope and description of the certification process, downgrading criteria and processes, and use

of PT samples;

• Requesting an annual program report that includes program highlights, training, continuing education efforts,

number of on-site evaluations performed, listing of laboratories certified by discipline or contaminant, and any

certification downgrading or upgrading actions along with reasons for those actions;

• Observing the state certification officers on-site audits of drinking water laboratories;

• Encouraging State and Regional laboratory auditorsto observe on-site audits of their own and other laboratories

as on-the-job training; 

• Sponsoring annual meetings of certification officers to discuss program issues, policies, and problems. Key

Regional, NERL, OGWDW, and State personnel should be invited to participate.

2. Requirements for Certification of Laboratories

In order to be eligible to analyze compliance samples under the Safe Drinking Water Act, Regional and Principal State

laboratories should meet the minimum criteria specified in this manual, which includes passing an on-site audit at least

once every three years, and satisfactorily analyzing a set of PT samples annually.

The Office of Ground Water and Drinking Water (OGWDW) will accept NELAP accreditation as equivalent to Drinking

Water certification, if all requirements of the drinking water program are met. 

3. Individual(s) Responsible for the Certification Program

The Technical Support Center, with the assistance from NERL-Ci is responsible for certifying the regional laboratories;

the Regions are responsible for certifying their States’ principal laboratory systems and Tribal Nations’ laboratories; and

the States are responsible for certifying private, municipal, non-principal state, and governments laboratories.

The certification program personnel in each Region should consist of a certification authority(s) (CA), the certification

program manager, and a certification team comprised of certification officers (COs) and technical experts. Additional

third party auditors and experts may be used. However, third parties has have no authority for certification decisions.

Third party auditing is discussed in Section 4.2.

The Certification Authority (CA) is the person who has signature authority for all certification decisions. This is the

Chief of the Technical Support Center and the Regional Administrator in the Regions. The RA may delegate this

authority to a lower level.

The Certification Program Manager (CPM) is the person responsible for managing the drinking water laboratory

certification activities in the Region. 

The Certification Officers (COs) are the regional and state personnel who have the responsibility of certifying

laboratories under their purview. 40 CFR 142.10(b)(3)(i) requires Primacy States to designate a person certified by the

Administrator as the official responsible for the State's certification program. This person would be the State certification

authority.
III-2

4. On-Site Laboratory Audit

4.1 On-Site Laboratory Audit Team

The certification program manager should establish one or more teams of certification officers and auditors to audit

laboratories. It is the responsibility of these teams to perform the on-site laboratory audits, review the laboratory PT data,

and make recommendations to the CA concerning the certification status of the laboratories. 

Team members should be experienced professionals, hold at least a bachelor's degree or equivalent education/experience

in the discipline (chemistry, radiochemistry, microbiology or a related field) for which they certify, and have recent

laboratory experience.

Team members should also have experience in laboratory evaluation and quality assurance, be familiar with the drinking

water regulations and data reduction and reporting techniques, be technically conversant with the analytical techniques

being evaluated, and be able to communicate effectively, both orally and in writing. 

The on-site team should include at least one person knowledgeable in each area being audited (e.g., inorganic and organic

chemistry, radiochemistry, and microbiology). COs need to successfully complete the appropriate EPA laboratory

certification course. In addition, there should be a mechanism for COs to receive periodic training regarding newly

promulgated regulations, newly adopted certification criteria, and new methods. This could be done by auditing the EPA

CO Training Course and/or attending the required annual Regional and State CO Meetings.

4.2 Third Party Auditors

Certification programs may employ third party auditors who meet all of the qualifications listed above. In areas where

experience does not exist within the certification team (e.g., asbestos), outside expert assistance may be obtained in the

needed areas to assist the on-site team. Outside experts who have not attended the EPA certification officer training

should be accompanied by a certification officer. Although these third parties may be used to assist EPA or State

certification officers, they have no authority for certification decisions and they may not make final certification

decisions. These decisions rest with the EPA or the State.

When using third party experts, it is critical to avoid conflicts of interest. A third party auditor who in any way stands

to benefit by the certification status of the laboratory audited may not be used.

5. Plans for Certification of Laboratories

The certification program manager should develop plans for certifying drinking water laboratories under her/his

authority. Written plans should include the following:

• Documentation of certification authority and certification officers and their education/experience;

• Schedules of laboratories to be audited;

• Specific types of analyses to be examined;

• Protocol to be followed;

• Strategy for assessing laboratory performance (e.g., PTs, data audits, etc.); 

• Plans for providing technical assistance to laboratories which need upgrading.

6. Principal State Laboratories

To receive and retain primacy, the State must (40 CFR 142.10(b)(4)) have available laboratory facilities capable of

performing analytical measurements for all the federally mandated contaminants specified in the State Primary Drinking

Water Regulations. This laboratory or laboratories are considered the Principal State Laboratory System and must be

certified by EPA.

7. Certification Process

The certification process begins when the laboratory director makes a formal request in writing to the certification

authority to be certified. This application may be one of the following:
III-3

• A request for first-time certification for microbiology, chemistry, or radiochemistry; 

• A request for certification to analyze additional or newly regulated contaminants; 

• A request to reapply for certification after correction of deficiencies which resulted in the

downgrading/revocation of certification status.

The response to a formal application for any of the above requests should be given within 30 days. At this time a

mutually agreeable date and time should be set for the on-site laboratory audit. 

Subsequent audits may be initiated by the CA or the laboratory.

A recommended protocol for conducting these audits is given in Appendix B. 

Drinking water laboratories should specifically state that they plan to analyze drinking water samples when they request

certification. If a laboratory has not been analyzing drinking water samples and does not plan to analyze drinking water

samples, OGWDW, the Region or State may choose not to expend the resources to renew their certification. 

8. Types of Certification

After review of PT sample results and an on-site visit, the certification authority should provide a written report within

45 days and classify the laboratory for each contaminant or group of contaminants according to the following rating

scheme:

8.1 Certified - a laboratory that meets the regulatory performance criteria as explained in this manual and all other

applicable regulatory requirements. 

8.2 Provisionally Certified - a laboratory that has deficiencies but demonstrates its ability to consistently produce

valid data within the acceptance limits specified in the NPDWR, and within the policy required by their certification

authority. A provisionally certified laboratory may analyze drinking water samples for compliance purposes, if the said

clients are notified of its downgraded status in writing, on any report. Provisional certification may not be given if the

evaluation team believes that the laboratory cannot perform an analysis within the acceptance limits specified in the

regulations.

8.3 Not Certified - a laboratory that possesses deficiencies and, in the opinion of the Certification Authority, cannot

consistently produce valid data.

8.4 Interim Certification - interim certification may be granted in certain circumstances when it is impossible or

unnecessary to perform an on-site audit. Interim certification status may be granted if, for example, the CA determines

that the laboratory has the appropriate instrumentation, is using the approved methods, has adequately trained personnel

to perform the analyses, and has satisfactorily analyzed PT samples, if available, for the contaminants in question. The

CO should perform an on-site audit as soon as possible but no later than three years. An example of a situation where

this type of certification is warranted would be a laboratory that has requested certification for the analysis of additional

analytes that involve a method for which it already has certification. The CO should review the laboratory's quality

control data before granting this type of certification.

9. Drinking Water Laboratories

For the purpose of certification, any laboratory which analyzes drinking water compliance samples is considered a

drinking water laboratory. This includes Federal laboratories that analyze compliance samples and any laboratories that

analyze compliance samples for Federal facilities. All such laboratories must (40 CFR 141.21, .23, .24, .25) be certified

by the State or EPA. If requested by the State, a Region may certify Federal laboratories in its Region.

The Region certifies individual laboratories on Tribal Nation lands, when requested by the tribal chairperson. These

laboratories must meet the criteria for certification as specified in the NPDWR.
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The Regions use the criteria, procedures, and mechanism as specified in the NPDWR and this manual in their decisions

to certify municipal or private drinking water laboratories in non-primacy states.

10. Other Considerations for Laboratory Certification

10.1 Laboratory Personnel

The laboratory should have sufficient supervisory and other personnel, with the necessary education, training, technical

knowledge, and experience for their assigned functions.

10.2 Laboratory Director/Manager or Technical Director

The laboratory director/manager should be a qualified professional with the technical education and experience, and

managerial capability commensurate with the size/type of the laboratory. The laboratory director/manager is ultimately

responsible for ensuring that all laboratory personnel have demonstrated proficiency for their assigned functions and that

all data reported by the laboratory meet the required quality assurance (QA) criteria and regulatory requirements.

10.3 Quality Assurance Manager

The QA manager should be independent from the laboratory management, if possible, and have direct access to the

highest level of management. The QA manager should have a bachelor's degree in science, training in quality assurance

principles commensurate with the size and sophistication of the laboratory, and at least one year of experience in quality

assurance. The QA manager should have at least a working knowledge of the statistics involved in quality control of

laboratory analysis and a basic understanding of the methods which the laboratory employs.

11. Laboratory Quality Assurance Plan

All laboratories analyzing drinking water compliance samples must adhere to any required QC procedures specified in

the methods. This is to ensure that routinely generated analytical data are scientifically valid and defensible, and are of

known and acceptable precision and accuracy. To accomplish these goals, each laboratory should (EPA Order 5360.1

A2) prepare a written description of its QA activities (a QA plan). It is the responsibility of the QA manager to keep the

QA plan up to date. All laboratory personnel need to be familiar with the contents of the QA plan. This plan should be

submitted to the auditors for review prior to the on-site visit or should be reviewed as part of the on-site visit. 

The laboratory QA plan should be a separately prepared text. However, documentation for many of the listed QA plan

items may be made by reference to appropriate sections of this manual, the laboratory's standard operating procedures,

(SOPs) or other literature (e.g., promulgated methods, Standard Methods for the Examination of Water and Wastewater,

etc.). The QA Plan should be updated at least annually (EPA Order 5360.1 A2).

At a minimum, the following items should be addressed in each QA plan:

11.1 Laboratory organization and responsibility

• include a chart or table showing the laboratory organization and lines of responsibility, including QA managers;

• list the key individuals who are responsible for ensuring the production of valid measurements and the routine

assessment of measurement systems for precision and accuracy (e.g., who is responsible for internal audits and

reviews of the implementation of the plan and its requirements);

• reference the job descriptions of the personnel and describe training to keep personnel updated on regulations

and methodology, and document that laboratory personnel have demonstrated proficiency for the methods they

perform.

11.2 Process used to identify clients' Data Quality Objectives 

11.3 SOPs with dates of last revision

C The laboratory should maintain SOPs that accurately reflect all phases of current laboratory activities

C keep a list of SOPs

C ensure that current copies of SOPs are in the laboratory and in the QA Managers files;

C ensure that SOPs are reviewed annually and revised as changes are made;

C ensure that SOPs have signature pages and revisions dated.
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11.4 Field sampling procedures

• describe the process used to identify sample collectors, sampling procedures and locations, required

preservation, proper containers, correct sample container cleaning procedures, sample holding times from

collection to analysis, and sample shipping and storage conditions;

• ensure that appropriate forms are legibly filled out in indelible ink or hard copies of electronic data are

available. See Chapters IV, V, and VI for specific items to be included;

• describe how samples are checked when they arrive for proper containers and temperature and how samples

are checked for proper preservation (e.g., pH, chlorine residual) before analysis;

• ensure that sampling protocol is written and available to samplers.

11.5 Laboratory sample receipt and handling procedures

• bound laboratory note books, if used, should be filled out in ink; entries dated and signed (A secure, password

protected, electronic data base is acceptable);

• store unprocessed and processed samples at the proper temperature, isolated from laboratory contaminants,

standards and highly contaminated samples and, sometimes, each other; holding times may not be exceeded;

• maintain integrity of all samples, (e.g., by tracking samples from receipt by laboratory through analysis to

disposal);

• require Chain-of-Custody procedures for samples likely to be the basis for an enforcement action (see Appendix

A);

• specify criteria for rejection of samples which do not meet shipping, holding time and/or preservation

requirements and procedures for notification of sample originators.

11.6 Instrument calibration procedures (may reference SOP)

• specify type of calibration used for each method and frequency of use; 

• describe calibration standards' source, age, storage, labeling;

• perform data comparability checks;

• use control charts and for radiochemistry, report counting errors with their confidence levels.

11. 7 Analytical procedures (may reference SOP)

• cite complete method manual;

• describe quality control procedures required by the methods that need to be followed.

11.8 Data reduction, validation, reporting and verification (may reference SOP)

• describe data reduction process: method of conversion of raw data to mg/L, picocuries/L, coliforms/100 mL,

etc.;

• describe data validation process;

• describe reporting procedures, include procedures and format;

• describe data verification process;

• for radiochemistry, describe reporting of counting uncertainties and confidence levels;

• describe procedure for data corrections.

11.9 Type of quality control (QC) checks and the frequency of their use (see Chapters IV, V and VI).(may reference

SOP)

Parameters for chemistry and radiochemistry should include or reference:

• instrument performance check standards;

• frequency and acceptability of method detection limit (MDL) calculations;

• frequency and acceptability of demonstration of low level capability;

• calibration, internal and surrogate standards;

• laboratory reagent blank, field reagent blank and trip blank;

• field and laboratory matrix replicates;

• quality control and proficiency testing samples;

• laboratory fortified blank and laboratory fortified sample matrix replicates;

• initial demonstration of method capability
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• use of control charts;

• qualitative identification/confirmation of contaminants.

Parameters for microbiology should include or reference:

• positive and negative culture controls;

• confirmation/verification of presumptive total coliform positive samples;

• sterility controls;

• proficiency testing and quality control samples.

11.10 List schedules of internal and external system and data quality audits and inter laboratory comparisons (may

reference SOP)

11.11 Preventive maintenance procedures and schedules

• describe location of instrument manuals and schedules and documentation of routine equipment maintenance;

• describe availability of instrument spare parts in the laboratory;

• list any maintenance contracts in place.

11.12 Corrective action contingencies

• describe response to obtaining unacceptable results from analysis of PT samples and from internal QC checks;

• name persons responsible for the various corrective actions;

• describe how corrective actions taken are documented;

11.13 Record keeping procedures

• describe procedures and documentation of those procedures; 

• list length of storage, media type (electronic or hard copy);

• describe security policy of electronic databases;

• all electronic data should have software support so it may be regenerated.

If a particular item is not relevant, the QA plan should state this and provide a brief explanation. A laboratory QA plan

should be responsive to the above items while remaining brief and easy to follow. Minimizing paperwork, while

improving dependability and quality of data, are the intended goals.

12. Chain-of-Custody Procedures

Certified laboratories, when requested to process a sample for possible legal action against a supplier, should use an

adequate chain-of-custody procedure. An example of such a procedure is found in Appendix A. The State or Region

should seek input from its attorney general's office to ensure that the laboratory's procedures are adequate. The procedure

used should be documented.

13. Requirements for Maintaining Certification Status

13.1 Proficiency Testing (PT) Samples

At least annually drinking water laboratories certified for chemical contaminants must satisfactorily analyze a PT sample

to maintain certification (40CFR 141.23(k)(3)(i),141.24(h)(17)(i)(A) and 141.89(a)(1)(i)). PT samples should be

analyzed in the same manner as routine samples. Laboratories must acquire the PT sample from a supplier acceptable

to the appropriate certification authority.

If the certified laboratory does not analyze the PT sample within the acceptance limits specified in the regulations, or

within policy described by their certifying authority, the certifying authority should follow the procedure discussed in

the section entitled, "Criteria and Procedures for Downgrading/Revoking Certification Status." 

If a laboratory wishes to be certified for a contaminant by more than one method, it must (40CFR

141.23(k)(3)(ii),141.24(h)(17)(i)(A) and 141.89(a)(1)(i)) analyze the PT samples by each method for which it wishes

to be certified. The methods listed on the laboratory's certification certificate must be the methods by which the PT

samples were analyzed.
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The laboratory should be able to provide documentation to the certification authority that the person(s) analyzing any

PT sample is a laboratory employee who routinely analyzes drinking water compliance samples.

13.2 Methodology

Laboratories must use the methods specified in the drinking water regulations at 40 CFR part 141 These methods are

listed in Chapters IV, V, VI, and Appendix G.

13.3 On-Site Evaluation

The CA should be satisfied that a laboratory is maintaining the required standard of quality for certification. Normally,

this should be based on a recommendation from a triennial on-site evaluation. However, if the laboratory undergoes a

major change or repeatedly fails a PT sample, the CA should consider conducting an evaluation before the usual three

year period has expired.

13.4 Notification of Certifying Authority (CA) of Major Changes

Certified laboratories should notify the appropriate CA (Regional Administrator or designee or the Chief, TSCOGWDW) in writing, within 30 days of major changes in personnel, equipment, or laboratory location. A major change

in personnel is defined as the loss or replacement of the laboratory supervisor or a situation in which a trained and

experienced analyst is no longer available to analyze a particular parameter for which certification has been granted.

The CA should discuss the situation with the laboratory supervisor and establish a schedule for the laboratory to address

major changes. If the CA determines that the laboratory can no longer produce valid data, the CA should follow the

procedure for revocation of certification.

14. Criteria and Procedures for Downgrading/Revoking Certification Status

14.1 Criteria for Downgrading Certification Status

A laboratory should be downgraded to "provisionally certified" status for a contaminant or group of contaminants for

any of the following reasons:

• Failure to analyze a PT sample at least annually within the acceptance limits specified in the regulations, or,

if there are no requirements specified in the regulations, within policy described by their certifying authority;

• Failure of a certified laboratory to notify the CA within 30 days of major changes (e.g., in personnel, equipment,

or laboratory location);

• Failure to satisfy the CA that the laboratory is maintaining the required standard of quality, based upon a EPA

on-site evaluation;

• Failure to report compliance data to the public water system or the State drinking water program in a timely

manner, thereby preventing compliance with Federal or State regulations and endangering public health. Data

which may cause the system to exceed an MCL should be reported as soon as possible. 

14.2 Procedures for Downgrading to "Provisionally Certified" Status

If a laboratory is subject to downgrading on the basis of the above indicated criteria, the CA should notify the laboratory

director or owner (by registered or certified mail) of its intent to downgrade within 14 days from becoming aware of the

situation warranting downgrading. The laboratory director should review the problems cited, and within 30 days of

receipt of the letter, send a letter to the CA specifying what immediate corrective actions are being taken and any

proposed actions that need the concurrence of the CA. The CA should consider the adequacy of the response and notify

the laboratory in writing (by registered or certified mail) of its certification status within 14 days of receipt of its

response. The CA should follow up to ensure that corrective actions have been taken.

If a laboratory fails to analyze a PT or other unknown sample within the acceptance limits, the CA should not downgrade

certification if the laboratory identifies and corrects the problem to the CA's satisfaction within 30 days of being notified

of the failure. If, after a review of the submitted information, the CA determines that the laboratory need not be

downgraded, then within 30 days of this decision, the CA should notify the laboratory that it is required to analyze
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another PT. If the laboratory analyzes this second unknown sample within the acceptance limits established by the EPA

or State, the laboratory should not be downgraded. If the laboratory fails to analyze this second unknown sample within

the established limits, the CA should downgrade the laboratory to "provisionally certified" status and notify the

laboratory within 14 days (by registered or certified mail). Laboratories should be downgraded only for the analyte

failed, except where EPA/State certifies a group of related analytes based on a limited number of analytes in the group.

(See Chapter 4, Section 7.2.1 for additional information.)

During any phase of this procedure, a laboratory may request that the EPA or State provide technical assistance to help

identify and resolve any problem.

After the CA notifies a laboratory, in writing, that it has been downgraded to "provisionally certified" status for

procedural, administrative, equipment or personnel deficiency, the laboratory should correct its problem within three

months. If the laboratory was downgraded to "provisionally certified" status because of a failure to analyze a PT sample

(or other unknown test sample) within the acceptance limits specified in the regulations, or within policy required by their

certifying authority, the laboratory should correct its problems and satisfactorily analyze another PT sample (or other

unknown sample) within one month of receipt of the second PT sample. A provisionally certified laboratory may

continue to analyze samples for compliance purposes, but should notify its clients of its downgraded status and provide

that information, in writing, on any report.

14.3 Criteria for Revoking Certification Status

A laboratory should be downgraded from certified, provisionally certified or interim certified status to "not certified"

for a particular contaminant analysis for the following reasons:

• Reporting PT data from another laboratory as its own; 

• Falsification of data or other deceptive practices;

• Failure to use the analytical methodology specified in the regulations;

• For provisionally certified laboratories, failure to successfully analyze a PT sample or any other unknown test

sample for a particular contaminant within the acceptance limits specified; 

• For provisionally certified laboratories, failure to satisfy the CA that the laboratory has corrected deviations

identified during an on-site evaluations;

• For provisionally certified laboratories, persistent failure to report compliance data to the public water system

or the State drinking water program in a timely manner thereby preventing compliance with Federal and/or State

regulations and endangering public health. Data which may cause the system to exceed an MCL should be

reported as soon as possible.

• Refusal to participate in an on-site evaluation conducted by the CA

14.4 Procedures for Revocation

The CA should notify the laboratory, in writing (by registered or certified mail) of the intent to revoke certification. If

the laboratory wishes to challenge this decision, a notice of appeal should be submitted in writing to the CA within 30

days of receipt of the notice of intent to revoke certification. If no notice of appeal is filed, certification should be

revoked.

The notice of appeal should be supported with an explanation of the reasons for the challenge and should be signed by

a responsible official from the laboratory such as the president/owner for a commercial laboratory, or the laboratory

supervisor in the case of a municipal laboratory or the laboratory director for a State or Regional laboratory.

Within 30 days of receipt of the appeal, the CA should make a decision and notify the laboratory in writing (by

registered or certified mail). Denial of the appeal should result in immediate revocation of the laboratory's certification.

Once certification is revoked, a laboratory may not analyze drinking water samples for compliance until its certification

has been reinstated.

If the appeal is determined to be valid, the CA should take appropriate measures to reevaluate the facility and notify the

laboratory, in writing (by registered or certified mail), of its decision within 30 days of the reevaluation.
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14.5 Upgrading or Reinstatement of Certification

Through a written request, a laboratory may seek upgrading or reinstatement of certification, when and if the laboratory

can demonstrate to the CA's satisfaction that the deficiencies which produced provisionally certified status or revocation

have been corrected. This may include an on-site evaluation, successful analysis of unknown samples or any other

measure the CA deems appropriate.

15. Record Keeping

The certification program manager should ensure that records for on-site laboratory assessments and certification

program reviews be maintained in an easily accessible central location for a period of 6 years to include the last two onsite audits, or longer if required by specific State regulations. This includes records/correspondence used to determine

compliance with the requirements in this manual. Records may include checklists, corrective action reports, final reports,

certificates, PT study results and related documents

16. Reciprocity

Reciprocity (mutually acceptable certification among Regions and/or primacy States) is strongly endorsed by EPA as

a highly desirable element in the certification program for drinking water laboratories.

States are encouraged to adopt provisions in their laws and regulations to permit reciprocity. Even though ultimate

responsibility for reciprocal certification resides with the Regions and primacy States, the States may ask for the

assistance of EPA in cases involving clarification of what should be considered in a reciprocal agreement. Such requests

should be submitted to the Region or OGWDW through the Region.

17. Training

Training is an integral part of the laboratory certification process for personnel conducting on-site evaluations of

laboratories on behalf of either the Regional Office or a primacy State.

EPA policy requires that all Regional Certification Officers initially pass the appropriate EPA laboratory certification

training courses for the discipline for which they certify (chemistry or microbiology). All laboratory auditors should be

experienced professionals, and have at least a bachelor's degree or equivalent education/experience in the discipline for

which they certify and recent laboratory experience in the field for which they audit laboratories. Third party auditors

(see Appendix D) also need to pass the EPA certification training course. Outside experts, retained for their knowledge

in a limited area (e.g., asbestos) are not required to pass the laboratory certification course if they are used as part of an

on-site audit team which includes a certification officer. Periodic training for both laboratory auditors and analysts

should be provided by the Regions. Certification officers should attend refresher training programs at least every five

years to keep their knowledge of the methods and the drinking water program current. It is highly recommended that

certification officers have recent bench experience in the methods for which they certify. OGWDW will notify

certification officers of major updates/changes to EPA's certification program. It is recommended that the States use

these same criteria in their certification programs. 

18. Alternate Test Procedures (ATPs)

EPA promulgates analytical methods for all regulated drinking water contaminants. A regulation for a particular

contaminant will include one or more methods that must be used to determine that contaminant. Subsequently, the

Agency may approve additional methods or modifications of EPA approved methods in another rule. EPA may also

authorize the use of alternate analytical methods as provided in 40 CFR 141.27, "With the written permission of the State,

concurred by the Administrator of the EPA, an alternate analytical technique may be employed. An alternate technique

may be accepted only if it is substantially equivalent to the prescribed test in both precision and accuracy as it relates

to the determination of compliance with any MCL."

Anyone can request that EPA approve a new method or modification of a method already approved by EPA, by

submitting EPA-specified data and other information to the Director, Analytical Methods Staff,(MS 4303T) Office of

Science and Technology, Office of Water, EPA, 1200 Pennsylvania Ave., NW, Washington DC 20460. EPA will

evaluate the material to determine whether the method or method modification meets EPA criteria. 
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In the case of “acceptable versions” of methods, (minor modifications to approved methods), a letter of approval will

be issued by OW. A list of these approved minor modifications can be found on the OW website at

http://www.epa.gov/OGWDW/methods.
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Chapter IV

Critical Elements for Chemistry

1. Personnel

1.1 Laboratory Supervisor

The laboratory supervisor should have at least a bachelor's degree with a major in chemistry or equivalent, and at least

one year of experience in the analysis of drinking water. The laboratory supervisor should have at least a working

knowledge of quality assurance principles. The laboratory supervisor has the responsibility to ensure that all laboratory

personnel have demonstrated their ability to satisfactorily perform the analyses to which they are assigned and that all

data reported by the laboratory meet the required quality assurance and regulatory criteria.

1.2 Laboratory Analyst

The laboratory analyst should have at least a bachelor's degree with a major in chemistry or equivalent, and at least one

year of experience in the analysis of drinking water. If the analyst is responsible for the operation of analytical

instrumentation, he or she should have completed specialized training offered by the manufacturer or another qualified

training facility or served a period of apprenticeship under an experienced analyst. The duration of this apprenticeship

should be proportional to the sophistication of the instrument. Data produced by analysts and instrument operators while

in the process of obtaining the required training or experience are acceptable only when reviewed and validated by a fully

qualified analyst or the laboratory supervisor.

Before beginning the analysis of compliance samples, the analyst must adhere to any required QC procedures specified

in the methods for blanks, precision, accuracy, sensitivity, specificity and satisfactory analysis on unknown samples. This

should be documented according to the laboratory’s QA Plan.

1.3 Technician

The laboratory technician should have at least a high school diploma or equivalent, complete a method training program

under an experienced analyst and have six months bench experience in the analysis of drinking water samples. 

Before beginning the analysis of compliance samples, the technician must adhere to any required QC procedures

specified in the methods for blanks, precision, accuracy, sensitivity, specificity and satisfactory analysis on unknown

samples. This should be documented according to the laboratory’s QA Plan.

1.4 Sampling Personnel

Personnel who collect samples should be trained in the proper collection technique for all types of samples which they

collect. Their technique should be reviewed by experienced sampling or laboratory personnel.

1.5 Waiver of Academic Training Requirement

The certification officer may waive the need for specified academic training, on a case-by-case basis, for highly

experienced analysts.

1.6 Training Records

Training records should be maintained for all personnel. These should include all job-related formal education and

training taken by the analyst which pertains to any aspect of his/her responsibilities, including but not limited to analytical

methodology, laboratory safety, sampling, quality assurance, data analysis, etc.

2. Laboratory Facilities

The analysis of compliance samples is to be conducted in a laboratory where the security and integrity of the samples

and the data can be maintained. The laboratory facilities should be clean, have adequate temperature and humidity

control, have adequate lighting at the bench top and should meet applicable OSHA standards. The laboratory must

adhere to any required QC procedures specified in the methods by having provisions for the proper storage and disposal

of chemical wastes; secondary containment for hazardous waste storage is recommended. The appropriate type of

exhaust hood is required where applicable.
IV- 2

There should be sufficient bench space for processing samples. Workbench space should be convenient to sink, water,

gas, vacuum and electrical sources free from surges. Instruments should be properly grounded. For safety reasons,

inorganic and organic facilities should be in separate rooms; organic analysis and sample extraction should also be

separated to prevent cross contamination. The analytical and sample storage areas should be isolated from all potential

sources of contamination. There should be sufficient storage space for the safe storage of chemicals, glassware and

portable equipment, sufficient floor and bench space for stationary equipment and areas for cleaning materials. 

3. Laboratory Equipment and Instrumentation

The laboratory is to have the instruments and equipment needed to perform the approved methods for which certification

has been requested. The checklist on pages 44 to 50 of this chapter provides more information on the necessary

equipment. All instruments are to be properly maintained and calibrated.

4. General Laboratory Practices

4.1 General

4.1.1 Chemicals/reagents: Chemicals and reagents used must meet any requirements specified in the methods.

If not specified, then "Analytical reagent grade" (AR) or American Chemical Society (ACS) grade chemicals or

better should be used for analyses in certified laboratories. Consult the currently promulgated editions of Standard

Methods for the Examination of Water and Wastewater, part 1070 for more detailed information on reagent grades.

 

4.2 Inorganic Contaminants

4.2.1 Reagent water: The laboratory must have a source of reagent water having a resistance value of at least 0.5

megohms (conductivity less than 2.0 micromhos/cm) at 25 C when required by the method. High quality water o

meeting such specifications may be purchased from commercial suppliers. Quality of reagent water is best

maintained by sealing it from the atmosphere. Quality checks to meet specifications above should be made and

documented at planned intervals based on use. Individual analytical methods may specify additional requirements

for the reagent water to be used. Inorganic methods require distilled or deionized water free of the analyte(s) of

interest and trace metals methods require ASTM Type 1 water.

4.2.2 Glassware preparation: Glassware cleaning requirements specified in the methods must be followed. If

no specifications are listed, then glassware should be washed in a warm detergent solution and thoroughly rinsed

first with tap water and then with reagent water. This cleaning procedure is sufficient for general analytical needs.

It is advantageous to maintain separate sets of suitably prepared glassware for the nitrate and mercury analyses due

to the potential for contamination from the laboratory environment. Table IV-1 summarizes the cleaning procedures

specified in the EPA methods.

4.3 Organic Contaminants

4.3.1 Reagent water: Reagent water for organic analysis must adhere to any required QC specified in the methods.

Most methods specify the reagent water not contain analytes of interest above their respective method detection

levels (MDLs). It may be necessary to treat water with activated carbon to eliminate all interferences. Reagent water

requirements of individual methods must be followed.

4.3.2 Glassware preparation: Glassware cleaning requirements specified in the methods must be followed.

Table IV-1 summarizes the cleaning procedures specified in the EPA methods.

4.4 Laboratory Safety

While safety criteria are not an aspect of laboratory certification, laboratory personnel should apply general and

customary safety practices as a part of good laboratory practices. Each laboratory is encouraged to have a safety plan

as part of their standard operating procedure which includes personnel safety, training and protection. Where safety

practices are required in an approved method (i.e., 515.1), they must be followed. See Standard Methods for the

Examination of Water and Wastewater, part 1090 for a discussion of laboratory safety.

4.5 Quality Assurance

Laboratories should maintain current Quality Assurance Plans as described in Chapter 3. All laboratory activities
IV- 3

including, but not limited to, sampling, test methods, instrument operation, data generation, data validation and corrective

action procedures should be described in the Plan. Plans need to be read by all personnel.

5. Analytical Methods

5.1 General

A list of promulgated methods for inorganic and organic contaminants can be found in Tables IV-2 and IV-3,

respectively. Methods manuals should be available to applicable personnel. Other methods cannot be used for

compliance samples unless approval has been granted by the Agency by obtaining an Alternate Test Procedure approval.

Allowed modification to the methods must be documented. Contact the appropriate certifying authority for the alternate

test procedure process (see Chapter 3, p 10). Table IV-4 lists the methods which must be used for the analysis of

disinfectant residuals. Recommended methods for Secondary contaminants are listed in Table IV-5. 

5.2 Analyses Approved by the State

Measurements for turbidity, pH, temperature, disinfectant residual, calcium, orthophosphate, silica, alkalinity, and

conductivity need not be made in certified laboratories, but may be performed by any persons acceptable to the State.

However, approved methodology must) be used (Tables IV-2 to IV-5). The State should institute a quality assurance

program to assure validity of data from these measurements.

5.2.1 Turbidity standards: Sealed liquid secondary turbidity standards purchased from the instrument

manufacturer or other sources should be calibrated against properly prepared and diluted formazin or styrene

divinylbenzene polymer primary standards and revised values assigned at least every four months in order to monitor

for any deterioration. This calibration should be documented. These standards should be replaced when they do

not fall within 15% of the initial assigned concentration of the standard. Solid turbidity standards composed of

plastic, glass, or other materials are not reliable and should not be used.

5.2.2 Residual chlorine standards: If visual comparison devices such as color wheels or sealed ampules are used

for determining free chlorine residual, the standards incorporated into such devices should be calibrated at least

every six months. These calibrations need to be documented. Directions for preparing temporary and permanent

type visual standards can be found in Method 4500-Cl-G, of the currently promulgated editions of Standard Methods

for the Examination of Water and Wastewater. By comparing standards and plotting such a comparison on graph

paper, a correction factor can be derived and applied to future results obtained on the now calibrated apparatus.

6. Sample Collection, Handling, and Preservation

The manner in which samples are collected and handled is critical to obtaining valid data. It is important that a written

sampling protocol with specific sampling instructions be available to and used by sample collectors and available for

inspection by the certification officer. (Appendix A, Chain-of-Custody).

6.1 Rejection of Samples

The laboratory’s rejection criteria should be documented in writing in the laboratory’s QA Plan or in an SOP. The

laboratory should reject any sample taken for compliance purposes which does not meet the criteria in 6.2 through 6.6.

The laboratory must (141.23(a)(4)(i))notify the authority requesting the analyses and ask for a resample. If resampling

is not possible and the sample is analyzed, the sample data should be clearly identified in the data package as being

unusable for its intended purpose. In addition, the inadmissibility of these sample data need to be clearly communicated

to all end data users. 

6.2 Sample Containers and Preservation

The type of sample container and the required preservative for each inorganic and organic chemical contaminant are

listed in Table IV-6. The laboratory must measure and record the temperature of the sample when it arrives when

temperature preservation is required by the method. The use of "blue ice" is discouraged because it generally does not

maintain the temperature of the sample at 4°C ±2°C or less. If blue ice is used, it should be frozen at the time of

sampling, the sample should be chilled before packing, and special notice taken at sample receipt to be certain the

required temperature (4°C) has been maintained.
IV- 4

6.3 Maximum Holding Times

Samples must be analyzed within the maximum holding times required by the method. These are listed in Table IV-6.

6.4 Sample Collection and Transport

There must be strict adherence to correct sampling procedures, sample handling, complete identification of the sample,

and prompt transfer of the sample to the laboratory when required by the method. When the laboratory is not responsible

for sample collection and transport, it must verify that the paperwork, preservatives, containers and holding times are

correct as required by the methods or reject the sample. The rejection criteria should (EPA Order 5360.1) be documented

in writing.

6.5 Sample Collector

The sample collector should be trained in sampling procedures and have complete written sampling instructions (SOPs)

for each type of sample to be collected. The samplers are to be able to demonstrate proper sampling technique.

6.6 Sample Report Form

The sample collection report form should contain, at a minimum, the ID, location, date and time of collection, collector's

name, preservative added and shipping requirements, container and volume, sample type, analysis, and any special

remarks concerning the sample. Indelible ink should be used. 

6.7 Sample Compositing

If samples are composited, the compositing must (40 CFR 141.23,24) be done in the laboratory. Samples may only be

composited if the laboratory detection limit is adequate for the number of samples being composited (up to a maximum

of five). For example, for inorganic samples, composite samples from a maximum of five samples are allowed if the

detection limit of the method used for analysis is less than one-fifth the MCL. If the concentration of any inorganic

chemical in the composite is greater than or equal to one-fifth of the MCL, then a followup sample must be taken within

14 days at each sampling point included in the composite. These samples must be analyzed for the contaminants which

exceeded one-fifth the MCL in the composite sample. [CFR 144.23(a)(4)] Compositing of VOCs is not recommended.

7. Quality Control

7.1 General Requirements

7.1.1 Availability of QA Documents: The laboratory's QA plan and appropriate Standard Operating Procedures

(SOPs) should be readily available to the analysts and for inspection by auditors. (see Chapter III's discussion of

Quality Assurance).

7.1.2 Availability of QC Information: All quality control information should be readily available for inspection

by auditors.

7.1.3 Balances and Weights: Balance range should be appropriate for the application for which it is to be used.

Drinking water chemistry laboratories should use balances that weigh to at least 0.0001 g. The balances should be

calibrated at least annually with ASTM Type I, Class 1 or 2 weights. (ASTM, 1916 Race St., Philadelphia, PA

19103) This may be done by laboratory personnel or under contract by a manufacturer's representative. We

strongly recommend that laboratories have a contract to calibrate balances due to the expense of the calibration

weights, and to serve as an outside QC check of the weights and balances. Weights meeting ASTM Type I, Class

1 or 2 specifications should be recertified at least every five years or if there is reason to believe damage (corrosion,

nicks) has occurred. 

Each day the mechanical or digital balance is used, a verification should be performed. The verification consists

of a check of a reference mass at approximately the same nominal mass to be determined. Verifications should be

done each weighing session unless it can be shown that fluctuations in the environment do not affect the calibration.

Weights meeting ASTM Type 1 specifications may be used. These should be calibrated annually against the

reference weights at time of balance calibration. The checks and their frequency should be as prescribed in the

laboratory's QA Plan. A record of all checks should be kept and be available for inspection. 
IV- 5

7.1.4 Color Standards: Wavelength settings on spectrophotometers should be verified at least annually with color

standards. The specific checks and their frequency should be as prescribed in the laboratory’s QA documents. A

record of these checks should be kept as prescribed in the laboratory’s QA documents and be available for

inspection. 

7.1.5 Temperature Measuring Devices Liquid bearing thermometers such as mercury or alcohol thermometers

need to be traceable to NIST calibration and verified at least annually and whenever the thermometer has been

exposed to temperature extremes. The correction factor should be indicated on the thermometer and the date the

thermometer was calibrated and the calibration factor should be kept as prescribed in the laboratory’s QA documents

and be available for inspection. The NIST thermometer should be recalibrated at least every five years or whenever

the thermometer has been exposed to temperature extremes.

Digital thermometers, thermocouples and other similar electronic temperature measuring devices should be

calibrated at least quarterly. The date the thermometer was calibrated and the calibration factor should be kept as

prescribed in the laboratory’s QA documents and be available for inspection.

When an infrared detection device is used to measure the temperature of samples, the device should be verified at

least every six months using a NIST certified thermometer over the full temperature range that the IR thermometer

will be used. This would include ambient (20-30°C), iced (4°C) and frozen (0 to -5°C). Each day of use a single

check of the IR should be made by checking the temperature of a bottle of water at the temperature of interest that

contains a calibrated thermometer. Agreement between the two should be within 0.5°C, or the device should be

recalibrated.

7.1.6 Traceability of Calibration: Calibrations of all measurement devices need to be traceable to national

standards whenever applicable.

7.2 Specific Requirements: The following are required for each analyte for which a laboratory is certified:

7.2.1 Proficiency Testing (PT) Samples: In order to receive and maintain full certification for an analyte, the

laboratory must (40CFR 141.23(k)(3)(i),141.24(h)(17)(i)(A) and 141.89(a)(1)(i)) analyze PT samples (if available)

acceptable to the Certifying Authority at least once every 12 months for each analyte and by each method used to

analyze compliance samples. Results from analysis of the PT sample must be within the acceptable limits

established by U.S. EPA. These acceptance limits are listed in Table IV-10, “MCL and Profeciency Testing Sample

Acceptance Criteria in the CFR, Primary and Secondary Drinking Water Regulations [§141.23(k)(3)(ii) and

141.24(f)(17) and (19)].” The laboratory should document the corrective actions taken when a PT sample is

analyzed unsuccessfully. A copy of this documentation should be available for review by the certification officer.

A make up PT sample must be successfully analyzed. If problems arise, the appropriate action to be taken is

specified in Chapter III, Implementation of Certification Program. 

Excluding vinyl chloride, the laboratory may be certified for all VOCs if they successfully analyze at least 80%

of the regulated VOCs (141.24(17)(f)(i)(B). The intention of this regulation is to allow some flexibility for

random misses because the VOC methods include 20 regulated analytes. A laboratory should not be certified

for an analyte which it fails repeatedly. This “80% rule” for VOCs has recently been made more difficult to

interpret since some PT providers are including THMs in the same vial as the VOCs. The 80% Rule does not

apply to the THMs.

The Stage 1 Disinfection By Products (DBP) Rule, which became effective in January 2002, regulates the sum

of five haloacetic acids (HAA5): monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, 

monobromoacetic acid and dibromoacetic acid. Laboratories are certified for HAA5, but successful analyses

of the HAA PT samples are based on the results for the individual compounds. The 80% Rule applies to the

HAA5s, so if four of the 5 HAA5s are successfully analyzed, the laboratory may be certified for HAA5. As

before, a laboratory should not be certified if the same analyte is failed repeatedly.
IV- 6

The DBP Rule also changed the way the trihalomethanes (THMs): chloroform, dichlorobromomethane,

chlorodibromomethane and bromoform PTs are evaluated. Laboratories are still certified for total THMs but

under the DBP Rule, each THM concentration must (141.131(b)(2) be reported, evaluated and passed

individually to pass the PT sample. The DBP Rule also states that if a laboratory fails one THM, it cannot be

certified for TTHMs, but must (141.131(b)(2)analyze another PT sample and pass all four of the THMs in a

PT sample to be certified to analyze compliance monitoring samples for total trihalomethanes.

The following table summarizes the 80% Rule.

 Analyte(s) PT Success Requirement

Vinyl Chloride 100%

20 VOCs 80% 1

4 THMs 100%

5 HAA5s 80% 1

 A lab should not maintain certification for analyte(s) which it repeatedly fails. 1

7.2.2 Quality Control Samples: At least once each quarter, the laboratory should analyze a quality control

sample for the analytes they are determining in that quarter. The sample should be prepared from a source other

than that from which their working standards are prepared. The sample should be in the same concentration

range as the drinking water calibration curve. If errors exceed limits required in the methods, corrective action

must be taken and documented, and a follow-up quality control sample analyzed as soon as possible to

demonstrate the problem has been corrected. 

7.2.3 Calibration Curve: Calibration requirements in the methods must be followed. If there are no

calibration requirements in the method, the following are guidelines to be used. At the beginning of each day

that samples are to be analyzed, a calibration curve covering the sample concentration range and all target

analytes should be generated according to the approved SOP. Depending on concentration ranges, the curve

should be composed of three or more points. Field measurements (e.g. pH and chlorine residual) need to be

made on instruments which have been properly calibrated as specified in the method or instrument manual and

checked each day of use. The less precise the measurement, the greater the number of concentrations which

should be included in the calibration curve. 

7.2.4 Calibration Check: The calibration for some methods is so time-consuming that 7.2.3 is impractical

on a daily basis. Where the determinative time is extensive such as Methods 508/508.1, 515.1, 524.2, 525.2,

etc. and the instrument is very stable, the calibration curve should be initially developed as specified in 7.2.3.

Thereafter, each day analyses are performed, this curve should be verified by analysis of at least one standard

for each of the target analytes at the expected concentration range. This verification should be done at both the

beginning and end of the analyses. All checks must be within the control limits required in the method or the

system is to be recalibrated as specified in 7.2.3. The concentration of the check standard should vary from day

to day across the range of analyte concentrations being measured. 

For some methods an initial conditioning injection is to be made to deactivate active sites that may have

developed overnight. Depending on the method, the blank may be appropriate for this.

Specific calibration requirements in the methods must be followed if different than the above. 

It is recommended that a calibration standard of one component of a multicomponent analyte (PCBs, toxaphene

or chlordane) also be analyzed each day or work shift. By rotating the analyte chosen, continuing calibration

data can be obtained on all the multicomponent analytes over a period of one to two weeks. If a positive for

a multcomponent analyte is found in a sample, a calibration check for that analyte should be performed as soon

as possible. 
IV- 7

7.2.5 Blanks: Requirements in the methods must be followed. A laboratory reagent blank should be carried

through the full analytical procedure with every sample batch. In general, results from laboratory reagent blanks

should not exceed the laboratory's Minimum Reporting Limit, the lowest concentration of standard used for

quantitation. (MRL).

7.2.6 Laboratory Fortified Blanks: Requirements in the methods must be followed. LFBs should be

analyzed at the level specified in the method. Some methods require that a laboratory fortified blank at ten times

the MDL or a mid level concentration be analyzed with each batch of samples. Precision and accuracy data

should be documented for this determination. In addition, the analyst should routinely verify the minimum

reporting limit for each analyte by analyzing a laboratory fortified blank at the minimum reporting level. 

7.2.7 Laboratory Fortified Sample Matrix: Laboratory fortified sample matrix requirements in the

methods must be met. If there are no laboratory fortified sample matrix requirements in the method, the

following are guidelines to be used. The laboratory should add a known quantity of analytes to a percentage

(to be described in the approved SOP) of the routine samples to determine sample matrix interference. The

fortified concentration should not be less than the concentration of the sample selected for fortification unless

specified by the method. If the sample concentration is unknown or less than detectable, the analyst should

choose an appropriate concentration (e.g., a percentage of the MCL or mid point in the calibration range). Over

time, samples from all routine sample sources should be fortified. The procedure should be described

in the SOP. If any of these checks are not within the criteria specified in the method or control limits specified

in 7.2.7, and the laboratory performance is in control, the result for that sample should be flagged to inform

the data user that the results are suspect due to matrix effects. 

7.2.8 Control Charts: Control charts for accuracy and precision, generated from laboratory fortified blanks

(LFBs) should be maintained and used by the laboratory. Until sufficient data are available from the laboratory,

usually a minimum of 20 to 30 test results on a specific analysis, the laboratory should use the control limits

specified in the methods. If there are no control limits specified in the method, the limits may be statistically

calibrated using the procedure below.

When sufficient data become available, the laboratory should develop LFB control charts from the mean percent

recovery (0) and the standard deviation (S) of the percent recovery for the QC checks specified above (see

Standard Methods for the Examination of Water and Wastewater, part 1020B, or similar QC reference texts

for further information). These data are used to establish upper and lower control limits as follows:

upper control limit = 0 + 3S (upper warning limit + 2S)

lower control limit = 0 - 3S (lower warning limit - 2S)

After each five to ten new recovery measurements, new control limits should be calculated using the most recent

20-30 data points. These calculated control limits should not exceed those established in the method. If any

of these control limits are tighter than the method specifications, the laboratory should use the tighter criteria.

7.2.9 Initial Demonstration of Capability: Requirements in the methods must be followed. Before

beginning the analysis of compliance samples, an initial demonstration of capability (IDC) must be performed

for each method as required in the method. The IDC includes a demonstration of the ability to achieve a low

background, the precision and accuracy required by the method, and determination of the method detection limit

(MDL)(see below). An IDC should be performed for each instrument. It is also recommended that an IDC be

performed by each analyst. In addition, it is recommended that the IDC also address the variability introduced

if more than one sample preparation technician is used. Precision, accuracy and MDL should be similar for

each technician. The analyst should recalculate IDCs when a change in the method, analyst or instrument is

made which could affect the precision or accuracy or sensitivity. Minor changes should prompt a check to

ascertain that the precision, accuracy and sensitivity have been maintained.
IV- 8

7.2.10 Quantitation of Multicomponent Organic Analytes (toxaphene, chlordane and PCBs) The

quantitation of multicomponent analytes requires professional judgment on the part of the analyst. This is

required due to the complex nature of the chromatography involved, sample weathering, degradation and

interferences that may be present in the samples. The pattern of peaks found in the sample should be examined

carefully and compared to a standard. The peaks in the sample that match the peak ratios in the standard can

be used in quantitation. Peaks that have obvious interferences (such as pesticides or phthalates or peaks

exhibiting poor peak shape) or appear to have been degraded or weathered should not be used for quantitation.

A representative number (5-9) of peaks is suggested. Peak area should be used for quantitation and the analyst

should ensure that the samples and standards have been integrated in the same manner. Quantitation can be

done by using the total peak area or height (comparing the area of the 5-9 peaks used for quantitation of the

sample to the area of the standard) or by calculating each peak separately (using area) and taking the average

concentration of the 5-9 peaks. Because of factors such as peak shape and baseline rise, the most accurate

quantitation is obtained when the concentration of the sample closely matches that of the standard (e.g., within

20% of the standard). See EPA Method 8081, Organochlorine Pesticides and PCBs as Aroclors by Gas

Chromatography: Capillary Column Technique, (EPA SW 846 Test Methods for Evaluating Solid Waste

Physical/Chemical Methods, Third Edition) for a more detailed discussion of quantitation of multicomponent

analytes.

Note: PCBs are qualitatively identified as Aroclors and measured for compliance purposes as

decachlorobiphenyl. Chlordane is regulated as technical chlordane, a mixture of at least 11 major components

and 30 minor ones.

7.2.11 MDL Calculation: Requirements in the methods must be followed. Most methods require initial

MDL calculations for all analytes and certification officers should require the laboratories to calculate their

detection limits for all regulated contaminants. If there is no procedure to determine the detection limits in

the method, it should be determined in accordance with the procedure given in 40 CFR 136, Appendix B. The

CFR, at141.24(f)(17)(ii)(C) requires an MDL of 0.0005 mg/L be attained for VOCs, and 141.89(a)(1)(iii)

requires an MDL of 0.001 mg/L be attained for lead if the lab will be processing source water composite

samples. For inorganics and SOCs, a method detection limit of 1/5 of the MCL must be attained for

compositing [CFR 141.23(a)(4)] and [CFR 141.24(f)(10)]. VOCs should not be composited. The SOC

detection limits listed at CFR (141.24(h)(18) are required to reduce monitoring (CFR 141.24(f)(11)(iv). Table

IV-8 lists the MCLs, MCLGs and MDLs, for VOCs which are in the drinking water regulations. Table IV-9

lists the SOC MCLs, MCLGs and Monitoring Triggers.

Sample preparation and analyses for the MDL calculation should be made over a period of at least three days

to include day-to-day variation as an additional source of error. The analyst should determine MDLs initially,

when any change is made which could affect the MDLs, or more frequently if required by the method.

(Inorganic methods may require MDLs to be determined differently, and in all cases the methods must be

followed.) In addition, the analyst must demonstrate low level capability on an ongoing basis through an MDL

determination or repeated low level analyses (MRL). 

The calculation of MDLs by the CFR procedure may not be adequate for toxaphene and chlordane because they

require pattern or peak profile recognition for identification. Presently, no standard procedure exists, so it is

recommended that the MDL be defined as the lowest concentration for which pattern recognition is possible.

Pattern recognition is used for qualitative identification of PCBs as Aroclors. Quantitation of PCBs is achieved

by conversion of PCBs to decachlorobiphenyl (DCB). 

7.2.12 Low Level Quantitation: The laboratory's minimum reporting limits (MRL) should be reported to the

client along with the data. The reporting limit must be below the MCL. Laboratories should run a LFB at

their MRL every analysis day and should not report contaminants at levels less than the level at which they

routinely analyze their lowest standard. While this is a scientifically sound practice, whether it is an acceptable

practice will depend on State and Federal reporting requirements. It is important for users of data to understand

the statistical and qualitative significance of the data. Laboratories may be required by the States to achieve

a specific MDL or quantitation limit more stringent than that required by EPA.
IV- 9

8. Records and Data Reporting

8.1 Legal Defensibility: Compliance monitoring data should be made legally defensible by keeping thorough and

accurate records. The QA plan and/or SOPs need to (EPA Order 5360.1) describe the policies and procedures used by

the facility for record integrity, retention and storage. If samples are expected to become part of a legal action, chain

of custody procedures should be used (See Appendix A).

8.2 Maintenance of Records: Public Water Systems are required to maintain records of chemical analyses of

compliance samples for 10 years (40 CFR 141.33) and lead and copper for 12 years (40 CFR 141.91). The laboratory

should maintain easily accessible records for five years or until the next certification data audit is complete, whichever

is longer. Changes in ownership, mergers, or closures of laboratories do not eliminate these requirements. The client

water system should be notified before disposing of records so they may request copies if needed. This includes all raw

data, calculations, and quality control data. These data files may be either hard copy, microfiche or electronic.

Electronic data should always be backed up by protected tape or disk or hard copy. If the laboratory changes its

computer hardware or software, it should make provisions for transferring old data to the new system so that it remains

retrievable within the time frames specified above. Data which is expected to become part of a legal action may need

to be maintained for a longer period of time. Check with your legal counsel.

8.3 Sampling Records: Data should be recorded in ink with any changes lined through such that the original entry

is visible. Data may also be kept electronically. Changes need to be initialed and dated. The following information

should be readily available:

8.3.1 Date, location (including name of utility and PWSS ID #), site within the system, time of sampling,

name, organization and phone number of the sampler, and analyses required;

8.3.2 Identification of the sample as to whether it is a routine distribution system sample, check sample, raw

or finished water sample, repeat or confirmation sample or other special purpose sample;

8.3.3 Date of receipt of the sample;

8.3.4 Sample volume/weight, container type, preservation and holding time and condition on receipt;

8.3.5 pH and disinfectant residual at time of sampling (if required) (from plant records);

8.3.6 Transportation and delivery of the sample (person/carrier, conditions).

8.4 Analytical Records Data should be recorded in ink with any changes lined through such that original entry is

visible. Changes need to be initialed and dated The following information should be readily available:

8.4.1 Laboratory and persons responsible for performing analysis;

8.4.2 Analytical techniques/methods used;

8.4.3 Date and time of analysis;

8.4.4 Results of sample and quality control analyses;

8.4.5 Calibration and standards information.

8.4.6 Analyst and technician Initial Demonstration of Capability documentation should be kept on file as

well as results of proficiency testing.

8.5 Reconstruction of Data: Adequate information should be available to allow the auditor to reconstruct the final

results for compliance samples and PT samples.
IV- 10

8.6 Computer Programs: Computer programs should be verified initially and periodically by manual calculations and

the calculations should be available for inspection. Access to computer programs and electronic data need to be limited

to appropriate personnel.

9. Action in Response to Noncompliant Laboratory Results

When a laboratory is responsible, either by contract or State policy, to report sample results which would indicate a

system is out of compliance, the laboratory must (141.23(a)(4)(i)) promptly notify the proper authority so that the

authority can request the water utility to resample from the same sampling location(s) immediately. See Chapter III.
IV- 11

Table IV-1 Glassware Cleaning Procedures - consult the method for complete details; do not over-heat volumetric

glassware

Method Washing Drying

502.2/504/504.1/524.2 Detergent wash, rinse with tap

and distilled water 

105°C for 1 hour

505 Detergent wash, rinse with tap

and reagent water 

400°C for 1 hour or rinse with

acetone

506 Rinse immediately with last

solvent used, wash with hot water

and detergent, rinse with tap

and reagent water

400°C for 1 hour or rinse with

acetone 

507/508 Rinse immediately with last

solvent used, wash with hot water

and detergent, rinse with tap and

reagent water

400°C for 1 hour or rinse with

acetone

508.1 Detergent wash, rinse with tap

and reagent water or solvent rinse

400°C for 2 hours

508A No specifications, suggest the

same as 515.1/515.2

no specification, suggest the

same as 515.1/515.2

515.1/515.2 Rinse immediately with last

solvent used, wash with hot water

and detergent, rinse with dilute

acid, tap and reagent water

400°C for 1 hour or rinse with

acetone

515.3 Rinse immediately with last

solvent used, wash with hot water

and detergent, rinse with tap and

reagent water

400°C for 1 hour or rinse with

acetone

515.4 Wash with tap water and

detergent, rinse with tap and

reagent water. A solvent rinse

may be necessary.

In place of solvent rinse, muffle

at 400°C for 2 hours. 

Heat volumetrics

at 120°C.

524.2 Not described in method Not described in method

525.2 Detergent wash, rinse with tap

and distilled water or solvent

rinse

air dry or muffle(no specs)

(suggest 400°C for 1 hour)

531.1/6610 Rinse immediately with last

solvent used, wash with hot water

and detergent, rinse with tap and

reagent water

450°C for 1 hour or rinse with

acetone
Method Washing Drying

IV- 12

547/548.1 Rinse immediately with last

solvent used, wash with hot water

and detergent, rinse with tap and

reagent water

400°C for several hours or rinse

with methanol

549.2 Rinse immediately with last

solvent used, wash with hot water

and detergent, rinse with tap and

reagent water

130°C for several hours or rinse

with methanol

550/550.1 Rinse immediately with last

solvent used, wash with hot water

and detergent, rinse with tap and

reagent water

400°C for 15-30 minutes or rinse

with acetone or pesticide quality

hexane

551.1 Wash with water and detergent,

rinse with tap and reagent water.

Rinse caps in acetone

Vials: 400°C for 30 minutes

Caps: 80°C for 1 hour

552.1, 552.2, 555 Rinse immediately with last

solvent used, wash with hot water

and detergent, rinse with tap and

reagent water

400°C for 1 hour or rinse 3 times

with acetone

1613 Rinse with solvent, sonicate with

detergent for 30 minutes, rinse

sequentially with methanol, hot

tap water, methanol, acetone and

methylene chloride

Air dry

Metals Wash with detergent, rinse with

tap water, soak 4 hours in 20%

(V/V) nitric acid or dilute

nitric(-8%)/

hydrochloric(-17%), rinse with

reagent water

Air dry

Inorganics Wash with detergent, rinse with

tap and reagent water (use

phosphate free detergent for

o-phosphate analysis)

Air dry
*ICP will not be an acceptable procedure after 2006 IV- 13

Table IV-2 Approved Methods for Primary Inorganic Chemicals, Parameters in the Lead and Copper Rule,

Sodium, and Turbidity [§141.23(k)(1)]

Contaminant Methodology EPA ASTM SM Other 1 2

Antimony ICP-MS 200.83

Hydride-AA D3697-92

AA-Platform 200.93

AA-Furnace 3113B

Arsenic ICP* 200.7 3120B 3

ICP-MS 200.83

AA-Platform 200.93

AA-Furnace D2972-93C 3113B

Hydride-AA D2972-93B 3114B

Asbestos T

EM 100.14

TEM 100.25

Barium

ICP 200.7 3120B 3

ICP-MS 200.83

AA-Direct 3111D

AA-Furnace 3113B

Beryllium

ICP 200.7 3120B 3

ICP-MS 200.83

AA-Platform 200.93

AA-Furnace D3645-93B 3113B

Bromate IC 300.16

Cadmium

 

ICP 200.7 

3

ICP-MS 200.83

AA-Platform 200.93

AA-Furnace 3113B 

Chlorite IC 300.07

IC 300.16
Contaminant Methodology EPA ASTM SM Other 1 2

IV- 14

Chromium

 

ICP 200.7 3120B 3

ICP-MS 200.83

AA-Platform 200.93

AA-Furnace 3113B

Cyanide Man. Distillation

followed by: 

 D2036-98A 4500-CN-C

 Spec., Amenable D2036-98B 4500-CN-G

 Spec.Manual D2036-98A 4500-CN-E I-3300-858

 Semi-auto 335.47

Ion Sel. Elec.(ISE) 4500CN-F

Lachat Kenda

Fluoride Ion Chromatography 300.0 D4327-91 4110B 7

Manual Distillation,

 SPADNS

4500F-B,D

Manual ISE D1179-93B 4500F-C

Automated ISE 380-75WE

9

Auto. Alizarin 4500F-E 129-71W9

Mercury Manual Cold Vapor 245.1 D3223-91 3112B 3

Auto. Cold Vapor 245.210

ICP-MS 200.83

Nitrate

 

Ion Chromatography 300.0 D4327-97 4110B B-1011 7 11

Au 3to Cd Reduction 353.2 D3867-90A 4500-NO -F 7

3

Ion Selective Elec. 4500-NO -D 60112

M 3an Cd Reduction D3867-90B 4500-NO -E

Nitrite

 

Ion Chromatography 300.0 D4327-97 4110B B-1011 7 11

Au 3to Cd Reduction 353.2 D3867-90A 4500-NO -F 7

M 3an Cd Reduction D3867-90B 4500-NO -E

Sp 2ectrophotometric 4500-NO -B
Contaminant Methodology EPA ASTM SM Other 1 2

IV- 15

Selenium Hydride-AA D3859-98A 3114B

ICP-MS 200.83

AA-Platform 200.93

AA-Furnace D3859-93B 3113B

Thallium ICP-MS 200.83

AA-Platform 200.9 3

Lead AA-Furnace D3559-96D 3113B

ICP-MS 200.83

AA-Platform 200.93

Copper AA-Furnace D1688-90C 3113B

AA-Direct D1688-90A 3111B 

ICP 200.7 3120B 3

ICP-MS 200.83

AA-Platform 200.93

pH Electrometric 150.1 D1293-84 4500-H -B 10 +

150.210

Conductivity Conductance D1125-91A 2510B

Calcium

EDTA titration 3500-Ca-B2a

EDTA titration D511-93A 3500-Ca-D2a

AA-Direct D511-93B 3111B

ICP 200.7 3120B 3

Alkalinity Titration D1067-92B 2320B

Elec. titration I-1030-858

Orthophosphate

unfiltered,

no digestion or

hydrolysis

Color, automated 

 ascorbic acid

365.1 4500-P-F 7

Color, ascorbic acid D515-88A 4500-P-E

Color, phosphomolybdate I-1601-858

AutoSegmented Flow I-2601-908

Auto discrete I-2598-858

Ion Chromatography 300.0 D4327-97 4110 7
Contaminant Methodology EPA ASTM SM Other 1 2

IV- 16

Silica Color, molybdate blue; I-1700-858

 auto seg. flow I-2700-858

Color D859-88

Molybdosilicate 4500-Si-D2a

Heteropoly blue 4500-Si-E2a

Auto. molybdate reactive

silica

4500-Si F2a

ICP 200.7 3120B 3

Temperature Thermometric 2550B

Sodium ICP 200.73

AA-Direct 3111B

Turbidity Nephelometric 180.1 2130B GLI Method 2 7 13

Hach 10133

Footnotes

Annual Book of ASTM Standards, Vols. 11.01 and 11.02, American Society for Testing and Materials, 1916 Race Street, 1

Philadelphia, PA 19103.

Standard Methods for the Examination of Water and Wastewater, 18 , 19 or 20th Edition, American Public Health Association, 2 th th

1015 Fifteenth Street NW, Washington, D.C. 20005. Except 3111B, 3111D, 3112B, 3113B, 3114B are not approved in the 20th

edition.

 Only approved in 20 edition 2a th

"Methods for the Determination of Metalsin EnvironmentalSamples - SupplementI,"EPA-600/R-94-111, May 1994. Available 3

at NTIS, PB 94-184942.

Method 100.1, "Analytical Method for Determination of Asbestos Fibers in Water," EPA-600/4-83-043, EPA, September 1983. 4

Available at NTIS, PB 83-260471.

Method 100.2, "Determination of Asbestos Structure Over 10-:m In Length in Drinking Water," EPA-600/R-94-134, June 1994. 5

Available at NTIS, PB 94-201902.

Methods for the Determination of Organic and Inorganic Compounds in Drinking Water - Volume 1,” document number EPA 6

815-R-00-014, August 2000.

"Methods for the Determination of Inorganic Substances in Environmental Samples," EPA-600/R-93-100, August 1993. 7

Available at NTIS, PB94-121811.

Available from Books and Open-File Reports Section, U.S. Geological Survey, Federal Center, Box 25425, Denver, CO 80225- 8

0425.

Industrial Method No. 129-71W, "Fluoride in Water and Wastewater," December 1972, and Method No. 380-75WE, "Fluoride 9

in Water and Wastewater," February 1976, Technicon Industrial Systems, Tarrytown, NY 10591. 

Methods 150.1, 150.2 and 245.2 are available fromUSEPA, NERL, Cincinnati, OH45268. The identicalmethods were formerly 10

in "Methods for Chemical Analysis of Water and Wastes," EPA-600/4-79-020, March 1983. 

Method B-1011, "Waters Test Method for Determination ofNitrite/Nitrate in Water Using Single Column Ion Chromatography," 11

Millipore Corporation, Waters Chromatography Division, 34 Maple Street, Milford, MA 01757.

Technical Bulletin 601 "Standard Method of Test for Nitrate in Drinking Water," July 1994, PN 221890-001, Thermo Orion, 12

500 Cummins Center, Beverly, MA 01915-9846. This method is identical to Orion WeWWG/5880, which is approved for nitrate

analysis. ATI Orion republished the method in 1994, and renumbered it as 601, because the 1985 manual "Orion Guide to Water

and Wastewater Analysis," which contained WeWWG/5880, is no longer available. 

GLI Method 2, "Turbidity," November 2, 1992, GLI International, 9020 W Dean Rd. Milwaukee, Wisconsin 53224. 13
IV- 17

Table IV-3 Approved Methods for Primary Organic Chemicals [§141.24(e)]

Contaminant Method (Revision Number) 1

Benzene 502.2(2.1), 524.2(4.1)

Carbon tetrachloride 502.2(2.1), 524.2(4.1), 551.1(1.0)

Chlorobenzene 502.2(2.1), 524.2(4.1)

1,2-Dichlorobenzene 502.2(2.1), 524.2(4.1)

1,4-Dichlorobenzene 502.2(2.1), 524.2(4.1)

1,2-Dichloroethane 502.2(2.1), 524.2(4.1)

cis-1,2-Dichloroethylene 502.2(2.1), 524.2(4.1)

trans-1,2-Dichloroethylene 502.2(2.1), 524.2(4.1)

Dichloromethane 502.2(2.1), 524.2(4.1)

1,2-Dichloropropane 502.2(2.1), 524.2(4.1)

Ethylbenzene 502.2(2.1), 524.2(4.1)

Styrene 502.2(2.1), 524.2(4.1)

Tetrachloroethylene 502.2(2.1), 524.2(4.1), 551.1(1.0)

1,1,1-Trichloroethane 502.2(2.1), 524.2(4.1), 551.1(1.0)

Trichloroethylene 502.2(2.1), 524.2(4.1), 551.1(1.0)

Toluene 502.2(2.1), 524.2(4.1)

1,2,4-Trichlorobenzene 502.2(2.1), 524.2(4.1)

1,1-Dichloroethylene 502.2(2.1), 524.2(4.1)

1,1,2-Trichloroethane 502.2(2.1), 524.2(4.1), 551.1(1.0)

Vinyl chloride 502.2(2.1), 524.2(4.1)

Xylenes (total) 502.2(2.1), 524.2(4.1)

2,3,7,8-TCDD (dioxin) 1613

2,4-D (as acids, salts and esters) 515.1(4.0), 515.2(1.1), 515.3(1.0), 555(1.0), D5317-93, 515.4(1.0)

Alachlor 505(2.1) , 507(2.1), 508.1(2.0), 525.2(2.0), 551.1(1.0) 1

Atrazine 505(2.1) , 507(2.1), 508.1(2.0), 525.2(2.0), 551.1(1.0) 1

Benzo(a)pyrene 525.2(2.0), 550, 550.1

Carbofuran 531.1(3.1), 6610, 531.2(1.0)

Chlordane 505(2.1), 508(3.1), 508.1(2.0), 525.2(2.0)
Contaminant Method (Revision Number) 1

IV- 18

Dalapon 515.1(4.0), 515.3(1.0), 552.1(1.0), 552.2(1.0), 515.4(1.0)

Di(2-ethylhexyl)adipate 506(1.1), 525.2(2.0)

Di(2-ethylhexyl)phthalate 506(1.1), 525.2(2.0)

Dibromochloropropane (DBCP) 504.1(1.1), 551.1(1.0)

Dinoseb 515.1(4.0),515.2(1.1), 515.3(1.0), 555(1.0), 515.4(1.0)

Diquat 549.2(1.0)

Endothall 548.1(1.0)

Endrin 505(2.1), 508(3.1), 508.1(2.0), 525.2(2.0), 551.1(1.0)

Ethylene dibromide (EDB) 504.1(1.1), 551.1(1.0)

Glyphosate 547, 6651

Heptachlor 505(2.1), 508(3.1), 508.1(2.0), 525.2(2.0), 551.1(1.0)

Heptachlor Epoxide 505(2.1), 508(3.1), 508.1(2.0), 525.2(2.0), 551.1(1.0)

Hexachlorobenzene 505(2.1), 508(3.1), 508.l(2.0), 525.2(2.0), 551.1(1.0)

Hexachlorocyclopentadiene 505(2.1), 508(3.1), 508.1(2.0), 525.2(2.0), 551.1(1.0)

Lindane 505(2.1), 508(3.1), 508.1(2.0), 525.2(2.0), 551.1(1.0)

Methoxychlor 505(2.1), 508(3.1), 508.1(2.0), 525.2(2.0), 551.1(1.0)

Oxamyl 531.1(3.1), 6610, 531.2(1.0)

PCBs (as decachlorobiphenyl)2

 (as Aroclors)

508A(1.0)

505(2.1), 508(3.1), 508.1(2.0), 525.2(2.0)

Pentachlorophenol 515.1(4.0), 515.2(1.1), 515.3(1.0), 525.2(2.0), 555(1.0), D5317-93,

515.4(1.0)

Picloram 515.1(4.0), 515.2(1.1), 515.3(1.0), 555(1.0), D5317-93, 515.4(1.0)

Simazine 505(2.1), 507(2.1), 508.1(2.0), 525.2(2.0), 551.1(1.0)

2,4,5-TP (Silvex) 515.1(4.0), 515.2(1.1), 515.3(1.0), 555(1.0), D5317-93, 515.4(1.0)

Toxaphene 505(2.1), 508(3.1), 508.1(2.0), 525.2(2.0)

HAA5 552.1(1.0), 552.2(1.0), SM6251 4

Total Trihalomethanes 502.2(2.1), 524.2(4.1), 551.1(1.0)

Footnotes

 Methods 508A, and 515.1 are in Methods for the Determination of Organic Compounds in Drinking Water, EPA- 1

600/4-88-039, December 1988, Revised, July 1991. Methods 547, 550, and 550.1 are in Methods for the Determination
IV- 19

of Organic Compounds in Drinking Water - Supplement I, EPA-600-4-90-020, July 1990. Methods 515.2, 524.2, 548.1,

552.1 and 555 are in Methods for the Determination of Organic Compounds in Drinking Water - Supplement II, EPA600/R-92-129. Methods 502.2, 504.1, 505, 506, 507, 508, 508.1, 515.1, 515.2, 524.2, 525.2, 531.1, 551.1, 552.2 are in

Methods for the Determination of Organic Compounds in Drinking Water - Supplement III, EPA 600/R-95/131. Methods

513.3 and 549.2 are in Methods for the Determination of Organic and Inorganic Compounds in Drinking Water - Volume

1, EPA 815-R-00-014, August 2000. Method 1613, Tetra-Through Octa- Chlorinated Dioxins and Furans by Isotopic

Dilution HRGC/HRMS, EPA-81/B-94-003, October 1994 These documents are available from the National Technical

Information Service, NTIS PB91-231480, PB91-146027, PB92-207703, PB2000-106981 and PB95-104774, U.S.

Department of Commerce, 5285 Port Royal Road, Springfield, Virginia 22161. The toll-free number is 800-553-6847.

Method 1613 is available from USEPA Office of Water Resource Center (RC-4100), 401 M. Street S.W., Washington,

D.C. 20460. The phone number is 202-260-7786. Method 6651 and 6610 are contained in the currently approved

editions of Standard Methods for the Examination of Water and Wastewater, American Public Health Association, 1015

Fifteenth Street NW, Washington, D.C. 20005.

 PCBs are qualitatively identified as Aroclors and measured for compliance purposes as decachlorobiphenyl using 2

Method 508A.

 A nitrogen-phosphorus detector should be substituted for the electron capture detector in Method 505 (or another 3

approved method should be used) to determine alachlor, atrazine and simazine, if lower detection limits are required.

The total of monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, monobromoacetic acid and dibromoacetic 4

acid.
IV- 20

Table IV-4 Approved Methods for Disinfectant Residuals

Public water systems need to measure residual disinfectant concentrations with one of the analytical methods in the

following table. The methods are contained in the 18 , 19 and 20 editions of Standard Methods for the Examination th th th

of Water and Wastewater. 

Residual1 Methodology SM3

Free Chlorine Amperometric Titration 2

DPD Ferrous Titrimetric

DPD Colorimetric

Syringaldahyde (FACTS)

 4500-Cl D

 D 1253-86

 4500-Cl F

 4500-Cl G

 4500-Cl H

Combined Chlorine

 (Chloramines)

Amperometric Titration

DPD Ferrous Titrimetric

DPD Colorimetric

 4500-Cl D

 D 1253-86

 4500-Cl F

 4500-Cl G

Total Chlorine Amperometric Titration 2

Amperometric Titration

(low level measurement)

DPD Ferrous Titrimetric

DPD Colorimetric

Iodometric Electrode

 4500-Cl D

D 1253-86

 4500-Cl E

 4500-Cl F

 4500-Cl G

 4500-Cl I

Chlorine Dioxide Amperometric Titration

DPD Method

Amperometric Titration

2

 4500-ClO C4

2

 4500-ClO D

2

 4500-ClO E

O 3zone Indigo Method 4500-O B

Footnotes

 If approved by the State, residual disinfectant concentrations for free chlorine and combined chlorine also may be 1

measured by using DPD colorimetric test kits. 

 Free and total chlorine residuals may be measured continuously by adapting a specified chlorine residual method for use 2

with a continuous monitoring instrument provided the chemistry, accuracy, and precision of the measurement remain the

same. Instruments used for continuous monitoring need to be calibrated with a grab sample measurement at least every

five days, or with protocol approved by the State.

Standard Methods for the Examination of Water and Wastewater, 18 , 19 or 20th Edition, American Public Health 3 th th

Association, 1015 Fifteenth Street NW, Washington, D.C. 20005.

2

 Method 4500-Cl0 is not approved for determining compliance at 141.131(c) because the other two methods are 4

superior.
IV- 21

Table IV-5 Recommended Methods for Secondary Drinking Water Contaminants

Analyses of aluminum, chloride, color, fluoride, foaming agents, iron, manganese, odor, silver, sulfate, total dissolved

solids (TDS) and zinc to determine compliance under §143.3 may be conducted with the methods in the following table.

Criteria for analyzing aluminum, iron, manganese, silver, and zinc samples with digestion or directly without digestion,

and other mandatory procedures are contained in Section IV of “Technical Notes on Drinking Water Methods”

EPA/600/R-94/173, October 1994. Measurement of pH may be conducted with one of the methods listed above in

Section I under "Methods for Inorganic Chemicals."

Contaminant EPA ASTM SM Other 1 2

Aluminum 200.7 3120B 3

200.8 3113B 3

200.9 3111D 3

Chloride 300.0 D4327-91 4110B 4

D512-89B 4500-Cl B,-D –

Color 2120B

Fluoride 300.0 D4327-91

D1179-93

4110 B

4500-F B, C, D, E - 

380-75WE

129-71W5

Foaming Agents 5540C

Iron 200.7 3120B 3

200.9 3111B 3

3113B

Manganese 200.7 3120B 3

200.8 3111B 3

200.9 3113B 3

Odor 2150B

Silver 200.7 3120B I-3720-85 3 6

200.8 3111B 3

200.9 3113B 3

Sulfate 300.0 D4327-91 4110B 4

375 4.2 D516-90 4500-SO -E,-F 4

4

 4500-SO -C,D

TDS 2540C

Zinc 200.7 3120B 3

200.8 3111B 3
IV- 22

Footnotes

Annual Book of ASTM Standards, Vols. 11.01 and 11.02, American Society for Testing and Materials, 1916 Race Street, 1

Philadelphia, PA 19103.

Standard Methods for the Examination of Water and Wastewater, 18 , 19 or 20th Edition, American Public Health 2 th th

Association, 1015 Fifteenth Street NW, Washington, D.C. 20005. Except 3111B, 3111D, 3112B, 3113B, 3114B are not

approved in the 20 edition. th

 "Methods for the Determination of Metals in Environmental Samples - Supplement I," EPA-600/R-94-111, May 1994. 3

Available at NTIS, PB94-184942.

 "Methods for the Determination of Inorganic Substances in Environmental Samples," EPA-600/R-93-100, August 1993. 4

Available at NTIS, PB94-121811.

 Industrial Method No. 129-71W, "Fluoride in Water and Wastewater," December 1972, and Method No. 380-75WE, 5

"Fluoride in Water and Wastewater," February 1976, Bran and Lubbe, 1025 Busch Parkway Buffalo Grove, IL 60089.

(Formerly Technicon Industrial Systems, Tarrytown, NY 10591)

Available from Books and Open-File Reports Section, U.S. Geological Survey, Federal Center, Box 25425, Denver, 6

CO 80225-0425.
IV- 23

 Table IV-6 Sample Containers, Preservation and Holding Times for Regulated Parameters

 Parameter/

 Method

 Preservative Sample Holding

 Time

 Extract Holding 

Time and Storage

Conditions

 Suggested

 Sample Size

Type of

Container

Metals

(except Hg)

HNO3 pH<2 6 months 1 L Plastic or Glass

M 3ercury HNO pH<2 28 days 100 mL Plastic or Glass

Alkalinity Cool, 4C 14 days 100 mL Plastic or Glass

Asbestos Cool, 4C 48 hours 1 L Plastic or Glass

Chloride none 28 days 100 mL Plastic or Glass

Residual 

Disinfectant

none immediately 200 mL Plastic or Glass

Color Cool, 4C 48 hours 100 mL Plastic or Glass

Conductivity Cool, 4C 28 days 100 mL Plastic or Glass

Cyanide Cool, 4C,

Ascorbic acid (if

chlorinated),

NaOH pH>12

14 days 1 L Plastic or Glass

Fluoride none 1 month 100 mL Plastic or Glass

Foaming Agents Cool, 4C 48 hours

Nitrate

 (chlorinated)

Cool, 4C

non-acidified

14 days 100 mL Plastic or Glass

Nitrate

(non chlorinated)

Cool, 4C, 

non-acidified

48 hours 100 mL Plastic or Glass

Nitrite Cool, 4C 48 hours 100 mL Plastic or Glass

Nitrate+ Nitrite H2SO4 pH<2 28 days 100 mL Plastic or Glass

Odor Cool, 4C 24 hours 200 mL Glass

pH none immediately 25 mL Plastic or Glass

o-Phosphate Cool, 4C 48 hours 100mL Plastic or Glass
 Parameter/

 Method

 Preservative Sample Holding

 Time

 Extract Holding 

Time and Storage

Conditions

 Suggested

 Sample Size

Type of

Container

IV- 24

Silica Cool, 4C 28 days 100 mL Plastic

Solids (TDS) Cool, 4C 7 days 100 mL Plastic or Glass

Sulfate Cool, 4C 28 days 100 mL Plastic or Glass

Temperature none immediately 1 L Plastic or Glass

Turbidity Cool, 4C 48 hours 100 mL Plastic or Glass

502.2 Sodium

Thiosulfate

or Ascorbic

Acid, 4C, HCl

pH<2

14 days 40-120 mL Glass with PTFE

Lined Septum

504.1 Sodium

Thiosulfate

Cool, 4C,

14 days 4C, 24 hours 40 mL Glass with PTFE

Lined Septum

505 Sodium

Thiosulfate

Cool, 4C

14 days

(7 days for

Heptachlor)

4C, 24 hours 40 mL Glass with PTFE

Lined Septum

506 Sodium

Thiosulfate

Cool, 4C, Dark

14 days 4C, dark

14 days

1 L Amber Glass with

 PTFE Lined Cap

507 Sodium

Thiosulfate

Cool, 4C, Dark

14 days(see

method for

exceptions)

4C, dark

14 days

1 L Amber Glass with 

PTFE Lined Cap

508 Sodium

Thiosulfate

Cool, 4C, Dark

7 days (see

method for

exceptions)

4C, dark

14 days

1 L Glass with PTFE

Lined Cap

508A Cool, 4C 14 days 30 days 1 L Amber Glass with 

PTFE Lined Cap

508.1 Sodium Sulfite

HCl pH<2

Cool, 4C

14 days (see

method for

exceptions)

30 days 1 L Glass

with PTFE

Lined Cap

515.1 Sodium

Thiosulfate

Cool, 4C, Dark

14 days 4C, dark

28 days

1 L Amber Glass with 

PTFE Lined Cap
 Parameter/

 Method

 Preservative Sample Holding

 Time

 Extract Holding 

Time and Storage

Conditions

 Suggested

 Sample Size

Type of

Container

IV- 25

515.2 Sodium

Thiosulfate or

Sodium Sulfite

HCl pH<2

Cool, 4C, Dark

14 days #4C, dark

14 days

1 L Amber Glass with

 PTFE Lined Cap

515.3 Sodium

Thiosulfate

Cool, 4C, Dark

14 days #4C, dark

14 days

50 mL Amber Glass with

 PTFE Lined Cap

515.4 Sodium Sulfite,

dark, cool #10C

fro first 48 hr. 

#6C thereafter

14 days #0C

21 days 

40 mL Amber glass with

PTFE lined

septum

524.2 Ascorbic Acid

or Sodium

Thiosulfate

HCl pH<2, Cool

4C

14 days 40-120 mL Glass with PTFE

Lined Septum

525.2 Sodium Sulfite,

Dark, 

Cool, 4C, HCl

pH<2

14 days (see 

method for 

exceptions)

#4C

 30 days

1 L Amber Glass with 

PTFE Lined Cap

531.1, 6610 Sodium

Thiosulfate,

Monochloroacet

ic acid, pH<3,

Cool, 4C

Cool 4C 28 days 60 mL Glass with PTFE

Lined Septum

531.2 Sodium

Thiosulfate,

Potassium

Dihydrogen

Citrate buffer to

pH 4, dark,

#10C for first 48

hr, #6C

thereafter

28 days 40 mL Glass with PTFE

Lined Septum

547 Sodium

Thiosulfate

Cool, 4C

14 days(18

mo.frozen)

60 mL Glass with PTFE

Lined Septum
 Parameter/

 Method

 Preservative Sample Holding

 Time

 Extract Holding 

Time and Storage

Conditions

 Suggested

 Sample Size

Type of

Container

IV- 26

548.1 Sodium

Thiosulfate

(HCl pH 1.5-2 if

high biological

activity)

Cool, 4C, Dark

7 days #4C

14 days

$ 250 mL Amber Glass with 

PTFE

Lined Septum

549.2 Sodium

Thiosulfate,

2 4 (H SO pH<2 if

biologically

active)

Cool, 4C, Dark

7 days 21 days $ 250mL High Density

Amber Plastic or

Silanized Amber

Glass

550, 550.1 Sodium

Thiosulfate

Cool, 4C, HCl

pH<2

7 days 4C, Dark

550, 30 days

550.1, 40 days

1 L Amber Glass with

 PTFE Lined Cap

551.1 Sodium Sulfite, 

Ammonium

Chloride,

pH 4.5-5.0 with

phosphate buffer

Cool, 4C

14 days $ 40 mL Glass with PTFE

Lined Septum

552.1 Ammonium

chloride

Cool, 4C, Dark

28 days #4C, dark

48 hours

250 mL Amber Glass with

 PTFE Lined Cap

552.2 Ammonium

chloride

Cool, 4C, Dark

14 days #4C, dark 7 days

#-10C 14 days

50mL Amber Glass with

 PTFE Lined Cap

555 Sodium Sulfite

HCl, pH#2

Dark, Cool 4C

14 days $ 100 mL Glass with PTFE

Lined cap

1613 Sodium

Thiosulfate

Cool, 0-4C,

Dark

Recommend

40 days

1 L Amber Glass with

 PTFE Lined Cap
IV- 27

Table IV-7 MCLs and Detection Limits Requirements in the CFR (mg/L) to Composite

Inorganics MCL* MCLG Detection Limit Required to

Composite [§141.23(a)(4)]

Asbestos 7 MFL 7 MFL 1.4 MFL

Bromate 0.010 NA

Chlorite 1.0 NA

Cyanide 0.2 0.2 0.04

Fluoride 4.0 0.8

Nitrate 10 10 2

Nitrite 1 1 0.2

* The monitoring trigger for the inorganics is the

 MCL except for both nitrate and nitrite, which are 1⁄2 the MCL

 Metals MCL * MCLG Detection Limit Required to

Composite [§141.23(a)(4)]

Antimony 0.006 0.006 0.001

Arsenic 0.01 - 0.01

Barium 2 2 0.4

Beryllium 0.004 0.004 0.0008

Cadmium 0.005 0.005 0.001

Chromium 0.1 0.1 0.02

Copper** 1.3 1.3 0.001

0.02 (for direct aspiration AA)

Lead** 0.015 zero 0.001

Mercury 0.002 0.002 0.0004

Selenium 0.05 0.05 0.01

Thallium 0.002 0.0005 0.0004

* The monitoring trigger for metals is the MCL unless compositing, then 1/5 MCL is required

**Action Level
IV- 28

TABLE IV-8 VOC MCLs and Detection Limit Requirements in the CFR (mg/L) for Compliance Monitoring

Volatile Organics MCL MCLG Required MDL *

THMs 0.08 NA NA

HAA5 0.06 NA NA

Benzene 0.005 zero 0.0005

Carbon tetrachloride 0.005 zero 0.0005

Chlorobenzene 0.1 0.1 0.0005

o-Dichlorobenzene 0.6 0.6 0.0005

p-Dichlorobenzene 0.075 0.075 0.0005

1.2-Dichloroethane 0.005 zero 0.0005

1,1-Dichloroethylene 0.007 0.007 0.0005

c-1,2-Dichloroethylene 0.07 0.07 0.0005

t-1,2-Dichloroethylene 0.1 0.1 0.0005

Dichloromethane 0.005 zero 0.0005

1,2-Dichloropropane 0.005 zero 0.0005

Ethylbenzene 0.7 0.7 0.0005

Styrene 0.1 0.1 0.0005

Tetrachloroethylene 0.005 zero 0.0005

Toluene 1 1 0.0005

1,2,4-Trichlorobenzene 0.07 0.07 0.0005

1,1,1-Trichloroethane 0.2 0.2 0.0005

1,1,2-Trichloroethane 0.005 0.003 0.0005

Trichloroethylene 0.005 zero 0.0005

Vinyl chloride 0.002 zero 0.0005

Xylenes 10 10 0.0005

 *A laboratory must be able to achieve an MDL of 0.0005 mg/L to be certified to analyze

 samples for compliance monitoring [§141.24(f)(17)(i)(E) and (ii)(C)]. This is also the 

 monitoring trigger for VOCs [§141.24(f)(11)].
IV- 29

TABLE IV-9 SOC MCLs and Detection Limit Requirements in the CFR to Reduce Monitoring (mg/L)

 SOCs MCL MCLG Monitoring Trigger*

Alachlor 0.002 zero 0.0002

Atrazine 0.003 0.003 0.0001

Benzo(a)pyrene 0.0002 zero 0.00002

Carbofuran 0.04 0.04 0.0009

Chlordane 0.002 zero 0.0002

2,4-D 0.07 0.07 0.0001

Di(2-ethylhexyl)adipate 0.4 0.4 0.0006

Di(2-ethylhexyl)phthalate 0.006 zero 0.0006

Dibromochloropropane (DBCP) 0.0002 zero 0.00002

Dalapon 0.2 0.2 0.001

Dinoseb 0.007 0.007 0.0002

Dioxin (2,3,7,8-TCDD) 3x10 zero 5x10 -8 -9

Diquat 0.02 0.02 0.0004

Endothall 0.1 0.1 0.009

Endrin 0.002 0.002 0.00001

Ethylenedibromide (EDB) 0.00005 zero 0.00001

Glyphosate 0.7 0.7 0.006

Heptachlor 0.0004 zero 0.00004

Heptachlor Epoxide 0.0002 zero 0.00002

Hexachlorobenzene 0.001 zero 0.0001

Hexachlorocyclopentadiene 0.05 0.05 0.0001

Lindane 0.0002 0.0002 0.00002

Methoxychlor 0.04 0.04 0.0001

Oxamyl 0.2 0.2 0.002

PCBs (as decachlorobiphenyl) 0.0005 zero 0.0001

Pentachlorophenol 0.001 zero 0.00004

Picloram 0.5 0.5 0.0001

Simazine 0.004 0.004 0.00007

Toxaphene 0.003 zero 0.001

2,4,5-TP (Silvex) 0.05 0.05 0.0002

*The monitoring triggers for SOCs listed in the regulation are also required for compositing but 

are not required by regulation for certification [§141.24(g)(7),(10)(i) and (18)].
 Recommended Level

1

IV- 30

Table IV-10 MCL and Proficiency Testing Sample Acceptance Criteria in the CFR 

 Primary and Secondary Drinking Water Regulations [§141.23(k)(3)(ii) and 141.24(f)(17) and (19)]

Regulated Parameter MCL/ [SMCL] MCLG Acceptance

METALS

Aluminum [0.05-0.2] -

Antimony 0.006 0.006 + 30%

Arsenic 0.01 -

Barium 2.0 2.0 + 15%

Beryllium 0.004 0.004 + 15%

Cadmium 0.005 0.005 + 20%

Calcium - -

Chromium 0.1 0.1 + 15%

Copper 1.3/90%

[1.0]

1.3 + 10%

Iron [0.3] - 

Lead 0.015/90% zero + 30%

Manganese [0.05] -

Mercury 0.002 0.002 + 30%

Selenium 0.05 0.05 + 20%

Silica - -

Silver [0.10]

Sodium 20 -

1

Thallium 0.002 0.0005 + 30%

Zinc [5.0] -
Regulated Parameter MCL/ [SMCL] MCLG Acceptance

IV- 31

INORGANICS

Alkalinity - -

Asbestos 7MF/L>10u 7MF/L>10u 2 Std Dev

Bromate 0.010 95% conf interval

around mean

Chloride [250] -

Chlorite 1.0 95% conf interval

around mean

Residual Disinfectant detectable -

Color [15cu] -

Conductivity - -

Corrosivity [non-corrosive] -

Cyanide 0.2 0.2 + 25%

Fluoride 4.0

[2.0]

 - + 10%

Foaming Agents [0.5] -

Nitrate (as N) 10 10 + 10%

Nitrite (as N) 1 1 + 15% 

Nitrate/Nitrite (as N) 10 10

Odor [3 t.o.n.] -

pH 6.5-8.5

[6.5-8.5]

 -

o-Phosphate - -

Solids(TDS) [500] -

Sulfate deferred

[250]

deferred

Temperature - -
Regulated Parameter MCL/ [SMCL] MCLG Acceptance

IV- 32

VOLATILES

Trihalomethanes(Total) 0.080 95% conf interval

around mean

HAA5 0.060 95% conf interval

around mean

Benzene 0.005 zero *

Carbon tetrachloride 0.005 zero *

Chlorobenzene 0.1 0.1 *

p-Dichlorobenzene 0.075

[0.005]

0.075 *

o-Dichlorobenzene 0.6 0.6 *

1,2-Dichloroethane 0.005 zero *

1,1-Dichloroethylene 0.007 0.007 *

c-1,2-Dichloroethylene 0.07 0.07 *

t-1,2-Dichloroethylene 0.1 0.1 *

Dichloromethane 0.005 zero *

1,2-Dichloropropane 0.005 zero *

Ethylbenzene 0.7 0.7 *

Styrene 0.1 0.1 *

Tetrachloroethylene 0.005 zero *

Toluene 1 1 *

1,2,4-Trichlorobenzene 0.07 0.07 *

1,1,1-Trichloroethane 0.2 0.2 *

1,1,2-Trichloroethane 0.005 0.003 *

Trichloroethylene 0.005 zero *

Vinyl chloride 0.002 zero ± 40%

Xylenes(Total) 10 10 *
Regulated Parameter MCL/ [SMCL] MCLG Acceptance

IV- 33

SYNTHETIC ORGANICS

Alachlor 0.002 zero + 45%

Aldicarb Postponed Postponed 2 Std Dev

Aldicarb Sulfoxide Postponed Postponed 2 Std Dev

Aldicarb Sulfone Postponed Postponed 2 Std Dev

Atrazine 0.003 0.003 + 45%

Carbofuran 0.04 0.04 + 45%

Chlordane 0.002 zero + 45%

2,4-D 0.07 0.07 + 50%

Dalapon 0.2 0.2 2 Std Dev

Dibromochloropropane(DBCP) 0.0002 zero + 40%

Dinoseb 0.007 0.007 2 Std Dev

Diquat 0.02 0.02 2 Std Dev

Endothall 0.1 0.1 2 Std Dev

Endrin 0.002 0.002 + 30%

Ethylenedibromide(EDB) 0.00005 zero + 40%

Glyphosate 0.7 0.7 2 Std Dev

Heptachlor 0.0004 zero + 45%

Heptachlor epoxide 0.0002 zero + 45%

Lindane 0.0002 0.0002 + 45%

Methoxychlor 0.04 0.04 + 45%

Oxamyl (Vydate) 0.2 0.2 2 Std Dev

Pentachlorophenol 0.001 zero + 50%
Regulated Parameter MCL/ [SMCL] MCLG Acceptance

IV- 34

Picloram 0.5 0.5 2 Std Dev

Simazine 0.004 0.004 2 Std Dev

Toxaphene 0.003 zero + 45%

2,4,5-TP(Silvex) 0.05 0.05 + 50%

Hexachlorobenzene 0.001 zero 2 Std Dev

Hexachlorocyclopentadiene 0.05 0.05 2 Std Dev

Benzo(a)pyrene 0.0002 zero 2 Std Dev

PCBs

(as decachlorobiphenyl)

0.0005 zero 0-200% 

2,3,7,8-TCDD( Dioxin) 3x10 zero 2 Std Dev -8

Acrylamide Treatment zero NA

Epichlorohydrin Treatment zero NA

Di(2-ethylhexyl)adipate 0.4 0.4 2 Std Dev

Di(2-ethylhexyl)phthalate 0.006 zero 2 Std Dev

 * the acceptance limits for VOCs are ±20% at $0.010mg/L and ±40% at <0.010mg/L

NA - Not Applicable
Table IV-11 Promulgated Organic Drinking Water Methods (As of January 2005) 

Method 

Number Method Title Revision 

IV- 35

502.2 Volatile Organic Compounds in Water By Purge and Trap Capillary 2.1 a

Column Gas Chromatography with Photoionizatio and Electrolytic

Conductivity Detectors in Series 

504.1 1,2-Dibromoethane (EDB), 1,2-Dibromo-3-Chloropropane (DBCP), 1.1 a

and 1,2,3-Trichloro-propane (123TCP) in Water by Microextraction and Gas Chromatography

505 Analysis of Organohalide Pesticides and Commercial Polychlorinated 2.1 a

Biphenyl Products in Water by Micro-Extraction and Gas Chromatography

506 Determination of Phthalate and Adipate Esters in Drinking Water by 1.1 a

Liquid-Liquid Extraction or Liquid-Solid Extraction and 

Gas Chromatography with Photoionization Detection 

507 Determination of Nitrogen and Phosphorus-Containing Pesticides 2.1 a

in Water by Gas Chromatography with a Nitrogen-Phosphorus Detector

508 Determination of Chlorinated Pesticides in Water by Gas 3.1 a

Chromatography with An Electron Capture Detector

508A Screening for Polychlorinated Biphenyls by Perchlorination and 1.0 b

Gas Chromatography

508.1 Determination of Chlorinated Pesticides, Herbicides, and Organohalides 2.0 a

by Liquid-Solid Extraction and Electron Capture Gas Chromatography

515.1 Determination of Chlorinated Acids in Water by Gas Chromatography 4.0 b

with an Electron Capture Detector 

515.2 Determination of Chlorinated Acids in Water Using Liquid-Solid 1.1 a

Extraction and Gas Chromatography With an Electron Capture Detector 

515.3 Determination of Chlorinated Acids in Drinking Water by Liquid-Liquid 1.0 e

Extraction, Derivatization and Gas Chromatography with Electron Capture

Detection

515.4 Determination of Chlorinated Acids in Drinking Water by Liquid-Liquid 1.0 f

Microextraction, Derivatization and Fast Gas Chromatography with 

Electron Capture Detection

524.2 Measurement of Purgeable Organic Compounds in Water by Capillary 4.1 a

Column Gas Chromatography/Mass Spectrometry

525.2 Determination of Organic Compounds in Drinking Water by Liquid-Solid 2.0 (a)

Extraction and Capillary Column Gas Chromatography/Mass Spectrometry

531.1 Measurement of N-Methylcarbamoyloximes and N-Methylcarbamates in 3.1 a

Water by Direct Aqueous Injection HPLC with Post Column Derivatization
Table IV-11 Promulgated Organic Drinking Water Methods (As of January 2005) 

Method 

Number Method Title Revision 

IV- 36

531.2 Measurement of N-Methylcarbamoyloximes and N-Methylcarbamates in 1.0 f

Water by Direct Aqueous Injection HPLC with Post Column Derivatization

547 Determination of Glyphosate in Drinking Water By Direct-Aqueous- c

Injection HPLC, Post-Column Derivatization, and Fluorescence Detection

548.1 Determination of Endothall in Drinking Water by Ion Exchange Extraction, 1.0 d

Acidic Methanol Methylation Gas Chromatography/Mass Spec.

549.2 Determination of Diquat and Paraquat in Drinking Water by Liquid-Solid 1.0 e

Extraction and High Performance Liquid Chromatography

with Ultraviolet Detection

550 Determination of Polycyclic Aromatic Hydro-carbons in Drinking Water c

by Liquid-Liquid Extraction and HPLC with Coupled Ultraviolet and 

Fluorescence Detection

550.1 Determination of Polycyclic Aromatic Hydro-carbons in Drinking Water c

by Liquid-Solid Extraction and HPLC with Coupled Ultraviolet and 

Fluorescence Detection 

551.1 Determination of Chlorination Disinfection Byproducts, Chlorinated Solvents, 1.0 a

and Halogenated Pesticides/Herbicides in Drinking Water by Liquid-Liquid 

Extraction and Gas Chromatography with Electron-Capture Detection

552.1 Determination of Haloacetic Acids and Dalapon in Drinking Water by 1.0 d

Ion Exchange Liquid-Solid Extraction and Gas Chromatography With 

Electron Capture Detection

552.2 Determination of Haloacetic Acids and Dalapon in Drinking Water by 1.0 a

Liquid-Liquid Extraction, Derivatization and Gas Chromatography With 

Electron Capture Detection 

 555 Determination of Chlorinated Acids in Water by High Performance Liquid 1.0 d

Chromatography with a Photodiode Array Ultraviolet Detector 
IV- 37

Footnotes

These methods are in the manual titled "Methods for the Determination of Organic Compounds in Drinking Water - a

Supplement III," document number EPA/600/R-95/131, August 1995. This manual is available from the National

Technical Information Service (NTIS*). Address your request to NTIS and ask for their order number PB95-261616,

cost is $101.00

These methods are in the manual titled "Methods for the Determination of Organic Compounds in Drinking Water," b

document number EPA/600/4-88/039, December 1988 (Revised July 1991). This manual is available from the

National Technical Information Service (NTIS*). Address your request to NTIS and ask for their order number

PB91-231480; cost is $77.50.

These methods are in the manual titled "Methods for the Determination of Organic Compounds in Drinking Water - c

Supplement I," document number EPA/600/4-90/020, July 1990. This manual is available from the National

Technical Information Service (NTIS*). Address your request to NTIS and ask for their order number PB91-146027;

cost is $58.50.

These methods are in the manual titled "Methods for the Determination of Organic Compounds in Drinking Water d

Supplement II," document number EPA/600/R-92/129, August 1992. This manual is available from the National

Technical Information Service (NTIS*). Address your request to NTIS and ask for their order number PB92-207703;

cost is $63.00.

These methods are in the manual titled, “Methods for the Determination of Organic and Inorganic Compounds in e

Drinking Water - Volume 1,” document number EPA 815-R-00-014, August 2000. This manual is available from the

National Technical Information Service (NTIS*). Address your request to NTIS and ask for their order number

PB2000-106981; cost is $71.50.

Stand alone methods. These manuals are available at the EPA web site at: f

http://www.epa.gov/ogwdw/methods/sourcalt.html

*National Technical Information Service (NTIS) 

 5285 Port Royal Road

 Springfield, VA 22161 

 Phone number: 800-553-6847

 Fax number: 703-0605-6900

There is a $5.00 handling charge for the total purchase.

**Microbiological and Chemical Exposure Assessment Research Division,

 Chemical Exposure Research Branch, Room 564

 U. S. Environmental Protection Agency

 26 West Martin Luther King Drive

 Cincinnati, OH 45268 

 Phone number: 513-569-7586

 Fax number: 513-569-7757 

 E-mail address: dwmethods-help@epamail.epa.gov
IV- 38
IV- 39

Example Checklists for On-Site Evaluation of 

Laboratories Analyzing Drinking Water
IV- 40

General Audit Information

Laboratory 

Street

City, State

Zip

Telephone No.

Fax No.

Audit Team Leader

Audit Team Members

Audit Team Affiliation

Date
IV- 41

Laboratory ___________________________________________ Evaluator _________________________

Location _____________________________________________ Date ______________________________

PHYSICAL FACILITY

Item Acceptable

Yes No

Comments

Environment

 Heating/Cooling/Humidity

 Lighting

 Ventilation/Exhaust hoods

 Cleanliness

 Electrical and water services 

Work Space

 Separation of incompatible testing areas

 Controlled access where appropriate

 Housekeeping

 Unencumbered access

 Adequate work space

Storage

 Chemicals properly stored and dated

 Standards properly stored, dated and

 labeled with concentration,

 preparer's name and solvent, origin,

 purity & traceability

Computers & automated equipment

Safety procedures
IV- 42

Laboratory ___________________________________________ Evaluator _________________________

Location _____________________________________________ Date ______________________________

PERSONNEL (Use additional paper if necessary.)

Position/ Title Name Education Level

Degree/Major*

Specialized

Training

Present

Specialty

Experience

Laboratory

Director

Manager

Supervisors

Instrument

 Operators

 AA 

 TEM

 HPLC

 GC

 ICP

 GC/MS

 

 IC

 Other analysts

 Yes No Comments

An organization chart available

QA manager has line authority

Personnel job descriptions and resumes

available

Personnel training documented

*If the major is not in chemistry, list hours of college level courses in chemistry. 
IV- 43

Laboratory ___________________________________________ Evaluator _________________________

Location _____________________________________________ Date ______________________________

QUALITY ASSURANCE AND DATA REPORTING

Item Comments Satisfactory

 Yes No

QA plan

Organization

Sampling

 SOPs available and used

 Preservation

 Containers

 Holding times

 Samplers trained

Sample Rejection

Laboratory sample handling

 Log in procedure

 Bound log book or secure

 computer log in

 Storage

 Tracking

Analytical Methods

 Written methods available

 Approved methods used

 SOPs available and used

Calibration

 Type and frequency

 Source of standards

 Data comparability

 Instrument tuning

Blanks

 Trip

 Field

 Method

Method Detection Limits

 Initial

 Frequency

 Acceptability

Precision and Accuracy

 Initial

 Frequency

 Acceptability

 Control charts

 Laboratory fortified blanks

 Matrix duplicates
Item Comments Satisfactory

 Yes No

IV- 44

Other QC Checks

 Performance check samples

 Internal and surrogate standards

 Matrix spikes and replicates

Qualitative Identification/

 Confirmation

Performance Evaluation

 Samples Analyzed

Data Reduction and Validation

 Calculations

 Transcription

 Significant Figures

 Validation

Preventive Maintenance

Records Retention

Corrective Action
IV- 45

Laboratory Evaluator 

Location Date 

Item No. of

Units

Method Manufacturer Model Satisfactory

Yes No

Analytical Balance

 0.1 mg readability

 Stable base

 ASTM type 1 or 2 weights 

 Service contracts

Magnetic Stirrer

 Variable speed, TFE coated stir bar

pH Meter

 Accuracy ±0.1 units

 Line or battery 

 Usable with specific ion electrodes

Conductivity Meter

 Readable in ohms or mhos

 Range of 2 ohms to 2 mhos

 Line or battery

Hot Plate - temp control

Centrifuge

 To 3000 rpm, Option of 4 x 50 mL

Color Standards

 To verify wavelengths photometers

 Should cover 200-800 nm

Refrigerator/Freezer

 Standard laboratory, explosion proof for organics

 Capable of maintaining nominal temperature of 4C
Item No. of

Units

Method Manufacturer Model Satisfactory

Yes No

IV- 46

Drying Oven

 Gravity or convection

 Controlled from room temp to 180°C or

 higher(±2°C)

Muffle Furnace

 To 450°C for cleaning organic glassware

Thermometer

 Mercury filled Celsius

 1°C or finer subdivision to 180°C

 NIST Certified or traceable

Glassware

 Borosilicate

 Volumetrics should be Class A

Spectrophotometer 

 Range 400 - 700 nm

 Band width - < 20 nm

 Use several size & shape cells

 Path length 1 - 5 cm

Cyanide, Fluoride

Mercury

Nitrate/Nitrite

Filter Photometer

 Range 400 - 700 nm

 Band width 10 -70 nm

 Use several size & shape cells

 Pathlength 1 - 5 cm

same as above

Amperometric Titrator Disinfectants

Specific Ion Meter

 Accuracy ± 1 mV

Cyanide

Fluoride

Nitrate
Item No. of

Units

Method Manufacturer Model Satisfactory

Yes No

IV- 47

Inductively Coupled Plasma (sequential,

simultaneous)

 Computer controlled

 Background correction

 Radio frequency generator

 Argon gas supply

 Mass Spectrometer

 Range 5-250 amu

 Resolution 1 amu peak

 width at 5% peak height

200.7, 3120B

200.8

Water Bath

 Electric or steam heat

 Controllable within 5°C to 100°C 

Mercury

Nitrate

Pesticides

Ion Chromatography

Conductivity detector, UV detector

 Suppressor column, Separator column

Fluoride, Chloride

Nitrate/Nitrite

Bromate, Chlorite

Atomic Absorption

Spectrophotometer

 Single channel, Single or double beam

 Grating monochrometer

 Photo multiplier detector

 Adjustable slits, Range 190-800 nm

 Readout system:

 Response time compatible with AA

 Able to detect positive interference for furnace 

 Chart recorder, CRT or hard copy printer 

Metals

Air/Acetylene

 commercial grade

Barium,

Copper
Item No. of

Units

Method Manufacturer Model Satisfactory

Yes No

IV- 48

Nitrous Oxide - comm. grade Barium

Graphite Furnace

 Argon or Nitrogen (commercial grade)

 Reach required temperatures

 Background corrector provision for offline analysis

 Pipets and tips

 microliter capacity with disposable tips

 5-100 microliters

 metal free tips

Antimony, Lead

Arsenic, Barium

Beryllium

Cadmium, Nickel

Chromium

Selenium

Thallium

Copper

Arsine Generator Arsenic, Selenium

Hydride Generator

 hydrogen, commercial grade

Antimony

Arsenic, Selenium

Mercury Analyzer

 Spectrophotometer

 Dedicated analyzer having a mercury lamp

 acceptable

 Adsorption cell:

 10 cm quartz cell with quartz end windows or

 11.5 cm plexiglass cell with 2.5 cm ID

 Air pump to deliver flow of at least

 1 L/min

 Aeration tube with coarse glass frit

 Flowmeter to measure air flow of 1 L/min

 Drying unit:

 6 in. tube with 20 grams magnesium

 perchlorate or heating device or lamp to

 prevent condensation on cell

Mercury
Item No. of

Units

Method Manufacturer Model Satisfactory

Yes No

IV- 49

Glassware

 Separatory funnels

 Kuderna Danish (K-D) concentrators

SOCs

Gas Chromatography

Split/splitless injection

 Oven temp. control ± 0.2°C

 Recorder, hard copy

 Oven temp. programmer

 Sub-ambient accessory

 Variable-constant differential flow control

Organics

 Electron Capture detector

 Linearized

Radiological Check

504.1, 505

508, 508.1

508A, 515.1

515.2, 551, 552.1

 Electrolytic Conductivity/Photoionization detector 502.2

506 (PID only)

 Nitrogen Phosphorus detector 507

Mass spectrometer

(quadrupole or ion trap)

 All glass enrichment device

 All glass transfer line

 Electron ionization at $70 eV

 Scanning 35-260 amu #2 sec

 Interfaced data system

524.2, 525.2

548.1

Purge & Trap system

 All glass purger

 5/25 mL sample size

502.2, 524.2
Item No. of

Units

Method Manufacturer Model Satisfactory

Yes No

IV- 50

High Performance Liquid Chromatography

 Constant flow

 Capable of injecting 

 20-500 μL

 Gradient system

 post-column reactor

 fluorescence detector 

 Absorption at 340nm and 308nm

 

 Gradient system

 UV detector at 254 nm

 fluorescence detector

 excitation at 280 nm

 detection at > 389 nm

 Isocratic system

 photodiode array detector

 excitation at 257 nm 

 detection at >308 nm

 Isocratic system

 post-column reactor

 fluorescence detector

 excitation at 340 nm

 detection at >455 nm

 Gradient system

 photodiode array/UV detector

 210-310 nm

531.1, 6610

550, 550.1

549.2

547

555
Item No. of

Units

Method Manufacturer Model Satisfactory

Yes No

IV- 51

Auto Analysis System

 multi-channel pump

 manifold, colorimeter

 

Cyanide, 

Nitrate/nitrite

Transmission Electron Microscope

 80 kV

 300-100,000X magnification

 1 nm resolution

 calibrate screen 

 SAED and ED

Asbestos
IV- 52

Laboratory Evaluator 

Location Date 

METHODOLOGY

Contaminant Method(s)

 Name/Number and revision

Reference 

 Cite source, year, page

Samples/Mo Satisfactory

Yes No

Antimony

Arsenic

Barium

Beryllium

Cadmium

Chromium

Copper

Lead

Mercury

Selenium

Thallium

Asbestos

Cyanide

Bromate

Chlorite

Fluoride
Contaminant Method(s)

 Name/Number and revision

Reference 

 Cite source, year, page

Samples/Mo Satisfactory

Yes No

IV- 53

Nitrate

Nitrite

Total THMs

HAA5

VOCs

Herbicides

Pesticides

EDB/DBCP

Dioxin

Other SOCs

PCBs

Carbamates

Diquat

Endothall

Glyphosate
IV- 54

Laboratory Evaluator 

Location Date 

SAMPLE COLLECTION

Item Comments Satisfactory 

Yes No 

Trained Sample Collector

Representative sampling

Complete sample form

Appropriate sampling

 and preservation

Samples exceeding

 holding times discarded

VOCs & THMs

 Hermetic seal
IV- 55

Laboratory Evaluator 

Location Date 

SAMPLE HANDLING AND PRESERVATION

Contaminant Container

material & size

Preservatives Holding Time

Sample Extract

Satisfactory

Yes No

Mercury

Metals

Asbestos

Cyanide

Fluoride

Nitrate

Nitrite

Bromate

Clorite

Total THMs

HAA5

VOCs

Herbicides

Pesticides

EDB/DBCP
Contaminant Container

material & size

Preservatives Holding Time

Sample Extract

Satisfactory

Yes No

IV- 56

Dioxin

Other SOCs

Carbamates

Diquat

Endothall

Glyphosate
V-1

Chapter V

Critical Elements for Microbiology

Note 1: This chapter uses the term "must" to refer to certification criteria that are required by the National Primary

Drinking Water Regulations. The term "should" is used for procedures that, while not specifically required by the

regulations, are considered good laboratory practices. To assure the validity of the data, it is critical that laboratories

observe both the regulatory and non-regulatory criteria. Certification Officers have the prerogative to refuse certification

if the quality control data are judged unsatisfactory or insufficient. 

Note 2: Quality control items, designated by a “QC,” necessitate written records. Each record should include analyst’s

initials and date(s). 

Note 3: References to Standard Methods for the Examination of Water and Wastewater are to the 18 , 19 , and 20th th th

editions (except where specifically noted).

1. Personnel

1.1 Supervisor/Consultant

The supervisor of the microbiology laboratory should have a bachelor's degree in microbiology, biology, or equivalent.

Supervisors who have a degree in a subject other than microbiology should have had at least one college-level microbiology

laboratory course in which environmental microbiology was covered. In addition, the supervisor should have a minimum

of two weeks training at a Federal agency, State agency, or academic institution in microbiological analysis of drinking

water or 80 hours of on-the-job training in water microbiology at a certified laboratory, or other training acceptable to the

State or EPA. If a supervisor is not available (and a waiver not granted per paragraph 1.3), a consultant having the same

qualifications may be substituted, as long as the laboratory can document that the consultant is acceptable to the State and

is present on-site frequently enough to satisfactorily perform a supervisor's duties.

The laboratory supervisor has the responsibility to ensure that all laboratory personnel have demonstrated their ability to

satisfactorily perform the analyses to which they are assigned and that all data reported by the laboratory meet the required

quality assurance and regulatory criteria.

1.2 Analyst (or equivalent job title)

The analyst should perform microbiological tests with minimal supervision and have at least a high school education. In

addition, the analyst should have a minimum of at least three months of bench experience in water, milk, or food

microbiology. The analyst should also have training acceptable to the State (or EPA for non-primacy States) in

microbiological analysis of drinking water and a minimum of 30 days of on-the-job training in drinking water microbiology

under an experienced analyst. Analysts should take advantage of workshops and training programs that may be available

from State regulatory agencies, professional societies, and manufacturers. Before analyzing compliance samples, the

analyst should demonstrate acceptable results on unknown samples.

1.3 Waiver of Academic Training

The certification authority may waive the need for the above specified academic training, on a case-by-case basis, for highly

experienced analysts. The certification authority may also waive the need for the above specified training, on a case-bycase basis, for supervisors of laboratories associated with drinking water systems that only analyze samples from that

system. If such a waiver for supervisor training is granted, the certification authority will prepare a written and signed

justification for such a waiver and have it available for inspection. Laboratories should also keep a copy of the waiver.

1.4 Personnel Records

 Personnel records that include academic background, specialized training courses completed, and types of microbiological

analyses conducted should be maintained on laboratory analysts.

2. Laboratory Facilities

Laboratory facilities should be clean, temperature and humidity controlled, and have adequate lighting at bench tops. The
V-2

laboratory should maintain effective separation between areas where activities are incompatible, minimize traffic flow and

ensure that contamination does not adversely affect data quality. Bench tops and floors should be of a material that is easily

cleaned and disinfected. Laboratory facilities should have sufficient bench-top area for processing samples; storage space

for media, glassware, and portable equipment; floor space for stationary equipment (incubators, water baths, refrigerators,

etc.); and associated area(s) for cleaning glassware and sterilizing materials. They should also have provisions for disposal

of microbiological waste. 

3. Laboratory Equipment and Supplies

The laboratory must have the equipment and supplies needed to perform the approved methods for which certification has

been requested.

3.1 pH Meter 

3.1.1 Accuracy and scale graduations should be within ±0.1 units.

3.1.2 pH buffer aliquots should be used only once.

3.1.3 Electrodes should be maintained according to the manufacturer's recommendations.

QC 3.1.4 pH meters should be standardized before each use period with pH 7.0 and either pH 4.0 or 10.0 standard buffers,

whichever range covers the desired pH of the media or reagent. The date and buffers used should be recorded in a

logbook, along with analyst’s initials.

QC 3.1.5 Record pH meter slope monthly, after calibration.

3.1.5.1 If the pH meter does not have a feature to automatically calculate the slope, but can provide the pH in

millivolts (mV), use the following formula to calculate the slope.

Slope (as %) = *mV at pH 7 - mV at pH 4* x 100/177

3.1.5.2 If the slope is below 95% or above 105%, the electrode or meter may need maintenance. Follow

manufacturer’s instructions for electrode maintenance and general cleaning. 

QC 3.1.6 Commercial buffer solution containers should be dated upon receipt and when opened. Buffers should be

discarded by the expiration date.

3.2 Balance (top loader or pan)

3.2.1 Balances should have readability of 0.1 g.

3.2.2 Balances should provide a sensitivity of at least 0.1 g for a load of 150 g, and 1 mg for a load of 10 g or less.

QC 3.2.3 Balances should be calibrated monthly using ASTM Class 1, 2, or 3 weights (minimum of three traceable

weights which bracket laboratory weighing needs, with a readability of 0.1 g.)(ASTM, 1916 Race St., Philadelphia,

PA 19103). Non-reference weights should be calibrated every six months with reference weights. Record calibrations

in a logbook with the initials of the individual performing the calibration. Correction values should be on file and used.

A reference weight should be re-certified every five years. Damaged or corroded weights should be replaced.

QC 3.2.4 Service contracts or internal maintenance protocols and maintenance records should be available. Maintenance,

calibration, and cleaning should be conducted at least annually by a qualified independent technician. In cases where

a laboratory is geographically isolated such that an annual visit from a technician is impractical, the certification officer

may modify or waive the need for a technician. 
V-3

3.3 Temperature Monitoring Device

3.3.1 Glass, dial, or electronic thermometers must be graduated in 0.5°C increments (0.2°C increments for tests which

are incubated at 44.5°C) or less, except as noted for hot air ovens (3.6.1) and refrigerators (3.9.1). The fluid column

in glass thermometers should not be separated. Dial thermometers that cannot be calibrated should not be used.

QC 3.3.2 The calibration of glass and electronic thermometers should be checked annually, and dial thermometers

quarterly, at the temperature used, against a National Institute of Standards and Technology (NIST)-traceable reference

thermometer or one that meets the requirements of NBS Monograph SP 250-23. The calibration factor and date of

calibration should be indicated on the thermometer. In addition, the laboratory should record in a QC record book the

following information:

C serial number of laboratory thermometer

C serial number of NIST-traceable thermometer (or other reference thermometer) 

C temperature of laboratory thermometer

C temperature of NIST-traceable thermometer (or other reference thermometer) 

C correction (or calibration) factor

C date of check 

C analyst’s initials

QC 3.3.3 If a thermometer differs by more than 1°C from the reference thermometer, it should be discarded. Reference

thermometers should be recalibrated at least every five years. Reference thermometer calibration documentation

should be maintained.

QC 3.3.4 Continuous recording devices that are used to monitor incubator temperature should be recalibrated at least

annually. A reference thermometer that meets the specifications described in paragraph 3.3.2 should be used for

calibration.

3.4 Incubator Unit

3.4.1 Incubator units must have an internal temperature monitoring device and maintain the temperature specified by

the method used, usually 35°±0.5°C and 44.5°±0.2°C. For non-portable incubators, thermometers should be placed

on the top and bottom shelves of the use area and immersed in liquid as directed by the manufacturer (except for

electronic thermometers). If an aluminum block incubator is used, culture dishes and tubes should fit snugly.

Laboratories which use the enzyme substrate tests with air-type incubators should note the product incubation details

indicated in paragraph 5.3.1.5.

QC 3.4.2 Calibration-corrected temperature should be recorded for each thermometer being used at least twice per day

during each day the incubator is in use, with readings separated by at least 4 hours. Documentation should include

the date and time of reading, temperature, and technician’s initials.

3.4.3 An incubation temperature of 44.5°±0.2°C can best be maintained with a circulating water bath equipped with

a gable cover.

3.5 Autoclave

3.5.1 The autoclave should have an internal heat source, a temperature gauge with a sensor on the exhaust, a pressure

gauge, and an operational safety valve. The autoclave should maintain a sterilization temperature during the sterilizing

cycle and complete an entire cycle (i.e., time between starting autoclave and removing items from autoclave) within

45 minutes when a 12-15 minute sterilization period is used. The autoclave should depressurize slowly enough to

ensure that media will not boil over and bubbles will not form in inverted tubes.

3.5.2 Because of safety concerns and difficulties with operational control, pressure cookers should not be used. 

QC 3.5.3 The date, contents, sterilization time and temperature, total time in autoclave, and analyst’s initials should be

recorded each time the autoclave is used. Copies of the service contract or internal maintenance protocol and

maintenance records should be kept. Maintenance should be conducted at least annually. A record of the most recent

service performed should be on file, available for inspection.
V-4

QC 3.5.4 A maximum-temperature-registering thermometer, electronic temperature readout device, or continuous

recording device should be used during each autoclave cycle to ensure that the proper temperature was reached, and

the temperature recorded. Overcrowding should be avoided. Spore strips or spore ampules should be used monthly

as bioindicators to confirm sterilization. (Since chemical indicators will respond to a wide range of times and

temperatures, i.e., a longer time at a lower temperature, as well as a shorter time at a higher temperature, a positive

result with the indicator does not necessarily show that sterilization has occurred.)

QC 3.5.5 Automatic timing mechanisms should be checked quarterly with a stopwatch or other accurate timepiece or time

signal, and the results recorded and initialed.

3.5.6 Autoclave door seals should be clean and free of caramelized media. Also, autoclave drain screens should be

cleaned frequently and debris removed.

3.6 Hot Air Oven

3.6.1 The oven should maintain a stable sterilization temperature of 170°-180°C for at least two hours. Overcrowding

should be avoided. The oven thermometer should be graduated in 10°C increments or less, with the bulb placed in

sand during use.

QC 3.6.2 The date, contents, sterilization time and temperature, and analyst's initials should be recorded. 

QC 3.6.3 Spore strips should be used monthly to confirm sterilization. Ampules are not recommended for hot air ovens

because they may explode or melt.

3.7 Colony Counter

A dark field colony counter should be used to count Heterotrophic Plate Count colonies.

3.8 Conductivity Meter

3.8.1 Meters should be suitable for checking laboratory reagent-grade water and readable in units of either

micromhos/cm or microsiemens/cm. 

QC 3.8.2 Calibrate the meter at least monthly, following the manufacturer’s recommendations and using an appropriate

certified and traceable low-level standard. If the meter cannot be calibrated with a commercial standard, the cell

constant should be determined at monthly intervals, using a method in Section 2510, "Conductivity," in Standard

Methods.

3.8.3 If an in-line unit cannot be calibrated, it should not be used to check reagent-grade water.

3.9 Refrigerator

3.9.1 Refrigerators should maintain a temperature of 1°-5°C. Calibrated thermometers should be graduated in at least

1°C increments and the thermometer bulb immersed in liquid.

QC 3.9.2 On days the refrigerator is in use, and the laboratory is staffed, the calibrated-corrected temperature should be

recorded at least once per day.

3.10 Inoculating Equipment

Sterile metal or disposable plastic loops, wood applicator sticks, sterile swabs, or sterile plastic disposable pipet tips

should be used. If wood applicator sticks are used, they should be sterilized by dry heat. The metal inoculating loops

and/or needles should be made of nickel alloy or platinum. (When performing an oxidase test, do not use nickel alloy

loops because they may interfere with the test).

3.11 Membrane Filtration Equipment (if MF procedure is used)

3.11.1 MF units must be stainless steel, glass, porcelain, or autoclavable plastic, not scratched or corroded, and must

not leak.
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QC 3.11.2 If graduation marks on clear glass or plastic funnels are used to measure sample volume, their accuracy should

be checked with a Class B graduated cylinder or better (or other Class B glassware), and a record of this calibration

check retained.

3.11.3 A 10X to 15X stereo microscope with a fluorescent light source must be used to count the target colonies (e.g.,

sheen colonies on M-Endo or Endo LES media).

3.11.4 Membrane filters must be approved by the manufacturer for total coliform water analysis. Approval is based

on data from tests for toxicity, recovery, retention, and absence of growth-promoting substances. Filters must be

gridmarked, 47 mm diameter, and 0.45 μm pore size, or alternate pore sizes if the manufacturer provides performance

data equal to or better than the 0.45 μm pore size. They should also be white, and of celluose ester. Membrane filters

and pads must be purchased presterilized or autoclaved for 10 minutes at 121°C before use.

QC 3.11.5 The lot number for membrane filters and the date received should be recorded. Ensure that membrane filters

are not brittle or distorted, and that manufacturer’s specification/certification sheet is available. 

3.11.6 Forceps used should be blunt and smooth-tipped without corrugations on the inner sides of tips.

3.12 Culture Dishes (loose or tight lids)

3.12.1 Presterilized plastic or sterilizable glass culture dishes should be used. To maintain sterility of glass culture

dishes, use stainless steel or aluminum canisters, or a wrap of heavy aluminum foil or char-resistant paper.

3.12.2 Loose-lid petri dishes should be incubated in a tight-fitting container, e.g., plastic vegetable crisper containing

a moistened paper towel to prevent dehydration of membrane filter and medium. 

3.12.3 Opened packs of disposable culture dishes should be resealed between use periods.

3.12.4 For membrane filter methods, culture dishes should be of an appropriate size to allow for the transfer of a single

membrane per plate.

3.13 Pipets

3.13.1 To sterilize and maintain sterility of glass pipets, stainless steel or aluminum canisters should be used, or

individual pipets should be wrapped in char-resistant paper or aluminum foil.

3.13.2 Pipets should have legible markings and should not be chipped or etched.

3.13.3 Opened packs of disposable sterile pipets should be resealed between use periods.

3.13.4 Pipets delivering volumes of 10 mL or less must be accurate to within a 2.5% tolerance. 

QC 3.13.5 Calibrated micropipetters may be used if tips are sterile. Micropipetters should be calibrated annually and

adjusted or replaced if the precision or accuracy is greater than 2.5%.

3.14 Glassware and Plasticware 

3.14.1 Glassware should be borosilicate glass or other corrosion-resistant glass and free of chips and cracks. Markings

on graduated cylinders and pipets must be legible. Plastic items should be clear and non-toxic to microorganisms.

QC 3.14.2 Graduated cylinders for measurement of sample volumes must be accurate to within a 2.5% tolerance. In lieu

of graduated cylinders, precalibrated containers that have clearly marked volumes accurate to within a 2.5% tolerance

may be used.

3.14.3 Culture tubes and containers containing fermentation medium should be of sufficient size to contain medium

plus sample without being more than three quarters full.
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3.14.4 Tube closures should be stainless steel, plastic, aluminum, or screw caps with non-toxic liners. Cotton plugs

and foam plugs should not be used. 

3.15 Sample Containers

3.15.1 Sample containers should be wide-mouth plastic or non-corrosive glass bottles with non-leaking ground glass

stoppers or caps with non-toxic liners that should withstand repeated sterilization, or sterile plastic bags containing

sodium thiosulfate. Other appropriate sample containers may be used. The capacity of sample containers should be

at least 120 mL (4 oz.) to allow at least a 1-inch head space. 

3.15.2 Glass stoppers must be covered with aluminum foil or char-resistant paper for sterilization.

3.15.3 Glass and plastic bottles that have not been presterilized should be sterilized by autoclaving. Glass bottles may

also be sterilized by dry heat. Empty containers should be moistened with several drops of water before autoclaving

to prevent an "air lock" sterilization failure.

3 2 2 3.15.4 If chlorinated water is to be analyzed, sufficient sodium thiosulfate (Na S O ) must be added to the sample

bottle before sterilization to neutralize any residual chlorine in the water sample. Dechlorination is addressed in

Section 9060A of Standard Methods. 

3.16 Ultraviolet lamp (if used)

3.16.1 A germicidal unit (254-nm) should be disconnected monthly and the lamp cleaned by wiping with a soft cloth

moistened with ethanol. A longwave unit (365-366-nm), used for fluorometric tests, should also be kept clean.

QC 3.16.2 A germicidal unit should be tested quarterly with a UV light meter or agar spread plate. The lamp should be

replaced if it emits less than 70% of its initial output or if an agar spread plate containing 200 to 250 microorganisms,

exposed to the UV light for two minutes, does not show a count reduction of 99%. Other methods may be used to test

a lamp if data demonstrate that they are as effective as the two suggested methods. (UV protective eye wear should

be used when checking the operation of a 254-nm lamp.)

3.17 Spectrophotometer or colorimeter (if used)

3.17.1 Wavelengths should be in the visible range—Spectronic 20 (Thermo Spectronic), or equivalent, with cell

holder for 1⁄2" diameter cuvettes (Model # 4015) or 13 mm × 100 mm cuvettes.

QC 3.17.2 A calibration standard and a method-specific blank should be analyzed every day the instrument is used, prior

to sample analysis. The calibration standard should give a reading in the desired absorbance range and should be

obtained from an outside source.

4. General Laboratory Practices

Although safety criteria are not covered in the laboratory certification program, laboratory personnel should be aware of

general and customary safety practices for laboratories. Each laboratory is encouraged to have a safety plan available.

Also, each laboratory should keep a copy, and follow the personal protection guidelines, of any material safety data sheet

accompanying the receipt of a toxic material.

4.1 Sterilization Procedures

4.1.1 Autoclaving times at 121°C are listed below. Except for membrane filters and pads and carbohydrate-containing

media, indicated times are minimum times and may necessitate adjustment depending upon volumes, containers, and

loads. Carbohydrate-based media should not be over-sterilized.
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Item Time (min)

Membrane filters & pads 10

Carbohydrate containing media 12-151

Contaminated test materials 302

Membrane filter assemblies 15

Sample collection bottles 15

Individual glassware 15

Dilution water blank 15

Rinse water (0.5 - 1 L) 15-30 

2

 except when otherwise specified by the manufacturer 1

 time depends upon water volume per container and autoclave load 2 

4.1.2 Autoclaved membrane filters and pads and all media should be removed immediately after completion of the

sterilization cycle.

4.1.3 Membrane filter equipment must be autoclaved before the beginning of a filtration series. A filtration series ends

when 30 minutes or longer elapses after a sample is filtered.

4.1.4 Ultraviolet light (254 nm) may be used to sanitize equipment (after initial autoclaving for sterilization), if all

supplies are presterilized. Ultraviolet light may be used to reduce bacterial carry-over between samples during a

filtration series. 

4.2 Sample Containers

QC At least one sample container should be selected at random from each batch of sterile sample bottles or other

containers (or lot of commercially available sample containers), and the sterility confirmed by adding approximately

25 mL of a sterile non-selective broth (e.g., tryptic soy, trypticase soy, or tryptone broth). The broth should be

incubated at 35°±0.5°C, and checked after 24 and 48 hours for growth. Record results. Resterilize entire batch if

growth is detected.

4.3 Reagent-Grade Water

4.3.1 Only satisfactorily tested reagent water from stills or deionization units may be used to prepare media, reagents,

and dilution/rinse water for performing microbial analyses.

QC 4.3.2 The quality of the reagent water should be tested and should meet the following criteria:

Parameter Limits Frequency 

Conductivity >0.5 megohms resistance or <2 micromhos/cm

(microsiemens/cm) at 25 C

°

Monthly4

Pb, Cd, Cr, Cu, Ni, Zn Not greater than 0.05 mg/L per contaminant. 

Collectively, no greater than 0.1 mg/L

Annually

Total Chlorine Residual <0.1 mg/L Monthly 1

Heterotrophic Plate Count < 500 CFU/mL Monthly 2 5
Parameter Limits Frequency 

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Bacteriological Quality of 

Reagent Water3

Ratio of growth rate 0.8 to 3.0 Annually

 DPD Method should be used. Not required if source water is not chlorinated. 1

 Pour Plate Method. See Standard Methods 9215B. 2

 See Standard Methods (18 or 19 eds.), Section 9020B, under Laboratory Supplies. This bacteriological quality test 3 th th

is not needed for Type II water or better, as defined in Standard Methods (18 and 19 eds), Section 1080C, or Medium th th

quality water or better, as defined in Standard Methods (20 ed.), Section 1080C. If Type II or Medium quality water or th

better is not available, and a glass still is used for reagent water, a silicon test that meets the specifications of Standard

Methods, Section 1080C (20 ed.) should also be accomplished. th

 Monthly, if meter is in-line or has a resistivity indicator light; otherwise, with each new batch of reagent water. 4

 CFU means colony-forming units (same as colonies, but is a more precise term). 5

4.4 Dilution/Rinse Water

4.4.1 Stock buffer solution or peptone water should be prepared, as specified in Standard Methods, Section 9050C.

4.4.2 Stock buffers should be autoclaved or filter-sterilized, and containers should be labeled and dated. Stock buffers

should be refrigerated. Stored stock buffers should be free from turbidity.

QC 4.4.3 Each batch (or lot, if commercially prepared) of dilution/rinse water should be checked for sterility by adding

50 mL of water to 50 mL of a double strength non-selective broth (e.g., tryptic soy, trypticase soy or tryptose broth).

Incubate at 35°±0.5°C, and check for growth after 24 and 48 hours. Record results. Discard batch if growth is

detected.

4.5 Glassware Washing

4.5.1 Distilled or deionized water should be used for final rinse.

4.5.2 Laboratory glassware should be washed with a detergent designed for laboratory use. 

QC 4.5.3 A glassware inhibitory residue test (Standard Methods, Section 9020B, under Laboratory Supplies) should be

performed before the initial use of a washing compound and whenever a different formulation of washing compound,

or washing procedure, is used. Record results. This test will ensure that glassware is free of toxic residue. 

QC 4.5.4 Each batch of dry glassware used for microbial analysis should be checked for pH reaction, especially if

glassware is soaked in alkali or acid (Standard Methods, Section 9020B, under Laboratory Supplies). Use 0.04%

bromthymol blue (or equivalent pH indicator) and observe color reaction. Clean glassware without an alkali or acid

residual should have a neutral color reaction (blue-green for bromthymol blue). Record results. This test will ensure

that glassware is at a neutral pH.

5. Analytical Methodology

5.1 General

5.1.1 For compliance samples, laboratories must use only the analytical methodology specified in the Total Coliform

Rule (40 CFR 141.21(f)), the Surface Water Treatment Rule (SWTR) (40 CFR 141.74(a)), and the Groundwater Rule

(TBD). For convenience, these regulations are reproduced in Appendix G.

5.1.2 A laboratory must be certified for all analytical methods that it uses for compliance purposes. At a minimum,

the laboratory must be certified for one total coliform method and one fecal coliform or E. coli method. A laboratory

should also be certified for a second total coliform method if one method cannot be used for some drinking waters

(e.g., where the water usually produces confluent growth on a plate). In addition, for laboratories that may enumerate

heterotrophic bacteria (as measured by the Heterotrophic Plate Count, HPC) for compliance with the Surface Water

Treatment Rule, the laboratory must be certified either for the Pour Plate Method or the SimPlate method for

heterotrophic bacteria.
V-9

5.1.3 Water samples should be shaken vigorously at least 25 times before analyzing.

QC 5.1.4 If dilution buffer is used, check the accuracy of the buffer volume in one dilution bottle in each batch or lot.

For a 90-mL or 99-mL volume, the tolerance should be ±2 mL.

5.1.5 Sample volume analyzed for total coliforms in drinking water must be 100 mL. To assure accuracy

and consistency within methods and between methods it is important that the laboratory obtain precise

measurement of the volume of sample to be analyzed. To ensure that the required volume of 100 mL is

analyzed, no matter which of the approved methods the laboratory will be employing for analysis, good

laboratory practice dictates that a sterile, calibrated measuring vessel be used for measurement of the sample

volume. It is inappropriate for a portion of the sample to be poured to waste in order to meet the required

sample volume, as this practice could easily result in laboratory error which could then require the sample to

be invalidated. 

5.1.6 Media (or defined substrate)

5.1.6.1 The use of dehydrated or prepared media manufactured commercially is strongly

recommended due to concern about quality control. Dehydrated media should be stored in a cool,

dry location, and discarded by manufacturer's expiration date. Caked or discolored dehydrated

media should be discarded.

QC 5.1.6.2 For media prepared in the laboratory, the date of preparation, type of medium, lot number,

sterilization time and temperature, final pH (after sterilization), and the technician's initials should be

recorded.

QC 5.1.6.3 For media prepared commercially, the date received, type of medium, lot number, and (if

identified by the manufacturer or method) pH verification for each lot should be recorded. Media

should be discarded by manufacturer's expiration date.

QC 5.1.6.4 Each new lot of dehydrated or prepared commercial medium and each batch of laboratoryprepared medium should be checked before use for sterility and with positive and negative culture

controls. Those laboratories using commercially prepared media with manufacturer shelf-lives of

greater than 90 days should run positive and negative controls each quarter, in addition to running

these controls and sterility checks on each new lot of media. Laboratories are encouraged to perform

positive and negative control tests on a more frequent basis. Control organisms (total coliforms, fecal

coliforms, and/or E. coli, as appropriate) can be stock cultures (periodically checked for purity) or

commercially available disks impregnated with the organism. Results should be recorded. The

following Table identifies a few positive and negative culture controls that laboratories might

consider, although other culture controls are also acceptable.
V-10

Control Cultures for Microbiological Tests

Group Positive Culture Control Negative Culture Control 9 9

Total

coliforms

Escherichia coli

Enterobacter aerogenes

Staphylococcus aureus1

Proteus vulgaris2

Pseudomonas aeruginosa1

Fecal

coliforms

Escherichia coli

Klebsiella pneumoniae

(thermotolerant)

Enterobacter aerogenes3

E. coli Escherichia coli (MUG-positive strain) Enterobacter aerogenes

Klebsiella pneumoniae (thermotolerant) 4

Enterococci Enterococcus faecalis 5

Enterococcus faecium

Staphylococcus aureus6

E. coli7

Serratia marcesens8

S. aureus, P. aeruginosa - not lactose fermenter 1

P. vulgaris - not lactose fermenter; uses hydrolyzed lactose, indicating “overcooked” medium 2

E. aerogenes - ferments lactose, but is not typically thermotolerant 3

K. pneumoniae - ferments lactose, but does not hydrolyze MUG 4

 Do not use closely related strains from genus Streptococcus as a positive control 5

S. aureus - sensitive to nalidixic acid in medium 6

E. coli - sensitive to sodium azide in medium 7

S. marcescens - will not hydrolyze fluorogenic compound in medium 8

 Examples of appropriate ATCC strains include the following: 9

Enterococcus faecalis ATCC 11700 Enterococcus faecium ATCC 6057

Enterobacter aerogenes ATCC 13048 Escherichia coli ATCC 8739 or

25922

Klebsiella pneumoniae (thermotolerant) ATCC 13883 Proteus vulgaris ATCC 13315

Pseudomonas aeruginosa ATCC 27853 Serratia marcesens ATCC 14756

Staphylococcus aureus ATCC 6538

5.1.6.5 If prepared medium is stored after sterilization, it should be maintained in the dark, avoiding

moisture loss, per the following Table. Prepared plates may be stored in sealed plastic bags or

containers. For either broth or agar media, each bag or container should include the date prepared or

an expiration date. If the medium is stored in a refrigerator, it should be warmed to room

temperature before use; tubes or plates that show growth and/or bubbles should be discarded. Liquid

media should be discarded if evaporation exceeds 10% of the original volume.

Maximum Holding Times and Temperatures for Prepared Media

Container Max storage temp. Max. storage time

Poured agar plates 1-5°C 2 weeks

Broth in tubes, bottles, or flasks

with loose-fitting closures 

1-30°C 2 weeks

Broth in tightly closed screw-cap

tubes, bottles, or flasks

1-30°C 3 months
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QC 5.1.7 Laboratories are encouraged to perform parallel testing between a newly approved test and another EPAapproved procedure for enumerating total coliforms for at least several months and/or over several seasons to

assess the effectiveness of the new test for the wide variety of water types submitted for analysis. During this

testing, spiking the samples occasionally with sewage or a pure culture may be necessary to ensure that some of

the tests are positive.

5.1.8 A list of approved analytical methods (or proposed methods, where noted), applicable regulations, and

section identifiers for each method is provided in the Table below.

Approved Methods Part Media Method

Citation1

TCR2

(Detect)

SWTR2

(Count)

GWR2

(Detect)

Total Coliforms

Fermentation broth

method 

5.2.2 LTBYBGLB Broth SM 9221B,C X X

5.2.3 P-A Broth Y BGLB

Broth SM 9221D X

Enzyme substrate

method

5.3.2 Colilert®, Colilert-18® SM 9223 X X

5.3.2 Colisure® SM 9223 X

5.3.2 Readycult® or Fluorocult

LMX® X

5.3.2 E*Colite® X

5.3.2 Colitag® X

Membrane filter

method 

5.4.2 M-Endo or LES-Endo Y

LTB, BGLB Broth SM 9222B,C X X

5.4.2 MI Medium SM 9222 X X

5.4.2 m-ColiBlue 24® X

5.4.2 Chromocult® X

5.4.2 Coliscan® X X

 Fecal Coliforms

Fermentation broth

method 

5.2.4 LTB or P/A broth Y EC

broth

(SM 9221B,D)

SM 9221E X X

5.2.4 A-1 broth SM 9221E X

Membrane filter

method 

5.2.4 M-Endo medium Y EC

broth

(SM 9222B)

SM 9221E X X

5.4.2 mFC SM 9222D X
Approved Methods Part Media Method

Citation1

TCR2

(Detect)

SWTR2

(Count)

GWR2

(Detect)

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Escherichia coli

Enzyme substrate

method

5.3.2 Colilert® or Colilert-18® SM 9223 X X

5.3.2 Colisure® SM 9223 X X

5.3.2 E*Colite® X X

5.3.2 Readycult® or Fluorocult

LMX® X

5.3.3 LTB, P/A broth, M-Endo

Y EC-MUG

(SM 9221B,D;

SM 9222B)

SM 9221F

X X

5.3.2 Colitag® X

Membrane filter

method 

5.4.2 MI Medium SM 9222 X X

5.4.2 m-ColiBlue24® X X

5.4.2 Chromocult® X

5.4.2 Coliscan® X

5.4.3 M-Endo or LES Endo Y

NA-MUG 

(SM 9222B) 6

SM 9222G X X

Enterococci3

Enzyme substrate

method 5.3.4 Enterolert

ASTM D6503-

99 X

Fermentation broth

method 5.2.5 Azide Dextrose YBEA/BHI SM 9230B X

Membrane filter

method 

5.4.4 mE YEIA

m-Enterococcus

SM 9230C X

5.4.4 mEI EPA 1600 X

Heterotrophic Bacteria

Pour plate method 5.5 Plate count agar SM 9215B X

Multiple enzyme

substrate

5.5 SimPlate® X

Pour plate, spread

plate, or membrane

filter methods

5.5 R2A X

4
Approved Methods Part Media Method

Citation1

TCR2

(Detect)

SWTR2

(Count)

GWR2

(Detect)

V-13

Male-Specific and Somatic Coliphage3

Agar plate method

5.6.2 Two-Step Enrichment EPA 1601 X

5.6.3 Single Agar Layer EPA 1602 X

SM = Standard Methods for the Examination of Water and Wastewater, 18 , 19 or 20 edition. 1 th th th

TCR=Total Coliform Rule (40 CFR 141.21 (f)), SWTR=Surface Water Treatment Rule (40 CFR 141.74 (a)). For 2 

convenience, analytical methods approved for the TCR and SWTR are reproduced in Appendix G.

 GWR = Based on proposed Groundwater Rule (65 FR 30194, dated 5/10/2000). Until the GWR is promulgated, 3

laboratories will not be certified for enterococci or coliphage methods.

 For possible use if system operates under a variance to the TCR. 4

5.2 Fermentation broth methods

5.2.1 General

5.2.1.1 The water level of the water bath should be above the upper level of the medium in the culture tubes.

5.2.1.2 A Dri-bath incubator is acceptable if the specified temperature requirement can be maintained in all tube

locations used.

5.2.2 Multiple Tube Fermentation Technique (for detecting total coliforms in drinking water and enumerating total

coliforms in source water)

5.2.2.1 For drinking water samples: Various testing configurations can be used (CFR141.21(f)(3), see Appendix

G), as long as a total sample volume of 100 mL is examined for each test. 

5.2.2.2 For source water samples: Laboratories must use at least 3 series of 5 tubes each with appropriate sample

dilutions of source water (e.g., 0.1 mL, 0.01 mL, 0.001 mL). 

5.2.2.3 Media

5.2.2.3.1 Lauryl tryptose broth (LTB) (also known as lauryl sulfate broth) must be used in the

presumptive test and 2% brilliant green lactose bile broth (BGLBB) in the confirmed test. Lactose broth

(LB) may be used in lieu of LTB (40 CFR 141.21(f)(3)) if the laboratory conducts at least 25 parallel

tests between this medium and LTB using the waters normally tested and this comparison demonstrates

that the false-positive rate and false-negative rate for total coliforms, using LB, is less than 10%. This

comparison should be documented and the records retained. The final pH must be 6.8±0.2 for LTB, and

7.2±0.2 for 2% BGLBB. 

5.2.2.3.2 The test medium concentration must be adjusted to compensate for the sample volume so that

the resulting medium after sample addition is single strength. Optionally, if a single 100-mL sample

volume is used, the inverted vialshould be replaced with an acid indicator (bromcresol purple) to prevent

problems associated with gas bubbles in large inverted tubes. The media must be autoclaved at 121°C

for 12-15 minutes.

5.2.2.3.3 Sterile medium in tubes must be examined to ensure that the inverted vials, if used, are free

of air bubbles and are at least one-half to two-thirds covered after the water sample is added.

5.2.2.4 After the medium is inoculated, it must be incubated at 35°±0.5°C for 24±2 hours. If no gas or acid is

detected, it must be incubated for another 24 hours (total incubation time 48±3 hours). 
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5.2.2.5 Each 24- and 48-hour tube that contains growth, acid, or gas must be confirmed using 2% BGLBB. A

completed test is not required.

5.2.2.6 For drinking water samples: Test each total coliform-positive sample for the presence of either fecal

coliforms or E. coli.

5.2.2.7 Invalidation of total coliform-negative samples 

5.2.2.7.1 For drinking water samples: All samples that produce a turbid culture (i.e., heavy growth) in

the absence of gas/acid production, in LTB or LB, must be invalidated. The laboratory must collect, or

request that the system collect, another sample within 24 hours from the same location as the original

invalidated sample. (Before invalidation, the laboratory may perform a confirmed test and/or a fecal

coliform/E. coli test on the total coliform-negative culture to check for coliform suppression. If the

confirmed test is total coliform-positive or if fecal coliforms/E. coli are detected, the sample must be

reported as such. A fecal coliform/E. coli- positive result is considered a total coliform-positive, fecal

coliform/E. coli-positive sample, even if the presumptive or confirmed total coliform test is negative.

If the follow-up test(s) is negative, the sample must be invalidated because high levels of non-coliform

bacteria in the presumptive tubes may have injured, killed, or suppressed the growth of any coliforms

in the sample.) 

5.2.2.7.2 For source water samples: All samples that produce a turbid culture (i.e., heavy growth) in

the absence of gas/acid production, in LTB or LB, should be invalidated. The laboratory should collect,

or request that the system collect, another sample from the same location as the original invalidated

sample. (Before invalidation, the laboratory may perform a confirmed test on the total coliform-negative

culture. If the confirmed test is total coliform-positive, the MPN should be reported. If the test is total

coliform-negative, the sample should be invalidated.) 

5.2.3 Presence-Absence (P-A) Coliform Test (for detecting total coliforms in drinking water)

5.2.3.1 Medium

5.2.3.1.1 Six-times formulation strength may be used. If the 6-times formulation is used, it must be

filter-sterilized rather than autoclaved.

5.2.3.1.2 The medium must be autoclaved for 12 minutes at 121°C. Total time in the autoclave should

be less than 30 minutes. Space should be allowed between bottles. The final pH must be 6.8±0.2.

5.2.3.1.3 If prepared medium is stored, it should be maintained in a culture bottle at 1°-30°C in the dark

for no longer than three months. If evaporation exceeds 10% of original volume, the medium should be

discarded. 

5.2.3.2 A 100-mL sample must be inoculated into a P-A culture bottle.

5.2.3.3 Medium must be incubated at 35°±0.5°C and observed for a yellow color (acid) after 24 and 48 hours.

5.2.3.4 Yellow cultures must be confirmed in BGLBB and a fecal coliform/E. coli test conducted.

5.2.3.5 All samples which produce a non-yellow turbid culture in P-A medium must be invalidated. The

laboratory must collect, or request that the system collect, another sample from the same location as the original

invalidated sample. (Before invalidation, the laboratory may perform a confirmed test on the total coliform

negative culture and/or a fecal coliform/E. coli test. If the confirmed test is total coliform-positive, the sample

must be reported as such. If the confirmed test is negative, the sample must be invalidated. A fecal coliform/E.

coli positive result is considered a total coliform-positive, fecal coliform/E. coli positive sample, even if the

presumptive and/or confirmed total coliform test is negative.) 
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5.2.4 Fecal Coliform Test (using EC Medium for fecal coliforms in drinking water or source water, or A-1 Medium

for fecal coliforms in source water only)

5.2.4.1 EC Medium

5.2.4.1.1 Use EC medium to test a total coliform-positive culture for fecal coliforms under the Total

Coliform Rule. The laboratory must transfer each total coliform-positive culture from a presumptive

tube/bottle, or each presumptive total coliform-positive colony unless a cotton swab is used, to at least

one tube containing EC Medium with an inverted vial, as specified by §141.21(f)(5)(See Appendix G).

5.2.4.1.2 EC Medium may be used to enumerate fecal coliforms in source water, in accordance with the

Surface Water Treatment Rule. Initially, conduct a MTF test (presumptive phase). Three sample

volumes of source water (e.g., 10, 1 and 0.1 mL), 5 or 10 tubes/sample volume, should be used. A

culture from each total coliform-positive tube should be transferred to a tube containing EC Medium

with an inverted vial.

5.2.4.1.3 Autoclave EC Medium for 12-15 minutes at 121°C. The final pH should be 6.9±0.2.

5.2.4.1.4 Inverted vials should be examined to ensure that they are free of air bubbles. The inverted vial

must be at least one-half to two-thirds covered after the sample is added. 

5.2.4.1.5 EC Medium must be incubated at 44.5°±0.2°C for 24±2 hours.

5.2.4.1.6 Any amount of gas detected in the inverted vial of a tube that has turbid growth must be

considered a fecal coliform-positive test, regardless of the result of any subsequent test on that culture.

5.2.4.2 A-1 Medium

5.2.4.2.1 A-1 medium may be used as an alternative to EC Medium to enumerate fecal coliforms in

source water, in accordance with the Surface Water Treatment Rule. A-1 Medium must not be used for

drinking water samples. Three sample volumes of source water (e.g., 10, 1 and 0.1 mL), 5 or 10

tubes/sample volume, should be used. Unlike EC Medium, A-1 Medium may be used for the direct

isolation of fecal coliforms from water. 

5.2.4.2.2 A-1 Medium must be sterilized by autoclaving at 121°C for 10 minutes. The final pH must be

6.9±0.1. 

5.2.4.2.3 Inverted vials should be examined to ensure that they are free of air bubbles. 

5.2.4.2.4 A-1 Medium must be incubated at 35°±0.5°C for three hours, then at 44.5°±0.2°C for 21±2

hours.

5.2.4.2.5 Loose-cap tubes should be stored in dark at room temperature not more than two weeks. A-1

Medium must not be held more than three months in tightly closed screw-cap tubes in the dark at 4°C.

5.2.4.3 Any amount of gas detected in the inverted vial of a tube that has turbid growth must be considered a fecal

coliform-positive test, regardless of the result of any subsequent test on that culture.

5.2.5 Azide dextrose medium (for detecting fecal streptococci in ground water)

5.2.5.1 For testing 100-mL samples, prepare triple strength (3X) formulation in a culture bottle and autoclave

at 121°C for 15 minutes. Final pH should be 7.2±0.2.
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5.2.5.2 Add a 100-mL water sample to the sterilized medium, and incubate at 35°±0.5°C. 

5.2.5.3 Check culture for turbidity after 24±2 hours. If turbidity is not observed, reincubate and check again

after a total incubation period of 48±3 hours.

5.2.5.4 A turbid culture may be confirmed as fecal streptococci by streaking a portion of the broth onto bile

esculin agar (BEA) or bile esculin azide agar (BEAA). (The confirmation medium in Standard Methods,

PSE Medium, is no longer commercially available.)

5.2.5.5 Before streaking, BEA and BEAA must be sterilized by autoclaving at 121°C for 15 minutes. Final pH

should be 6.6±0.2 for BEA and 7.1±0.2 for BEAA.

5.2.5.6 After streaking, BEA and BEAA plates must be incubated at 35°±0.5°C for 48 hours.

5.2.5.7 Brownish-black colonies on BEA or BEAA with brown halos confirm the presence of fecal streptococci.

If required, an enterococci test can be performed on one or more fecal streptococci colonies by

transferring them to brain heart infusion broth supplemented with 6.5% NaCl, and incubating the culture

at 35°±0.5°C for 48 hours. Growth indicates the presence of enterococci. 

5.3 Enzyme (Chromogenic/fluorogenic) substrate tests 

5.3.1 General

5.3.1.1 For detecting total coliforms and E. coli in drinking water samples, a laboratory may use the MMO-MUG

test (Colilert), Colisure test, E*Colite test, Readycult Coliforms 100 Presence/Absence Test (or Fluorocult LMX

Broth test), or Colitag test. These tests may be available in various configurations. For enumerating total

coliforms in source waters, a laboratory may use the Colilert test. If a laboratory uses a fermentation method to

detect total coliforms in drinking water, and the sample is total coliform-positive, the laboratory may transfer the

positive culture to the EC+MUG test to detect E. coli, but not to any other enzyme substrate test medium in this

section.

5.3.1.2 Media

5.3.1.2.1 Media must not be prepared from basic ingredients, but rather purchased from a commercially

available source.

5.3.1.2.2. The media must be protected from light. 

5.3.1.2.3 Some lots of enzyme substrate media have been known to fluoresce. Therefore, each lot of

medium should be checked before use with a 365-366-nm ultraviolet light with a 6-watt bulb. For

checking Colilert, Colilert-18, Colisure, Readycult/Fluorocult LMX, and Colitag media, a packet of

medium should be dissolved in sterile water in a non-fluorescing vessel. If the medium exhibits faint

fluorescence, the laboratory should use another lot that does not fluoresce. 

5.3.1.2.4 If the samples plus a medium exhibit an inappropriate color change before incubation, it should

be discarded and another lot of medium used. The laboratory should notify the medium vendor and

request another water sample from the water system. Before incubation, Colilert, Colilert-18, and

Colitag should appear colorless to a slight tinge of color, while Colisure and E*Colite are yellow and

Readycult/Fluorocult is slightly yellow.

5.3.1.3 Glass and plastic bottles and test tubes should be tested before use with a 365-366-nm ultraviolet light

source with a 6-watt bulb to ensure they do not fluoresce. If they fluoresce, use another lot of containers that do

not fluoresce.
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5.3.1.4 If a Whirl-Pak® bag is used to incubate the Colilert or Colitag medium or any other medium which

changes to a yellow color to indicate a positive result, use a type that has a barrier (e.g., B01417) to prevent

gaseous emissions to other Whirl-Pak® bags during incubation. 

QC 5.3.1.5 Incubators, especially small, low wattage air-type incubators, may not bring a cold 100-mL water

sample(s) to the specified incubation temperature for several hours. The problem may cause false-negative results

with the enzyme substrate tests and possibly other tests as well. Therefore, laboratories with air-type incubators

should observe the following instructions for chromogenic/fluorogenic substrate tests: 

Test Pre-incubation sample instructions1,2

Colilert (Presence/Absence) Specified 24-hour incubation time includes time it takes to bring sample

temperature up to 35°C1

Colilert Quanti-Tray Specified 24-hour incubation time includes time it takes to bring sample

temperature up to 35°C 

Colilert-18 (Presence/Absence) Prewarm sample in 35°C water bath for 20 minutes or 44.5°C for

7-10 minutes

Colilert-18 Quanti-Tray Allow sample to equilibrate to room temperature (20-30°C) before beginning

18-hour incubation time

Colisure Allow sample to equilibrate to room temperature (20-30°C) before beginning

24-hour incubation time

Readycult Coliforms 100

Presence/Absence Test and

Fluorocult LMX Broth

Specified 24-hour incubation time includes time it takes to bring sample

temperature up to 35°±0.5°C 

Colitag Specified 24-hour incubation time includes time it takes to bring sample

temperature up to 35°±0.5°C 

 If the laboratory plans to put a large load into a small incubator, samples should be brought to room temperature before 1

incubation.

 Information based on manufacturer’s instructions. 2

5.3.1.6 If a water bath is used, the water level should be above the upper level of the medium.

5.3.1.7 For E. coli testing, the laboratory must place all total coliform-positive samples under an ultraviolet lamp

(365-366 nm, 6-watt) in a darkened area. If E. coli is present, the medium will emit a blue fluorescence.

5.3.1.8 The enzyme substrate tests should not be used to confirm a presumptive total coliform-positive culture

in fermentation broth (e.g., LTB, LB, P-A coliform test) or on a membrane filter. The high densities of noncoliforms or turbidity in the inoculum may either suppress coliforms or overload the enzyme substrate test

suppressant reagent system and cause false-positive results.

5.3.1.9 Any sample that produces an atypical color change (e.g., greenish-black or black) in the absence of a

yellow color should be invalidated. The laboratory must collect, or request that the system collect, another sample

from the same location as the original invalidated sample. The laboratory should use another method to test the

second sample. According to the manufacturer of Colilert, water with high iron or manganese levels in the

presence of hydrogen sulfide may cause a greenish-black or black color. This greenish-black color does not occur

when using Readycult, Colisure, or Colitag, according to their manufacturers.

5.3.1.10 Any reference comparator provided by the manufacturer should be discarded by the manufacturer’s

expiration date. 
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5.3.2 Criteria for specific media

5.3.2.1 For the Colilert test, samples must be incubated at 35°±0.5°C for 24 hours. A yellow color in the medium

equal to or greater than the reference comparator indicates that the sample is total coliform-positive. If the sample

is yellow, but lighter than the comparator, it must be incubated for another four hours (do not incubate more than

28 hours total). If the color is still lighter than the reference comparator at 28 hours, the sample should be

reported as negative. A coliform-positive sample that fluoresces under a UV light indicates the presence of E.

coli. Laboratories that use the Colilert-18 test must incubate samples for 18 hours (up to 22 hours if sample after

18 hours is yellow, but is lighter than the comparator).

5.3.2.1.1 For enumerating total coliforms in source water with the Colilert test, a 5- or 10-tube

configuration, Quanti-Tray, or Quanti-Tray 2000 may be used for each sample dilution tested. Dilution

water (if used) may be sterile deionized or sterile distilled water, but not buffered water.

QC 5.3.2.1.2 If the Quanti-Tray or Quanti-Tray 2000 test is used, the sealer should be checked monthly by adding

a dye (e.g., bromcresol purple) to the water. If dye is observed outside the wells, either perform maintenance or

use another sealer. 

5.3.2.2 For the Colisure test, samples must be incubated at 35°±0.5°C for 24 hours. If an examination of the

results at 24 hours is not convenient, then results may be examined at any time up to 48 hours. If the medium

changes from a yellow color to a red/magenta color, the sample is total coliform-positive. A coliform-positive

sample that fluoresces under a UV light indicates the presence of E. coli.

5.3.2.3 For the E*Colite test, samples must be incubated at 35°±0.5°C for 28 hours. If total coliforms are present,

the medium changes from a yellow color to a blue or blue-green color, or a blue color in the corners of the bag.

If E. coli is present, medium will fluoresce under a UV light. If no fluorescence is observed, re-incubate for an

additional 20 hours (for a total incubation time of 48 hours) and again check for fluorescence. If medium becomes

red in color, assume that a faulty seal has allowed the bactericide (in the third compartment of the bag) to leak into

the compartment containing the medium. In this case, discard the sample, and request another sample.

5.3.2.4 For the Readycult Coliforms 100 Presence-Absence test, the contents of a snap pack should be added to

a 100-mL water sample, followed by incubation at 35°±05/C for 24±1 hours. If coliforms are present, the medium

changes color from a slightly yellow color to blue-green. In addition, if E. coli is present, the medium will emit

a bright light-blue fluorescence when subjected to a long wave (365-366 nm) ultraviolet (UV) light. If

confirmation of E. coli is desired, Kovac’s indole reagent should be added to the broth; the immediate formation

of a red ring confirms the presence of E. coli.

5.3.2.5 Fluorocult LMX broth is identical to Readycult, except that it is a dehydrated culture medium in

granulated form packed primarily in a 500 g plastic bottle. For testing a 100-mL water sample, suspend 34 g of

Fluorocult LMX in 1L purified water and boil to dissolve completely. Transfer 100-mL aliquots to 250-mL

bottles and autoclave for 15 min at 121°C. Cool to room temperature, add the 100-mL water sample, and

incubate. Do not add E. coli/Coliform Supplement to the medium.

5.3.2.6 For the Colitag test, samples must be incubated at 35E±0.5EC for 24±2 hours. During incubation,

trimethylamine-N-oxide in the Colitag medium causes the pH of the medium to increase from 6.2 to 6.8-7.2. A

yellow color in the medium indicates the presence of total coliforms. A coliform-positive sample that fluoresces

under a UV light indicates the presence of E. coli.

5.3.3 EC Medium + MUG Test (for detection of E. coli)

5.3.3.1 If EC medium + MUG is used, a total coliform-positive culture must be transferred from a presumptive

tube/bottle or colony to EC medium + MUG, as specified by §141.21(f)(5)(See Appendix G).
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5.3.3.2 MUG may be added to EC Medium before autoclaving. EC Medium+MUG is also available

commercially. The final MUG concentration must be 50 μg/mL. The final pH should be 6.9±0.2.

5.3.3.3 The inverted vial may be omitted, because gas production is not relevant to the E. coli test. 

5.3.3.4 The medium must be incubated at 44.5°±0.2°C for 24±2 hours, and tested for fluorescence.

5.3.4 Enterolert test (for detection of enterococci in ground water)

5.3.4.1 Medium should be stored in the dark at 4-30°C until use.

5.3.4.2 Add Enterolert reagent to 100-mL water sample, and incubate at 41°± 0.5°C for 24-28 hours.

Fluorescence under a UV lamp indicates the presence of enterococci.

5.3.4.3 The development of fluorescence after 28 hours is not a valid test for enterococci.

5.4 Membrane Filter (MF) methods

5.4.1 General

5.4.1.1 For source water samples (SWTR): To optimize counting, appropriate sample dilutions must be used to

yield 20 to 80 total coliform colonies or 20-60 fecal coliform colonies for at least one dilution or volume. 

QC 5.4.1.2 At least one membrane filter and filtration unit sterility check should be conducted at the beginning and

the end of each filtration series by filtering 20-30 mL of dilution water through the membrane filter and testing

for growth. If the control indicates contamination, all data from affected samples must be rejected and an

immediate resampling should be requested. A filtration series ends when 30 minutes or more elapse between

sample filtrations.

5.4.1.3 Each filtration funnel must be rinsed after each sample filtration with two or three 20-30 mL portions of

sterile rinse water to ensure that entire sample is rinsed off the funnel before the filter is removed. After the filter

is removed, the funnel may be rinsed again with two or three 20-30 mL portions of sterile rinse water or exposed

to UV light with a 254-nm wavelength for at least two minutes to prevent carry-over between samples, especially

for surface water samples..

5.4.1.4 Absorbent pads must be saturated with a liquid medium (at least 2 mL of broth) and excess medium

removed by "decanting" the plate. 

5.4.2 MF method for detecting total coliforms and E. coli in drinking water, enumerating total coliforms or fecal

coliforms in source water, and detecting E. coli in ground water

5.4.2.1 Media for total coliforms, fecal coliforms, and E. coli

5.4.2.1.1 M-Endo Medium agar or broth (also known as M-Endo broth MF and M-Coliform Broth) or

LES Endo agar (also known as M-Endo Agar LES) for detecting total coliforms in drinking water or

enumerating total coliforms in source water. Medium may be used in the single step or enrichment

techniques. Ensure that ethanol used in the rehydration procedure is not denatured. Medium should be

prepared in a sterile flask and brought just to the boiling point with a boiling water bath or, if constantly

attended, a hot plate with a stir bar. The medium must not be boiled. Final pH should be 7.2±0.2 for

M-Endo Agar LES and 7.2±0.1 for M-Endo medium.

5.4.2.1.2 m-ColiBlue24 medium for detecting total coliforms and E. coli in drinking water. Ampules
V-20

of broth should be inverted 2-3 times to mix contents before breaking. Then contents should be poured

evenly over absorbent pad. Unopened refrigerated ampules may be stored in the dark until the expiration

date, but should be discarded earlier if growth is observed. The final pH of medium should be 7.0±0.2.

5.4.2.1.3 MI Medium (with or without agar) for detecting total coliforms and E. coli in drinking water

or enumerating total coliforms in source water. Do not autoclave commercially made, presterilized

bottled MI agar or broth. Melt bottled agar in a boiling water bath (or by other processes recommended

by the manufacturer). As soon as complete melting has occurred, cool slightly and pour immediately

into sterile plates. Care should be taken to prevent overheating the agar, as excessive heat destroys the

effectiveness of the antibiotic, cefsulodin. If dehydrated culture medium is used, it should be prepared

and autoclaved according to the manufacturer’s instructions. Cool the agar, add freshly prepared, filtersterilized cefsulodin, and pour immediately into sterile plates. The final pH of MI agar should be

6.95+0.20; the final pH of MI broth should be 7.05+0.20. The preparation and use of MI agar and MI

broth is described in the article, “New medium for the simultaneous detection of total coliform and

Escherichia coli in water” by Brenner, K.P., et al., 1993, Applied and Environmental Microbiology

59:3534-3544. EPA Method 1604, which can be found online at www.epa.gov/microbes, is identical.

5.4.2.1.4 Chromocult® Coliform Agar for detecting total coliforms and E. coli in drinking water. Do

not autoclave or overheat. The final pH should be 6.8+0.2. If a heavy background of heterotrophic

bacteria is expected (especially Pseudomonas and Aeromonas spp.), add cefsulodin solution to the

cooled (45°-50°C) medium (dissolve 10 mg cefsulodin in 2 mL deionized or distilled water, and add

solution to 1L of medium). Check with the manufacturer, EMD Chemicals, Inc., at

www.emdchemicals.com, or call (800) 222-0342 for additional information on the performance of this

test with various filter types. 

5.4.2.1.5 Coliscan® for detecting total coliforms and E. coli in drinking water or enumerating total

coliforms in source water. Coliscan is available as a dry powder agar mix or as a presterilized bottled

agar. For reconstitution and antibiotic addition, follow the protocol of the manufacture (Micrology

Laboratories, LLC). Do not overheat the antibiotic, cefsulodin. The final pH of Coliscan agar should

be 7.00+0.20.

5.4.2.1.6 m-FC broth (with or without agar) for enumerating fecal coliforms in source water. Do not

autoclave. Bring medium just to the boiling point. The final pH should be 7.4±0.2.

5.4.2.1.7 When stored, prepared medium should be refrigerated. Petri dishes containing medium should

be stored in a plastic bag or tightly closed container, and used within two weeks. Before use, refrigerated

sterilized medium should be brought to room temperature. Plates with laboratory prepared broth

medium must be discarded after 96 hours, poured MF agar plates discarded after two weeks, and

ampuled M-Endo broth and other prepared media discarded in accordance with the manufacturer's

expiration date. Broth, plates, or ampules should be discarded earlier if growth or (for M-Endo agar)

surface sheen is observed. Record date and time prepared.

5.4.2.2 Incubation conditions and colony color of inoculated medium

Medium Incubation Total coliforms E. coli 1

M-Endo medium or

M-Endo agar LES

35°±0.5°C for 22-24 hrs Metallic (golden) sheen

colonies (presumptive)

N/A

m-ColiBlue24 35°±0.5°C for 24 hrs Red colonies Blue to purple colonies 

MI 35°±0.5°C for 24±2 hrs Fluorescent colonies under

UV light

Blue colonies under normal

light
Medium Incubation Total coliforms E. coli 1

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Chromocult 36°±1°C for 24±1 hrs Salmon to red colonies Dark-blue to violet colonies2

Coliscan 32°-37°C for 24-28 hrs Pink-magenta colonies Purple-blue colonies

m-FC 44.5°±0.2°C for 24±2 hrs N/A Blue colonies (fecal

coliforms)

Without the presence of E. coli. If an E. coli colony is present, as indicated by the last column, it should be counted as 1

a total coliform-positive colony.

 If confirmation of E. coli is desired, add one drop of Kovac’s reagent to each dark-blue to violet colony; the formation 2

of a cherry-red color within seconds confirms the presence of E. coli.

5.4.2.3 Invalidation of a total coliform-negative drinking water sample: All samples resulting in confluent or

TNTC (too numerous to count) growth must be invalidated unless total coliforms are detected. If no total

coliforms are detected, record as "confluent growth" or "TNTC" and request an additional sample from the same

sampling site. Confluent growth is defined as a continuous bacterial growth covering the entire membrane filter

without evidence of total coliform-type colonies. TNTC is defined as greater than 200 colonies on the membrane

filter in the absence of detectable coliforms. Laboratories must not invalidate samples when the membrane filter

contains at least one coliform-type colony (i.e., sheen colony for M-Endo medium, red or blue colony for mColiBlue 24 agar, fluorescent or blue colony for MI agar, salmon to red or dark-blue to violet colonies for

Chromocult Coliform agar, pink/magenta or blue/purple colony for Coliscan). (Before invalidation, the laboratory

may perform a verification test on the total coliform-negative culture, i.e., on confluent or TNTC growth, and a

fecal coliform/E. coli test. If the verification test is total coliform-positive, the sample must be reported as total

coliform-positive. If the test is total coliform-negative, the sample must be invalidated. A fecal coliform/E. colipositive result is considered a total coliform-positive, fecal coliform/E. coli-positive sample, even if the initial

and/or verification total coliform test is negative.) 

5.4.2.4 Invalidation of source water samples (SWTR): Laboratories must invalidate any sample which results in

confluent growth or TNTC, even when total coliform or fecal coliform colonies are present, because coliform

density must be determined. 

5.4.2.5 For drinking water samples (to verify colonies on Endo-type medium): At least five typical sheen

colonies and five nontypical colonies must be verified using either single strength lactose broth (LB) or lauryl

tryptose broth (LTB) and then single strength 2% brilliant green lactose bile broth (BGLBB). Alternatively, sheen

colonies may be verified using a cytochrome oxidase and ß-galactosidase procedure. Individual colonies can be

transferred with a sterile needle or loop, or applicator stick. If no sheen colonies are observed, verify up to five

red questionable sheen colonies and/or red non-sheen colonies representing different morphological types.

Alternatively, wipe the entire surface of the membrane filter with a sterile cotton swab, and inoculate the

verification media (LTB, then BGLBB).

5.4.2.6 For drinking water samples: Total coliform-positive colonies must be tested for E. coli or fecal coliforms.

The membrane filter tests approved by EPA to date do not require additional media for such a test, except for

those using Endo-type medium (M-Endo medium or M-Endo agar LES). EPA has approved several options for

testing a total coliform-positive colony on Endo-type medium for E. coli or fecal coliforms. When EC Medium

(for fecal coliforms) or EC Medium + MUG (for E. coli) is used, the colonies must be transferred by employing

one of the options specified by the Total Coliform Rule at 40 CFR 141.21(f)(5)(See Appendix G). For the swab

technique, a single swab can be used to inoculate a presumptive total coliform-positive culture into up to three

different media (e.g., EC or EC-MUG Medium, LTB, and BGLBB, in that order). If Nutrient Agar + MUG is

used, refer to paragraph 5.4.3.

5.4.2.7 For source water samples: Initial total coliform counts must be adjusted based upon verified data, as in

Standard Methods, Section 9222B(5).
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QC 5.4.2.8 For source water samples (SWTR): If two or more analysts are available, each analyst should count total

coliforms or fecal coliform colonies on the same membrane monthly. Colony counts should agree within 10%.

5.4.3 Nutrient Agar + MUG Test (for detection of E. coli in drinking water or ground water)

5.4.3.1 Medium must be autoclaved at 121°C for 15 minutes. MUG may be added to Nutrient Agar before

autoclaving. Nutrient Agar + MUG is also available commercially. The final MUG concentration must be 100

μg/mL. The final pH should be 6.8±0.2.

QC 5.4.3.2 Positive and negative controls should be tested as stated in paragraph 5.1.6.4. Filter or spot-inoculate

control cultures onto a membrane filter on M-Endo agar LES or M-Endo broth or agar, and incubate at 35°±0.5°

C for 24 hours. Then transfer the filter to Nutrient Agar + MUG and incubate at 35°C for another four hours.

The results should be read and recorded.

5.4.3.3 The membrane filter containing coliform colony(ies) must be transferred from the total coliform medium

to the surface of Nutrient Agar + MUG medium. Each sheen colony should be marked with a permanent marker

on the lid. Also, the lid and the base should be marked with a line to realign the lid should it be removed. (A

portion of the colony may be transferred with a needle to the total coliform verification test before transfer to

Nutrient Agar + MUG or after the 4-hour incubation time. Another method is to swab the entire membrane filter

surface with a sterile cotton swab after the 4-hour incubation time on Nutrient Agar + MUG medium, and transfer

to a total coliform verification test.)

5.4.3.4 Inoculated medium must be incubated at 35°±0.5C° for four hours.

5.4.3.5 Check the fluorescence using an ultraviolet lamp (365-366 nm) with a 6-watt bulb in a darkened area.

Any amount of fluorescence in a halo around a sheen colony should be considered positive for E. coli.

5.4.4 MF method for detecting enterococci/fecal streptococci in ground water

5.4.4.1 Media

5.4.4.1.1 For mE agar (SM 9230C) for the detection of enterococci: Prepare basal mE agar. Then

autoclave and cool in a 44-46°C water bath. Dissolve 0.48 g nalidixic acid and 0.4 mL 10 N NaOH into

10 mL of reagent-grade distilled water and mix. Filter-sterilize the solution, and add 5.2 mL per liter

of basal mE agar. For triphenyl tetrazolium chloride (TTC), add 0.25 g of TTC to 25 mL of reagentgrade water, and warm to dissolve. Filter-sterilize the solution, and add 15 mL per liter of basal mE

agar. Final pH should be 7.1± 0.2.

5.4.4.1.2 For m-Enterococcus agar (SM 9230C) for the detection of fecal streptococci (not enterococci):

Heat to dissolve ingredients, but do not autoclave. Dispense into sterile petri plates (9 X 50 mm) (about

4 mL), and allow to solidify. Final pH should be 7.2±0.2.

5.4.4.1.3 For mEI agar (EPA Method 1600) for the detection of enterococci: Add 0.75 g indoxyl-$-Dglucoside to 1L of basal mE agar, and proceed according to paragraph 5.4.4.1.1, except that the

preparation of TTC is as follows: Add 0.1 g of TTC to 10 mL of reagent-grade distilled water, and warm

to dissolve. Filter-sterilize the solution, and add 2 mL per liter of medium. Final pH should be 7.1±0.2.

5.4.4.2 After filtering a 100-mL sample, place membrane in a petri dish on one of the agar media listed above.

Serial dilutions should not normally be necessary for detecting enterococci in ground water.

5.4.4.3 If m-Enterococcus agar is used, incubate inverted plate at 35°±0.5°C for 48 hours and, using

magnification and a fluorescent lamp, count all light and dark red colonies as fecal streptococci. 
V-23

5.4.4.4 If mE agar is used, incubate inverted plate for 48 hours at 41°±0.5°C, and then transfer filter to EIA

medium. Incubate at 41°± 0.5°C for 20-30 minutes and, using magnification and a fluorescent lamp, examine the

colonies. Pink to red colonies on mE agar with a black or reddish brown precipitate on the underside of filter on

EIA indicates the presence of enterococci.

5.4.4.5 If mEI agar is used, incubate inverted plate for 24 hours at 41°±0.5°C. Using magnification and small

fluorescent lamp, examine both the top and bottom of the plate for colonies with a blue halo. A colony with a blue

halo, regardless of colony color, indicates presence of enterococci. 

5.5 Heterotrophic Plate Count (for enumerating heterotrophic bacteria in drinking water)

5.5.1 The Pour Plate Method (Standard Methods 9215B) or the SimPlate Method must be used for determining

compliance with 40 CFR 141.74(a)(1) (also listed in Appendix G) and should also be used for testing reagent grade

water. For systems that have been granted a variance from the Total Coliform Rule's maximum contaminant level (see

variance criteria in the preamble of FR 56:1556-1557, January 15, 1991), any method in Standard Methods, Section

9215, Heterotrophic Plate Count, may be used with R2A medium, for enumerating heterotrophic bacteria in drinking

water. 

5.5.2 Media 

Method Medium Final pH

Pour Plate Plate count agar, also known as tryptone

glucose yeast agar

7.0 ± 0.2

Pour Plate R2A agar 7.2 ± 0.2 

Spread Plate R2A agar 7.2 ± 0.2 

Membrane Filter R2A agar 7.2 ± 0.2 

SimPlate Multiple enzyme substrate 7.2 ± 0.2 

5.5.3 (For Pour Plate Method) Melted agar must be tempered at 44°-46°C in waterbath before pouring. Melted agar

should be held no longer than three hours. Sterile agar medium should not be melted more than once.

5.5.4 (For Spread Plate Method) 15 mL of R2A agar medium (or other medium) should be poured into a petri dish

(100 x 15 mm or 90 x 15 mm) and allowed to solidify. 

5.5.5 Refrigerated medium may be stored in bottles or in screw-capped tubes for up to six months, or in petri dishes

for up to two weeks. Prepared petri dishes with R2A medium may be stored for up to one week.

5.5.6 For most potable water samples, countable plates can be obtained by plating 1.0 mL and/or 0.1 mL volumes of

the undiluted sample (dilutions may not be necessary for SimPlate, which has a counting range up to 738/mL). At least

duplicate plates per dilution should be used. 

5.5.7 (For Pour Plate Method) The sample must be aseptically pipetted onto the bottom of a sterile petri dish. Then

at least 10-12 mL mL of tempered melted (44°-46°C) agar must be added to each petri dish. The sample and melted

agar must be mixed carefully to avoid spillage. After agar plates have solidified on a level surface, the plates must

be inverted and incubated at 35°±0.5°C for 48±3 hours. Plates should be stacked no more than four high and arranged

in the incubator to allow proper air circulation and to maintain uniform incubation temperature. Avoid excessive

humidity in the incubator to reduce the possibility of spreader formation on the agar medium. Also avoid excessive

drying of the agar medium; agar medium in plates should not lose more than 15% by weight during 48 hours of

incubation. 
V-24

5.5.8 (For Spread Plate Method) 0.1 or 0.5 mL of the sample (or dilution) should be pipetted onto the surface of the

predried agar in the plate, and then spread over the entire surface of the agar using a sterile bent glass rod. The

inoculum should be absorbed completely by the agar before the plate is inverted and incubated. The plate should be

incubated at 20°-28°C for 5-7 days.

5.5.9 (For Membrane Filter Technique) The volume to be filtered should yield between 20-200 colonies. The filter

is transferred to a petri dish containing 5 mL of solidified R2A medium, and incubated at 20°-28°C for 5-7 days. If

plates with loose-fitting lids are used, plates should be placed in a plastic box with a close fitting lid containing

moistened paper towels. Paper towels should be rewetted as necessary to maintain moisture. Colonies should be

counted using a stereoscopic microscope at 10-15X amplification. 

5.5.10 (For SimPlate Method)

5.5.10.1 Unit Dose (for a single sample). A10-mL volume of test sample is added to a test tube containing

dehydrated SimPlate medium. Then the dissolved medium should be poured onto the center of a plate containing

84 small wells (provided by the manufacturer, IDEXX Laboratories, Inc.). Alternatively, 9 mL of sterile diluent

(D.I. water, distilled water, or buffered water [Standard Methods, 9050 C,1a]) can be added to the tube, followed

by 1-mL sample. Then follow the procedure as indicated above for the 10-mL sample. The mixture should be

distributed evenly to the 84 wells on the plate, and the excess liquid drained into an absorbent pad on the plate.

The plate should then be inverted (the fluid in each well is held in place by surface tension), and incubated for

45-72 hours at 35°±0.5°C. Bacterial density is determined by counting the number of wells that fluoresce under

a 365-366 nm UV light, and converting this value to a Most Probable Number using the Unit Dose MPN table

provided by the manufacturer. If 10-mL sample is used, read the Unit Dose MPN/mL directly. If a 1-mL sample

is used, then correct the MPN/mL value by multiplying it by 10.

5.5.10.2 Multiple Dose (for 10 samples of 1 mL each): A 100-mL sterile diluent should be added to the

dehydrated SimPlate medium to reconstitute, and shaken to dissolve. Then a 1.0-mL test sample should be

pipetted to the center of a plate containing 84 small wells, followed by 9.0 mL of the reconstituted medium.

Gently swirl plate to mix the sample and medium, and distribute the mixture evenly to the 84 wells on the plate.

Then continue with the procedure indicated in paragraph 5.5.10.1 above, except that the Multi-Dose table supplied

by the manufacturer should be used to determine the MPN/mL. If a dilution is made during sample preparation,

then multiply the MPN/mL value by the dilution factor.

5.5.11 (For Pour Plate and Spread Plate Techniques) Colonies should be counted manually using a dark-field colony

counter. In determining sample count, laboratories must only count plates having 30 to 300 colonies, except for plates

inoculated with 1.0 mL of undiluted sample. Counts less than 30 for such plates are acceptable. (Fully automatic

colony counters are not suitable because of the size and small number of colonies observed when potable water is

analyzed for heterotrophic bacteria.)

QC 5.5.12 Each batch or flask of agar should be checked for sterility by pouring a final control plate. Data should be

rejected if control is contaminated.

5.6 Coliphage (Draft Method 1601 and 1602, proposed Ground Water Rule) 

Note : EPA Method 1601 and 1602 are performance-based methods for detecting the presence of male-specific (F ) and +

somatic coliphage in ground water and other waters. (Performance-based method: In recognition of the variety of

situations to which some methods may be applied, and in recognition of continuing technological advances, some methods

are performance-based. A performance-based method permits laboratories to modify or omit steps or procedures, provided

that all performance requirements set forth in the validated methods are met. Any steps that may not be modified or omitted

must be specified in the method.)

5.6.1 EPA Method 1601: Male-specific (F ) and Somatic Coliphage in Water by Two-Step Enrichment

+

Procedure

Method Summary: A 100-mL (or 1-L water sample) is supplemented with magnesium chloride, log-phase host
V-25

bact amp eria (E. coli F for male-specific coliphage and E. coli CN-13 for somatic coliphage), and Tryptic Soy Broth

(TSB) as an enrichment step for coliphage. After an overnight incubation, samples are “spotted” onto a lawn of host

bacteria specific for each type of coliphage, incubated, and examined for circular lysis zones, which indicate the

presence of coliphage.

5.6.1.1 Media

5.6.1.1.1 Antibiotic stocks— Antibiotics must always be added to medium after the medium has been

autoclaved. Store frozen at -20°C for up to one year. Thaw at room temperature or rapidly in a water bath

up to 37°C and mix well prior to use. Please note: Antibiotics may be toxic. Wear suitable protective

clothing, gloves, and eye/face protection and use in a chemical fume hood.

5.6.1.1.2 10X Tryptic Soy Broth (TSB)—Store at 1°-5°C until use. 

5.6.1.1.3 1.5% Tryptic Soy Agar (TSA)—If not used immediately after adding antibiotic and letting the

plated medium solidify, store the plates inverted at 1°-5°C for up to 2 weeks.

5.6.1.1.4 0.7% TSA top agar tubes with appropriate antibiotics—Dispense 5 mL per sterile 10-mL tube,

label, and keep at 45°- 48°C until use. Tubes must be used the day they are prepared.

5.6.1.1.5 Spot plates—Condensation may accumulate at the edges of stored spot plates and may drip over

agar surface if tilted, ruining the spot pattern. If the stored spot plates have condensation, incubate plates

for approximately 10 minutes to reduce condensation prior to inoculation. Spot plates may be used that

day or stored at 1°-5°C for up to four days.

5.6.1.2 Coliphage stock

5.6.1.2.1 MS2 (ATCC#15597-B1, male-specific) and phi-X 174 stock coliphage (ATCC#13706-B1,

somatic)—May be stored at 2-8°C for up to 5 years. Refer to http://www.atcc.org for initial preparation

of pure coliphage stock. 

5.6.1.2.2 Analysis of raw sewage filtrate should begin within 24 hours of collection.

5.6.1.2.3 Allow the raw sewage to settle at 1°-5°C for 1 to 3 hours. This will make the filtration process

easier.

5.6.1.2.4 Hold the assembly over a 15-mL polypropylene tube with screw-cap or snap-cap, insert the

plunger into the syringe barrel, and push the sewage through the filter into the sterile tube. If filter clogs,

change it as necessary and continue to filter sewage until at least 10 mL of filtered sewage is obtained in

the 15-mL polypropylene tube (filtration may require use of numerous filters).

5.6.1.2.5 If filtrate is stored more than 24 hours, it must be re-titered before use.

5.6.1.3 Host bacteria stock cultures

5.6.1.3.1 Frozen host bacteria stock cultures—After preparation, freeze host bacteria stock cultures at -

70°C, if possible. Cultures can be frozen at -20°C if the laboratory does not have the capability to freeze

samples at -70°C. Host bacteria stored at -70°C may be retained for up to one year. If stored at -20°C, the

host bacteria may be retained for up to two months.

5.6.1.3.2 Overnight host bacteria stock cultures—After preparation, chill on wet ice or at 1°-5°C until

ready for use.

5.6.1.3.3 Log-phase host bacteria stock cultures—After preparation, chill on wet ice or at

1°-5°C to slow replication until ready for use. The suspension may be stored up to 48 hours. However,

the best results occur when cultures are used immediately (within 6 hours). Store remaining bacterial host

culture at 1°-5°C overnight to inoculate flasks for the preparation of new log-phase bacterial hosts.
V-26

5.6.1.4 General QC

5.6.1.4.1 Initial demonstration of capability (IDC). The laboratory must demonstrate the ability to

generate acceptable performance with this method by performing an IDC test before analyzing any field

samples. The IDC test consists of ten reagent water samples spiked with enumerated sewage or equivalent

at 1-2 PFU per sample for each coliphage type used, according to the IDC Table below. A minimum

number of samples must be positive, depending on coliphage type used (see IDC Table). Spike samples

in “bulk” at concentrations in the Table. Tests must be accompanied by a method blank for each coliphage

type.

5.6.1.4.2 Method blanks. The laboratory must analyze method blanks (reagent water sample containing

no coliphage) to demonstrate freedom from contamination. For each coliphage type used, prepare and

analyze a sterile reagent water sample containing no coliphage using the same procedure used for analysis

of the field or QC samples. At a minimum, the laboratory must analyze one method blank for each spot

plate used for field samples. In an effort to determine if cross-contamination is an issue, the sterile method

blank should be spotted onto the lawn of host bacteria immediately following the positive control spot.

5.6.1.4.3 Positive controls. The laboratory must analyze positive controls to ensure that stock coliphage

suspensions, host bacterial cultures, and growth media are performing properly. For each coliphage type

used, a 100-mL reagent water sample must be spiked with 20 PFU from sewage filtrate or 60 PFU from

a pure coliphage stock culture. The laboratory must inoculate one positive control spot for each spot plate

used for field samples. If multiple spot plates are inoculated with samples on the same day, a single

enriched positive control sample may be used to inoculate multiple spot plates on that day. 

5.6.1.4.4 Matrix spikes (MS). To assess method performance in a given source water matrix, the

laboratory must analyze one set of MS samples for each coliphage type when samples are first received

from a ground water source for which the laboratory has never before analyzed samples. For each

coliphage type analyzed, three field samples are spiked with 1-2 PFU. At a minimum, one out of the three

MS samples for each coliphage type must be positive for method performance to be considered acceptable

for that ground water source. If the MS results are unacceptable, and the ODC sample and positive control

sample results associated with this batch of samples are acceptable, a matrix interference may be causing

the poor results. In addition, the laboratory must analyze one set of MS samples on an ongoing basis after

every 20 field sample for each ground water source. (For example, when a laboratory receives the first th

sample from a source, the laboratory must obtain additional aliquots of the field samples to be used for the

MS test. When the laboratory receives the 20 field sample from this site, additional aliquots of this sample th

must be collected and spiked.) MS samples should be collected at the same time as routine field samples.

Spike samples in “bulk” at the concentrations indicated in the MS and ODC Table below.

5.6.1.4.5 Ongoing demonstration of capability (ODC). The laboratory must demonstrate that the

analytical system is in control on an ongoing basis through analysis of ODC samples. For each coliphage

type used, three reagent water samples are spiked with 1-2 PFU. The ODC test samples are analyzed

exactly like field samples, and at a minimum, one out of three ODC test samples must be positive for each

coliphage type used. If not, method performance is unacceptable, and analysis of field samples must be

stopped. Identify and correct the problem and demonstrate acceptable performance through analysis of

another ODC test before continuing with the analysis of field samples. The laboratory must analyze one

set of ODC samples after every 20 field and MS samples or one per week, whichever occurs more

frequently. Spike samples in “bulk” at the concentrations indicated in the MS and ODC Table below.

5.6.1.4.6 Performance studies. The laboratory should periodically analyze an external QC sample, such

as a performance testing sample, when available. The laboratory should also participate in available

interlaboratory performance studies conducted by local, State, and federal agencies or commercial

organizations. The laboratory should review results, correct unsatisfactory performance, and record

corrective actions.
V-27

 Initial demonstration of laboratory capability (IDC) for Method 1601

Coliphage

type

Sample

size1

Target spike

concentration

(PFU per

sample)

“Bulk”

volume to be

spiked

Bulk spike

concentration 

(PFU per bulk

volume)

Minimum number

of positive samples

out of 10

F 100-mL 1.3 1000 mL 13 5

+

Somatic 100-mL 1.5 1000 mL 15 5

A 100-mL sample is required under the Ground Water Rule. However, for other purposes, this test may be used with a 1

1-L sample volume. Because IDC samples should be analyzed just like field samples, including sample volumes, the IDC

analyses should be performed at the 1-L sample volume when the laboratory is evaluating 1-L samples. (The IDC

procedure for 1-L samples is provided in the protocol to Method 1601, Table 1.)

MS and ODC sample spiking requirements for ongoing evaluation of Method 1601 performance

Coliphage

type

Sample

size1

Target spike

concentration

(PFU per

sample)

Number of samples

that must be spiked

($1 must be

positive)

“Bulk”

volume 

to be spiked

Bulk spike

concentration

(PFU per bulk

volume)

F 100-mL 1.3 3 300-mL 3.9 +

Somatic 100-mL 1.5 3 300-mL 4.5

 A 100-mL sample is required under the Ground Water Rule. However, for other purposes, this test may be used with a 1

1-L sample volume. Because ODC and MS samples should be analyzed just like field samples, including sample volumes,

the ODC and MS analyses should be performed at the 1-L sample volume when the laboratory is evaluating 1-L samples.

(The MS and ODC procedure for 1-L samples is provided in the protocol to Method 1601, Tables 2.) 

5.6.2 EPA Method 1602: Male-specific (F ) and Somatic Coliphage in Water by Single Layer Agar (SAL)

+

Procedure

Method Summary: Method 1602 is a performance-based method for detecting or enumerating male-specific (F ) and +

somatic coliphage in ground water and other waters. A 100-mL ground water sample is assayed by adding magnesium

amp chloride and host bacteria (E. coli F for F coliphage and E. coli CN-13 for somatic coliphage), and then adding the +

sample/host bacteria mixture to 100 mL of double-strength molten Tryptic Soy Agar containing the appropriate antibiotic.

The sample is thoroughly mixed and the total volume is poured into 5 to 10 plates (dependent on plate size). After an

overnight incubation, any circular lysis zones (plaques) indicate the presence of coliphage.

5.6.2.1. Media--Please refer to Section 5.6.1 for antibiotic stocks, 10X Tryptic Soy Broth (TSB), 1.5% Tryptic

Soy Agar (TSA), 0.7% TSA top agar tubes with appropriate antibiotics, and spot plates.

5.6.2.1.1 Double Strength Tryptic Soy Agar (2X TSA)—Medium may become darker after autoclaving

but this should not affect media performance.

5.6.2.1.2 2X TSA with appropriate antibiotics—Keep molten at 45°-48°C in water bath until use. Agar

must be used the day of preparation.

5.6.2.2 Coliphage stock—Please refer to Section 5.6.1.2 for coliphage stock.

5.6.2.3 Host bacteria stock cultures — Please refer to Section 5.6.1.3 for host bacteria stock cultures.

5.6.2.4 General QC

5.6.2.4.1 Initial precision and recovery (IPR). The laboratory must demonstrate the ability to perform

this method acceptably by performing an IPR test before analyzing any field samples. Four reagent water
V-28

samples for each coliphage type are required for the IPR test. IPR samples must be spiked in bulk to yield

a target spike concentration of 80 PFU per sample. IPR samples must be spiked with enumerated sewage

r

filtrate or equivalent. The relative standard deviation of the recovery (RSD ) and the average percent

recovery (0) based on all four sample results for each coliphage type should meet the acceptance criteria

in the QC acceptance table below.

5.6.2.4.2 Method blanks. The laboratory must analyze method blanks (reagent water sample containing

no coliphage) to demonstrate freedom from contamination. The laboratory must analyze one method blank

with each analytical batch. For each coliphage type used, prepare and analyze a sterile reagent water

sample containing no coliphage using the same procedure as used for analysis of the field or QC samples.

An analytical batch is defined as all samples analyzed during a single day, up to a maximum of 20 samples

(field samples and matrix spike samples) per coliphage type.

5.6.2.4.3 Matrix spikes (MS). To assess method performance in a given matrix, the laboratory must

analyze one set of MS samples for each coliphage type when samples are first received from a ground

water source for which the laboratory has never before analyzed samples. The MS analysis is performed

on an additional (second) sample aliquot collected from the ground water source at the same time as the

routine field sample. If the laboratory routinely analyzes samples from one or more ground water sources,

one MS analysis must be performed per 20 field samples. For example, when a laboratory receives the first

sample from a source, the laboratory must obtain a second aliquot of this sample to be used for the MS.

When the laboratory receives the 20 sample from this site, a separate aliquot of this 20 sample must be th th

collected and spiked. Compare the coliphage recovery with the corresponding limits in the QC Table

below. If the recovery for coliphage falls outside its limit, method performance is unacceptable for that

sample. If the results for the OPR sample associated with this batch of samples are within their respective

control limits, a matrix interference may be causing poor recovery. If the results for the OPR are not within

their control limits, method performance is unacceptable (see Section 5.6.2.4.4). The problem should be

identified and corrected, and the matrix spike and associated field sample(s) should be qualified. The

recovery should be maintained on a control chart and updated on a regular basis. 

5.6.2.4.4 Ongoing precision and recovery (OPR). The laboratory must, on an ongoing basis,

demonstrate acceptable performance through analysis of an OPR sample. For each coliphage type used,

a reagent water sample is spiked with approximately 80 PFU. The OPR is analyzed exactly like a field

sample. The laboratory must analyze one OPR sample for each analytical batch. An analytical batch is

defined as all samples analyzed during a single day, up to a maximum of 20 samples (field samples and

matrix spike samples) per coliphage type used. Please note: the OPR serves as the positive control for

Method 1602. Compare the OPR percent recovery (R) with the corresponding limits for ongoing precision

and recovery in the QC Table below. If R meets the acceptance criteria, system performance is acceptable

and analysis of samples may continue. If R falls outside the range for recovery, method performance is

unacceptable, and analysis of field samples must be stopped. Identify and correct the problem and

demonstrate acceptable performance through successful analysis of another OPR test before continuing

with the analysis of field samples.

5.6.2.4.5 Performance studies. The laboratory should periodically analyze an external QC sample, such

as a performance testing sample, when available. The laboratory also should participate in available

interlaboratory performance studies conducted by local, state, and federal agencies or commercial

organizations. The laboratory should review results, correct unsatisfactory performance, and record

corrective actions.
V-29

Quality control acceptance criteria for Method 1602

Performance test

Male-specific

acceptance

criteria

Somatic

acceptance

criteria

Initial precision and recovery (IPR)

• Mean percent recovery 

• Precision (as maximum relative standard deviation)

9% - 130% 86% - 177%

46% 23%

Ongoing precision and recovery (OPR) as percent recovery 4% - 135% 79% - 183%

Matrix spike (MS)

• MS percent recovery Detect - 120% 48% - 291%

Matrix spike, matrix spike duplicate (MS/MSD)

• Mean percent recovery for MS/MSD Detect - 120% 48% - 291%

• Precision (as maximum relative percent difference of MS/MSD) 57% 28%

6. Sample Collection, Handling, and Preservation

Paragraphs 6.1-6.5 are applicable to those laboratories that collect samples. However, all laboratories should make an

effort to ensure proper sample collection; all laboratories are responsible for paragraph 6.6. 

6.1 Sample Collector

The sample collector should be trained in aseptic sampling procedures and, if required, approved by the appropriate

regulatory authority or its designated representative.

6.2 Sampling

6.2.1 (For TCR) Samples must be representative of the water distribution system. Water taps used for sampling

should be free of aerators, strainers, hose attachments, mixing type faucets, and purification devices. Cold water taps

should be used. The service line must be cleared before sampling by maintaining a steady water flow for at least two

minutes (until a steady water temperature is achieved). At least 100 mL of sample must be collected, allowing at least

a 1-inch air space to facilitate mixing of the sample by shaking. Immediately after collection, a sample information

form should be completed (see paragraph 6.5). See Section 3.15.4 regarding sample dechlorination. If a sample bottle

is filled too full to allow for proper mixing, do not pour off and discard a portion of the sample. Rather, pour the entire

sample into a larger sterile container, mix properly, and proceed with the analysis.

6.2.2 (For SWTR) Source water samples must be representative of the source of supply, collected not too far from

the point of intake, but at a reasonable distance from the bank or shore. The sample volume should be sufficient to

perform all the tests required.

6.2.3 (For coliphage analysis under GWR) A 100-mL sample volume is required for the assay. Collection of an

additional 100-mL water sample would allow for sample re-analysis, if necessary (e.g., if the positive or negative

controls fail). To ensure sufficient sample volume, an additional 50-mL water sample should be collected. 

6.2.4 (For E. coli and enterococci under GWR) A 100-mL sample volume is required for the assay. 

6.3 Sample Icing

6.3.1 (Bacterial samples) Samplers are encouraged, but not required, to hold drinking water samples at <10°C during

transit to the laboratory. Source water samples required by the Surface Water Treatment Rule (SWTR) must be held
V-30

at <10°C during transit (see Standard Methods, Section 9060B). Laboratories should reject samples that have been

frozen. 

6.3.2 (For coliphage analysis under GWR) Ship samples at <10°C using wet ice, Blue Ice®, or similar products to

maintain temperature, and store at 1°-5°C. Samples should not be frozen.

QC 6.3.3 For SWTR samples and coliphage samples, sample temperature upon receipt should be recorded. A sample that

has a temperature upon receipt of >10°C , whether iced or not, should be flagged unless the time since sample

collection has been less than two hours.

6.4 Sample Holding/Travel Time

6.4.1 For the analysis of total coliforms in drinking water, the time between sample collection and the placement of

sample in the incubator must not exceed 30 hours (per regulation at 40 CFR 141.21(f)(3)). All samples received in

the laboratory should be analyzed on the day of receipt. If the laboratory receives the sample late in the day, the

sample may be refrigerated overnight as long as analysis begins within 30 hours of sample collection.

6.4.2 The time from sample collection to placement of the sample in the incubator for total coliforms and fecal

coliforms in surface water sources, and heterotrophic bacteria in drinking water, must not exceed eight hours (per

regulation at 40 CFR 141.74(a)(1)). 

6.4.3 (For coliphage analysis) The time between sample collection and the placement of sample in the incubator must

not exceed 48 hours. The time from sewage sample collection to analysis of QC spiking suspensions may not exceed

24 hours, unless re-titered and titer has not decreased by more than 50%. If titer has not decreased by more than 50%,

the sample can be stored for up to 72 hours.

6.4.4 (For E. coli and enterococci under GWR) The time between sample collection and the placement of sample in

the incubator must not exceed 30 hours.

6.5 Sample Information Form

After collection, the sampler should enter on a sample information form, in indelible ink, the following information:

• Name of system (public water system site identification number, if available)

• Sample identification (if any)

• Sample site location 

• Sample type (e.g., routine distribution system sample, repeat sample, raw or process water, other special purpose sample)

• Date and time of collection

• Analysis requested

• Disinfectant residual

• Name of sampler

• Any remarks

6.6 Chain-of-Custody

Sample collectors and laboratories must follow applicable State regulations pertaining to chain-of-custody. An example

of such a plan is provided in Appendix A.

7. Quality Assurance

7.1 A written QA plan should be prepared and followed (see Chapter III). The QA plan should be available for inspection

by the certification officer. As specified by the QA plan, a laboratory that performs its own calibration of equipment or

supplies (e.g., thermometers) should have a Standard Operating Procedure available for review. If a laboratory wishes to

perform additional QA beyond those in this manual, the laboratory may refer to Standard Methods, Section 9020, Quality

Assurance (Quality Assurance/Quality Control, in 20 ed.). th

7.2 States are encouraged to establish proficiency testing (PT) as part of their drinking water certification program for

microbiology. A laboratory should successfully analyze at least one set of PT samples once every 12 months, for each

method for which it is certified. 
V-31

For methods used to test the presence or absence of an organism in a sample, each PT set should contain ten samples, all

shipped at the same time in either a lyophilized, dehydrated, or aqueous state. The set should include samples, in various

combinations, that contain total coliforms, fecal coliforms, E. coli, non-coliforms, and at least one blank. Each set should

be used only with a single analytical method. To be acceptable, a laboratory should correctly analyze a minimum of nine

of the ten samples, with no false-negative result (i.e., a single false-positive result may be acceptable).

Because even methods based upon the same principle (e.g., membrane filtration) may be quite dissimilar, a Region or State

should consider certifying a laboratory only for those specific methods for which the laboratory has successfully analyzed

a set of PT samples. The Table below reflects this approach, and identifies the few methods that may be sufficiently similar

to allow a laboratory to be certified for more than one method upon successful completion of a single set of PT samples.

Method Category Specific Method1

Fermentation broth method LTB or P-A broth, followed by BGLB and either EC or EC-MUG

Fermentation broth method A-1 broth (fecal coliform, SWTR only)

Enzyme substrate method Colilert or Colilert 18

Enzyme substrate method Colisure

Enzyme substrate method Readycult or Fluorocult LMX

Enzyme substrate method E*Colite

Enzyme substrate method Colitag

Membrane filter method M-Endo or LES Endo, followed by BGLB and either EC, EC-MUG,

or NA-MUG

Membrane filter method MI Medium

Membrane filter method Coliscan

Membrane filter method m-ColiBlue24

Membrane filter method Chromocult

Membrane filter method mFC agar (fecal coliform, SWTR only)

HPC method PCA

HPC method SimPlate

Separate set of proficiency test samples recommended for each cell. A single set of PT samples would cover every method within the 1 

same cell.

8. Records and Data Reporting

8.1 Legal Defensibility: Compliance monitoring data should be made legally defensible by keeping thorough and accurate

records. The QA plan and/or SOPs should describe the policies and procedures used by the facility for record retention

and storage. If samples are expected to become part of a legal action, chain-of-custody procedures should be used (See

Appendix A).

8.2 Maintenance of Records: Public water systems are required to maintain records of microbiological analyses of

compliance samples for five years (40 CFR 141.33). The laboratory should maintain easily accessible records for five years

or until the next certification data audit is complete, whichever is longer. A change in ownership, merger, or closure of

a laboratory does not cancel this requirement. The client water system should be notified before disposing of records so

they may request copies if needed. This includes all raw data, calculations, and quality control data. These data files may

be either hard copy, microfiche or electronic. Electronic data should always be backed up by protected tape or disk or hard
V-32

copy. If the laboratory changes its computer hardware or software, it should make provisions for transferring old data to

the new system so that it remains retrievable within the time frames specified above. Data which is expected to become

part of a legal action will probably need to be maintained for a longer period of time. Check with your legal counsel. See

Good Automated Laboratory Practices, EPA 2185, Office of Information Management, Research Triangle Park, NC

27711, 8/10/95.

8.3 Sampling Records: Data should be recorded in ink with any changes lined through such that original entry is visible.

Changes should be initialed and dated. The following information should be readily available in a summary or other

record(s):

8.3.1 Sample information form, from 6.5 above

8.3.2 Date and time of sample receipt by the laboratory

8.3.3 Name of laboratory person receiving the sample

8.3.4 Any deficiency in the condition of the sample. A sample should be invalidated for the following reasons:

- Time between sample collection and receipt by laboratory has been exceeded

- Presence of disinfectant in sample is noticed (e.g., odor)

- Evidence of freezing

- Use of a container not approved by the laboratory for the purpose intended

- Insufficient sample volume (e.g., <100 mL)

- Presence of interfering contaminant, if noticed (e.g., hydrocarbons, cleansers, heavy metals, etc.)

- Sample temperature exceeds the maximum allowable

8.4 Analytical Records: Data should be recorded in ink with any changes lined through such that original entry is visible.

Changes should be initialed and dated. The following information should be readily available in a summary or other

record(s):

8.4.1 Laboratory sample identification

8.4.2 Date and time analysis begins

8.4.3 Laboratory and a signature or initials of person(s) performing analysis

8.4.4 Analytical technique or method used

8.4.5 All items marked QC

8.4.6 Results of analyses

8.5 Preventive Maintenance

Laboratories should maintain preventive maintenance and repair activities records for all instruments and equipment

(including pH meters, analytical balances, incubators, refrigerators, autoclaves, and water baths). Records should be kept

for five years in a manner that allows for easy inspection.

9. Action Response to Laboratory Results

9.1 Testing Total Coliform-Positive Cultures

For the Total Coliform Rule, laboratories must test all total coliform-positive cultures for the presence of either fecal

coliforms or E. coli.

9.2 Notification of Positive Results

9.2.1 For the Total Coliform Rule, laboratories must promptly notify the proper authority of a positive total coliform,

fecal coliform, or E. coli result, so that appropriate follow-up actions (e.g., collection of repeat samples) can be

conducted (see 40 CFR 141.21(b) and (e), and 141.31, etc.). 

9.2.2 If any sample is fecal coliform- or E. coli-positive, “the system must notify the State by the end of the day when

the system is notified of the test result, unless the system is notified of the result after the State office is closed, in

which case the system must notify the State before the end of the next business day.” (40 CFR 141.21(e)(1)). 
V-33

9.2.3 A total coliform-positive result is based on the confirmed phase if the Multiple Tube Fermentation Technique

or Presence-Absence (P-A) Coliform Test is used, or the verified test for the Membrane Filter Technique if M-Endo

medium or LES Endo agar is used. No requirement exists to confirm a total coliform-positive result using Colilert,

Colisure, MI agar, E*Colite, MI agar, m-ColiBlue24, Chromocult, Readycult/Fluorocult, Coliscan, or Colitag test.

Also, no requirement exists to confirm a positive fecal coliform or E. coli test. In those rare cases where a presumptive

total coliform-positive culture does not confirm/verify as such, but is found to be fecal coliform or E. coli-positive,

the sample is considered total coliform-positive and fecal coliform/E. coli-positive.

9.3 Notification of Total Coliform Interference 

For the Total Coliform Rule, the laboratory must promptly notify the proper authority (usually the water system) when

results indicate that non-coliforms may have interfered with the total coliform analysis, as described in 40 CFR

141.21(c)(2).
V-34

Example Checklists for On-site Evaluation of

Laboratories Analyzing Drinking Water
MICRO CHECKLISTS

V-35

General Audit Information

Laboratory:

Mailing Address (mailing address of owner if different):

Street

City, State, Zip code

Audit Location (if different):

Telephone:

Fax:

E-mail:

Other:

Audit Organization:

Auditors/Signatures:

Audit Date(s):
MICRO CHECKLISTS

V-36

Laboratory Personnel

Position/Title Name Education Level

Degree/Major

Specialized

Training

Present

Specialty

Experience,

including 

# yrs at current

position

Laboratory

Supervisor

Laboratory

Consultant

Primary Analyst

Analyst 2

Analyst 3

Analyst 4

Others
MICRO CHECKLISTS

V-37

Element Number Yes No NA Comments

1. PERSONNEL

Supervisor/Consultant 1.1

Does the supervisor of the microbiology laboratory have a bachelor’s

degree in microbiology, biology, or equivalent? 

Has a supervisor with a degree in a subject other than those listed

above had at least one college-level microbiology laboratory course

in which environmental microbiology was covered? 

In addition, has the supervisor had a minimum of two weeks training

at a Federal or State agency or academic institution in

microbiological analysis of drinking water or 80 hours of on-the-job

training in water microbiology at a certified laboratory, or other

training acceptable to the State or EPA?

If a supervisor is not available, and a waiver has not been granted as

per Section 1.3, is a consultant with the same qualifications

substituted? 

Can the laboratory document that the consultant is acceptable to the

State, and present on-site frequently enough to satisfactorily perform

a supervisor’s duties?

Can the laboratory supervisor demonstrate that all laboratory

personnel have the ability to satisfactorily perform the analyses to

which they are assigned?

Can the laboratory supervisor demonstrate that all data reported by

the laboratory meets the required quality assurance and regulatory

criteria?

Analyst (or equivalent job title) 1.2

Does the analyst have at least a high school education, a minimum of

three months bench experience in water, milk or food microbiology,

training in microbiological analysis of drinking water acceptable to

the State (or EPA), and a minimum of 30 days on-the-job training

under an experienced analyst?

Has the analyst demonstrated acceptable results on unknown samples

before analyzing compliance samples?

Waiver of Academic Training 1.3 

Has the certification authority waived the need for the above

specified academic training for highly experienced analysts in this

laboratory?

Has the certification authority waived the need for the above

specified training for supervisors of laboratories associated with

drinking water systems that only analyze samples from that system?
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-38

If yes to either of the above, does the laboratory have a copy of that

written and signed waiver available for inspection?

Personnel Records 1.4

Does the laboratory maintain personnel records on laboratory

analysts that include academic background, specialized training

courses completed, and types of microbiological analyses conducted?

2. LABORATORY FACILITIES

Does the laboratory have facilities that are clean and temperature and

humidity controlled, and with adequate lighting at the bench tops?

Does the laboratory maintain effective separation of incompatible

testing areas?

Does the laboratory control access where appropriate, and minimize

traffic flow through the work areas?

Does the laboratory ensure that contamination does not adversely

affect data quality?

Does the laboratory have bench tops and floors that are easily

cleaned and disinfected?

Does the laboratory have sufficient space for processing samples;

storage space for media, glassware, and portable equipment; floor

space for stationary equipment; and areas for cleaning glassware and

sterilizing materials?

Does the laboratory have provisions for disposal of microbiological

wastes?

3. LABORATORY EQUIPMENT AND SUPPLIES

Does the laboratory have the equipment and supplies needed to

perform the approved methods for which certification has been

requested?

pH meter 3.1 

Are accuracy and scale graduations within ±0.1 units? 3.1.1

Are pH buffer aliquots used only once? 3.1.2

Are electrodes maintained according to the manufacturer’s

recommendations?

3.1.3

QC Are pH meters standardized before each use period with pH 7.0

and either 4.0 or 10.0 standard buffers, whichever covers the desired

pH of the media or reagent?

3.1.4
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-39

QC Are both the date and buffers used recorded in a logbook along

with the analyst’s initials?

QC Is the pH slope recorded monthly, after calibration? 3.1.5

QC If the pH meter does not have a feature to automatically

calculate the slope, but can provide in the pH in millivolts, is the

formula in Section 3.1.5.1 used to calculate the slope?

3.1.5.1

QC If the slope is below 95% or above 105%, are the

manufacturer’s instructions followed for meter or electrode

maintenance and general cleaning?

3.1.5.2

QC Are commercial pH buffer solutions dated when received and

when opened? 

3.1.6

QC Are pH buffer solutions discarded by the expiration date?

Balance (top loader or pan) 3.2

Does the balance have a readability of 0.1 g? 3.2.1

Does the balance have a sensitivity of at least 0.1 g for a load of 150

g, and 1 mg for a load of 10 g or less?

3.2.2

QC Are the balances calibrated monthly using ASTM Class 1, 2, or

3 weights (minimum 3 traceable weights which bracket laboratory

weighing needs, with a readability of 0.1 g)? 

3.2.3

QC Are non-reference weights calibrated every six months with

reference weights?

QC Are calibrations recorded in a logbook with the initials of the

individual performing the calibration?

QC Are correction values on file and used?

QC Are reference weights re-certified every five years?

QC Are damaged or corroded weights replaced?

QC Are service contracts or internal maintenance protocols and

maintenance records available?

3.2.4

QC Is maintenance, calibration, and cleaning conducted at least

annually by a qualified independent technician, unless the need is

modified or waived by the certification officer?

Temperature Monitoring Device 3.3

Are glass, dial, or electronic thermometers graduated in 0.5/C

increments (0.2/C increments for tests which are incubated at

44.5/C) or less, except as noted for hot air ovens (Section 3.6.1) and

refrigerators (Section 3.9.1)?

3.3.1
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-40

Does observation of glass thermometers indicate no separation in

fluid columns?

Are only dial thermometers which can be adjusted used?

QC Are glass and electronic thermometers calibrated annually and

dial thermometers quarterly at the temperature used, against a NISTtraceable reference thermometer or one that meets the requirements

of NBS Monograph SP 250-23?

3.3.2

QC Are both the calibration factor and calibration date indicated on

the thermometer?

QC Is the following calibration information recorded in a QC

record book?

S Serial number of the laboratory thermometer

S Serial number of the NIST-traceable thermometer (or other

reference thermometer)

S Temperature of the laboratory thermometer

S Temperature of the NIST-traceable thermometer (or other

reference thermometer)

S Correction (or calibration) factor

S Date of check

S Analyst’s initials 

QC Is the thermometer discarded if it differs by more than 1/C from

the reference thermometer?

3.3.3

QC Are reference thermometers recalibrated at least every five

years?

QC Is reference thermometer calibration documentation

maintained?

QC Are continuous recording devices used to monitor incubator

temperature recalibrated at least annually, using a reference

thermometer that meets the specifications noted in Section 3.3.2?

3.3.4

Incubator Unit 3.4

Do incubator units have an internal temperature monitoring device

and maintain a temperature specified by the method used, usually

35°±0.5°C and 44.5°±0.2°C?

3.4.1

For non-portable incubators, are thermometers placed on top and

bottom shelves of the use area and immersed in liquid as directed by

the manufacturer (except for electronic thermometers)?

When aluminum block incubators are used, do culture dishes and

tubes fit snugly?
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-41

QC Is the calibration-corrected temperature recorded for each

thermometer being used at least twice per day during each day the

incubator is in use?

3.4.2

QC Are these readings separated by at least four hours?

QC Does the documentation include the date and time of reading,

temperature, and technician’s initials?

If a circulating water bath is used, is it equipped with a gable cover

to ensure an incubation temperature of 44.5°±0.2°C?

Autoclave 3.5

Does the autoclave have an internal heat source, a temperature gauge

with a sensor on the exhaust, a pressure gauge, and an operational

safety valve?

3.5.1

Can the autoclave maintain a sterilization temperature during the

sterilizing cycle and complete an entire cycle within 45 minutes when

a 12-15 minute sterilization period is used? 

Does the autoclave depressurize slowly enough to ensure that media

will not boil over and bubbles will not form in inverted tubes?

QC Is the following information recorded each time the autoclave is

used?

S Date

S Contents

S Sterilization time and temperature

S Total time in the autoclave

S Analyst’s initials

3.5.3

QC Are copies of the service contracts or internal maintenance

protocols and maintenance records kept?

QC Is maintenance conducted at least annually?

QC Is a record of the most recent service performed on file and

available for inspection?

QC Is a maximum-temperature-registering thermometer, electronic

temperature readout device, or continuous recording device used

each autoclave cycle to ensure that the proper temperature was

reached?

3.5.4

QC Is the temperature recorded?

QC Is overcrowding avoided?

QC Are spore strips or spore ampules used monthly as

bioindicators to confirm sterilization?
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-42

QC Are automatic timing mechanisms checked quarterly with a

stopwatch or other accurate timepiece or time signal, and the results

recorded and initialed?

3.5.5

Are autoclave door seals clean and free of caramelized media? 3.5.6

Are autoclave drain screens cleaned frequently and debris removed?

Hot Air Oven 3.6

Does the oven maintain a stable sterilization temperature of 170/-

180/C for at least two hours?

3.6.1

Is overcrowding avoided?

Is the oven thermometer graduated in 10/C increments or less, with

the bulb placed in sand during use?

QC Is the following information recorded for each cycle?

S Date

S Contents

S Sterilization time and temperature

S Analyst’s initials

3.6.2

QC Are spore strips used monthly to confirm sterilization? 3.6.3

Colony Counter 3.7

Is a dark field colony counter used to count Heterotrophic Plate

Count colonies?

Conductivity Meter 3.8

Are meters suitable for checking laboratory reagent-grade water and

readable in units of either micromhos/cm or microsiemens/cm? 

3.8.1

QC Is the meter calibrated at least monthly, following the

manufacturer’s recommendations and using an appropriate certified

and traceable low-level standard? 

3.8.2

QC If the meter cannot be calibrated as noted above, is the cell

constant determined at monthly intervals using a method in Standard

Methods, Section 2510?

Is an in-line unit that cannot be calibrated used to check reagentgrade water?

3.8.3

Refrigerator 3.9

Does the refrigerator maintain a temperature of 1/-5/C? 3.9.1

Is the refrigerator thermometer graduated in at least 1/C increments

and the thermometer bulb immersed in liquid?
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-43

QC On days the refrigerator is in use, and the laboratory is staffed,

is the calibrated-corrected temperature recorded at least once per

day?

3.9.2

Inoculating Equipment 3.10

Are sterile metal or disposable plastic loops, wood applicator sticks,

sterile swabs, or sterile plastic disposable pipet tips used?

Are wood applicator sticks, if used, sterilized by dry heat?

Are metal inoculating loops and/or needles made of nickel alloy or

platinum? 

Membrane Filtration (MF) Equipment 3.11

Are MF units made of stainless steel, glass, porcelain, or

autoclavable plastic? 

3.11.1

Are they scratched, corroded, or leaking?

QC If graduations on clear or plastic funnels are used to measure

sample volume, is their accuracy checked with a Class B graduated

cylinder or better (or other Class B glassware) and a record of this

calibration check retained?

3.11.2

Is a 10x to 15x stereo microscope with a fluorescent light source

used to count sheen colonies?

3.11.3

Are the membrane filters approved by the manufacturer for total

coliform water analysis?

3.11.4

Are membrane filters to be used cellulose ester, white, gridmarked,

47 mm diameter, and 0.45 :m pore size?

If alternate pore sizes are used, does the manufacturer provide

performance data equal to or better than the 0.45 :m pore size?

Are membrane filters and pads purchased presterilized or autoclaved

for 10 minutes at 121/C before use?

QC Is the lot number for membrane filters and the date received

recorded?

3.11.5

QC Are the membranes checked to see that they are not brittle or

distorted?

QC Are the manufacturer’s specification/certification sheets

available?

Are the forceps blunt and smooth-tipped without corrugations on the

inner sides of the tips?

3.11.6

Culture Dishes (loose or tight lids) 3.12
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-44

Are presterilized plastic or sterilizable glass culture dishes used? 3.12.1

Is the sterility of the glass culture dishes maintained by placement in

stainless steel or aluminum canisters or a wrap of heavy aluminum

foil or char-resistant paper?

Are loose-lid petri dishes incubated in a tight-fitting container with a

moistened paper towel?

3.12.2

Are opened packs of disposable culture dishes resealed between use

periods?

3.12.3

For membrane filter methods, are culture dishes of an appropriate

size to allow the transfer of a single membrane per plate?

3.12.4

Pipets 3.13

Are glass pipets sterilized and maintained in stainless steel or

aluminum canisters or wrapped individually in char-resistant paper or

aluminum foil?

3.13.1

Do pipets have legible markings and are they not chipped or etched? 3.13.2

Are opened packs of disposable sterile pipets resealed between use

periods?

3.13.3

Are pipets delivering volumes of 10 mL or less accurate to within a

2.5% tolerance?

3.13.4

Are calibrated micropipetters used with sterile tips? 3.13.5

Are micropipetters calibrated annually and adjusted or replaced if the

precision or accuracy is greater than 2.5%?

Glassware and Plasticware 3.14

Is the glassware made of borosilicate glass, or other corrosionresistant glass, and free of chips and cracks?

3.14.1

Are markings on graduated cylinders and pipets legible?

Are plastic items clear and nontoxic to microorganisms?

QC Are the graduated cylinders used for measurement of sample

volumes, or other precalibrated containers that have clearly marked

volumes used in lieu of graduated cylinders, accurate to within a

2.5% tolerance?

3.14.2

Are culture tubes and containers containing fermentation medium of

sufficient size to contain medium plus sample without being more

than three quarters full?

3.14.3

Are tube closures made of stainless steel, plastic, aluminum, or screw

caps with nontoxic liners?

3.14.4
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-45

Are cotton or foam plugs used?

Sample Containers 3.15

Are sample containers wide-mouth plastic or non-corrosive glass

bottles with non-leaking ground glass stoppers or caps with nontoxic

liners, sterile plastic bags containing sodium thiosulfate, or other

appropriate sample containers?

3.15.1

Is sample container capacity at least 120 mL (4 oz) to allow at least a

1-inch head space?

Are glass stoppers covered with aluminum foil or char-resistant

paper for sterilization?

3.15.2

Are unsterilized glass and plastic bottles sterilized by autoclaving or,

alternatively, by dry oven for glass bottles?

3.15.3

Are empty containers moistened with several drops of water before

autoclaving to prevent an “air lock” sterilization failure?

If chlorinated water is to be analyzed, is sufficient sodium thiosulfate

added to the sample bottles before sterilization to neutralize any

residual chlorine in the water sample?

3.15.4

Ultraviolet Lamp (if used) 3.16

Is the germicidal unit disconnected monthly and the lamp cleaned by

wiping with soft cloth moistened with ethanol?

3.16.1

Is the longwave unit used for fluorometric tests kept clean?

QC Is the germicidal unit tested quarterly with a UV light meter or

agar spread plate? 

3.16.2

QC Is the lamp replaced if it emits less than 70% of its initial

output or if an agar spread plate containing 200 to 250

microorganisms, exposed to the UV light for two minutes, does not

show a count reduction of 99%?

Spectrophotometer or colorimeter (if used) 3.17

Are wavelengths in the visible range? 3.17.1

QC Is a calibration standard and a method-specific blank analyzed

every day the instrument is used, prior to sample analysis?

3.17.2

QC Is this calibration standard obtained from an outside source?

QC Does the calibration standard give a reading in the desired

absorbance range?
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-46

4. GENERAL LABORATORY PRACTICES

Are laboratory personnel aware of general and customary safety

practices for laboratories? 

Does the laboratory have a safety plan available?

Does the laboratory keep a copy, and follow the personal protection

guidelines, of any material safety data sheet accompanying the

receipt of a toxic material?

Sterilization Procedures 4.1

Does the laboratory follow the minimum times for autoclaving the

materials listed below at 121/C?

S Membrane filters and pads 10 min

S Carbohydrate containing media 12-15 min1

S Contaminated test materials 30 min2

S Membrane filter assemblies 15 min

S Sample collection containers 15 min

S Individual glassware 15 min

S Dilution water blank 15 min

S Rinse water (0.5 - 1 L) 15-30 min2

 except where otherwise specified by the manufacturer 1

 time depends upon water volume per container and autoclave load 2

4.1.1

Are autoclaved membrane filters and pads and all media removed

immediately after completion of the sterilization cycle?

4.1.2

Is membrane filter equipment autoclaved before the beginning of a

filtration series?

4.1.3

If a UV light (254 nm) is used to sanitize equipment after initial

autoclaving for sterilization, are all supplies presterilized?

4.1.4

Sample Containers 4.2

QC Is at least one sample container selected at random from each

batch of sterile sample bottles, or other containers (or lot of

commercially available sample containers), and the sterility

confirmed by adding 25 mL of a sterile non-selective broth,

incubating at 35/±0.5/C, and checking for growth after 24 and 48

hours?

QC Are these results recorded?

QC If growth is detected, is the entire batch resterilized? 

 Reagent-Grade Water 4.3
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-47

Does the laboratory only use satisfactorily tested reagent water from

stills or deionization units to prepare media, reagents, and

dilution/rinse water for performing microbial analyses?

4.3.1

QC Is the quality of reagent water tested and does it meet the

following criteria?

Conductivity >0.5 megohms resistance Monthly*

or <2 micromhos/cm

(microsiemens/cm) at 25/C

Pb, Cd, Cr, Not greater than 0.05 mg/L Annually

Cu, Ni, Zn per contaminant. Collectively 

no greater than 0.1 mg/L 

Total chlorine <0.1 mg/L Monthly

residual*

Heterotrophic <500/mL CFU/mL* Monthly

plate count*

Bacteriological Ratio of growth rate Annually

quality of 0.8 to 3.0

reagent water*

*See Section 4.3.2 for footnotes

4.3.2

 Dilution/Rinse Water 4.4

Is stock buffer solution or peptone water prepared as specified in

Standard Methods, Section 9050C?

4.4.1

Are stock buffers autoclaved or filter-sterilized? 4.4.2

Are these containers labeled, dated, and refrigerated?

Are stored stock buffers free from turbidity?

QC Is each batch (or lot, if commercially prepared) of dilution/rinse

water checked for sterility by adding 50 mL of water to 50 mL

double strength non-selective broth, incubating at 35/± 0.5/C, and

checking for growth after 24 hours and 48 hours?

4.4.3

QC Are these results recorded?

QC Is the batch/lot discarded if growth is detected?

 Glassware Washing 4.5

Is distilled or deionized water used for the final rinse? 4.5.1

Is laboratory glassware washed with a detergent designed for

laboratory use?

4.5.2
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-48

QC Is the glassware inhibitory residue test performed before the

initial use of a washing compound and whenever a different

formulation, or washing procedure is used?

4.5.3

QC Are these results recorded?

QC Is each batch of dry glassware used for microbial analysis spotchecked for pH reaction using 0.04% bromthymol blue (or

equivalent pH indicator) and the color reaction recorded? 

4.5.4

5. ANALYTICAL METHODOLOGY

General 5.1

For compliance samples, does the laboratory use only the analytical

methodologies specified in the Total Coliform Rule (TCR), the

Surface Water Treatment Rule (SWTR), and the Groundwater Rule

(GWR)? 

5.1.1

Is the laboratory certified for all analytical methods it uses for

compliance purposes?

5.1.2

At a minimum, is the laboratory certified for one total coliform

method and one fecal coliform or E. coli method?

Is the laboratory certified for a second total coliform method if one

method cannot be used for some drinking waters?

For a laboratory that enumerates heterotrophic bacteria for

compliance with the SWTR, is the laboratory certified for either the

Pour Plate Method or the SimPlate method for heterotrophic

bacteria?

Are water samples shaken vigorously at least 25 times before

analyzing?

5.1.3

QC If dilution buffer is used, does the laboratory check the buffer

volume in one dilution bottle of each batch or lot? 

5.1.4

QC For a 90-mL or 99-mL volume, is the tolerance ±2 mL?

Does the laboratory analyze a 100-mL sample volume for total

coliforms in drinking water?

5.1.5

Media (or defined substrate) 5.1.6

Are dehydrated media stored in a cool dry location and discarded by

the manufacturer’s expiration date?

5.1.6.1

Is caked or discolored dehydrated media discarded?
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-49

QC For media prepared in the laboratory is the following

information recorded?

S Date of preparation

S Type of medium

S Lot number

S Sterilization time and temperature

S Final pH (after sterilization)

S Technician’s initials

5.1.6.2

QC For media prepared commercially is the following recorded for

each lot?

S Date received

S Type of medium

S Lot number

S pH verification

5.1.6.3

QC Are media prepared commercially discarded by manufacturer’s

expiration date?

QC Is each new lot of dehydrated or prepared commercial medium

and each batch of laboratory-prepared medium checked before use

for sterility and with positive and negative culture controls?

5.1.6.4

QC Are these results recorded?

QC For laboratories using commercially prepared media with

manufacturer shelf-lives of greater than 90 days, are positive and

negative controls run each quarter, in addition to that noted above?

QC Are these results recorded?

QC For control organisms, are stock cultures periodically checked

for purity and the results recorded, or are commercially available

disks impregnated with the organism used?

If prepared medium is stored after sterilization, is it maintained in the

dark as follows?

S poured plates 1/-5/C 2 weeks

S broth in containers with 1/-30/C 2 weeks

loose-fitting closures

S broth in tightly closed 1/-30/C 3 months

containers 

5.1.6.5

QC Does the laboratory perform parallel testing between a newly

approved test and another EPA-approved procedure for enumerating

total coliforms for at least several months and/or several seasons to

assess the effectiveness of the new test for the wide variety of water

types submitted for analysis? Recommended.

5.1.7

Does the laboratory perform the approved methods listed in this

section for the TCR, SWTR, and/or GWR?

5.1.8
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-50

Fermentation broth methods 5.2

General 5.2.1

Is the water level of the water bath above the upper level of the

medium in the culture tubes?

5.2.1.1

If a Dri-bath incubator is used, is the specified temperature

requirement maintained in all tube locations used?

5.2.1.2

Multiple Tube Fermentation Technique (for detecting total coliforms

in drinking water and enumerating total coliforms in source water)

5.2.2

For drinking water samples, is the total sample volume of 100 mL

used for each test? 

5.2.2.1

For source water samples, are at least 3 series of five tubes each with

appropriate sample dilutions used?

5.2.2.2

Media 5.2.2.3

Is lauryl tryptose broth (LTB) used in the presumptive test and 2%

brilliant green lactose bile broth (BGLBB) in the confirmed test?

5.2.2.3.1

If lactose broth (LB) is used in lieu of LTB, has the laboratory

conducted at least 25 parallel tests between this medium and LTB

using the waters normally tested?

Has this comparison demonstrated that the false-positive rate and

false-negative rate for total coliforms, using LB, is less than 10%?

Is this comparison information documented and the records retained?

Is the final pH of LTB medium 6.8 ± 0.2?

Is the final pH of 2% BGLBB 7.2 ± 0.2?

Is the test medium concentration adjusted to compensate for the

sample volume so that the resulting medium after sample addition is

single strength?

5.2.2.3.2

If a single 100-mL sample volume is used, is the inverted vial

replaced with an acid indicator (bromcresol purple)?

Is the medium autoclaved at 121/C for 12-15 minutes?

Is the sterile medium in tubes examined to ensure that the inverted

vials, if used, are free of air bubbles and are at least one-half to twothirds covered after the water sample is added?

5.2.2.3.3

Is the inoculated medium incubated at 35/±0.5/C for 24±2 hours? 5.2.2.4

If no gas or acid detected, is the inoculated medium incubated for

another 24 hours for a total incubation time of 48±3 hours?
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-51

Is each 24- and 48-hour tube that has growth or is gas-positive or

acid-positive confirmed using 2% BGLBB?

5.2.2.5

For drinking water samples, is each total coliform-positive sample

tested for the presence of either fecal coliforms or E. coli?

5.2.2.6

Invalidation of total coliform-negative samples 5.2.2.7

For drinking water samples, are all samples that produce a turbid

culture (i.e., heavy growth) in the absence of gas/acid production, in

LTB or LB, invalidated?

5.2.2.7.1

Does the laboratory then collect, or request that the system collect,

another sample within 24 hours from the same location as the

original invalidated sample?

Although not required before invalidation, does the laboratory

perform a confirmed test and/or a fecal coliform/E. coli test on the

total coliform-negative culture to check for coliform suppression?

And if the confirmed test is total coliform-positive or fecal

coliform/E. coli-positive, does the laboratory report the sample as

such? 

If the follow-up test is total coliform-negative, does the laboratory

invalidate the sample?

For source water samples, are all samples that produce a turbid

culture (i.e., heavy growth) in the absence of gas/acid production in

LTB or LB invalidated? 

5.2.2.7.2

Does the laboratory collect, or request that the system collect,

another sample from the same location as the original invalidated

sample?

Although not required before invalidation, does the laboratory

perform a confirmed test on the total coliform-negative culture and,

if the confirmed test is total coliform-positive, is the MPN reported?

If the confirmed test is total coliform-negative, is the sample

invalidated?

Presence-Absence (P-A) Coliform Test (for detecting total coliforms

in drinking water)

5.2.3

Medium 5.2.3.1

When six-times formulation strength medium is used, is it filtersterilized rather than autoclaved?

5.2.3.1.1

Is the medium autoclaved for 12 minutes at 121/C? 5.2.3.1.2

Is the total time in the autoclave less than 30 minutes?
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-52

Are the bottles placed in the autoclave with space between them?

Is the final pH of the medium 6.8±0.2?

If the prepared medium is stored, is it maintained in a culture bottle

at 1°-30/C in the dark for no more than 3 months?

5.2.3.1.3

Is the stored medium discarded if evaporation exceeds 10% of

original volume?

Is a 100-mL sample inoculated into a P-A culture bottle? 5.2.3.2

Is the sample/medium incubated at 35/±0.5/C and observed for

yellow color (acid) after 24 and 48 hours?

5.2.3.3

Are yellow cultures confirmed in BGLBB and a fecal coliform/E.

coli test conducted?

5.2.3.4

Are all samples which produce a non-yellow turbid culture in P-A

medium invalidated?

5.2.3.5

Does the laboratory collect, or request that the system collect,

another sample from the same location as the original invalidated

sample?

Although not required before invalidation, does the laboratory

perform a confirmed test on the total coliform-negative culture

and/or a fecal coliform/E. coli test and, if the confirmed test is total

coliform-positive, is the sample reported as such?

If the confirmed test is total coliform-negative, is the sample

invalidated?

Fecal Coliform Test (using EC Medium for fecal coliforms in

drinking or source water, or A-1 Medium for fecal coliforms in

source water only)

5.2.4

EC Medium 5.2.4.1

Is EC medium used to test a total coliform-positive culture for fecal

coliforms under the Total Coliform Rule?

5.2.4.1.1

Is each total coliform-positive culture transferred from a presumptive

tube/bottle, or each presumptive total coliform-positive colony

(unless a cotton swab is used), to at least one tube containing EC

Medium with an inverted vial?

Is EC medium used to enumerate fecal coliforms in source water, in

accordance with the SWTR?

5.2.4.1.2

When conducting a MTF test, are three sample volumes of source

water with five or ten tubes/sample volume used?
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-53

Is a culture from each total coliform-positive tube transferred to a

tube containing EC Medium with an inverted vial?

Is EC Medium autoclaved at 121/C for 12-15 minutes? 5.2.4.1.3

Is the final pH of EC medium 6.9±0.2?

Are the inverted vials examined to ensure that they are free of air

bubbles and at least one-half to two-thirds covered after the sample is

added?

5.2.4.1.4

Is EC Medium incubated at 44.5/±0.2/C for 24±2 hours? 5.2.4.1.5

Is any amount of gas detected in the inverted vial of a tube that has

turbid growth considered a fecal coliform-positive test, regardless of

the result of any subsequent test on that culture?

5.2.4.1.6

A-1 Medium 5.2.4.2

If A-1 Medium is used, is it used to enumerate only fecal coliforms

in source water, in accordance with SWTR, and not for drinking

water samples?

5.2.4.2.1

Are three sample volumes of source water used in a five- or tentube/sample volume format?

Is A-1 Medium autoclaved at 121/C for 10 minutes? 5.2.4.2.2

For A-1 Medium, is the final pH 6.9±0.1?

Are inverted tubes examined to ensure that they are free of air

bubbles?

5.2.4.2.3

Is A-1 Medium incubated at 35/±0.5/C for three hours, and then

incubated at 44.5/±0.2 /C for 21±2 hours?

5.2.4.2.4

Are loose-cap tubes stored in the dark at room temperature for no

longer than two weeks, or in tightly closed screw-cap tubes in the

dark at <30/C for no longer than three months?

5.2.4.2.5

Is any amount of gas detected in the inverted vial of a tube with

turbid growth considered a fecal coliform-positive test?

5.2.4.3

Azide dextrose medium (for detecting fecal streptococci in ground

water)

5.2.5

For testing 100-mL samples, is triple strength (3X) formulation in a

culture bottle prepared and then autoclaved at 121/C for 15 minutes?

5.2.5.1

Is medium final pH 7.2±0.2?

Is a 100-mL water sample added to the sterilized medium and

incubated at 35/±0.5/C?

5.2.5.2
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-54

Is the culture checked for turbidity after 24±2 hours? 5.2.5.3

If turbidity is not observed, is the culture reincubated and checked

again after a total incubation period of 48±3 hours?

Are turbid cultures confirmed as fecal streptococci by streaking a

portion of the broth onto bile esculin agar (BEA) or bile esculin

azide agar (BEAA)?

5.2.5.4

Are BEA and BEAA autoclaved at 121/C for 15 minutes? 5.2.5.5

Is the final pH 6.6±0.2 for BEA and 7.1±0.2 for BEAA?

After streaking, are plates incubated at 35/±0.5/C for 48 hours? 5.2.5.6

Are the brownish-black colonies with brown halos on BEA or BEAA

used as confirming the presence of fecal streptococci?

5.2.5.7

If required, does the laboratory perform an enterococci test by

transferring one or more fecal streptococci colonies to brain heart

infusion broth supplemented with 6.5% NaCl and incubating the

culture at 35/±0.5C for 48 hrs?

Enzyme (chromogenic/fluorogenic) substrate tests 5.3

General 5.3.1

For detecting total coliforms and E. coli in drinking water by an

enzyme substrate test, does the laboratory use one of the following: 

MMO-MUG test (Colilert), Colisure test, E*Colite test, Readycult

Coliforms 100 Presence/Absence Test, Fluorocult LMX test, or

Colitag test?

5.3.1.1

For enumerating total coliforms in source waters by an enzyme

substrate test, does the laboratory use the Colilert test?

If a laboratory uses a fermentation method to detect total coliforms in

drinking water, and the sample is total coliform-positive, does the

laboratory transfer the positive culture to the EC+MUG test to detect

E. coli, but not to any other enzyme substrate test medium in Section

5.3?

Media 5.3.1.2

Does the laboratory purchase media from a commercially available

source only, and not prepare media from basic ingredients?

5.3.1.2.1

Are media kept protected from light? 5.3.1.2.2

Is each lot of medium checked for fluorescence before use with a

365-366-nm ultraviolet light with a six watt bulb?

5.3.1.2.3
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-55

If medium exhibits faint fluorescence, is another lot used that does

not fluoresce? 

If samples plus medium exhibit color changes before incubation, is

the medium discarded and another lot of medium used?

5.3.1.2.4

Are glass and plastic bottles and test tubes checked before use with a

365-366-nm ultraviolet light source with a 6-watt bulb to ensure that

they do not fluoresce?

5.3.1.3

If they fluoresce, does the laboratory use another lot of containers

that does not fluoresce?

 If a Whirl-Pak® bag is used to incubate the Colilert or Colitag

medium or any other medium which changes to a yellow color to

indicate a positive result, is a type used that has a barrier (e.g.,

B01417) to prevent gaseous emissions to other Whirl-Pak® bags

during incubation? 

5.3.1.4

QC If a small air-type incubator is used, are samples brought to

room temperature before incubation?

5.3.1.5

If a water bath is used, is the water level above the upper level of the

medium?

5.3.1.6

For E. coli testing, are all total coliform-positive samples placed

under a UV lamp (365-366 nm) in a darkened area?

5.3.1.7

Does the laboratory refrain from using the enzyme substrate test to

confirm a presumptive total coliform-positive culture in a

fermentation broth or on a membrane filter?

5.3.1.8

Does the laboratory invalidate any sample that produces an atypical

color change (in the absence of a yellow color) and then collect, or

request that the system collect, another sample from the same

location as the original invalidated sample?

5.3.1.9

Does the laboratory use another method to test the second sample?

Is the reference comparator provided by the manufacturer discarded

by the manufacturer’s expiration date?

5.3.1.10

Criteria for specific media 5.3.2

For the Colilert test, are samples incubated at 35/±0.5/C for 24

hours?

5.3.2.1

Is a sample with a yellow color in the medium equal to or greater

than reference comparator recorded as total coliform-positive?

Is a sample with a yellow color lighter than comparator incubated for

another four hours but no longer than 28 hours total?
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-56

Is a sample with a yellow color lighter than the comparator after 28

hours of incubation recorded as total coliform-negative? 

Are coliform-positive samples that fluoresce under a UV light

marked as E. coli-positive?

For the Colilert-18 test, are samples incubated for 18 hours (up to 22

hours if the sample after 18 hours is yellow, but lighter than the

comparator)?

For enumerating total coliforms in source waters, does the laboratory

use the Colilert test, a 5- or 10-tube configuration, Quanti-Tray, or

Quanti-Tray 2000 for each sample dilution tested?

5.3.2.1.1

When dilution water is used, is it either sterile deionized or sterile

distilled water, not buffered water?

QC If the Quanti-Tray or Quanti-Tray 2000 test is used, is the

sealer checked monthly by adding a dye to the water?

5.3.2.1.2

For the Colisure test, are samples incubated at 35/±0.5/C for 24-48

hours?

5.3.2.2

If the medium changes from a yellow color to a red/magenta color, is

the sample noted as total coliform-positive?

Is a coliform-positive sample that fluoresces under a UV light

marked as E. coli-positive?

For the E*Colite test, is the sample incubated at 35/±0.5/C for 28

hours?

5.3.2.3

If the medium changes from a yellow color to a blue or blue-green

color, or a blue color in the corners of the bag, is the sample marked

as total coliform-positive?

If the medium fluoresces under a UV light, is the sample considered

as E. coli-positive?

If fluorescence is not observed, is the sample reincubated for an

additional 20 hours (for a total incubation time of 48 hours) and

checked again for fluorescence?

If the medium becomes red in color, is the sample discarded and

another sample requested?

For the Readycult Coliforms 100 Presence-Absence test, are the

contents of a snap pack added to a 100-mL sample and then

incubated at 35/±0.5/C for 24±1 hours?

5.3.2.4

If the medium changes color from a slightly yellow color to bluegreen, is the sample marked as coliform-positive?
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-57

If the medium fluoresces a bright light-blue color when subjected to

long wave UV (365-366 nm) light, is the sample marked as E. colipositive?

For the Fluorocult LMX test, is the medium added to purified water,

mixed, and the mixture then boiled to dissolve the medium

completely in the water? 

5.3.2.5

Are 100-mL aliquots transferred to 250-mL bottles and then

autoclaved for 15 minutes?

Are the autoclaved bottles cooled before adding the 100-mL water

sample?

Is the E. coli/Coliform Supplement not added to the medium?

Is the sample then incubated at 35/±0.5/C for 24±1 hours?

If the medium changes color from a slightly yellow color to bluegreen, is the sample marked as coliform-positive?

If the medium fluoresces a bright light-blue color when subjected to

long wave UV (365-366 nm) light, is the sample marked as E. colipositive?

For the Colitag test, are samples incubated at 35E±0.5EC for 24±2

hours?

5.3.2.6

If the medium changes to a yellow color, is the sample marked as

coliform-positive?

If the medium fluoresces under a UV light, is the sample marked as

E. coli-positive?

EC Medium + MUG (for detection of E. coli) 5.3.3

If EC medium + MUG is used, is a total coliform-positive culture

transferred from a presumptive tube/bottle or colony to this medium? 

5.3.3.1

Is the final pH of EC medium + MUG 6.9±0.2? 5.3.3.2

Is the medium plus sample incubated at 44.5/±0.2/C for 24±2 hours

and then tested for fluorescence?

5.3.3.4

Enterolert test (for detection of enterococci in ground water) 5.3.4

Is the medium stored in the dark at 4/-30/C until used? 5.3.4.1

Is Enterolert reagent added to a 100-mL sample and the

sample/medium incubated at 41/±0.5/C for 24-28 hours?

5.3.4.2
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-58

Is fluorescence under a UV lamp used to indicate the presence of

enterococci?

Membrane Filter (MF) methods 5.4

General 5.4.1

For source water samples (SWTR), do dilutions yield 20 to 80 total

coliform colonies or 20 to 60 fecal coliforms for at least one dilution

or volume?

5.4.1.1

QC Is at least one membrane filter and filtration unit sterility check

conducted at the beginning and the end of each filtration series by

filtering 20-30 mL of dilution water through the membrane filter and

testing for growth?

5.4.1.2

QC If the control indicates contamination, does the laboratory

reject all data from affected samples and request an immediate

resampling?

QC Does the laboratory consider a filtration series as ended when

30 minutes or more has elapsed between sample filtrations?

Are filtration funnels rinsed after each sample filtration with two or

three 20-30 mL portions of sterile rinse water to ensure that the entire

sample is rinsed off the funnel onto the filter?

5.4.1.3

Are absorbent pads saturated with at least 2 mL of broth and the

excess medium removed by “decanting” the plate?

5.4.1.4

MF method for detecting total coliforms and E. coli in drinking

water, enumerating total coliforms or fecal coliforms in source water,

and detecting E. coli in ground water

5.4.2

Media for total coliforms, fecal coliforms, and E. coli 5.4.2.1

If either M-Endo agar or broth or M-Endo agar LES is used to detect

total coliforms in drinking water or enumerating total coliforms in

source water, is either the single step or the enrichment technique

used?

5.4.2.1.1

Is denatured ethanol used in the rehydration procedure?

Is the medium prepared in a sterile flask?

Is a boiling water bath or a constantly attended hot plate with a stir

bar used to bring the medium just to the boiling point but not boiled?

Is the final for M-Endo medium pH 7.2±0.1 and the final pH for MEndo agar LES 7.2±0.2?

Is M-Endo medium or M-Endo agar LES incubated at 35°±0.5°C for

22-24 hrs? 

(5.4.2.2)
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-59

Are colonies with a metallic (golden) sheen recorded as presumptive

total coliforms?

(5.4.2.2)

If m-ColiBlue24 medium is used to detect total coliforms and E. coli

in drinking water, are the ampules of broth inverted 2-3 times to mix

contents before breaking and the contents then poured evenly over an

absorbent pad?

5.4.2.1.2

Are unopened refrigerated ampules stored in the dark?

Are unopened ampules discarded before the expiration date, or

earlier if contamination is observed?

Is the medium final pH 7.0±0.2?

Is m-ColiBlue24 incubated at 35°±0.5°C for 24 hrs? (5.4.2.2)

Are red colonies recorded as total coliforms, and blue to purple

colonies recorded as E. coli?

(5.4.2.2)

If MI medium (with or without agar) is used to detect total coliforms

and E. coli in drinking water or enumerate total coliforms in source

water, is commercially prepared presterilized bottled MI agar or

broth not autoclaved?

5.4.2.1.3

Is this presterilaized bottled agar medium melted in a boiling water

bath (or by other processes recommended by the manufacturer), and

care taken not to overheat the agar? 

Is the medium then cooled slightly and poured immediately into

sterile plates?

If dehydrated culture medium is used, is it prepared and autoclaved

according to manufacturer’s instructions?

Is this agar medium cooled before adding freshly prepared, filtersterilized cefsulodin, and then poured immediately into sterile plates?

Is the final pH of MI agar 6.95±0.20 and the final pH of MI broth

7.05±0.20?

Is MI medium incubated at 35°±0.5°C for 24±2 hrs? (5.4.2.2)

Are fluorescent colonies under UV light recorded as total coliforms,

and blue colonies under normal light recorded as E. coli?

(5.4.2.2)

If Chromocult® Coliform Agar is used to detect total coliforms and

E. coli in drinking water, is the agar medium autoclaved or

overheated?

5.4.2.1.4

Is the final pH of this medium 6.8±0.2?
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-60

If a heavy background of heterotrophic bacteria is expected, is

cefsulodin solution added to 1L of cooled (45/-50/C) medium as a

solution of 10 mg cefsulodin dissolved in 2-mL deionized or distilled

water?

Is Chromocult incubated at 36°±1°C for 24±1 hrs? (5.4.2.2)

Are salmon to red colonies recorded as total coliforms, and dark-blue

to violet colonies recorded as E. coli?

(5.4.2.2)

If Coliscan® is used to detect total coliforms and E. coli in drinking

water or enumerate total coliforms in source water, is the

manufacturer’s protocol for reconstitution and antibiotic addition

followed?

5.4.2.1.5

Is the antibiotic, cefsulodin, overheated?

Is the final pH of Coliscan agar 7.00±0.20?

Is Coliscan incubated at 32°-37°C for 24-28 hrs? (5.4.2.2)

Are pink-magenta colonies recorded as total coliforms, and purpleblue colonies recorded as E. coli?

(5.4.2.2)

If m-FC broth, with or without agar, is used to enumerate fecal

coliforms in source water, is the medium autoclaved? 

5.4.2.1.6

Is m-FC broth just brought to the boiling point?

Is the final pH of m-FC medium 7.4±0.2?

Is m-FC broth incubated at 44.5°±0.2°C for 24±2 hrs? (5.4.2.2)

Are blue colonies recorded as fecal coliforms? (5.4.2.2)

Is the prepared medium refrigerated when stored and brought to

room temperature before use?

5.4.2.1.7

Are petri dishes containing medium stored in a plastic bag or tightly

closed container, and used within 2 weeks?

Are plates with laboratory-prepared broth medium discarded after 96

hours, poured agar plates after 2 weeks, and ampuled broth discarded

before the manufacturer’s expiration date?

Are the date and time of medium preparation recorded?

For invalidation of a total coliform-negative drinking water sample,

are all samples resulting in confluent growth or TNTC growth

invalidated unless at least one total coliform colony is detected?

5.4.2.3
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-61

If no coliforms are detected, is the sample recorded as “confluent

growth” or “TNTC” and an additional sample requested from the

same sampling site?

Does the laboratory perform a verification test on the total coliformnegative culture before invalidation?

If the verification test is total coliform-positive, does the laboratory

report the sample as total coliform-positive?

If the verification test is total coliform-negative, is the sample

invalidated?

For invalidation of source water samples (SWTR), where coliform

density must be determined, does the laboratory invalidate any

sample that results in confluent growth or TNTC, even when total

coliform or fecal coliform colonies are present?

5.4.2.4

For drinking water samples on M-Endo type media, are all sheen

colonies, up to a maximum of five, verified by using either LB or

LTB and then 2% BGLBB or, alternatively, by using a cytochrome

oxidase and $-galactosidase procedure?

5.4.2.5

If no sheen colonies are observed, are up to five red questionable

sheen colonies and/or red non-sheen colonies representing different

morphological types verified?

For drinking water samples, are total coliform-positive colonies

tested for E. coli or fecal coliforms?

5.4.2.6

When EC Medium or EC + MUG is used, are colonies transferred by

employing one of the options specified by the Total Coliform Rule?

When the swab technique is used, is a single swab used to inoculate a

presumptive total coliform-positive sample into EC or EC+MUG

first, LTB second, and BGLBB third?

For source water samples, are the initial total coliform counts

adjusted based upon verified data?

5.4.2.7

QC For source water samples when two or more analysts are

available, does each analyst count the total coliform or fecal coliform

colonies on the same membrane monthly and do the counts agree

within 10%?

5.4.2.8

Nutrient Agar + MUG Test (for detection of E. coli in drinking

water or ground water)

5.4.3

Is the medium autoclaved at 121/C for 15 minutes? 5.4.3.1

Is the final MUG concentration 100 :g/L?

Is the final pH of NA + MUG 6.8±0.2?
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-62

QC Are positive and negative culture controls tested as stated in

5.1.6.4?

5.4.3.2

QC Are culture controls filtered or spot-inoculated onto a

membrane filter on M-Endo broth or agar, or M-Endo agar LES, and

incubated at 35/±0.5/C for 24 hours?

QC Is the filter then transferred to NA + MUG and incubated at 

35/±0.5/C for another four hours?

QC Are these results read and recorded?

Is the membrane filter containing total coliform colonies transferred

to the surface of the Nutrient Agar + MUG medium?

5.4.3.3

Is the presence of each sheen colony marked on the petri dish lid

with permanent marker, and the lid and base marked to realign the lid

when removed?

For the total coliform verification test, is a portion of each colony

transferred with needle before the MF transfer or after the four-hour

NA + MUG incubation time?

Alternatively, is the membrane filter surface swabbed with a sterile

cotton swab after the four-hour incubation time on NA + MUG and

then transferred to a total coliform verification test?

Is the inoculated NA + MUG medium incubated at 35/±0.5/C for

four hours?

5.4.3.4

Is fluorescence checked by using a UV lamp (365-366 nm) with a

six-watt bulb in a darkened room and any fluorescence in the halo

around a sheen colony considered positive for E. coli?

5.4.3.5

MF method for detecting enterococci/fecal streptococci in ground

water

5.4.4

Media 5.4.4.1

When mE agar is used for the detection of enterococci, is basal mE

agar prepared, autoclaved, and cooled before the addition of

nalidixic acid (or its sodium salt) and triphenyl tetrazolium chloride,

both of which are added separately to the medium and mixed?

5.4.4.1.1

Is the final pH of mE agar 7.1±0.2?

When m-Enterococcus agar is used for the detection of fecal

streptococci (not enterococci), is the medium heated, not autoclaved,

to dissolve the ingredients?

5.4.4.1.2

Is the final pH of m-Enterococcus agar 7.2±0.2?
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-63

When mEI agar is used for the detection of enterococci, is 0.75g

indoxyl-$-D-glucoside added to 1L basal mE agar and then prepared

according to 5.4.4.1.1 except that only 0.02 g/L triphenyl tetrazolium

chloride is added?

5.4.4.1.3

Is the final pH of mEI agar 7.1±0.2?

Is a 100-mL sample filtered and the membrane placed on one of the

agar media previously listed?

5.4.4.2

If m-Enterococcus agar is used, are the plates incubated in an

inverted position at 35/±0.5/C for 48 hours? 

5.4.4.3

Using magnification and a fluorescent lamp, are all light and dark red

colonies counted as fecal streptococci?

If mE agar is used, are the plates incubated in an inverted position for

48 hours at 41/±0.5/C?

5.4.4.4

Is the membrane filter then transferred to EIA medium and incubated

at 41/±0.5/C for 20-30 minutes?

Using magnification and a fluorescent lamp, are all pink to red

colonies on mE agar with a black or reddish brown precipitate on the

underside of the filter on EIA agar counted as enterococci?

If mEI agar is used, are plates incubated in an inverted position for

24 hours at 41/±0.5/C?

5.4.4.5

Using magnification and a fluorescent lamp, is the plate examined,

top and bottom, for colonies with a blue halo, and any colony with a

blue halo (regardless of colony color) considered as positive for

enterococci?

Heterotrophic Plate Count (for enumerating heterotrophic bacteria

in drinking water)

5.5

Does the laboratory use the Pour Plate Method or the SimPlate

Method for enumerating heterotrophic bacteria in drinking water and

for testing reagent grade water?

5.5.1

For systems granted a variance from the TCR’s maximum

contaminant level, does the laboratory use R2A medium with a

method in Standard Methods, Section 9215 for enumerating

heterotrophic bacteria in drinking water?

Media 5.5.2

Is the final pH recorded for plate count agar pH 7.0±0.2, R2A agar

7.2±0.2, and SimPlate 7.2±0.2?

For the Pour Plate Method, is melted agar tempered at 44/-46/C in a

water bath and maintained no more than 3 hours before pouring?

5.5.3
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-64

Is this sterile medium melted only once?

For the Spread Plate Method, is 15 mL of R2A medium (or other

medium) poured into a sterile petri dish and allowed to solidify?

5.5.4

Is refrigerated medium in bottles or screw-capped tubes stored for no

longer than six months, or in petri dishes for no longer than 2 weeks

(one week for prepared petri dishes with R2A medium)?

5.5.5

For countable plates of most potable water samples, are 1.0 mL

and/or 0.1 mL volumes of the undiluted sample plated? 

5.5.6

Are at least duplicate plates prepared per dilution tested?

For the Pour Plate Method, is the sample pipetted aseptically onto

the bottom of a sterile petri dish and then at least 10-12 mL tempered

melted agar added?

5.5.7

Is the sample and melted agar mixed, avoiding spillage?

After the agar plates have solidified on a level surface, are they

inverted and incubated at 35/±0.5/C for 48±3 hours?

Are plates stacked no more than four high and arranged in the

incubator to allow proper air circulation and to maintain a uniform

incubation temperature?

Does the laboratory ensure that incubator does not have excess

humidity and that the plates do not lose more than 15% by weight

during the 48 hours of incubation?

For the Spread Plate Method, is 0.1 or 0.5 mL of the sample (or

dilution) pipetted onto the surface of the predried agar in the plate

and then spread over the entire surface using a sterile bent glass rod?

5.5.8

Is the inoculum absorbed completely before incubating?

Are the plates incubated in an inverted position at 20/-28/C for 5-7

days?

For the Membrane Filter Technique, does the filtered volume yield

between 20-200 colonies?

5.5.9

Is the filter transferred to a petri dish containing 5 mL solidified R2A

medium and then incubated at 20/-28/C for 5-7 days?

Are plates with loose-fitting lids placed in a plastic box with a closefitting lid and moistened paper towels, and rewetted as necessary?

Are colonies counted using a stereoscopic microscope at 10-15X

magnification?

SimPlate Method 5.5.10
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-65

For a single sample Unit Dose, is a 10-mL test sample added to a test

tube containing dehydrated SimPlate medium and then poured onto

the center of a plate containing 84 small wells?

5.5.10.1

Alternatively, is 9-mL of sterile diluent added to the test tube

containing the dehydrated medium, followed by a 1-mL sample, and

the medium plus sample then poured onto the center of a plate

containing 84 small wells?

Is this mixture distributed evenly to the 84 wells and is the excess

liquid drained into the absorbent pad on the plate?

Is the plate inverted and incubated at 35/±0.5/C for 45-72 hours?

Is bacterial density determined by counting the number of wells that

fluoresce under a 365-366-nm UV light, and converting this value to

a Most Probable Number/mL using the manufacturer’s Unit Dose

MPN table?

If a 10-mL sample is used, is the Unit Dose MPN/mL read directly

or, if a 1-mL sample is used, is the MPN/mL value corrected by

multiplying it by 10?

For the Multiple Dose for 10 samples of 1 mL each, is a 100-mL

sterile diluent added to the dehydrated SimPlate medium and shaken

to dissolve?

5.5.10.2

Is a 1.0-mL test sample then pipetted to the center of a plate,

followed by 9 mL of the reconstituted medium?

Is the plate then gently swirled to mix and distribute the sample and

medium mixture evenly to the 84 wells, with the excess liquid then

being drained into the absorbent pad on the plate?

Is the plate inverted and incubated at 35/±0.5/C for 45-72 hours?

Is bacterial density determined by counting the number of wells that

fluoresce under a 365-366-nm UV light, and converting this value to

a Most Probable Number/mL using the manufacturer’s Multi-Dose

MPN table?

If sample dilutions were made during sample preparation, is the

MPN/mL value multiplied by the dilution factor?

For the Pour Plate and Spread Plate Techniques, are colonies

counted manually using a dark field colony counter?

5.5.11

Are only plates having 30 to 300 colonies counted, except for plates

inoculated with 1.0 mL of undiluted sample where counts of less than

30 are acceptable?
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-66

QC Is each batch or flask of agar checked for sterility by pouring a

final control plate?

5.5.12

QC Does the laboratory reject data if the control is contaminated?

Coliphage (Draft Method 1601 and 1602 for the GWR) 5.6

EPA Method 1601: Male-specific (F+) and Somatic Coliphage in

Water by Two-Step Enrichment

5.6.1

Is the 100-mL (or 1-L) water sample supplemented with magnesium

chloride, log-phase host bacteria, and tryptic soy broth (TSB) as an

enrichment step for coliphage?

After incubation overnight, are samples spotted onto a lawn of host

bacteria, incubated, and then examined for circular lysis zones?

Media 5.6.1.1

For antibiotic stocks, are antibiotics always added to the medium

after the medium has been autoclaved?

5.6.1.1.1

Are antibiotic stocks stored frozen at -20/C for no longer than one

year?

Are antibiotic stocks thawed at room temperature or rapidly in a

water bath up to 37/C and mixed well before using?

Is 10X tryptic soy broth (TSB) stored at 1/-5/C until used? 5.6.1.1.2

Are 1.5% tryptic soy agar (TSA) plates after antibiotic

supplementation and solidification stored inverted at 1/-5/C for no

longer than two weeks, if not used immediately?

5.6.1.1.3

Are 0.7% TSA top agar tubes with appropriate antibiotics, dispensed

as 5-mL per sterile 10-mL tube, labeled, and kept at 45/-48/C until

used?

5.6.1.1.4

Are these tubes used the day of preparation?

For spot plates that develop condensation during storage, are plates

incubated for approximately 10 minutes to reduce condensation prior

to inoculation?

5.6.1.1.5

Are spot plates used that day or stored at 1/-5/C for up to four days?

Coliphage stock 5.6.1.2

Are MS2 (ATCC#15597-B1 male-specific) and phi-X 174 stock

coliphage (ATCC#13706-B1, somatic) stored at 2/-8/C for up to five

years?

5.6.1.2.1
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-67

Does analysis of raw sewage filtrate begin within 24 hours of

collection?

5.6.1.2.2

Is at least 10 mL of filtered sewage obtained? 5.6.1.2.4

If the filtrate is stored more than 24 hours, is it re-titered before use? 5.6.1.2.5

Host bacteria stock cultures 5.6.1.3

After preparation, are host bacteria stock cultures held at a

temperature between -20/C and -70/C?

5.6.1.3.1

Are bacteria stored no longer than two months at -20/C or no longer

than one year at -70/C?

Are prepared overnight host bacteria stock cultures chilled on wet ice

or at 1/-5/C until ready to use?

5.6.1.3.2

After preparation, are log-phase host bacteria stock cultures chilled

on wet ice or at 1/-5/C to slow replication until ready for use?

5.6.1.3.3

When stored, are these suspensions stored no more than 48 hours?

Are remaining bacterial host cultures stored at 1/-5/C overnight to

inoculate flasks for the preparation of new log-phase bacterial hosts?

General QC 5.6.1.4

Initial demonstration of capability (IDC) 5.6.1.4.1

QC Did the laboratory demonstrated ability to generate acceptable

performance with this method by performing an IDC test before

analyzing any field samples?

QC Did the IDC test consist of ten reagent water samples spiked

with enumerated sewage filtrate or equivalent at 1-2 PFU mL per

sample for every sample for each coliphage type used, according to

the IDC Table?

QC Were these IDC tests accompanied by a method blank for each

coliphage type used?

Method Blanks 5.6.1.4.2

QC Is a method blank (reagent water sample containing no

coliphage) analyzed to demonstrate freedom from contamination?

For each coliphage type used, is a sterile reagent water sample

prepared and analyzed using the same procedure used for analysis of

field and QC samples?

QC Is at least one method blank analyzed for every spot plate used

for field samples?
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-68

Positive Controls 5.6.1.4.3

QC Are positive controls analyzed to ensure that stock coliphage

suspensions, host bacterial cultures, and growth media are

performing properly?

QC For each coliphage type used, is a 100 mL reagent water

sample spiked with 20 PFU from sewage filtrate or 60 PFU from a

pure coliphage stock culture?

QC Is one positive control analyzed for every spot plate used for

field samples?

QC If multiple spot plates are inoculated with samples on the same

day, is a single enriched positive control sample used to inoculate

multiple spot plates that day?

Matrix spikes (MS) 5.6.1.4.4

QC Is method performance assessed by analyzing one set of MS

samples for each coliphage type used when samples are first received

from a new ground water source?

QC For each coliphage type analyzed, are three field samples

spiked with 1-2 PFU, with at least one out of the three MS samples

being positive for each coliphage type?

QC Is one set of MS samples analyzed on an ongoing basis after

every 20 field sample for each ground water source? th

QC Are these MS samples collected at the same time as routine

field samples?

QC Are these samples spiked in “bulk” at the concentrations

indicated in the MS and ODC Sample Spiking Table?

Ongoing demonstration of capability (ODC) 5.6.1.4.5

QC Does the laboratory demonstrate that the analytical system is in

control on an ongoing basis through analysis of ODC samples?

QC Are three reagent water samples spiked with 1-2 PFU for each

coliphage type used?

QC Are ODC test samples analyzed exactly like field samples?

QC Is a minimum of one out of three ODC samples positive for

each coliphage type used?

QC Is one set of ODC samples analyzed after every 20 field and

MS samples or one per week, whichever occurs more frequently?
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-69

QC Are samples spiked in “bulk” at the concentrations indicated in

the MS and ODC Sample Spiking Table?

Performance studies 5.6.1.4.6

QC Does the laboratory periodically analyze external QC samples

when available?

QC Does the laboratory participate in available interlaboratory

performance studies?

QC Does the laboratory review results, correct unsatisfactory

performance, and record corrective actions?

EPA Method 1602: Male-specific (F+) and Somatic Coliphage in

Water by Single Agar Layer (SAL) Procedure

5.6.2

Is the 100-mL ground water sample supplemented with magnesium

chloride and host bacteria, and then added to 100-mL of doublestrength molten tryptic soy agar containing the appropriate

antibiotic?

Is the sample thoroughly mixed and the total volume then poured

into five to ten plates?

After overnight incubation, are circular lysis zones recorded as

coliphage?

Media 5.6.2.1

For antibiotic stocks, are antibiotics always added to the medium

after the medium has been autoclaved?

Are antibiotic stocks stored frozen at -20/C for up to one year?

Are antibiotic stocks thawed at room temperature or rapidly in a

water bath up to 37/C and then mixed well before using?

Is 10X TSB stored at 1/-5/C until used?

Are 1.5% TSA plates, after antibiotic supplementation and

solidification, stored inverted at 1/-5/C for no longer than two weeks,

if not used immediately?

Are 0.7% TSA top agar tubes with appropriate antibiotics, dispensed

as 5-mL per sterile 10-mL tube, labeled, and kept at 45/-48/C until

used?

Are these tubes used the day they are prepared?

Are spot plates that develop condensation during storage, incubated

for approximately 10 minutes to reduce condensation prior to

inoculation?
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-70

Are these plates used that day or stored at 1/-5/C for up to four days?

Is 2X TSA, with appropriate antibiotics, kept molten at 45/-48/C in a

water bath?

5.6.2.1.2

Is this agar used only on the day of preparation?

Coliphage Stock 5.6.2.2

Are MS2 (ATCC#15597-B1 male-specific) and phi-X 174 stock

coliphage (ATCC#13706-B1, somatic) stored at 2/-8/C for up to five

years?

Does analysis of raw sewage filtrate begin within 24 hours of

collection?

Is at least 10 mL of filtered sewage obtained?

If the filtrate is stored more than 24 hours, is it re-titered before use?

Host bacteria stock cultures 5.6.2.3

After preparation, are host bacteria stock cultures held at a

temperature between -20/C and -70/C?

Are bacteria stored no longer than two months at -20/C, or no longer

than one year at -70/C?

After preparation, are overnight host bacteria stock cultures chilled

on wet ice or at 1/-5/C until ready for use?

After preparation, are log-phase host bacteria stock cultures chilled

on wet ice or at 1/-5/C to slow replication until ready to use?

When stored, are these suspensions stored no longer than 48 hours?

Are remaining bacterial host cultures stored at 1/-5/C overnight to

inoculate flasks for the preparation of new log-phase bacterial hosts?

General QC 5.6.2.4

Initial precision and recovery (IPR) 5.6.2.4.1

QC Has the laboratory demonstrated the ability to perform this

method acceptably by performing an IPR test before analyzing any

field samples?

QC Does the IPR Test consist of four reagent water samples tested

for each coliphage type used, spiked with enumerated sewage filtrate

or equivalent to yield a target spike concentration of 80 PFU per

sample?
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-71

QC Does the relative standard deviation of the recovery (RSD), and

the average percent recovery (0) based on all four sample results for

each coliphage type used, meet the acceptance criteria in the QC

Acceptance Criteria table?

Method blanks 5.6.2.4.2

QC Is one method blank (reagent water sample containing no

coliphage) analyzed with each analytical batch to demonstrate

freedom from contamination?

QC For each coliphage type used, are sterile reagent water samples

prepared and analyzed using the same procedures used for analysis

of the field and QC samples?

QC Is an analytical batch defined as all samples analyzed during a

single day, up to a maximum of 20 samples (field samples and matrix

spike samples) per coliphage type used?

Matrix spikes (MS) 5.6.2.4.3

QC Is method performance assessed by analyzing one set of MS

samples for each coliphage type used when samples are first received

from a new ground water source?

QC Is one set of MS samples routinely analyzed after every 20th

field sample for each ground water source?

QC If the recovery for coliphage falls outside its limit (see the QC

Acceptance Criteria table), is method performance considered

unacceptable?

QC If OPR results are not within control limits, is the problem

identified and corrected and the data qualified?

QC Does the laboratory maintain a control chart on recovery and

update it on a regular basis?

Ongoing precision and recovery (OPR) 5.6.2.4.4

QC Does the laboratory demonstrate acceptable performance

through analysis of an OPR sample on an ongoing basis?

QC For each coliphage type used, is a reagent water sample spiked

with approximately 80 PFU?

QC Is the OPR sample analyzed exactly like a field sample?

QC Is one OPR sample analyzed for each analytical batch?

QC Does the laboratory compare the OPR percent recovery (R)

with the corresponding limits in the QC Acceptance Criteria table?
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-72

QC If R falls outside the range for recovery, is analysis stopped

until the problem is identified, corrected, and another OPR test is

successfully performed?

Performance studies 5.6.2.4.5

QC Does the laboratory periodically analyze an external QC sample

when available?

QC Does the laboratory participate in available interlaboratory

performance studies?

QC Does the laboratory review these results, correct unsatisfactory

performance, and record corrective actions?

6. SAMPLE COLLECTION, HANDLING, AND PRESERVATION

Sample Collector 6.1

Is the sample collector trained in aseptic sampling procedures and, if

required, approved by the appropriate regulatory authority or its

designated representative?

Sampling 6.2

Are the drinking water samples collected under the Total Coliform

Rule representative of the water distribution system?

6.2.1

Are the water taps used for sampling free of aerators, strainers, hose

attachments, mixing type faucets, and purification devices?

Are only cold water taps used?

Are service lines cleared before sampling by maintaining a steady

water flow for at least 2 minutes or until a steady water temperature

is reached?

Is at least a 100-mL sample volume collected, allowing at least a 1-

inch air space in the container to facilitate mixing of the sample by

shaking?

Is a sample information form completed immediately after sample

collection?

If a sample bottle is filled too full to allow for proper mixing, is the

entire sample poured into a larger sterile container and mixed before

proceeding with the analysis?

For the SWTR, are the source water samples representative of the

source of supply and collected not too far from the intake point, but

at a reasonable distance from the bank or shore?

6.2.2

Is the sample volume sufficient to perform all the tests required?
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-73

For the analysis of coliphage, E. coli, or enterococci under the GWR,

is at least a 100-mL sample volume collected?

6.2.3

6.2.4

Sample Icing 6.3

For drinking water bacterial samples, is the sampler encouraged to

hold samples at <10/C during transit to the laboratory?

6.3.1

For source water bacterial samples, are samples held at <10/C during

transit to the laboratory?

Does the laboratory reject samples that have been frozen?

For coliphage analysis under the GWR, are samples shipped at

<10/C, stored at 1/-5/C, and not frozen?

6.3.2

QC For SWTR samples and coliphage samples, does the laboratory

record sample temperature upon receipt?

6.3.3

QC Does the laboratory flag samples that have a temperature upon

receipt of >10/C, whether iced or not, unless the time since the

sample collection is less than two hours?

Sample Holding/Travel Time 6.4

For the analysis of total coliforms in drinking water, does the time

between sample collection and placement of the sample in the

incubator not exceed 30 hours?

6.4.1

Are all samples analyzed on the day of receipt?

Are samples received late in the day refrigerated overnight only if

analysis can begin within 30 hours of collection?

For total coliforms and fecal coliforms in surface water sources, and

for heterotrophic bacteria in drinking water, is the time from sample

collection to placement in the incubator less than eight hours?

6.4.2

For coliphage analysis, is the time from sample collection to

placement of sample in the incubator less than 48 hours?

6.4.3

For coliphage analysis, is the time from sewage sample collection to

analysis of QC spiking suspension less than 24 hours, unless retitered and the titer has not decreased by more than 50%?

If the titer has not decreased by more than 50%, is the sample stored

no longer than 72 hours?

For E. coli and enterococci analysis under the GWR, is the time

between sample collection and the placement of sample in the

incubator less than 30 hours?

6.4.4

Sample Information Form 6.5
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-74

After collection, does the sampler enter the following information, in

indelible ink, on sample information form?

S Name of system (PWSS identification number if available)

S Sample identification (if any)

S Sample site location

S Sample type (e.g., a routine distribution, repeat, raw or process, or

other special purpose)

S Date and time of collection

S Analysis requested

S Disinfectant residual

S Name of sampler

S Any remarks 

Chain-of-Custody 6.6

Are applicable State regulations pertaining to chain-of-custody

followed by sample collectors and the laboratory?

7. QUALITY ASSURANCE

Does the laboratory have a written QA Plan prepared and available

for inspection?

7.1

Does the laboratory follow the written QA Plan?

Does the laboratory have a Standard Operating Procedure available

for review pertaining to its own calibration of equipment or supplies?

Does the laboratory successfully analyze at least one set of PT

samples once every 12 months for each method for which it is

certified?

7.2

For methods used to test the presence or absence of an organism in a

sample, does the laboratory analyze each PT sample set using a

single analytical method only? 

8. RECORDS AND DATA REPORTING

Legal Defensibility 8.1

Are compliance monitoring data being maintained by the laboratory

both thorough and accurate, and thus legally defensible?

Does the laboratory’s QA plan and/or SOPs describe the policies and

procedures used by the facility for record retention and storage?

If samples are expected to become part of legal action, does the

laboratory follow chain-of-custody procedures?

Maintenance of Records 8.2

Does the public water system maintain records of microbiological

analyses for five years? 
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-75

Does the laboratory maintain easily accessible records for five years

or until the next certification data audit is completed, whichever is

longer?

Does the laboratory notify the client water system before disposing

of records so they may request copies if needed?

Does the laboratory backup all electronic data by protected tape,

disk, or hard copy?

When the laboratory changes its computer hardware or software, are

provisions in place for transferring old data to the new system so that

data remain retrievable within the specified time frames?

Sampling Records 8.3

Are all data recorded in ink, with any changes lined through such that

the original entry is visible?

Are changes initialed and dated?

Does the laboratory have the following sample information readily

available?

S Date and time of sample receipt by the laboratory

S Name of the laboratory person receiving the sample

S Information on any deficiency in the condition of the sample

8.3.1-4

Are samples invalidated for the following reasons?

S Time between sample collection and receipt by laboratory

exceeded

S Presence of disinfectant in sample noticed, e.g., odor

S Evidence of freezing

S Use of a container not approved by the laboratory for the purpose

intended

S Insufficient sample volume, e.g., <100 mL

S Presence of interfering contaminants noticed, e.g., hydrocarbons,

cleansers, heavy metals, etc.

S Sample temperature exceeding the maximum allowable

8.3.4

Analytical Records 8.4

Are all recorded data in ink with any changes lined through such that

original entry is visible?

Are these changes initialed and dated?
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-76

Are the following readily available?

S Laboratory sample identification information

S Information concerning date and time analysis begins

S Name of the laboratory and a signature or initials of the person(s)

performing analysis

S Information concerning the analytical technique or method used 

S Information concerning all items marked “QC”

S Results of the analyses 

8.4.1-6

Preventive Maintenance 8.5

Does the laboratory maintain preventive maintenance and repair

records for all instruments and equipment?

Are these records kept for five years in a manner that allows for easy

inspection?

9. ACTION RESPONSE TO LABORATORY RESULTS

Testing Total Coliform-Positive Cultures 9.1

For the Total Coliform Rule, does the laboratory test all total

coliform-positive cultures for the presence of either fecal coliforms

or E. coli?

Notification of Positive Results 9.2

For Total Coliform Rule, does the laboratory promptly notify the

proper authority of a positive total coliform, fecal coliform, or E. coli

result, so that appropriate follow-up actions can be conducted?

9.2.1

For the Total Coliform Rule, if a sample is fecal coliform- or E. colipositive, does the system notify the State as soon as it is notified of

the test result, i.e., at the end of that day or, if the State office is

closed, by the end of the next business day?

9.2.2

Does the laboratory base a total coliform-positive result on the

confirmed phase if the Multiple Tube Fermentation Technique or

Presence-Absence Coliform Test is used, or the verified test for the

Membrane Filtration Technique if M-Endo medium or M-Endo LES

agar is used?

9.2.3

If a presumptive total coliform-positive culture does not

confirm/verify as such, but is found to be fecal coliform or E. colipositive, is the sample considered total coliform-positive and fecal

coliform/E. coli-positive?

Notification of Total Coliform Interference 9.3
MICRO CHECKLISTS

Element Number Yes No NA Comments

V-77

For the Total Coliform Rule, does the laboratory promptly notify the

proper authority when results indicate non-coliforms may have

interfered with total coliform analysis?
VI-1

Chapter VI

Critical Elements for Radiochemistry

1. Personnel

1.1 Laboratory Supervisor

At a minimum, the laboratory supervisor should have a bachelor’s degree in chemistry or an equivalent degree, and one

year of experience in the measurement of radioactive analytes in drinking water. The laboratory supervisor is required to

have a working knowledge of Quality Assurance (QA) and Quality Control (QC) principles and apply it to all radiochemical

practices and procedures conducted in his or her laboratory. The laboratory supervisor is responsible for ensuring that all

laboratory personnel have demonstrated their ability to satisfactorily perform the analyses to which they are assigned and

that all data reported by the laboratory meet the required quality assurance criteria.

1.2 Laboratory Analyst

At a minimum, the laboratory analyst should have a bachelor’s degree in chemistry or an equivalent degree, and one year

of experience in the measurement of drinking water for radiochemical parameters. If the analyst is responsible for the

operation of analytical instrumentation, he or she is required to have completed specialized training offered by the

manufacturer, another qualified training facility, or served a period of apprenticeship under an experienced analyst. The

duration of this apprenticeship is proportional to the sophistication of the instrument. Completion of this apprenticeship

period for instrumentation should be documented and maintained in a training file.

1.3 Technician

At a minimum, the laboratory technician should have a high school diploma or its equivalent. Prior to working

independently on drinking water samples, technicians should have at least 6 months bench experience in drinking water

analyses, and have completed method training programs in the methods they will use on a daily basis. Their completed

method training should be recorded in a training file. 

1.4 Sampling Personnel

If the laboratory also conducts field sampling activities, the laboratory personnel who collect samples should have training

in the proper collection technique for all the types of samples they collect. Their technique will be reviewed by experienced

sampling personnel prior to independently collecting drinking water compliance monitoring samples. Both training and

technique reviews should be documented in sampling personnel’s training files.

1.5 Initial and Ongoing Demonstrations of Proficiency for Analysts and Technicians

Before beginning the analysis of compliance samples, all analysts, and technicians must demonstrate the ability to conduct

their measurements with acceptable accuracy, precision, and freedom from interferences and demonstrate this proficiency

annually. These demonstrations of proficiency should be recorded in each analyst’s and technician’s training file. The

bench sheets and instrument printouts made during these demonstrations of proficiency should be retained and be available

for inspection. 

Demonstrations of Proficiency must be done, by conducting an MDL study as described in 40 CFR part 136, Appendix

B, or by the following alternate procedure. The analyst should prepare and measure a sample set of at least four reagent

blanks and four laboratory fortified blanks that have the radioanalyte of interest added to them at a known concentration

for the relevant method. To demonstrate proficiency at quantitation levels appropriate for drinking water samples, the

activity level added to the laboratory fortified blanks should be between the radioanalyte’s MCL and its required detection

limit. To be deemed an acceptable demonstration of proficiency, the mean recoveries and the standard deviation of the

recoveries of the replicate measurements should be consistent with the requirements for accuracy and precision described

in Section 7.7, and reagent blank measurements must have a mean result below the detection limit for each analyte

measured with the method.

Continuing Demonstrations of Proficiencies may be done by repeating the studies described above annually, or can consist

of documenting batch QC samples the analyst has processed during the year since the last Demonstration of Proficiency.

The data for at least four reagent blanks in different batches and measured on non-consecutive days is used to assess

sensitivity, and data from four laboratory fortified blanks not in the same batch and in sample batches measured on nonconsecutive days, is used to assess accuracy and precision. The amount of the analyte added to the sample preparation

batch laboratory fortified blanks should follow the guidance for the studies described in the preceding paragraph, and use

the acceptance criteria found for them in Section 7.7.
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All Initial and Ongoing Demonstrations of Proficiency should be recorded in a training file specific for each analyst and

technician. Records related to methods training and external training relevant to laboratory operations should also be

documented in the training file.

1.6 Data Produced by Analysts and Technicians in Training

Data produced by analysts and technicians who have not completed their training, or do not have a current demonstration

of proficiency on record, as well as instrument operators still in the process of obtaining the required training or

experience, are acceptable only when reviewed and validated by a fully qualified analyst, or the laboratory supervisor.

They should provide a permanent record of their review and the data’s acceptability by placing their signature and date in

ink on any bench sheets, calculation sheets, or reports generated by staff who lack sufficient training or a current

demonstration of proficiency.

1.7 Waiver of Academic Training

The certification officer may waive the need for specified academic training, on a case-by-case basis, for highly experienced

analysts.

2. Laboratory Facilities

2.1 General 

The analysis of compliance samples must be conducted in a laboratory where the security and integrity of the samples and

the data can be maintained. The laboratory facilities should be clean, have adequate temperature and humidity control and

adequate lighting at the bench top. The laboratory must have provisions for the proper disposal of chemical and radiological

wastes, including liquid scintillation cocktail mixtures. Since many radiochemistry procedures can involve routine use

of strong mineral acids, sulfides and/or organic liquids, the appropriate type of exhaust hoods are required. These hoods

must be able to provide the analyst or technician with a sufficient hood velocity to remove hazardous fumes from the

working area, and adequate bench space inside them to perform sample preparation activities safely and without concern

of cross-contaminating samples. Hood velocities should be checked annually.

Analytical and sample storage areas must be isolated from all potential sources of contamination. Any sample having an

emission rate in excess of 0.5 mrem/hr must be stored in a secured location away from drinking water samples. There

should be sufficient storage space for chemicals, glassware, and portable equipment; sufficient floor and bench space for

stationary equipment; and areas for cleaning materials.

2.2 Instrumentation

Instruments should be properly grounded. Counting instruments must be located in a room other than one in which samples

and standards are being prepared, or where other types of chemical analyses are being performed. An uninterrupted power

supply (UPS) should be available for all radiation measurement instruments and their associated computers used for data

acquisition and reduction.

2.3 Preparation of Standards

In areas where radioactive standards are being prepared, care should be taken to minimize contamination of surfaces, other

samples, and personnel. Bench surfaces of an impervious material covered with adsorbent paper, or plastic or fiberglass

trays lined with adsorbent paper are acceptable.

3. Laboratory Equipment and Instrumentation

The laboratory is required to have the equipment, supplies and instrumentation necessary to perform the approved methods

for which it is certified.

3.1 Radiation Counting Instruments

Specific types of radiation counting systems are necessary to conduct the measurements as described in the regulations.

All measurement instruments used to measure radioactivity in compliance monitoring samples must have a documented

history of stability with regard to their ability to make measurements accurately, with acceptable precision, and sufficiently

free from interfering background radiation so the detection limits can be met. The following subsections specify

instrumentation that many of the approved methods use for their measurements, when calibration or recalibration is

required, and stability checks that are documented and recorded to monitor the laboratories ability to make measurements

reliably.
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3.1.1 Liquid Scintillation Counting (LSC) system: An LSC system is essential if the laboratory is to be certified

for the measurement of tritium in drinking water samples. It is recommended that the liquid scintillation system

have spectral analysis capabilities to establish proper regions of energy discrimination. The system should have

adequate sensitivity to meet the detection limit of the methods.

Prior to their use to measure compliance monitoring samples, LSCs are initially calibrated for efficiency using

NIST-traceable sources, and the background measured. Afterwards, an efficiency check source and an instrument

blank to check backgrounds are measured on each day of use prior to any sample measurements to verify the

instrument is ready to measure samples. These prior-to-use efficiency check source and the instrument blank

measurements are recorded and used to establish control limits for the instrument’s performance. During periods

when LSCs are idle, the efficiency calibration and background should be checked weekly to confirm the LSC’s

ready status for sample measurements. 

Warning limits and control limits should be calculated from the efficiency and background checks. If any

efficiency check source measurement or instrument blank measurement exceeds its control limits, the LSC is

placed out of service until the reason for the out of control measurement can be identified and corrected.

3.1.2 Gas-flow proportional counting system: A gas-flow proportional counting system may be used for the

measurement of gross alpha and gross beta activities, radium-226, radium-228, strontium-89, strontium-90,

radioactive cesium, iodine-131, and uranium as described in the references in 40 CFR part 141.25(a). The detector

may be either a "windowless" (internal proportional counter) or a "thin window" type. A combination of shielding

and a cosmic (guard) detector operated in anticoincidence with the main detector should be used to achieve low

background beta-counting capability. The alpha and beta background count of the system should be low enough

so that the sensitivity of the radioanalysis of water samples will meet the requirement of 40 CFR part 141.25(c)

with reasonable counting time (not more than 1,000 minutes).

Solids absorption curves that relate the attenuation of alpha or beta particle emission with increasing sample mass

in the planchet are essentially initial calibrations for specific analytes measured with gas proportional counters.

They are either remeasured and a new curve generated, or the current accuracy of the original curve reverified

on a regular basis. EPA requires this be done annually.

EPA recommends the following as an acceptable approach to solids absorption curve reverification. Solids

absorption standards are stored in a desiccator, isolated from other samples after their initial use if the laboratory

plans to use them later to reverify their solids absorption curves. When solids absorption curves need to be

reverified, at least three of the original solids absorption standards made to produce the solids absorption curve

currently in use for data reduction are selected for remeasurement. They should span the range of weights used

in the original solids absorption curve. For the reverification measurement to be acceptable, the original

measurement of the solids standard should occur within the range defined by the uncertainty of the reverification

measurement calculated at the 95 percent confidence level. If any of the verification checks are not within this

acceptability range when compared to their original measurements, all solids absorption standards must be

remeasured for both sample weight and activity, then used to generate a new solids absorption curve to use for

data reduction. If a solids absorption standard weight is not within five percent of its previously recorded weight,

then it is possible the sample has been contaminated, or suffered loss of sample, and therefore is discarded and

a new solids absorption standard manufactured to replace it. If more than one solids absorption standard weight

is not within this acceptance range, then the currently used solids absorption curve may be verified as described

below, or an entirely new set of solids absorption standards can be manufactured to measure and generate a new

solids absorption curve to use for data reduction.

For short lived radioanalytes, or if the original solids absorption standards are suspected of being compromised,

at least three new solids absorption standards (one each representative of the low, medium and high weight ranges

of the original solids absorption curve) can be manufactured and used as solids absorption verification standards.

For the verification measurement to be acceptable, the efficiency for the verification standard calculated from the

solids absorption curve presently in use should occur within the range defined by the uncertainty of the efficiency

verification measurement calculated at the 95 percent confidence level. If any verification standard’s measured

efficiency is not within this acceptance range, then additional solids absorption standards are manufactured that

span the range of sample weights used at the laboratory. The complete set of solids standards is then measured

to produce a new solids absorption curve to use for data reduction.
VI-4

For stability checks, an efficiency calibration check source and the instrument background is measured and

recorded prior to measuring samples on each day of use to verify the instrument is ready to make sample

measurements. During periods when gas proportional counters are idle, the efficiency calibration should be

checked daily and the background checked weekly to confirm the ready status of the instrument for sample

measurements. 

If a gas proportional counter measures samples sequentially with an automatic sample changer, it is possible both

the size of the batch and long count times creates a sample counting set that requires several days to complete.

In these cases, measurements of sample preparation batch Quality Control (QC) samples may substitute for the

instrument checks. After 24 hours of continuously measuring samples, a measurement of any preparation batch

QC sample with a known amount of the analyte added to it (See section 7.7) may be substituted for the calibration

check source measurement, and any QC sample measurement expected to have no activity in it (reagent blanks)

may substitute for an instrument blank measurement.

Warning limits and control limits for instrument stability measurements should be calculated. If instrument control

measurements exceed their control limits, the proportional counter is placed out of service until the reason for the

change in efficiency or background can be determined and corrected. If the out of control measurement is a sample

batch QC sample substituting for an instrument control measurement, the entire batch is remeasured after the

reason for the out of control event is identified and addressed. If a gas proportional counter is moved, serviced,

or had an interruption in either gas flow or electrical power, the plateau voltage for both alpha and beta is verified,

its crosstalk factors remeasured, and the solids absorption curves for each analyte reverified or regenerated prior

to measuring any compliance monitoring samples.

3.1.3 Alpha scintillation counting system: For measurement of gross alpha activities and radium-226, a

scintillation system designed for alpha counting may be substituted for the gas-flow proportional counter described

above. In such a system, a Mylar disc coated with a phosphor (silver-activated zinc sulfide) is either placed

directly on the sample or on the face of a photo-multiplier tube, enclosed within a light-tight container, along with

the appropriate electronics (high voltage supply, pre-amplifier, amplifier, timer, and scalar). Solids absorption

curves for each analyte measured by alpha scintillation counting systems are generated or reverified at least

annually as described above for gas proportional counters. 

Photo Multiplier Tube (PMT) efficiency and background checks are measured and recorded prior to the

measurement of a set of compliance monitoring samples, or at least weekly to verify the alpha scintillation

system’s ready status to measure samples. Warning limits and control limits for both types of instrument stability

measurements should be calculated. If any PMT efficiency or background check exceeds its control limits, the

instrument is placed out of service until such time the reason for the out of control measurement can be identified

and corrected.

3.1.4 Scintillation cell system: An alpha scintillation system designed to accept alpha scintillation flasks ("Lucas

cells") should be used for the specific measurement of radium-226 by the radon emanation method. The detection

system consists of a light-tight enclosure capable of accepting Lucas cells, a photomultiplier tube detector

(phototube), and the appropriate electronics (high voltage supply, amplifier, timers, and scalars). The Lucas cells

needed for this measurement may either be purchased from commercial suppliers or constructed by the laboratory.

Lucas cell efficiency calibration constants are checked and recorded at least quarterly or every 10 uses (whichever

occurs more frequently) using a standardized solution of Ra-226 and a bubbler exclusively dedicated to this

activity. All cell constants are measured relative to a specific scintillation cell counting system. Also, each Lucas

cell should have acceptability limits for results that are calculated from the previous cell constants determined for

each specific cell. If a laboratory has more than one scintillation cell counting system, the efficiency of each

counting system should be determined using a calibrated alpha scintillation source. Their efficiencies are checked

at least weekly to verify the ready status of the system to measure samples, or prior to use (whichever is more

frequent). Cell backgrounds also are measured and recorded prior to the measurement of compliance monitoring

samples. Control limits for both efficiency and background checks are calculated. 

If a cell constant or efficiency check exceeds the determined control limits, the affected Lucas cell or counting

system must not be used until the out of control parameter is investigated. If, after remeasuring the check source,

the change in cell constant or counting system efficiency is confirmed, the laboratory supervisor may allow them
VI-5

to be used, using the new cell constant or counting system efficiency when reducing counting data from

compliance monitoring sample measurements. If the out of control check is the background of the Lucas cell, then

the cell must be placed out of service until the excess background decays to levels that allow the required

detection limits be achieved within reasonable counting times.

3.1.5 Gamma spectrometer systems: Either a solid-state, lithium-drifted germanium detector or a high-purity

intrinsic germanium detector connected to a multichannel analyzer is needed if the laboratory is to be certified

for analyses of photon emitting radionuclides.

A system with a lithium-drifted germanium, or a high-purity intrinsic germanium detector may be used for

measurement of photon emitting radionuclides if the efficiency of the detector is adequate to meet the detection

limits listed at 40 CFR part 141.25(c). These detectors should be shielded with a minimum of 10 cm of iron or

equivalent. In addition to appropriate electronics, the multichannel analyzer should contain a memory of not less

than 4096 channels and at least one readout device.

Upon receipt, after servicing or if the instrument’s settings are changed for any reason, gamma spectrometry

systems must be efficiency and energy calibrated, and the background of the detector cave measured for a

sufficient time so that any potentially interfering photo-peaks from naturally occurring gamma emitters in the

count room environment can be identified, as well as the gross gamma activity. Calibration sources must be in

the same geometry the laboratory chooses to measure drinking water samples, and contain NIST-traceable sources

for several gamma emitters whose photo-peaks span the width of the gamma spectral window the instrument is

set to detect. The cave background must be remeasured at least monthly, and the background gross gamma

activity recorded.

A calibration check source is measured prior to sample measurements on each day of use. If gamma detectors

are idle, the calibration check source is measured at least weekly to verify the detector’s ready status for measuring

compliance monitoring samples.

Calibration check sources for intrinsic germanium gamma spectrometers must contain a minimum of two photopeaks. One photo-peak is located at energies below the maximum efficiency energy of the detector (known as

the “knee” of the detector energy vs efficiency curve) where the curve has a positive slope, and another above the

detector efficiency knee, where the efficiency curve has a negative slope. Each peak must be evaluated for the

detector efficiency at that energy, the channel number of the peak centroid, and the Full Width at Half Maximum

(FWHM).

Warning limits and control limits for the efficiency, the channel number of the peak centroid, and the FWHM

of each photo-peak in the check source, and the gross total activity of the detector cave background measurement

is calculated. If any monitored parameter in the efficiency check source measurement exceeds their control limits,

the gamma detector must be recalibrated. If the gross gamma activity of the cave background check exceeds its

control limit, the source of the increased background must be identified, removed if possible, and the cave

background remeasured prior to analyzing compliance monitoring samples. 

3.1.6 Alpha Spectrometer Systems: Alpha spectrometry systems should be used for uranium measurements if

specific uranium isotope activities are of interest. Alpha spectrometry systems consist of a solid state device (a

silicon surface barrier detector, a passively implanted planar silicon detector, etc.) mounted in a vacuum chamber

and connected to a multichannel analyzer system. 

The detectors in alpha spectrometry systems are energy and efficiency calibrated, and the background of its

sample chamber measured when it is placed into service. Energy calibrations are checked weekly, and efficiency

calibrations checked monthly to verify the alpha spectrometry system’s stability. The sample chamber background

is checked at least monthly, and should be checked after each day of sample measurements to monitor for alpha

recoil contamination from samples.

The alpha peaks in the check source are evaluated for the detector efficiency, the channel number of the peak

centroid, and the FWHM. These alpha peak properties are recorded and control limits calculated. The

background activity check is for total alpha activity for each detector sample chamber and is also recorded and

used to set control limits. If any monitored alpha peak property is out of control, the detector is to be recalibrated.
VI-6

If a background check is out of control, the detector and chamber is placed out of service until the source of the

background increase is identified and removed. If excess alpha background cannot be removed, the detector is

placed out of service until the contamination decays to a level that allows the required detection limits to be

achieved within reasonable count times for the analyte being interfered with by the contamination.

3.1.7 Other Instrumentation: Other radiation measurement devices as mentioned in legislation or referred to

in the approved methods can also be used for measuring radioanalytes in drinking water. Their initial calibration

periods and checks should be consistent with the methods they are used for and the manufacturer’s

recommendations. 

4. General Laboratory Practices

4.1 Chemicals/reagents

Chemicals and reagents should meet the specifications in the methods. If not specified, then "Analytical Reagent Grade"

(AR) or American Chemical Society (ACS) grade chemicals or better should be used. Radioactive standards are to be

certified by the National Institute of Standards and Technology (NIST) or traceable to a NIST-certified source. 

4.2 Reagent Water

The laboratory should have a source of reagent water meeting the requirements of being an ASTM Type 1,2, or 3 reagent

water, having a minimum resistivity of 10 megohms/cm at 25 C. It should be monitored daily by measuring the reagent o

water’s conductivity or resistivity and documented.

Radioactive components have been known to break through reagent water manufacturing units before an increase in

resistivity is noted. To monitor the background radioactivity of the reagent water, it is to be screened for radioactivity after

each time the treatment unit is serviced, and periodically thereafter depending on volume of reagent water used at the

laboratory between unit servicing.

4.3 Glassware/Plasticware

Specific requirements in the methods for the cleaning of glassware should be followed. The purpose of these requirements

are to minimize the possibility that glassware can contaminate samples, and should include acid rinsing. Acid rinsing not

only mobilizes any metals remaining adhering to their surfaces, but also hydrates the outer silica layer on glassware which

inhibits contamination with radioactive materials. If there are no specifications for cleaning glassware in the method, then

glassware should first be washed in detergent solution, then thoroughly rinsed in tap water followed by a second rinse in

a dilute acid solution, and finally rinsed with reagent water and dried. 

Laboratory glassware should also be checked periodically for cracks, scratches, and abrasions. If they are found to be

damaged, they are to be discarded. Scratched glassware increases the likelihood of sample contamination or losses due

to the increase of surface area that is exposed to the sample. 

4.4 Safety

Guidelines in the Laboratory Safety Manual, the Chemical Hygiene Plan, or the Standard Operating Procedures (SOPs)

should include safety training and protection information specific to a radiochemistry laboratory. This should include

identifying what operations of the laboratory use hazardous materials, identify same, and determine the health risks that

are possible if someone is exposed to them. This should also include what workers should do to protect themselves from

exposure and possible injury. For radiochemistry laboratories, this should include when and how radiation shielding is used

to protect analysts and technicians from harmful levels of radioactivity. It should also define when circumstances warrant

the use of protective equipment, and should include the use of gloves, laboratory coats, eye protection and appropriate

pipetting techniques to avoid exposure and possible injury from chemicals and radioactive substances.

5. Analytical Methods

The approved methods cited at 40 CFR part 141.25(a) and (b) must be used for the analysis of drinking water compliance

samples. These are listed in Table VI-1. 

5.1 Standard Operating Procedures

A laboratory-specific Standard Operating Procedure (SOP) should be written for each method used by the laboratory for

measuring regulated radio-analytes in compliance monitoring samples. These SOPs should be consistent with a referenced

approved method. Any EPA-approved modifications should be noted.
VI-7

6. Sample Collection, Handling, and Preservation

Sample containers, preservatives, and holding times specified in the methods should be followed. Table VI-2 lists critical

elements for sample handling, including preservation. Sample preservatives provided by the laboratory should be screened

for radioactive content by lot number prior to their use in the laboratory and documented. Samples preserved with reagents

not provided by the laboratory are to be accompanied by a blank, radioactive free sample that is preserved in the same

manner as the submitted sample.

6.1 Composited Samples

If deemed acceptable by the state, samples may be composited by the utility or the laboratory as an alternate sampling

protocol, provided that all sample aliquots are properly preserved at the time of collection. Since the required compliance

monitoring measurement is “total activity” samples are not filtered before preservation, as it must represent the maximum

potential exposure from drinking water. Samples must be drawn on a quarterly basis and where compositing is not done

by the laboratory, there must be documentation submitted with the composite sample detailing on what days each aliquot

was obtained, its volume, and when it was preserved. A sample of the preservative should accompany the composited

sample to the laboratory to determine the contribution of radioactivity, if any, from the addition of the preservative to the

sample. Analysis of composite samples must be completed within one year after the first sample is collected or within

normal holding times if the compositing period is less than 90 days. Where possible, the laboratory should be responsible

for managing the compositing of samples.

7. Quality Assurance

Laboratory Quality Assurance systems are all the protocols, policies and procedures implemented at a laboratory to ensure

the generation of data of known quality. Comprehensive programs should encompass every aspect in the sample processing

path through a laboratory, from sample receipt, storage, measurement, to producing the final report for the sample

measurements as well as record keeping for all sampling information, raw data and reports for samples. It should also

address laboratory support functions, such as specifying procedures to train and qualify technicians, instrument maintenance

and calibration procedures, support equipment maintenance, purchasing supplies, and problem solving protocols. Each

area discussed should address providing documented evidence of compliance with the specific areas of the Quality

Assurance system. Specific areas for Quality Assurance systems are addressed in Chapter III, and areas specifically

relevant in radiochemistry laboratories are discussed in the sections below.

7.1 General Requirements

7.1.1 Availability of Records and Documents: The analytical methods references, Quality Assurance Manual,

and SOPs are to be readily available to the analysts. All QC data and records are to be available for inspection

by the certification officer.

7.2 Balance and Weights

Radiochemistry laboratories should have balances with the appropriate ranges for their operations. Since weights are used

to calculate gravimetric recoveries used in the calculation of analytical results for some methods, balances used to weigh

samples should have a documented history of accuracy and precision. At a minimum, the laboratory should have an

analytical balance with at least a .01 mg sensitivity to measure sample weights. Also, a top loading balance should be

available to weigh out chemicals for reagent preparation. Balances must be re-calibrated at least annually with ASTM Type

1 weights. At least once every 31 calendar days, the analytical balance’s range appropriate for sample measurements

should be checked using three traceable Type 1, Class 1, or Class 2 weights that span the range of sample weighing needs.

Prior to use, the calibration of the balance should be checked with the ASTM Type 1, Class 1, or Class 2 weight closest

in weight to most sample measurements. Most commonly this is the 10 gram weight because tared steel planchets often

weigh between 8 and 10 grams. These calibration checks should be recorded in a permanent log. Weights used for

calibration checks should be recertified every five years.

7.3 Method Sensitivity Studies

The laboratory should determine the standard analytical conditions for each method for measuring compliance monitoring

samples that can produce detection limits that are equal to or less than those specified in 40 CFR part 141.25(c)(1) Table

B, and 40 CFR part 141.25(c)(2) Table C. These procedures should be consistent with Appendix C, of “Prescribed

Procedures for the Measurement of Radioactivity in Drinking Water (EPA-600/4-80-032). Once the method standard

analytical conditions are determined, the laboratories should then institute a monitoring program to ensure the sensitivity
VI-8

of each method used to analyze compliance monitoring samples does not exceed the detection limits defined in the CFR

references cited above.

Method background levels, as measured from a reagent blank, monitor for potential contamination from several potential

sources of interferences in the sample preparation laboratory in addition to the background of the detector used for its

measurement. Consequently, sensitivity monitoring for radiochemical methods used to analyze drinking water should use

the results of the reagent blank. The activity and uncertainty of each reagent blank prepared with sample preparation

batches should be calculated in the same concentration units as the detection limits listed in the regulations referred to

above, recorded, then the activity plotted on a control chart to provide a record that will serve as a continuous

contamination and sensitivity monitor for the method as it is performed at the laboratory.

7.4 Proficiency Test (PT) Studies

To be certified for the analysis of a radio-analyte in drinking water, a laboratory must successfully participate in at least

one PT study for the analyte for which they are seeking certification. To maintain certification for an analyte, the laboratory

must pass one study per year thereafter. Scoring criteria for PT samples can be found in the CFR as cited above. The

laboratory may choose to participate in either type of study from a provider acceptable to the state or EPA that is described

below.

7.4.1 Mixed Alpha and Mixed Beta/Gamma PT Studies: These PT study samples contain either a mixture of

known alpha emitting radionuclides whose concentrations are unknown to the laboratory, or a mixture of known

beta/gamma emitting radioanalytes whose concentrations are also unknown to the laboratory.

7.4.2 Other PT Studies: These are PT studies whose samples contain a single analyte at an unknown

concentration, or varied number of analytes at unknown concentrations that are often associated with each other

in drinking water. These studies are distributed several times per year. If the analyte the laboratory wishes to be

certified for is not contained in a Mixed PT sample, they must successfully analyze it at least once a year by

participating in one of these other PT studies that contain the radioanalyte for which the laboratory wishes

certification.

7.4.3 Additional Proficiency Testing Studies: The laboratories may also participate in a second PT study each

year for each analyte for which they seek certification. Voluntary interlaboratory round robin studies could meet

this recommendation. These additional PT studies should be performed in the half of the year that the blind or

analyte-specific PT study the laboratory participates in for annual certification is not performed. This will provide

a biannual check of the laboratory’s performance.

 

7.5 Operating Manuals

Operating manuals and calibration protocols for counting instruments should be available to analysts and technicians. These

documents should be stored near the instruments for easy reference.

7.6 Maintenance of Records

Calibration data and maintenance records on all radiation instruments and analytical balances should be maintained in a

permanent record. If this is a hard copy record, it should be maintained in a permanently bound notebook and all entries

made in ink. If it is an electronic record, the information should be stored on a computer system that is password protected,

and backed up weekly with a copy of the back up data stored offsite to be considered a permanent record. 

7.7 Sample Measurement Quality Control Requirements

Just as the stability of the measurement instruments should be monitored to verify the measurements they produce will have

reasonable accuracy, precision and freedom from interferences, similar controls should be used to monitor sample

preparation activities. In order to assess the effect of sample preparation activities on the accuracy, precision, and freedom

from interferences of drinking water compliance monitoring measurements, the laboratory includes the QC samples listed

below at the specified frequencies for each preparation batch of samples that are measured for the radioanalytes for which

the laboratory is certified, unless otherwise noted for some methods. A preparation batch of samples are those samples

prepared sequentially together within a 24 hour period. The number of compliance monitoring samples in a preparation

batch should not exceed 20 (excluding the QC samples described below).
VI-9

7.7.1 Assessment of preparation batch precision: The laboratory should assess the precision of each sample

preparation batch. This can be done in one of two alternative ways. First, the laboratory may measure a second

aliquot of a sample for a duplicate measurement (DUP). Alternately, if nondetects for a method are frequent, the

laboratory may elect to measure a matrix spike (MS)/matrix spike duplicate (MSD) pair instead. An MS is a

second aliquot of a sample that has a known amount of the radioanalyte being measured added to it. An MSD is

a third aliquot of the same sample that has the same amount of radioanalyte added to it as the MS. Regardless

of sample choice, the DUP or MS/MSD pair must be prepared with the other samples with a frequency no less

than 1 every 10 samples or less.

With the exception of gross alpha particle activity, gross beta particle activity, and tritium measurements, if there

are insufficient sample volumes for a duplicated sample or a duplicate matrix spike sample in a set of samples,

two aliquots from the laboratory’s tap water may substitute for the sample aliquots and be used for the duplicated

sample.

Precision shall be assessed by calculating the relative percent difference (RPD) for either the sample and its

duplicate, or for the MS/MSD pair. An RPD result for either precision assessment measurement that exceeds its

calculated control limit, which, ideally should be 20 percent or less, indicates the precision of the sample

preparation batch is questionable, and data reported from these results should be flagged as having questionable

precision.

If a duplicated sample measurement has an activity that is less than 5 times the radioanalyte’s detection limit, and

exceeds 20 percent RPD when compared to the first measurement for the sample, it may be reevaluated using the

two measurement’s replicate error ratio (RER). The RER of two measurements made from the same sample is

an assessment of whether they are within two standard deviations of their aggregate measurement uncertainty of

each other. The RER is calculated using the following formula;

RER = |A - a b B| / SQRT( s + s ) < 2 2 2

where;

 A = Net Activity of the first measurement

 B = Net Activity of the second measurement made from a different aliquot from the same

sample.

 sA = The uncertainty of the first measurement

B

 s = The uncertainty of the second measurement

 If the RER is less than or equal to two, then the two measurements are within 2 standard deviations of each other,

and so are acceptable. If the RER exceeds 2, it is unacceptable since it means there is greater than 2 standard

deviations of difference between the two measurements.

If precision assessments exceed their limits, calculations and procedures should be examined and samples

recounted. If the precision assessments are still unsatisfactory after the samples are recounted, then all sample

results in the preparation batch are to be reported with a qualifier to indicate the measurement has questionable

precision. If the client requires unqualified results, then all the samples in the discarded sample preparation batch

are then re-measured using new aliquots of the sample if hold times and sufficient volume is available or

resampled and reanalyzed.

7.7.2 Assessment of Preparation Batch Accuracy: Matrix Spike (MS) samples are prepared to monitor how

sample preparation procedures impact the accuracy of measurements of the samples in a sample preparation batch.

A MS sample is prepared by adding a known amount of the radioanalyte being measured to a second aliquot of

a sample. The MS sample has radioanalyte activities added to them that are approximately 10 times the

anticipated level of the sample activity, or the detection limit, as appropriate for the measured radioanalyte. MS

samples should be prepared and processed with the samples at a frequency of one MS per sample preparation

batch.

MS performance is assessed using the percent recovery of the known activity of radioanalyte added to the sample.
VI-10

The percent recovery should be within the control limits calculated from previous MS measurements, which

should ideally be + 20 percent of the amount of activity added to the MS sample. For gross alpha particle activity,

gross beta particle activity measurements, and Ra-228 methods of analyses, where experience has shown lower

accuracies can be expected, control limits calculated from previous MS results should be within + 30 percent of

the amount of activity added to the MS for the accuracy relative to the sample matrices to be considered

acceptable.

MS samples need not be included in preparation batches for certain methods. Since there is no way currently to

quantitatively add an inert gas to a liquid, MS samples will not be required for radon in water measurements.

Also, gamma screens of drinking water need not include MS samples in their preparation batches of samples.

Density, as expressed by the average atomic weight, or Z, of the material surrounding a gamma detector is the

principle factor in scattering or absorbing gamma rays. With respect to this property, there is little difference

between deionized water and drinking water sample matrices. Consequently, the same information with respect

to accuracy can be assessed with the laboratory fortified blank.(LFB).

With the exception of gross alpha particle activity, gross beta particle activity, and tritium measurements, if there

are insufficient sample volumes for a matrix spike in a set of samples, two aliquots from the laboratory’s tap water

may substitute for the sample aliquots and used for the unspiked/spiked MS sample pair.

If MS assessments of accuracy exceed their calculated control limits, calculations and procedures should be

examined and samples recounted. If the assessment is still unsatisfactory after the samples are recounted, then

all sample results in the batch should be flagged as possibly biased low or high (as the result indicates) due to

matrix effects. If the LFB assessment of accuracy independent of matrix effects is also unsatisfactory in the same

preparation batch (see below), then all the samples in the discarded sample preparation batch are remeasured using

new aliquots of the sample if hold times and sufficient volume is available. 

7.7.3 Assessing the preparation batch accuracy independent of matrix effects: In order to assess the method’s

preparation batch accuracy independent of matrix effects, an (LFB) shall be prepared with each preparation batch

of samples. The LFB is prepared by adding a known amount of the radioanalyte being measured to an aliquot

of deionized water that is prepared using the same procedures used to measure the samples in the preparation

batch. LFBs are prepared at a frequency of one per preparation batch. 

LFB accuracy is assessed using the percent recovery of the known activity of radioanalyte added to the sample.

The percent recovery should be within the control limits calculated from previous results, which should ideally

be within ± 10 percent of the amount of activity added to the LFB sample. For gross alpha particle activity, gross

beta particle activity, and Ra-228 measurements, where lower accuracies can be expected, the calculated control

limits should not exceed + 20 percent recovery of the amount of activity added to it. 

If LFB assessments exceed their limits, calculations and procedures should be examined and samples recounted.

If the LFB assessment is still unsatisfactory after the samples are recounted, the batch must be considered

contaminated. All samples in the sample preparation batch must then be discarded. After the source of

contamination is identified and addressed, all the samples in the discarded sample preparation batch must then

be remeasured using new aliquots of the sample if hold times and sufficient volume is available.

7.7.4 Assessing instrument drift during sample measurements: While most radiation measurements systems

are noteworthy for their stability, sudden changes can occur due to instrument component failure, loss of gas

pressure, vacuum, or contamination of a detector or sample chamber from a high activity sample can possibly

occur while samples are being counted. Instrument drift in detector efficiency and background must be checked

both before and after measuring compliance monitoring samples to verify both the calibration and background

did not change significantly while the samples were being measured.

A sample measurement, or “counting” batch is a second type of sample batch that can be defined in radiochemical

methods of analysis. A sample measurement batch includes those samples that are measured within 24 hours of

each other, and their measurements occur in between the detector efficiency and instrument background checks

made prior to and at the end of measuring a set of compliance monitoring samples. Often a sample measurement
VI-11

batch and a sample preparation batch consist of the same number of samples. In some cases, however, it may take

several days to measure all the samples in a sample preparation batch. In these instances, efficiency and instrument

background checks should not wait until the last sample in a preparation batch has been measured. To do so could

possibly result in several days of lost counting time if they are out of control. Instead, a sample count order must

be arranged so an efficiency check and an instrument background check is to be made at least every 24 hours

while samples are being counted. 

To facilitate these daily checks, the sample order may be arranged so a sample spiked with a known activity of

the radioanalyte being measured (i.e an LFB, MS, or MSD sample) and a sample not expected to have measurable

activity in it (RB) is counted at the end of a 24 hour period, or if the total sample measurement batch counting

time does not exceed 24 hours, after all the other samples in the sample preparation batch have been measured.

If these QC samples meet their acceptance criteria, then it may be assumed little or no change in the efficiency

or background occurred while samples were being measured. If however, sample counting requirements prevent

these samples from being counted at the end of each 24 hours sample measurement period or as the last samples

measurements of the sample preparation batch, the efficiency calibration check source used for the efficiency

calibration checks, and an instrument blank must be measured in their place. Both of these measurements must

be within the control ranges produced from their previous measurements. 

If either the QC samples or the instrument efficiency or background check exceed their calculated control limits,

the instrument is placed out of service until the source of the out of control condition is identified and corrected.

If the failed instrument QC check occurs with a measurement made at the end of measuring a sample measurement

batch, then all the samples in the measurement batch are recounted after the source of the out of control condition

is identified and corrected..

7.8 Instrument and Method Performance Charts/Records

Quality control performance records or control charts should be maintained for each instrument and method used by the

laboratory for compliance monitoring sample measurements. For instruments, the initial calibrations and all efficiency

calibration and instrument background checks should be maintained in a permanent record. The efficiency calibration and

instrument background checks should have control limits calculated, and be control charted separately to monitor the

instruments ready status to measure samples. For each method used for compliance monitoring measurements, the

assessments of sample preparation batch precision (DUP or MSD), accuracy (MS and MSD), and interferences (RB) should

be recorded separately from sample results in a permanent record. Laboratory-specific performance warning and control

limits for each parameter monitored for both instruments and methods should be recalculated every 20 measurements for

each QC parameter. If a QC result exceeds its calculated control limit, all measurements using the associated method or

instrument must cease until the source of the exceedance is identified and corrected. 

7.9 Quality Assurance (QA) Plan 

The laboratory should prepare and follow a written QA plan (see Chapter III).

8. Records and Data Reporting

8.1 Legal Defensibility

Laboratories are to retain sufficient data and documentation for compliance monitoring samples so that their receipt and

any measurement made for this purpose can be reproduced if validation of the data is required. The QA plan and/or SOPs

should describe the policies and procedures used by the facility for record retention and storage. Since all compliance

monitoring measurements are used to interpret and enforce legal requirements, chain of custody procedures should be used

(See Appendix A).

8.2 Maintenance of Records

Public water systems are required to maintain records of radionuclide analyses of compliance samples for 10 years (40 CFR

part 141.33). The laboratory should maintain easily accessible records for 10 years. The client water system should be

notified before disposing of records so they may request copies if needed. This includes all raw data, calculations, and QC

data. These data files may be either hard copy, microfiche, or electronic. Electronic data should always be backed up by

protected tape, disk, or hard copy. If the laboratory changes its computer hardware or software, it should make provisions

for transferring old data to the new system so that it remains retrievable within the time frames specified above. Data which

is expected to become part of a legal action should be maintained for a longer period of time. 
VI-12

8.3 Sampling Records

Data should be recorded in ink with any changes lined through such that original entry is visible. Changes should be

initialed and dated. The following information should be readily available in a summary or other record(s):

8.3.1 Date, location (including name of utility and PWSID), site within the system, time of sampling, name,

organization and phone number of the sampler, and analyses required.

8.3.2 Identification of the sample as to whether it is a routine distribution system sample, check sample, raw

or finished water sample, repeat or confirmation sample or other special purpose sample.

8.3.3 Date of receipt of the sample.

8.3.4 Sample volume/weight, container type, preservation and holding time and condition on receipt.

8.3.5 pH and disinfectant residual at time of sampling (from plant records).

8.3.6 Transportation and delivery of the sample (person/carrier, conditions).

8.4 Analytical Records

Data must be recorded in ink with any changes lined through such that original entry is visible. Changes must be initialed

and dated. The following information must be readily available:

8.4.1 Laboratory and persons responsible for performing analysis.

8.4.2 Analytical techniques/methods used.

8.4.3 Date and time of analysis.

8.4.4 Results of sample and quality control analyses.

8.4.5 Calibration and standards information.

8.4.6 Counting data;

8.4.7 Results of analyses, including the activity, uncertainty of the measurement, and the method detection

limit.

8.5 Data Reporting

Reports to drinking water suppliers and programs should contain sufficient information to establish the validity of the

reported results for the required analyses. It should be designed as a summary form that contains the sampling and

analytical information described above. At a minimum, these reports should contain the following information:

8.5.1 Name and location of the water supply, including its PWSID.

8.5.2 Location in the water supply where the sample was taken.

8.5.3 Date and time of sampling.

8.5.4 The name of the person responsible for taking the sample.

8.5.5 Sample receipt date at the laboratory.

8.5.6 Date and time of analysis.

8.5.7 The method used for the analysis.

8.5.8 The laboratory and the initials of the person responsible for performing the analysis.
VI-13

8.5.9 The analytical result, including the calculated uncertainty of the measurement.

8.5.10 If the result is below the calculated detection limit for that sample, the result should be reported as less

than the calculated detection limit.

8.6 Computer programs

Computer programs should be verified initially and periodically by manual calculations and the calculations should be

available for inspection. Access to computer programs and electronic data should be limited to appropriate personnel.

9. Action Response to Laboratory Results

When a laboratory is responsible, either by contract or state policy, to report sample results which would cause a system

to be out of compliance, the proper authority must be promptly notified and a request made for resampling from the same

sampling point.
VI-14

Table VI-1 Methods for Radionuclide Analysis CFR 141.25 

Contaminant Methodology Reference (method or page number)

EPA EPA EPA EPA EPA SM ASTM USGS DOE Other 1 2 3 4 5 5 6 7 8

Naturally

occurring

Gross alpha and 11

beta

Evaporation 900.0 p 1 00-01 p 1 302, 7110 B . . . . . . . . . . . . . R-1120-76 .......

Gross alpha Co-precipitation . . . . . .

11

....... 00-02 ........ 7110 C ............. .......... .......

Radium 226 Radon emanation,

Radiochemical

903.1

903.0

p 16

p 13

Ra-04

Ra-03

p 19

........

7500-Ra C

304, 305,

7500-Ra B

D 3454-97

D 2460-97

R-1141-76

R-1140-76

Ra-04

.......

N.Y.9

Radium 228 Radiochemical 904.0 p 24 Ra-05 p 19 304, 7500-Ra

D ...........

............. R-1142-76 . . . . . . . N.Y.9

N. J.10

Uranium Radiochemical 12

Fluorometric

Alpha spectrometry

Laser

Phosphorimetry

ICP-MS

908.0

908.1

......

......

200.813

.......

.......

.......

.......

......

......

00-07

......

........

........

p 33

........

7500-U B

7500-U C (17th

Ed.)

7500-U C (18 ,

th

19 , or 20 th th

Ed.)

.............

3125

.............

D 2907-97

D 3972-97

D 5174-97

D5673-03

..........

R-1180-76

R-1181-76

R-1182-76

..........

.......

U-04

U-02

.......

Man-made

Radioactive

cesium

Radiochemical

Gamma ray

spectrometry

901.0

901.1

p 4

.......

......

......

........

p 92

7500-Cs B

7120 

D-2459-72

D 3649-91

R-1111-76

R-1110-76

.......

4.5.2.3

Radioactive iodine Radiochemical

Gamma ray

spectrometry

902.0

901.1

p 6

p 9

.......

......

......

........

p 92

7500-I B

7500-I C,

7500-I D

7120 (19th Ed.)

D 3649-91

D 4785-93

..........

..........

.......

4.5.2.3

Radioactive

Strontium 89, 90

Radiochemical 905.0 p 29 Sr-04 p. 65 303, 7500-Sr B ............. R-1160-76 Sr-01

Sr-02

Tritium Liquid scintillation 906.0 p 34 H-02 p. 87 306, 7500-3H

B ...........

D 4107-91 R-1171-76 . . . . . . .

Gamma emitters Gamma ray

Spectrometry

901.1

902.0

901.0

....... ...... p 92 7120

7500-Cs B

7500-I B

D 3649-91

D 4785-93

R-1110-76 GA-01-

R
VI-15

The procedures shall be performed in accordance with the documents listed below. The incorporation by reference of the following documents was approved by the

Director of the Federal Register in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies of the documents may be obtained from the sources listed below.

Information regarding obtaining these documents can be obtained from the Safe Drinking Water Hotline at 800-426-4791. Documents may be inspected at EPA’s

Drinking Water Docket, 401 M Street, SW., Washington, DC 20460 (Telephone: 202-260-3027); or at the Office of Federal Register, 800 North Capitol Street, NW.,

Suite 700, Washington, DC.

1. “Prescribed Procedures for Measurement of Radioactivity in Drinking Water", EPA 600/4-80-032 , August 1980. Available at U.S. Department of Commerce,

National Technical Information Service ( NTIS), 5285 Port Royal Road, Springfield, VA 22161 (Telephone 800-553-6847) , PB 80-224744.

2. “Interim Radiochemical Methodology for Drinking Water”, EPA 600/4-75-008(revised), March 1976. Available at NTIS, ibid. PB 253258.

3. "Radiochemistry Procedures Manual", EPA 520/5-84-006, December 1987. Available at NTIS, ibid. PB 84-215581.

4. "Radiochemical Analytical Procedures for Analysis of Environmental Samples", March 1979. Available at NTIS, ibid. EMSL LV 053917.

5. "Standard Methods for the Examination of Water and Wastewater", 13th, 17th, 18th, 19th Editions, or 20th edition. 1971, 1989, 1992, 1995, 1998. Available

at American Public Health Association, 1015 Fifteenth Street N.W., Washington, D.C. 20005. Methods 302, 303, 304, 305, and 306 are only in the 13th

edition. Methods 7110B, 7110C, 7500-Ra B, 7500Ra C, 7500-Ra D, 7500-U B, 7500-Cs B, 7500-I B, 7500-I C, 7500-I D, 7500-Sr B, 7500-3H B are in the

17th, 18th, 19th and 20th editions. Method 7500-U C Fluorometric Uranium is only in the 17th edition, and 7500-U C Alpha spectrometry is only in the 18th

19th and 20th editions. Method 7120 is only in the 19th and 20th editions. 

6. Annual Book of ASTM Standards, Vol. 11.02, 1999. Available at American Society for Testing and Materials, 100 Barr Harbor Drive, West Conshohocken,

PA 19428.

7. "Methods for Determination of Radioactive Substances in Water and Fluvial Sediments", Chapter A5 in Book 5 of Techniques of Water-Resources

Investigations of the United States Geological Survey, 1977. Available at U.S. Geological Survey (USGS) Information Services, Box 25286, Federal Center,

Denver, CO 80225-0425. 

8. "EML Procedures Manual”, 28 (1997) or 27 (1990) Edition, Volume I and II. Available at the Environmental Measurements Laboratory, U.S. Department th th

of Energy (DOE), 376 Hudson Street, New York, NY 10014-3621. 

9. "Determination of Ra-226 and Ra-228 (Ra-02)”, January 1980, Revised June 1982. Available at Radiological Sciences Institute Center for Laboratories and

Research, New York State Department of Health, Empire State Plaza, Albany, NY 12201.

10. "Determination of Radium 228 in Drinking Water", August 1980. Available at State of New Jersey, Department of Environmental Protection, Division of

Environmental Quality, Bureau of Radiation and Inorganic Analytical Services, 9 Ewing Street, Trenton, NJ 08625.

11. Natural uranium and thorium-230 are approved as gross alpha calibration standards for gross alpha with co-precipitation and evaporation methods; americium241 is approved with co-precipitation methods. 

12. If uranium (U) is determined by mass, a 0.67 pCi/:g of uranium conversion factor must be used. This conservative factor is based on the 1:1 activity ratio of

U-234 to U-238 that is characteristic of naturally occurring uranium. 

13. "Methods for the Determination of Metals in Environmental Samples - Supplement I," EPA-600/R-94-111, May 1994. Available at NTIS, PB 94-184942.
VI-16

Table VI-2: Sample Handling, Preservation, and Instrumentation

Parameter Preservative Container Maximum 1 2

Holding

Time3

Instrumentation4

G 3ross Alpha Conc. HCl or HNO to pH <2 P or G 6 mo A, B, or G 5

G 3ross beta Conc. HCl or HNO to pH <2 P or G 6 mo A or G 5

Stront 3ium-89 Conc. HCl or HNO to pH <2 P or G 6 mo A or G 5

Stront 3ium-90 Conc. HCl or HNO to pH <2 P or G 6 mo A or G 5

R 3adium-226 Conc. HCl or HNO to pH <2 P or G 6 mo A, B, D or G 5

R 3adium-228 Conc. HCl or HNO to pH <2 P or G 6 mo A or G 5

Cesium-134 Conc. HCl to 

pH <25

 P or G 6 mo A, C or G

Iodine-131 None P or G 8 da A, C or G

Tritium None G 6 mo E

U 3ranium Conc. HCl or HNO to pH <2 P or G 6 mo A, B, F, H 5

Photon 3 emitters Conc. HCl or HNO to pH <2 P or G 6 mo C

5

It is recommended that the preservative be added to the sample at the time of collection unless suspended solids activity 1

is to be measured. If the sample has to be shipped to a laboratory or storage area unpreserved, acidification of the sample

(in its original container) may be delayed for a period not to exceed 5 days. A minimum of 16 hours must elapse between

acidification and analysis. 

P = Plastic, hard or soft; G = Glass, hard or soft. 2

Holding time is defined as the period from time of sampling to time of analysis. In all cases, samples should be analyzed 3

as soon after collection as possible. If a composite sample is prepared, a holding time cannot exceed 12 months.

A = Low background proportional system; B = Alpha and beta scintillation system; C = Gamma spectrometer [Ge(Hp) 4

or Ge(Li)]; D = Scintillation cell system; E = Liquid scintillation system (section C.2.a); F = Fluorometer ( section C.1.1);

G = Low background alpha and beta counting system other than gas-flow proportional; H=Alpha spectrometry system.

If HCl is used to acidify samples which are to be analyzed for gross alpha or gross beta activities, the acid salts must be 5

converted to nitrate salts before transfer of the samples to planchets. 
VI-17

Example Checklists for On-Site Evaluation of

Laboratories Analyzing Drinking Water for Radiochemistry
VI-18

General Audit Information

Laboratory____________________________________________________

Street________________________________________________________

City______________________________________State_______________

Survey By_____________________________________________________

Affiliation___________________________________________________

Date________________________________Telephone No._____________
VI-19

Laboratory_______________________________Date_________________

Location_________________________________Evaluator____________

PERSONNEL

Position/Title Name Academic

Training

Present 

Specialty

Years

Experience

(chemistry)

Years

Experience

(radiochemistry)

Laboratory Director

Quality Assurance

Officer

Section/Division

Chief/Director 

(if applicable)

Supervisory Analyst

Chemical Analyst(s)

Chemical

Technician(s)

Computer Support

Technician

Electronics

Support Technician
VI-20

Laboratory_______________________________Date_________________

Location_________________________________Evaluator____________

LABORATORY FACILITIES 

Item Available Comments

Yes No

Laboratory 

Electrical outlets

 120V ac. grounded

Distilled or deionized water

or ASTM type 1, 2, or 3

Exhaust Hood

Vacuum source

Counting Room - separate from wet 

chemistry, sample and standards

preparation area

Regulated power supply

Reagents
VI-21

Laboratory_______________________________Date_________________

Location_________________________________Evaluator____________

GENERAL LABORATORY EQUIPMENT AND INSTRUMENTS

Item No.of

Units

Manufacturer Model Satisfactory

Yes No

Analytical Balance

 0.1 mg sensitivity

 stable base

 ASTM type 1 or 2 weights or better

pH meter

 ±0.5 units

 readability ±0.1 units

 line or battery

Conductivity meter

 Readable in ohms or mhos

 Range of 2 ohms or mhos

 Line or battery

Drying oven

 gravity or convection

 controlled from room

 temp to 180°C or higher

 (±2°C) 

Infrared lamp

 may be substituted for drying oven

Desiccator

 Glass or plastic

Hot plate

 temperature control

Refrigerator

Magnetic Stirrer

 variable speed

 Teflon coated stir bar

Balance, top loading

Glassware

Thermometers

Muffle furnace to 450°C

Centrifuge

 to 3000 rpm

 to hold 4 x 50 mL
VI-22

Laboratory_______________________________Date_________________

Location_________________________________Evaluator____________

ALL INSTRUMENTATION

Item Yes No Comments

Are operating manuals readily

available to the operator

Are calibration protocols available

to the operator

Are calibrations kept in a

permanent control chart

Are permanent service maintenance

records kept
VI-23

Laboratory_______________________________Date_________________

Location_________________________________Evaluator____________

THIN WINDOW GAS-FLOW PROPORTIONAL COUNTER

Instrument

 number Manufacturer Model Year

Sample Changing

Manual Automatic Capacity

Counting Gas Window 

Density

(g/cm )

2

Instrument Background

Operating

Voltage

Alpha 

cpm

Operatin

g

Voltage

Beta

cpm

Calibration Standard 

Type:

 Alpha

 Beta

Supplier:

 Alpha

 Beta

Calibration Frequency Service Maintenance Frequency Condition 1 2 3

D W M Other Q S A Other G R N

WINDOWLESS GAS-FLOW PROPORTIONAL COUNTER

Instrument

 number

Manufacturer Model Year Sample Changing

Manual Automatic Capacity

Counting Gas Window 

Density

(g/cm )

2

Instrument Background

Operating

Voltage

Alpha 

cpm

Operating

Voltage

Beta

cpm

Calibration

Standard

Type:

 Alpha

 Beta

Supplier:

 Alpha

 Beta

Calibration Frequency Service Maintenance Frequency Condition 1 2 3

D W M Other Q S A Other G R N

1. Daily, Weekly, Monthly.

2. Quarterly, Semiannually, Annually.

3. Good, operating but needs Repair, Not operating
VI-24

Laboratory______________________________Date_____________________

Location________________________________Evaluator________________

LIQUID SCINTILLATION COUNTER

Instrument

 number

Manufacturer Model Year Sample Changing

Manual Auto Capacity

Calibration Standard

Type:

Supplier:

Calibration Frequency Service Maintenance Frequency Condition 1 2 3

D W M Other Q S A Other G R N

ALPHA SCINTILLATION COUNTER

Instrument

number

Manufacturer Model Year

Sample Changing

Manual Auto Capacity

Calibration Standard

Type:

 

Supplier:

 

Calibration Frequency Service Maintenance Frequency Condition 1 2 3

D W M Other Q S A Other G R N

RADON-GAS COUNTING SYSTEM

System number Gas counting cells/system

 Manufacturer of gas

counting cell

Counting Instrument

 Make Model Year

Calibration Frequency Service Maintenance Frequency Condition 1 2 3

D W M Other Q S A Other G R N

1. Daily, Weekly, Monthly.

2. Quarterly, Semiannually, Annually.

3. Good, operating but needs Repair, Not operating
VI-25

Laboratory______________________________Date_____________________

Location________________________________Evaluator________________

GAMMA SPECTROMETER SYSTEM

Detector System System Number

Type Make Model Year Size

Analyzer System

Make Model Year Channels

Calibration

Standard

 Type

 Supplier

Calibration Frequency Service Maintenance Frequency Condition

D W M Other Q S A Other G R N

OTHER APPROVED DETECTOR

Detector System System Number

Type Make Model Year Size

Analyzer System

Make Model Year Channels

Calibration

Standard

 Type

 Supplier

Calibration Frequency Service Maintenance Frequency Condition 1 2 3

D W M Other Q S A Other G R N

1. Daily, Weekly, Monthly.

2. Quarterly, Semiannually, Annually.

3. Good, operating but needs Repair, Not operating
VI-26

Laboratory______________________________Date_____________________

Location________________________________Evaluator________________

SAMPLE HANDLING AND PRESERVATION

Parameter Container 

Used

Preservative Used Comments Satisfactory

Yes No

Gross Alpha Activity

Gross Beta Activity

Strontium-89

Strontium-90

Radium-226

Radium-228

Cesium-134

Iodine-131 NONE

Tritium NONE

Uranium

Photon Emitters

 a.

 b.

 c.

 d.

 e.
VI-27

Laboratory______________________________Date_____________________

Location________________________________Evaluator________________

METHODOLOGY

Parameter

Sample

Load/M o

 Method Used - Cite Edition, Year, and Page 1

Satisfactory

EPA SMI ASTM USGS DOE Other Yes No

Gross Alpha

Activity

Gross Beta

Activity

Strontium-89

Strontium-90

Radium-226

Radium-228

Cesium-134

Iodine-131

Tritium

Uranium

Photon

Emitters

Identify:

 a.

 b.

 c.

 d.

 e.

 1 - Methods used must be referenced in the National Primary DrinkingWaterRegulations (40 CFR 141.25)
VI-28

Laboratory______________________________Date_______________

Location________________________________Evaluator___________

QUALITY CONTROL

Item Performance Testing Studies

 A B 1 2

Other PE Studies

 A B 1 2

Participation in

performance 

evaluation and 

Blind PE

studies

Reporting Period:

_______________

 to

_______________

Gross Alpha Gross Alpha

Gross Beta Gross Beta

Sr-89 Sr-89

Sr-90 Sr-90

Ra-226 Ra-226

Ra-228 Ra-228

Uranium Uranium

Cs-134 Cs-134

Cs-137 Cs-137

Co-60 Co-60

Ba-133

Written QA Plan 

implemented and 

available for review

Zn-65

Tritium

I-131

Frequency Yes No Comment Satisfactory

 Yes No

Duplicate analyses

Spikes

Reagent Blanks

Laboratory Fortified Blanks

Failed PE studies

Control charts

Calibration and 

Maintenance records

 1 - Scheduled frequency of participation by the laboratory, times per year.

2 - Number of acceptable performance results in the past year, where an acceptable result is a normalized deviation from the known value of #3.0 sigma
VI-29

Laboratory______________________________Date_____________________

Location________________________________Evaluator________________

DATA REPORTING

Item Comments: systems used, frequency, etc.

Records kept for 10 years 

 Actual laboratory reports

 Tabular Summary

Information included

 Date

 Place of sampling

 Time of sampling

 Sampler

 Date of sample receipt

 Date of analysis

 Type of analysis

Laboratory & person 

responsible

Other reported data

Other reported data

Method(s) used

Results

Appendices
A-1

Appendix A

Chain-of-Custody Evaluations

A. Introduction

Written procedures for sample handling should be available and followed whenever samples are collected, transferred,

stored, analyzed or destroyed. For the purposes of litigation, it is necessary to have an accurate written record to trace the

possession and handling of samples from collection through reporting. The procedures defined here represent a means to

satisfy this requirement.

A sample is in someone’s “custody” if:

1. It is in one’s actual physical possession;

2. It is in one’s view, after being in one’s physical possession;

3. It is one’s physical possession and then locked up so that no one can tamper with it;

4. It is kept in a secured area, restricted to authorized personnel only.

B. Sample Collection, Handling and Identification

1. It is important that a minimum number of persons be involved in sample collection and handling. Guidelines established

in standard manuals for sample collection preservation and handling should be used (e.g., EPA NPDES Compliance

Sampling Inspection Manual, MCD 51, Standard Methods for Examination of Water and Wastewater). Field records

should be completed at the time the sample is collected and should be signed or initialed, including the date and time,

by the sample collector(s). Field records should contain the following information:

a. Unique sample or log number;

b. Date and time;

c. Source of sample (including name, location and sample type);

d. Preservative used;

e. Analyses required;

f. Name of collector(s);

g. Pertinent field data (pH, DO, Cl residual, etc.); 

h. Serial number on seals and transportation cases;

i. Comments.

2. Each sample is identified by affixing a pressure sensitive gummed label or standardized tag on the container(s). This

label should contain the sample number, source of sample, preservative used, and the collector(s’) initials. The analysis

required should be identified. Where a label is not available, the sample information should be written on the sample

container with an indelible marking pen. An example of a sample identification tag is illustrated in Figure A-1.

3. The closed sample container should then be placed in a transportation case along with the chain-of-custody record form,

pertinent field records, and analysis request form. The transportation case should then be sealed and labeled. All

records should be filled out legibly in waterproof pen. The use of locked or sealed chests will eliminate the need for

close control of individual sample containers. However, there will undoubtedly be occasions when the use of a chest

will be inconvenient. On these occasions, the sampler should place a seal around the cap of the individual sample

container which would indicate tampering if removed.
A-2

C. Transfer of Custody and Shipment

1. When transferring the possession of the samples, the transferee must sign and record the date and time on the chain-ofcustody record. Custody transfers, if made to a sample custodian in the field, should account for each individual sample,

although samples may be transferred as a group. Every person who takes custody must fill in the appropriate section

of the chain-of-custody record.

2. The field custodian (or field sampler if a custodian has not been assigned) is responsible for properly packaging

and dispatching samples to the appropriate laboratory for analysis. This responsibility includes filling out, dating,

and signing the appropriate portion of the chain-of-custody record. A recommended chain-of-custody format is

illustrated in Figure A-2.

3. All packages sent to the laboratory should be accompanied by the chain-of-custody record and other pertinent

forms. A copy of these forms should be retained by the field custodian (either carbon or photocopy).

4. Mailed packages can be registered with return receipt requested. If packages are sent by common carrier, receipts

should be retained as part of the permanent chain-of-custody documentation.

5. Samples to be transported must be packed to prevent breakage. If samples are shipped by mail or by other common

carrier, the shipper must comply with any applicable Department of Transportation regulations. (Most water

samples are exempt unless quantities of preservatives used are greater than certain levels.) The package must be

sealed or locked to prevent tampering. Any evidence of tampering should be readily detected if adequate sealing

devices are used.

6. If the field sampler delivers samples to the laboratory, custody may be relinquished to laboratory personnel. If

appropriate personnel are not present to receive the samples, they should be locked in a designated area of the

laboratory to prevent tampering. The person delivering the samples should make a log entry stating where and how

the samples were delivered and secured. Laboratory personnel may then receive custody by noting in a logbook,

the absence of evidence of tampering, unlocking the secured area, and signing the custody sheet.

D. Laboratory Sample Control Procedures

Sample control procedures are necessary in the laboratory from the time of sample receipt to the time the sample is

discarded. The following procedures are recommended for the laboratory:

1. A specific person must be designated as custodian and an alternate designated to act as custodian in the custodian’s

absence. All incoming samples must be received by the custodian, who must indicate receipt by signing the

accompanying custody/control forms and who must retain the signed forms as permanent records.

2. The custodian must maintain a permanent logbook to record, for each sample, the person delivering the sample,

the person receiving the sample, date and time received, source of sample, date the sample was taken, sample

identification log number, how transmitted to the laboratory, and condition received (sealed, unsealed, broken

container, or other pertinent remarks). This log should also show the movement of each sample within the

laboratory; i.e., who removed the sample from the custody area, when it was removed, when it was returned, and

when it was destroyed. A standardized format should be established for logbook entries.

3. A clean, dry, isolated room, building, and/or refrigerated space that can be securely locked from the outside must

be designated as a “custody room.”

4. The custodian must ensure that heat-sensitive samples, light-sensitive samples, radioactive samples, or other sample

materials having unusual physical characteristics, or requiring special handling, are properly stored and maintained

prior to analysis.

5. Distribution of samples to the analyst performing the analysis must be made by the custodian.

6. The laboratory area must be maintained as a secured area, restricted to authorized personnel only.
A-3

7. Laboratory personnel are responsible for the care and custody of the sample once it is received by them and must

be prepared to testify that the sample was in their possession and view or secured in the laboratory at all times from

the moment it was received from the custodian until the time that the analyses are completed.

8. Once the sample analyses are completed, the unused portion of the sample, together with all identifying labels, must

be returned to the custodian. The returned tagged sample must be retained in the custody room until permission

to destroy the sample is received by the custodian.

9. Samples will be destroyed only upon the order of the responsible laboratory official when it is certain that the

information is no longer required or the samples have deteriorated. (For example, standard procedures should

include discarding samples after the maximum holding time has elapsed.) The same procedure is true for sample

tags. The logbook should show when each sample was discarded or if any sample tag was destroyed.

10. Procedures should be established for internal audits of sample control information. Records should be examined to

determine traceability, completeness, and accuracy. 
A-4
A-5

Figure A-2 Chain-of-Custody Record

Survey Samplers: Signature

Station

Number 

Station Location Date Time Sample Type Seq. No. No. Of

Containers

Analysis

Required

 Water Air

Comp Grab.

Relinquished by: Signature Received by: Signature Date/Time

Relinquished by: Signature Received by: Signature Date/Time

Relinquished by: Signature Received by: Signature Date/Time

Relinquished by: Signature Received by Mobile Laboratory for Field analysis:

Signature

Date/Time

Dispatched by: Date/Time Received for Laboratory by: Signature Date/Time

Method of Shipment:

Distribution: Orig. --Accompany Shipment, 1 Copy--Survey Coordinator Field Files 
B-1

Appendix B

Recommended Protocol for Regions Conducting On-Site Laboratory Evaluations

Before conducting the on-site evaluation, the Region should:

C Plan all the required activities to be completed during the assessment.

C Hold a pre-evaluation conference with appropriate laboratory and field activity representatives to establish a schedule that

would have a minimum impact on the laboratory activities.

C Request and review appropriate records.

C Request that a variety of tests be scheduled during the on-site evaluation.

C Arrange for the laboratory staff to be available during the on-site visit.

During the on-site visit, the team shall:

C Conduct an opening conference or entrance interview.

C Evaluate the procedures and equipment used for those specific analyses for which the laboratory has requested certification,

using the criteria in this manual.

C Review the records and written standard operating procedures for compliance with the required sampling frequency, sample

collection, sample holding times, and if appropriate, resample notification.

C Perform a data audit on at least one sample and one PE sample for at least one method but preferably for each method the

laboratory performs.

C Insure that the laboratory has a QA plan in effect by:

- Determining if the laboratory has written procedures (QA plan or equivalent) for conducting its quality

assurance program.

- Examining the quality assurance data to determine if the quality assurance program is being implemented.

C Complete the on-site checklists and other evaluation forms during the visit (see Chapters IV, V, and VI).

C Conduct a closing conference or exit interview in which the auditors review the results of the evaluation with the director of

the laboratory, the director of State water supply activities, and appropriate staff members. The review should:

- Discuss any deviations in the observed procedures and records.

- Recommend changes in equipment and supply needs, staffing requirements, and facility improvements, if

necessary.

- Discuss possible assistance the Region can provide the laboratory. 

- Discuss a time frame for corrective actions and response.

Evaluation Report for Principal State Laboratories and Laboratories in Non-Primacy States

After an on-site inspection, the evaluation team should prepare a narrative report and action memorandum. This report should

contain all information pertinent to the evaluation and also recommend the certification status for all analyses evaluated. The

report should then be forwarded for evaluation to the Certification Program Manager for review. After reviewing and, if

necessary, revising the report, it should be forwarded to the Certification Authority for signature.
B-2

The Certification Authority should decide the certification status of the laboratory within time constraints on page III-7 and notify

the State. The State should be sent the complete report. If the report indicates that the laboratory should not be certified for an

analysis, the Certification Authority should give the specific reasons.

The narrative report should be attached to each copy of the completed evaluation form. It should include the general headings

and information listed below.

Title Page

The title page should contain the following:

Title: Report of an on-site evaluation of the

 (name of laboratory)

At: (city, state, and zip code)

On: (date)

By: (name, title, organization, and

 address of the certification team)

Certification Status

List either "Certified", "Provisionally Certified", "Administratively/Interim Certified", or "Not Certified" for each

contaminant evaluated or if applicable (for VOCs, for instance) for each class of compounds evaluated.

List of Deviations

List each deviation by item number used on the evaluation checklists. Describe the exact deviation and recommended changes.

Remarks

Recommend improvements which, while not affecting certification status, would improve laboratory operation. Other remarks

might include reasons for failing the on-site evaluation, special recognition for outstanding performance, and description of

unusual tests.

List of Personnel

List name and title of personnel along with the individual tests that each normally performs. Also, identify the critical laboratory

personnel.

Signature

Team members should sign the report.

Distribution

Copies of this report should be distributed to the State requesting the evaluation. For local laboratories in non-primacy States,

reports should be distributed to appropriate Regional personnel.

Annually, each Region should submit to OGWDW a listing of laboratories in the Region having U.S. EPA certification. The

listing should include the names and location of each laboratory, and its certification status for all regulated contaminants. In

addition, Regions should notify OGWDW of all changes in status soon after they occur so that OGWDW can maintain an updated

list of certification status.
C-1

Appendix C

Definitions and Abbreviations

ASTM: American Society of Testing and Materials

AWWA: American Water Works Association

NERL-Ci: U.S. EPA National Exposure Research Laboratory in Cincinnati, Ohio (ORD).

NPDWR: National Primary Drinking Water Regulations.

OGWDW: U.S. EPA Office of Ground Water and Drinking Water.

ORD: U.S. EPA Office of Research and Development.

SDWA: The Safe Drinking Water Act as amended (42 U.S.C. 300f et seq.). 

Accuracy: A measure of the closeness of an individual measurement or the average of a number of measurements to the true

value. Accuracy includes a combination of random error (precision) and systematic error (bias) components which are due to

sampling and analytical operations. Refer to Standard Methods, Data Quality Section for a more detailed explanation.

Administrator: The administrator of the U.S. EPA or her/his authorized representative. See 40 CFR 142.2. 

Agency: The U.S. EPA. See 40 CFR 142.2. 

Auditor - A person who evaluates laboratories to determine if they meet the criteria to be certified. This person should be an

experienced professional, who has effective communication skills, experience in quality assurance, the analytical techniques being

evaluated, and familiarity with the drinking water regulations and this manual.

Bachelor Degree or Equivalent: A college degree with an equivalent 30 semester hours in a specific discipline. Equivalent

is at least four years of experience in a specific scientific discipline.

Bias: The systematic or persistent distortion of a measurement process which causes errors in one direction.

Certification Authority: (CA) The person or designee who has the authority to certify laboratories conducting drinking water

analyses and to certify the officials of the State responsible for the State's certification program in accordance with Section 1412

of the Safe Drinking Water Act. This authority is delegated to the Regional Administrator but may be redelegated.

Certification Program Manager: (CPM) The person responsible for managing the certification program which includes

tracking the certification status of the State laboratories, ensuring that the regional and State certification officers are qualified

and reviewing the certification evaluation reports. 

Certification Officer: (CO) A State or Federal laboratory auditor who has passed the NERL certification officers training course

(limited at this time to chemistry and microbiology). This person provides information to the CA or CPM for the purpose of

making decisions on the certification status of a laboratory. 

CFR: Code of Federal Regulations - A compilation of regulations is revised each time a regulation is promulgated. It is

published every year in July.
C-2

Confirmation: Verification of the presence of a component through the use of an analytical technique based on a different

scientific principle from the original method (e.g., second column, alternate wavelength or detector, etc.)

Conflict of Interest: a personal interest or relationship, as defined by law or regulation, that conflicts with the faithful

performance of your official duty.

Data Audit: A qualitative and quantitative evaluation of the documentation and procedures associated with measurements to

verify that the resulting data are acceptable.

Data Quality Objectives: qualitative and quantitative specifications used to design a study that will limit uncertainty to an

acceptable level.

Data Reduction: The process of transforming the number of data items by arithmetic or statistical calculations, standard curves,

concentration factors, etc. and collation into a more useful form. Data reduction is irreversible and generally results in the loss

of detail.

Detection: Any concentration of an analyte which equals or exceeds the laboratory's detection limit. For VOCs, detection limit

is defined as 0.0005 mg/L.

Drinking Water Laboratory: A laboratory that analyses samples as part of compliance monitoring for a public water supply.

Holding time: The allowed time from when a sample was taken (or extracted) until it must be analyzed.

IDC: Initial Demonstration of Capability - before analyzing compliance samples an analytical team must demonstrate acceptable

precision, accuracy, sensitivity, and specificity for the method to be used.

LRB - Laboratory Reagent Blank: (Method blank) An aliquot of reagent water or other blank matrix that is treated exactly

as a sample to determine if method analytes or other interferences are present.

LFB - Laboratory Fortified Blank: (Spike) An aliquot of reagent water or other blank matrix to which known quantities of

the method analytes are added in the laboratory. The LFB is analyzed exactly like a sample to determine whether the method

is in control. 

MCL: Maximum contaminant level means the maximum permissible level of a contaminant in water which is delivered to any

user of a public water system. See 40 CFR Part 141.2.

MCLG: Maximum contaminant level goal means the maximum level of a contaminant in drinking water at which no known or

anticipated adverse effect on the health of persons would occur, and which allows an adequate margin of safety. Maximum

contaminant level goals are nonenforceable health goals. See 40 CFR 141.2.

Method Detection Limit: (MDL) the minimum concentration of a substance that can be measured and reported with 99%

confidence that the analyte concentration is greater than zero. The MDL is determined from analysis of a sample in a given matrix

containing this analyte. See 40 CFR 136 App. B. 

Method Reporting Limit: (MRL) the lowest concentration of standard used for calibration

Monitoring Trigger: The concentration of a regulated contaminant which triggers additional monitoring. (See Detection Limit

141.24(h)(iii)(18))

NELAC: National Environmental Laboratory Accreditation Conference - a voluntary organization of State, Federal and other

groups to establish mutually acceptable standards for accrediting environmental laboratories. 
C-3

Precision: The measure of mutual agreement among individual measurements.

Primacy: Primary responsibility for administration and enforcement of primary drinking water regulations and related

requirements applicable to public water systems within a State.

Principal State Laboratory System: All facilities, whether part of the State laboratory or contracted by the State, producing

data for the State and certified by the EPA, fulfilling the requirements for Primacy as listed in the 40 CFR 142.10(b)(4). 

Profeciency Testing Samples (PTs): A sample provided to a laboratory for the purpose of demonstrating that the laboratory

can successfully analyze the sample within specified acceptance limits specified in the regulations. The qualitative and/or

quantitative composition of the reference material is unknown to the laboratory at the time of the analysis. See 40 CFR Part

141.2.

Public Water System: 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.

 See 40 CFR Part 141.2.

Quality Assurance: An integrated system of management 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.

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 the users; operational techniques and activities that are used to fulfill requirements for

quality.

QA Plan: A comprehensive plan detailing the aspects of quality assurance needed to adequately fulfill the data needs of a

program. This document is required before the laboratory is certified.

Regulatory Level: A concentration of a contaminant which is cited in the Federal Regulations (e.g., MCL, detect, etc.)

Shall: Denotes a mandatory requirement.

Should: Denotes a guideline or recommendation.

Standard Operating Procedure: A written document which details the method of an operation, analysis or action whose

techniques and procedures are thoroughly prescribed and which is officially approved as the method for performing certain

routine or repetitive tasks.

Third Party Auditor: Person or persons, not affiliated with a Region or State, who is designated by the Region or State to audit

a laboratory. This person must pass the certification training course prior to auditing any laboratory unless he or she is a part of

an audit team which includes a Regional/State certification officer. The third party auditor must also meet the

educational/experience requirements specified in this manual. The certification decision remains with the Region.

Third Party Expert: Any person not designated as a certification officer or auditor, who is requested by the Region to assist

in the audit of a laboratory because of his or her expertise in a particular area (e.g., asbestos). This person is not required to take

the certification officers' course if he or she is part of an audit team which includes a certification officer.

Tribal Nation: Areas which for regulatory purposes are treated as independent States. On these lands, the Indian tribe has a

Federally recognized governing body carrying out government duties and powers.

"Unregulated" Contaminants: Contaminants for which monitoring is required but which have no MCL.
C-4
D-1

Appendix D

 UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

 WASHINGTON, D.C. 20460 

January 16,1997

MEMORANDUM

SUBJECT: The Use of "Third-Parties" in the Drinking Water Laboratory Certification

Program

FROM: Cynthia Dougherty, Director

Office of Ground Water and Drinking Water 

TO: Water Supply Representatives, Regions I-X

Certification Authorities, Regions I-X

Quality Assurance Officers, Regions I-X

Regional Laboratories, Regions I-X

Purpose

This memorandum updates and clarifies the guidance memorandum from Michael Cook

dated December 5, 1989 on "Third-Party Certification for Laboratories in Primacy

States." 

Action

Under 40 CFR 142.10(b) (3), if a State does not perform all analytical

measurements in its own laboratory, it must establish and maintain a program for

the certification of laboratories as a condition for receiving and maintaining

authority to administer the Safe Drinking Water Act in lieu of EPA (primacy).

This memorandum notifies States with primacy that they may contract with other

organizations (third parties) to assist the State in fulfilling this requirement.

The authority for making certification decisions however, must remain with the

State.

Discussion

Several States have asked USEPA its position on the use of third-parties, i.e.,

private sector organizations which assist the States with their certification

program. OGWDW realizes that dwindling State resources may necessitate assistance

from third-parties in the State certification programs. Consistent with the

regulatory requirement at 40 CFR 142.10(b), providing for the "establishment and

maintenance of a State program for the certification of laboratories," the State

must retain ultimate authority to decide whether individual laboratories will be

certified: this decision may not be abdicated to the third party.

This Office will not pass judgment on any specific third-party program. It is

the responsibility of each primacy State to assess the qualifications of the

third-party. In assessing whether to choose a particular third-party, the State

should consider, as a minimum, the following items which are described in the
D-2

Manual for the Certification of Laboratories Analyzing Drinking Water:

o Ability to provide technical assistance and training

o Availability of records for review by the State

o Quality assurance program

o Freedom from conflicts of interest

o EPA policy, which provides that the auditor should pass an

appropriate course on how to audit in the discipline for which he or

she will be auditing.

o Experience of the auditor. 

The auditor should be an experienced professional with at least

a bachelor's degree or equivalent education/experience in the

discipline for which he or she audits.

The auditor should have recent laboratory experience

Any State certification program using third party assistance should meet the

requirements in the Manual for the Certification of Laboratories Analyzing

Drinking Water just as it would if it were using State employees to perform these

functions. The Regions should assist the State and third-party agent to assure

that the certification program meets EPA guidelines. 

Regions and States should be sensitive to potential conflict-of-interest problems

between a third-parties and evaluated laboratories. For instance, inspectors

employed by firms that provide analytical services in the drinking water area

should not be put in the position of passing judgement on their competitors.

Further Information 

If you have questions or need additional information or assistance, please

contact the OGWDW Technical Support Center at 513-569-7904.
E-1

Appendix E

Required Analytical Capability for Principal State Laboratory Systems

INORGANICS

(40 CFR 141.23)

Asbestos

Cyanide

Fluoride

Nitrate

Nitrite

Antimony

Arsenic

Barium

Beryllium

Cadmium

Chromium

Mercury

Selenium

Thallium

(40 CFR 141.89)

Copper

Lead

Conductivity

Calcium

Alkalinity

Orthophosphate

Silica

VOLATILE ORGANICS

(40 CFR 141.24)

THMs

Benzene

Carbon tetrachloride

Chlorobenzene

o-Dichlorobenzene

p-Dichlorobenzene

1,2-Dichloroethane

1,1-Dichloroethylene

cis-1,2-Dichloroethylene

trans-1,2-Dichloroethylene

Dichloromethane

1,2-Dichloropropane

Ethylbenzene

Styrene

Tetrachloroethene

Toluene

1,2,4-Trichlorobenzene

1,1,1-Trichloroethane

1,1,2-Trichloroethane

Trichloroethene

Vinyl chloride

Xylenes

DBPs

HAA5

Bromate

Chlorite

SOCs

(40 CFR 141.24)

Alachlor

Atrazine

Benzo(a)pyrene

Carbofuran

Chlordane

2,4-D

Di(2-ethylhexyl)adipate

Di(2-ethylhexyl)phthalate

Dibromochloropropane

Dalapon

Dinoseb

Dioxin (2,3,7,8-TCDD)

Diquat

Endothall

Endrin

Ethylenedibromide

Glyphosate

Heptachlor

Heptachlor epoxide

Hexachlorobenzene

Hexachlorocyclopentadiene

Lindane

Methoxychlor

Oxamyl

PCBs (as decachlorobiphenyl)

Pentachlorophenol

Picloram

Simazine

Toxaphene

2,4,5-TP

RADIONUCLIDES

(40 CFR 141.25)

Gross Alpha

 Uranium

Gross Beta 

 Cesium-134

 Strontium-89

 Iodine-131

 Strontium-90

 Tritium

 Other beta/photon emitters

Radium-226/228

M ICROORGANISM S

(40 CFR 141.21)

Total coliforms

Escherichia coli or fecal

coliforms

Heterotrophic bacteria
F-1

Appendix F
F-2
G-1

Appendix G

Analytical Methods for Microbiology

Note: Information in brackets is not yet included in the Code of Federal Regulations 

1. Total Coliform Rule (40 CFR 141.21(f))

(f) Analytical methodology. 

 (1) The standard sample volume required for total coliform analysis, regardless of analytical method used, is 100 mL.

 (2) Public water systems need only determine the presence or absence of total coliforms; a determination of total

coliform density is not required.

(3) Public water systems must conduct total coliform analyses in accordance with one of the analytical methods in the

following table. 

Organisms Methodology Citation 12 1

Total

Coliforms2

Total Coliform Fermentation Technique .................................

 3,4,5

Total Coliform Membrane Filter Technique ...............................

6

Presence-Absence (P-A) Coliform Test .....................................

5,7 

ONPG-MUG Test ........................................................................ 

8

Colisure Test9

E*Colite Test ® 10

m-ColiBlue24 Test ® 11 

Readycult Coliforms 100 ® 13

Membrane Filter Technique using Chromocult Coliform Agar ® 14

Colitag Test15

9221 A, B 

9222 A, B, C 

9221 D 

9223 

 

The procedures shall be done in accordance with the documents listed below. The incorporation by reference of the

following documents listed in footnotes 1, 6, 8, 9, 10, 11, 13 and 14 was approved by the Director of the Federal Register

in accordance with 5 U.S.C. 552(a) and 1 CFR Part 51. Copies of the documents may be obtained from the sources listed

below. Information regarding obtaining these documents can be obtained from the Safe Drinking Water Hotline at 800-

426-4791. Documents may be inspected at EPA’s Drinking Water Docket, 1301 Constitution Avenue, NW., EPA West,

Room B102, Washington, DC 20460 (Telephone 202-566-2426) or at the Office of Federal Register, 800 North Capitol

Street, NW, Suite 700, Washington, DC 20408.

Standard Methods for the Examination of Water and Wastewater, 18 edition (1992), 19 edition (1995), or 20 edition 1 th th th

(1998). American Public Health Association, 1015 Fifteenth Street NW, Washington, D.C. 20005. The cited methods

published in any of these three editions may be used.

The time from sample collection to initiation of analysis may not exceed 30 hours. Systems are encouraged but not 2

required to hold samples below 10 deg. C during transit.

Lactose broth, as commercially available, may be used in lieu of lauryl tryptose broth, if the system conducts at least 25 3

parallel tests between this medium and lauryl tryptose broth using the water normally tested, and this comparison

demonstrates that the false-positive rate and false-negative rate for total coliform[s], using lactose broth, is less than 10

percent.

If inverted tubes are used to detect gas production, the media should cover these tubes at least one-half to two-thirds after 4

the sample is added.

No requirement exists to run the completed phase on 10 percent of all total coliform-positive confirmed tubes. 5

MI agar also may be used. Preparation and use of MI agar is set forth in the article, ``New medium for the simultaneous 6

detection of total coliform[s] and Escherichia coli in water'' by Brenner, K.P., et al., 1993, Appl. Environ. Microbiol.

59:3534-3544. Also available from the Office of Water Resource Center (RC-4100T), 1200 Pennsylvania Avenue, NW,

Washington, DC 20460, EPA/600/J-99/225. Verification of colonies is not required.
G-2

 Six-times formulation strength may be used if the medium is filter-sterilized rather than autoclaved. 7

The ONPG-MUG Test is also known as the Autoanalysis Colilert System. 8 

A description of the Colisure Test, Feb 28, 1994, may be obtained from IDEXX Laboratories, Inc., One IDEXX Drive, 9

Westbrook, Maine 04092. The Colisure Test may be read after an incubation time of 24 hours.

A description of the E*Colite Test, ``Presence/Absence for Coliforms and E. Coli in Water,'' Dec 21, 1997, is available 10 ® 

from Charm Sciences, Inc., 36 Franklin Street, Malden, MA 02148-4120.

A description of the m-ColiBlue24 Test, Aug 17, 1999, is available from the Hach Company, 100 Dayton Avenue, 11 ®

Ames, IA 50010.

EPA strongly recommends that laboratories evaluate the false-positive and negative rates for the method(s) they use for 12

monitoring total coliforms. EPA also encourages laboratories to establish false-positive and false-negative rates within

their own laboratory and sample matrix (drinking water or source water) with the intent that if the method they choose

has an unacceptable false-positive or negative rate, another method can be used. The Agency suggests that laboratories

perform these studies on a minimum of 5% of all total coliform-positive samples, except for those methods where

verification/confirmation is already required, e.g., the M-Endo and LES Endo Membrane Filter Tests, Standard Total

Coliform Fermentation Technique, and Presence-Absence Coliform Test. Methods for establishing false-positive and

negative-rates may be based on lactose fermentation, the rapid test for $-galactosidase and cytochrome oxidase, multi-test

identification systems, or equivalent confirmation tests. False-positive and false- negative information is often available

in published studies and/or from the manufacturer(s).

 The Readycult® Coliforms 100 Presence/Absence Test is described in the document, “Readycult® Coliforms 100 13

Presence/Absence Test for Detection and Identification of Coliform Bacteria and Escherichia coli in Finished Waters,”

(November 2000, Version 1.0) and is available from EM Science [now EMD Chemicals, Inc.], an affiliate of Merck

KGgA, Darmstadt Germany), 480 S. Democrat Road, Gibbstown, NJ 08027-1297. Telephone number is (800) 222-0342,

E-Mail address is: adellenbusch@emscience.com. [E-Mail address is now adellenbusch@emdchemicals.com. Website

is www.emdchemicals.com]

 Membrane Filter Technique using Chromocult® Coliform Agar is described in the document, “Chromocult® Coliform 14

Agar Presence/Absence Membrane Filter Test Method for Identification of Coliform Bacteria and Escherichia coli in

Finished Waters,” November 2000, Version 1.0, available from EM Science [now EMD Chemicals, Inc.] (an affiliate of

Merck KGgA, Darmstadt Germany), 480 S. Democrat Road, Gibbstown, NJ 08027-1297. Telephone number is (800)

222-0342, E-Mail address is: adellenbusch@emscience.com. [E-Mail address is now adellenbusch@emdchemicals.com.

Website is www.emdchemicals.com]

Colitag® product for the determination of the presence/absence of total coliforms and E. coli is described in ``Colitag® 15

Product as a Test for Detection and Identification of Coliforms and E. coli Bacteria in Drinking Water and Source Water

as Required in National Primary Drinking Water Regulations,'' August 2001, available from CPI International, Inc., 5580

Skylane Blvd., Santa Rosa, CA, 95403, telephone (800) 878-7654, Fax (707) 545-7901, Internet address

http://www.cpiinternational.com. 

 (4) [Reserved]

 (5) Public water systems must conduct fecal coliform analysis in accordance with the following procedure. When the

MTF Technique or Presence-Absence (PA) Coliform Test is used to test for total coliforms, shake the lactose-positive

presumptive tube or P-A vigorously and transfer the growth with a sterile 3-mm loop or sterile applicator stick into

brilliant green lactose bile broth and EC medium to determine the presence of total and fecal coliforms, respectively. For

EPA-approved analytical methods which use a membrane filter, transfer the total coliform-positive culture by one of the

following methods: remove the membrane containing the total coliform colonies from the substrate with a sterile forceps

and carefully curl and insert the membrane into a tube of EC medium (the laboratory may first remove a small portion of

selected colonies for verification), swab the entire membrane filter surface with a sterile cotton swab and transfer the

inoculum to EC medium (do not leave the cotton swab in the EC medium), or inoculate individual total coliform-positive

colonies into EC Medium. Gently shake the inoculated tubes of EC medium to insure adequate mixing and incubate in

a waterbath at 44.5± 0.2° C for 24 ± 2 hours. Gas production of any amount in the inner fermentation tube of the EC

medium indicates a positive fecal coliform test. The preparation of EC medium is described in Method 9221E (paragraph

1a) in Standard Methods for the Examination of Water and Wastewater, 18th edition (1992), 19th edition (1995), and

20th edition (1998); the cited method in any one of these three editions may be used. Public water systems need only

determine the presence or absence of fecal coliforms; a determination of fecal coliform density is not required.

 (6) Public water systems must conduct analysis of Escherichia coli in accordance with one of the following analytical
G-3

methods:

 (i) EC medium supplemented with 50 μg/mL of 4-methylumbelliferyl-beta-D-glucuronide (MUG) (final

concentration), as described in Method 9222G in Standard Methods for the Examination of Water and Wastewater,19th

edition (1995) and 20th edition (1998). Either edition may be used. Alternatively, the 18th edition (1992) may be used

if at least 10 mL of EC medium, as described in paragraph (f)(5) of this section, is supplemented with 50 μg/mL of MUG

before autoclaving. The inner inverted fermentation tube may be omitted. If the 18 edition is used, apply the procedure th

in paragraph (f)(5) of this section for transferring a total coliform-positive culture to EC medium supplemented with MUG,

incubate the tube at 44.5 ± 0.2°C for 24 ± 2 hours, and then observe fluorescence with an ultraviolet light (366 nm) in the

dark. If fluorescence is visible, E. coli are present. 

 (ii) Nutrient agar supplemented with 100 μg/mL 4-methylumbelliferyl-beta-D-glucuronide (MUG) (final

concentration), as described in Method 9222G in Standard Methods for the Examination of Water and Wastewater,19th

edition (1995) and 20th edition (1998). Either edition may be used for determining if a total coliform-positive sample,

as determined by a membrane filter technique, contains E. coli. Alternatively, the 18 edition (1992) may be used if the th

membrane filter containing a total coliform-positive colony(ies) is transferred to nutrient agar, as described in Method

9221B (paragraph 3) of Standard Methods (18th edition), supplemented with100 μg/mL of MUG. If the 18th edition is

used, incubate the agar plate at 35°C for 4 hours and then observe the colony(ies) under ultraviolet light (366 nm) in the

dark for fluorescence. If fluorescence is visible, E. coli are present. 

 (iii) Minimal Medium ONPG-MUG (MMO-MUG) Test, as set forth in the article ``National Field Evaluation

of a Defined Substrate Method for the Simultaneous Detection of Total Coliforms and Escherichia coli from Drinking

Water: Comparison with Presence-Absence Techniques'' (Edberg et al.), Applied and Environmental Microbiology,

Volume 55, pp. 1003-1008, April 1989. (Note: The Autoanalysis Colilert System is an MMO-MUG test). 

If the MMO-MUG test is total coliform-positive after a 24-hour incubation, test the medium for fluorescence with a

366-nm ultraviolet light (preferably with a 6-watt lamp) in the dark. If fluorescence is observed, the sample is E.

coli-positive. If fluorescence is questionable (cannot be definitively read) after 24 hours incubation, incubate the culture

for an additional four hours (but not to exceed 28 hours total), and again test the medium for fluorescence. The

MMO-MUG 

Test with hepes buffer in lieu of phosphate buffer is the only approved formulation for the detection of E. coli.

 (iv) The Colisure Test. A description of the Colisure Test may be obtained from the Millipore Corporation,

Technical Services Department, 80 Ashby Road, Bedford, MA 01730. [Note: Manufacturer is now IDEXX Laboratories.

See footnote 9 to the table in paragraph (f)(3) of this section.]

 (v) The membrane filter method with MI agar, a description of which is cited in footnote 6 to the table in paragraph

(f)(3) of this section.

 (vi) E*Colite Test, a description of which is cited in footnote 10 to the table at paragraph (f)(3) of this section. ®

(vii) m-ColiBlue24 Test, a description of which is cited in footnote 11 to the table in paragraph (f)(3) of this ®

section.

 (viii) Readycult® Coliforms 100 Presence/Absence Test, a description of which is cited in footnote 13 to the table

at paragraph (f)(3) of this section.

 (ix) Membrane Filter Technique using Chromocult Coliform Agar, a description of which is cited in footnote ®

14 to the table at paragraph (f)(3) of this section.

 (7) As an option to paragraph (f)(6)(iii) of this section, a system with a total coliform-positive, MUG-negative,

MMO-MUG test may further analyze the culture for the presence of E. coli by transferring a 0.1 mL, 28-hour MMO-MUG

culture to EC Medium + MUG with a pipet. The 

formulation and incubation conditions of EC Medium + MUG, and observation of the results are described in paragraph

(f)(6)(i) of this section.
G-4

 (8) The following materials are incorporated by reference in this section with the approval of the Director of the

Federal Register in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies of the analytical methods cited in

Standard Methods for the Examination of Water and Wastewater (18th, 19th, and 20th editions) may be obtained from

the American Public Health Association et al.; 1015 Fifteenth Street NW., Washington, DC 200052605. Copies of the

MMO-MUG Test as set forth in the article ``National Field Evaluation of a Defined Substrate Method for the

Simultaneous Enumeration of Total Coliforms and Escherichia coli from Drinking Water: Comparison with the Standard

Multiple Tube Fermentation Method'' (Edberg et al.) may be obtained from the American Water Works Association

Research Foundation, 6666 West Quincy Avenue, Denver, CO 80235. A description of the Colisure Test may be obtained

from the Millipore Corp., Technical Services Department, 80 Ashby Road, Bedford, MA 01730 [Note: Now a description

of the Colisure Test may now be obtained from IDEXX Laboratories, Inc., One IDEXX Drive, Westbrook, Maine

04092.]. Copies may be inspected at EPA's Drinking Water Docket; 401 M St., SW.; Washington, DC 20460 [Note:

current location of EPA's Drinking Water Docket is 1301 Constitution Avenue, NW., EPA West, Room B102,

Washington, DC 20460], or at the Office of the Federal Register; 800 North Capitol Street, NW., Suite 700, Washington,

DC.

2. Surface Water Treatment Rule (40 CFR 141.74(a))

 (a) Analytical requirements. Only the analytical method(s) specified in this paragraph, or otherwise approved by EPA,

may be used to demonstrate compliance with §§141.71, 141.72 and 141.73. Measurements for pH, turbidity, temperature

and residual disinfectant 

concentrations must be conducted by a person approved by the State. Measurement for total coliforms, fecal coliforms

and HPC must be conducted by a laboratory certified by the State or EPA to do such analysis. Until laboratory

certification criteria are developed for the 

analysis of fecal coliforms and HPC, any laboratory certified for total coliforms analysis by the State or EPA is deemed

certified for fecal coliforms and HPC analysis. The following procedures shall be conducted in accordance with the

publications listed in the following section. 

This incorporation by reference was approved by the Director of the Federal Register in accordance with 5 U.S.C. 552(a)

and 1 CFR part 51. Copies of the methods published in Standard Methods for the Examination of Water and Wastewater

may be obtained from the American Public Health Association et al., 1015 Fifteenth Street, NW., Washington, DC 20005;

copies of the Minimal Medium ONPG-MUG Method as set forth in the article ``National Field Evaluation of a Defined

Substrate Method for the Simultaneous Enumeration of Total Coliforms and Escherichia coli from Drinking Water:

Comparison with the Standard Multiple Tube Fermentation Method'' (Edberg et al.), Applied and Environmental

Microbiology, Volume 

54, pp. 1595-1601, June 1988 (as amended under Erratum, Applied and Environmental Microbiology, Volume 54, p.

3197, December, 1988), may be obtained from the American Water Works Association Research Foundation, 6666 West

Quincy Avenue, Denver, Colorado, 80235; and copies of the Indigo Method as set forth in the article ``Determination of

Ozone in Water by the Indigo Method'' (Bader and Hoigne), may be obtained from Ozone Science & Engineering,

Pergamon Press Ltd., Fairview Park, Elmsford, New York 10523. Copies may be inspected at the U.S. Environmental

Protection Agency, Room EB15, 401 M St., SW., Washington, DC 20460 [Note: current location of EPA's Drinking

Water Docket is 1301 Constitution Avenue, NW., EPA West, Room B102, Washington, DC 20460;] or at the Office of

the Federal Register, 800 North Capitol Street, NW., suite 700, Washington, DC.

 (1) Public water systems must conduct analysis of pH and temperature in accordance with one of the methods listed

at §141.23(k)(1). Public water systems must conduct analysis of total coliforms, fecal coliforms, heterotrophic bacteria,

and turbidity in accordance with one of the following analytical methods and by using analytical test procedures contained

in Technical Notes on Drinking Water Methods, EPA-600/R-94-173, October 1994, which is available at NTIS

PB95-104766.
G-5

Organism Methodology Citation1

Total Coliform .............

 2

Fecal Coliforms ...........

2

Heterotrophic bacteria ..

2

Turbidity ....................... 

Total Coliform Fermentation Technique3, 4, 5

Total Coliform Membrane Filter Technique6

ONPG-MUG Test 7

Fecal Coliform Procedure8

Fecal Coliform Filter Procedure

Pour Plate Method

SimPlate11

Nephelometric Method

Nephelometric Method

Great Lakes Instruments

Hach FilterTrak

9221 A. B. C

9222 A, B, C

9223

9221 E

9222 D

9215 B

2130 B

180.19

Method 210

1013312

The procedures shall be done in accordance with the documents listed below. The incorporation by reference

of the following documents listed in footnotes 1, 6, 7, 9-12 was approved by the Director of the Federal Register in

accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies of the documents may be obtained from the sources listed

below. Information regarding obtaining these documents can be obtained from the Safe Drinking Water Hotline at

800-426-4791. Documents may be inspected at EPA's Drinking Water Docket, 1301 Constitution Avenue, NW., EPA

West, Room B102, Washington, DC 20460 (Telephone: 202-566-2426); or at the Office of the Federal Register, 800

North Capitol Street, NW, Suite 700,Washington, D.C. 20408.

Except where noted, all methods refer to Standard Methods for the Examination of Water and Wastewater, 18 edition 1 th

(1992), 19 edition (1995), or 20 edition (1998), American Public Health Association, 1015 Fifteenth Street, NW,

th th

Washington, D.C. 20005. The cited methods published in any of these three editions may be used.

The time from sample collection to initiation of analysis may not exceed 8 hours. Systems must hold samples below 10 2

deg. C during transit.

Lactose broth, as commercially available, may be used in lieu of lauryl tryptose broth, if the system conducts at least 25 3

parallel tests between this medium and lauryl tryptose broth using the water normally tested, and this comparison

demonstrates that the false- positive rate and false-negative rate for total coliform, using lactose broth, is less than 10

percent.

Media should cover inverted tubes at least one-half to two-thirds after the sample is added. 4

No requirement exists to run the completed phase on 10 percent of all total coliform-positive confirmed tubes. 5

MI agar also may be used. Preparation and use of MI agar is set forth in the article, ``New medium for the simultaneous 6

detection of total coliform[s] and Escherichia coli in water'' by Brenner, K.P., et al., 1993, Appl. Environ. Microbiol.

59:3534-3544. Also available from the Office of Water Resource Center (RC-4100T), 1200 Pennsylvania Ave., NW.,

Washington, DC 20460, EPA 600/J-99/225. Verification of colonies is not required.

The ONPG-MUG Test is also known as the Autoanalysis Colilert System. 7 

A-1 Broth may be held up to three months in a tightly closed screw cap tube at 4 deg. C. 8

``Methods for the Determination of Inorganic Substances in Environmental Samples'', EPA/600/R-93/100, August 1993. 9 

Available at NTIS, PB94-121811.

GLI Method 2, ``Turbidity'', November 2, 1992, Great Lakes Instruments, Inc., 8855 North 55th Street, Milwaukee,

10 

Wisconsin 53223.

 A description of the SimPlate method, “IDEXX SimPlate TM HPC Test Method for Heterotrophs in Water”, November 11

2000, can be obtained from IDEXX Laboratories, Inc., One IDEXX Drive, Westbrook, Maine 04092, telephone (800)

321-0207.

 A description of the Hach FilterTrak Method 10133, “Determination of Turbidity by Laser Nephelometry”, January 12

2000, Revision 2.0, can be obtained from Hach Co., P.O. Box 389, Loveland, Colorado 80539-0389. Phone: 800-227-

4224.

3. Ground Water Rule (to be added after rule promulgation)