Ground Water Rule Source Water 

Monitoring Methods Guidance 

Office of Water (4607M) 

EPA 815-R-07-019 

Revised March 2008 

Prepared for: 

U.S. Environmental Protection Agency 

Office of Ground Water and Drinking Water 

1200 Pennsylvania Ave, N.W. 

Washington, D.C. 20460 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Disclaimer 

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

reviewed and approved this guidance manual for publication. This report was prepared by the CSC Microbiology 

and Biochemistry Studies Group under subcontract to The Cadmus Group contract 68-C-02-026. Neither the 

United States Government nor any of its employees, contractors, or their employees make any warranty, expressed 

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

any information, apparatus, product, or process discussed in this guidance manual, or represents that its use by such 

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

constitute endorsement or recommendation for use. 

The statutory provisions and EPA regulations described in this document contain legally binding requirements. 

While EPA has made every effort to ensure the accuracy of the discussion in this guidance, the obligations of the 

regulated community are determined by statutes, regulations, or other legally binding requirements. In the event of 

a conflict between the discussion in this document and any statute or regulation, this document would not be 

controlling. 

Questions regarding this document should be addressed to: 

Michael Finn 

U.S. EPA Office of Ground Water and Drinking Water
 

Standards and Risk Management Division
 

1200 Pennsylvania Avenue, N.W. 4607M
 

Washington, DC 20460
 

Finn.Michael@epa.gov
 

202-564-5261
 

202-564-3767 (facsimile)
 

Acknowledgments 

American Water Works Association 

Association of Metropolitan Water Agencies 

Association of State Drinking Water Administrators 

Rick Danielson - Biovir Laboratories 

Carol F Smith - South Carolina Department of Health and Environmental Control 

Maureen Smith - Antelope Valley East Kern Water Agency 

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Table of Contents 

1. Introduction ..................................................................................................................................... 1-1

1.1 Background .......................................................................................................................... 1-1

1.2 Document Objectives and Organization ............................................................................... 1-2

2. Ground Water Rule (GWR) Summary and Source Water Monitoring Methods Requirements .......... 2-1

2.1 Ground Water Rule Summary .............................................................................................. 2-1

2.2 Source Water Monitoring Methods Requirements................................................................ 2-4

3. Basis for Ground Water Monitoring for Fecal Indicators ........................................................... 3-1

3.1 Indicators Evaluated ............................................................................................................. 3-1

3.2 Bacterial Indicators ............................................................................................................... 3-2

3.2.1 E. coli ................................................................................................................................... 3-2

3.2.2 Enterococci ........................................................................................................................... 3-2

3.3 Viral Indicators: Coliphage ................................................................................................... 3-3

4. Determining the Appropriate Fecal Indicator for Source Water Monitoring .......................... 4-1

4.1 Literature Overview .............................................................................................................. 4-1

4.2 Viral and Bacterial Transport ............................................................................................... 4-1

4.2.1 Sand Aquifers ....................................................................................................................... 4-2

4.2.2 High Population Density Combined with On-site Wastewater Treatment Systems. ............. 4-2

4.2.3 Other Aquifers ...................................................................................................................... 4-3

4.2.4 Non-Fecal Contamination and Proximity of Contamination to the Well .............................. 4-3

4.3 One Versus Two or More Fecal Indicators ........................................................................... 4-3

4.4 Tropical/Subtropical Environments ...................................................................................... 4-4

4.5 Use of Historical Source Water Monitoring Data ................................................................. 4-4

5. Collecting and Shipping Ground Water Samples ......................................................................... 5-1

5.1 Sample Collection Location and Monitoring Frequency ...................................................... 5-1

5.1.1 Triggered Source Water Monitoring ..................................................................................... 5-1

5.1.2 Assessment Source Water Monitoring .................................................................................. 5-2

5.2 Sample Containers and Volume ........................................................................................... 5-2

5.3 Shipping Regulations and Documentation ............................................................................ 5-3

5.4 Holding Time........................................................................................................................ 5-4

5.5 Holding Temperature and Temperature Monitoring ............................................................. 5-4

6. Understanding Ground Water Rule Fecal Indicator Methods .................................................... 6-1

6.1 Selection of Method Format ................................................................................................. 6-1

6.1.1 Presence/Absence ................................................................................................................. 6-1

6.1.2 Most Probable Number (MPN) ............................................................................................ 6-1

6.1.3 Direct Plating ........................................................................................................................ 6-2

6.2 E. coli Methods Approved for Ground Water Rule Monitoring ........................................... 6-2

6.2.1 Colilert (Standard Methods 9223) ........................................................................................ 6-2

6.2.2 Colisure® (Standard Methods 9223) ..................................................................................... 6-3

6.2.3 E*Colite ................................................................................................................................ 6-4

6.2.4 EC-MUG (Standard Methods 9221B/9221D 9221F) ....................................................... 6-4

6.2.5 NA-MUG (Standard Methods 9222B/9222C  9222G) ..................................................... 6-4

6.2.6 MI Medium (EPA Method 1604) ......................................................................................... 6-4
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6.2.7 m-ColiBlue24®

..................................................................................................................... 6-4

6.3 Enterococci Methods Approved for Ground Water Rule Monitoring ................................... 6-5

6.3.1 Multiple-Tube Technique (Standard Methods 9230B) ......................................................... 6-5

6.3.2 mE-EIA (Standard Methods 9230C) .................................................................................... 6-6

6.3.3 mEI (EPA Method 1600)...................................................................................................... 6-6

6.3.4 EnterolertTM (Budnick, G.E. et al., 1996) ............................................................................. 6-6

6.4 Coliphage Methods Approved for Ground Water Rule Monitoring ..................................... 6-7

6.4.1 Two-Step Enrichment (Method 1601) .................................................................................. 6-7

6.4.2 Single Agar Layer (SAL) (Method 1602) ............................................................................. 6-7

7. Evaluating Fecal Indicator Data .................................................................................................... 7-1

7.1 Data Recording at the Laboratory ......................................................................................... 7-1

7.1.1 Sample Identification Information ........................................................................................ 7-1

7.1.2 Primary Data ......................................................................................................................... 7-1

7.1.3 Sample Results ..................................................................................................................... 7-2

7.2 Data Archiving ..................................................................................................................... 7-2

7.3 Evaluating Fecal Indicator Data ............................................................................................ 7-2

7.3.1 Data Completeness Check .................................................................................................... 7-2

7.3.2 Evaluation of Data Against Method Quality Control Requirements ..................................... 7-3

8. Frequently Asked Questions ........................................................................................................... 8-1

8.1 Collecting and Shipping Ground Water Samples ................................................................. 8-1

8.2 Understanding Ground Water Rule Fecal Indicator Analyses .............................................. 8-1

8.3 Evaluating Fecal Indicator Data ............................................................................................ 8-2

9. References ........................................................................................................................................... 1
 

 

 

 

 

 

List of Exhibits 

Exhibit 2.1 Summary of System GWR Requirements................................................................ 2-3
 

Exhibit 6.1: E. coli Methods Approved for Use under the Ground Water Rule.......................... 6-3
 

Exhibit 6.2: Enterococci Methods Approved for Use under the GWR ....................................... 6-6
 

Exhibit 6.3 Coliphage Methods Approved for Use under the GWR ........................................... 6-7
 

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1. Introduction
 

1.1 Background 

The 1996 Amendments to the Safe Drinking Water Act (SDWA) required the United States 

Environmental Protection Agency (EPA) to promulgate a Ground Water Rule (GWR) that assures public 

health protection for people served by ground water sources. More than 147,000 public water systems in 

the United States use ground water as their primary water source (GWSs); these GWSs serve more than 

100 million people. Ground water occurrence studies and outbreak data show that pathogenic viruses and 

bacteria can occur in GWSs and that people may become ill due to exposure to contaminated ground 

water. Pathogens found in GWSs may include enteric viruses such as Echovirus, Coxsackieviruses, 

Hepatitis A and E, Rotavirus and Noroviruses (i.e., Norwalk-like viruses) and enteric bacterial pathogens 

such as Escherichia coli, Salmonella, Campylobacter and Shigella. Ingestion of these pathogens can cause 

gastroenteritis or serious illnesses such as hemolytic uremic syndrome, meningitis, hepatitis, or 

myocarditis. Health implications in sensitive subpopulations (e.g., children, elderly, immunocompromised) may be severe and may cause death. The primary goal of the GWR is to improve public 

health by identifying public ground water systems that are susceptible to fecal contamination and ensure 

that these systems take corrective action to eliminate the source of contamination or to remove or inactivate 

pathogens in the drinking water they provide to the public. 

The control of microbial contaminants in drinking water supplies using ground water is 

complicated, as there are a substantial number of microbial contaminants of concern and pathways of 

contamination, and no single approach for controlling pathogens is universally applicable. The risk of fecal 

contamination of ground water sources and the subsequent threat to public health is addressed under the 

GWR through implementation of a risk-targeted approach. This risk-targeted approach uses the following 

elements to identify and mitigate potential fecal contamination of ground water sources: 

•	 Periodic sanitary surveys of GWSs requiring the evaluation of eight critical elements and the 

identification of significant deficiencies (discussed in Section 2.1.1) 

•	 Triggered source water monitoring of systems that do not achieve 4-log inactivation or removal of 

viruses (discussed in Section 2.1.2) 

•	 Corrective actions to eliminate significant deficiencies and fecal contamination (discussed in 

Section 2.1.3) 

•	 Compliance monitoring to ensure that disinfection treatment for drinking water is reliably 

operated where it is used and achieves a 4-log inactivation or removal of viruses (discussed in 

Section 2.1.4) 

In addition, assessment source water monitoring is recommended for systems that are determined by 

the State to be high risk systems (discussed in Section 2.1.2). 

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1.2 Document Objectives and Organization 

The main objective of this document is to provide guidance on triggered and assessment source 

water monitoring issues such as selection of fecal indicators, sample collection and shipping, source water 

monitoring methods, laboratory quality assurance (QA) and quality control (QC), and evaluation of fecal 

indicator data. This manual also provides an overview of GWR requirements and includes frequently 

asked questions regarding source water monitoring. Several appendices provide example forms that may 

assist in performing the procedures described in this guidance manual. However, it is important to note that 

these forms are provided as guidance and are NOT required for compliance with GWR requirements. 

Regarding the selection of fecal indicators, this manual provides guidance, when possible, based 

on aquifer type, historical data, environmental elements, and whether assessment monitoring is required by 

the State. The guidance provided in this manual is definitive to the extent that the available literature 

provided sufficient data for EPA to evaluate and make recommendations on a national level. An overview 

of literature pertaining to the selection of fecal indicators is provided in Section 4.1. 

This document is organized into nine chapters and 10 appendices. A description of each 

remaining chapter and the appendices is provided below. 

Chapter 2—Ground Water Rule (GWR) Summary and Source Water Monitoring Methods 

Requirements: Summarizes GWR components and method requirements. 

Chapter 3—Basis for Ground Water Monitoring for Fecal Indicators: Discusses the rationale 

for indicator monitoring and describes the indicators considered and selected for GWR 

monitoring. 

Chapter 4—Determining the Appropriate Fecal Indicator for Source Water Monitoring: 

Provides guidance on determination of the most appropriate fecal indicator. 

Chapter 5—Collecting and Shipping Ground Water Samples: Provides guidance on GWR 

monitoring sample collection and shipping procedures. 

Chapter 6—Understanding Ground Water Rule Fecal Indicator Methods: Summarizes and 

discusses the analytical methods approved for use under the GWR. 

Chapter 7—Evaluating Fecal Indicator Data: Provides information on how to evaluate fecal 

indicator data including guidance regarding reporting, archiving, and evaluating data. 

Chapter 8—Frequently Asked Questions: Provides answers to frequently asked questions 

pertaining to GWR requirements, collecting and shipping samples, indicator analyses, and data 

evaluation. 

Chapter 9—References: Provides a list of the references cited within the manual. 

Appendix A—Glossary: Provides definitions, acronyms, and abbreviations cited within the 

manual. 

Appendix B—Procedure for Collecting Ground Water Samples for E. coli and Enterococci 

Analyses: Provides detailed sampling guidance for collection of E. coli and enterococci samples. 

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Appendix C—Procedure for Collecting Ground Water Samples for Coliphage Analyses: 

Provides detailed sampling guidance for collection of coliphage samples. 

Appendix D—E. coli Method Bench Sheets: Provides example bench sheets for the E. coli 

methods approved for use under the GWR. 

Appendix E—Enterococci Method Bench Sheets: Provides example bench sheets for the 

enterococci methods approved for use under the GWR. 

Appendix F—Coliphage Method Bench Sheets: Provides example bench sheets for the 

coliphage methods approved for use under the GWR. 

Appendix G—Ground Water Rule Source Water Monitoring Quality Control Checklist for 

Presence Absence and Most Probable Number E. coli or Enterococci Sample Results: 

Provides a checklist of quality control procedures and descriptions for the evaluation of E. coli and 

enterococci data from presence/absence or most probable number method formats. 

Appendix H—Ground Water Rule Source Water Monitoring Quality Control Checklist for 

Membrane Filtration E. coli or Enterococci Sample Results: Provides a checklist of quality 

control procedures and descriptions for the evaluation of E. coli and enterococci data from 

membrane filtration method formats. 

Appendix I—Ground Water Rule Source Water Monitoring Quality Control Checklist for 

Method 1601: Two-Step Enrichment Coliphage Sample Results: Provides a checklist of 

quality control procedures and descriptions for the evaluation of data from Method 1601. 

Appendix J—Ground Water Rule Source Water Monitoring Quality Control Checklist for 

Method 1602: Single Agar Layer (SAL) Coliphage Sample Results: Provides a checklist of 

quality control procedures and descriptions for the evaluation of data from Method 1602. 

1.3 Other Guidance Manuals Available 

Several additional guidance manuals are under development which EPA expects to be published to 

help water systems comply with the requirements of the Ground Water Rule. 

– Complying with the Ground Water Rule: Small Entity Compliance Guide (EPA 815-R-07­

018, July 2007) 

Consecutive System Guide for the Ground Water Rule (EPA 815-R-07-020, July 2007) 

Ground Water Rule Corrective Action Guidance Manual 

Ground Water Rule Source Water Assessment Guidance Manual

–

–

–

– Ground Water Rule Sanitary Survey Guidance Manual 

– The Ground Water Rule Implementation Guidance 

Further information about the status of these guidance documents is available on EPA’s 

website (http://www.epa.gov/safewater/disinfection/gwr/compliancehelp.html). 

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2. Ground Water Rule (GWR) Summary and Source Water 

Monitoring Methods Requirements 

The GWR applies to all public water systems that use ground water, except public water systems that 

combine all of their ground water with surface water or with ground water under the direct influence of surface 

water prior to treatment under subpart H (“Filtration and Disinfection”). The GWR also applies to consecutive 

systems receiving finished ground water. Ground water systems (GWSs) must comply, unless otherwise noted, 

with the GWR beginning December 1, 2009. 

This chapter provides an overview of the general requirements of the final ground water rule (2.1) and 

analytical method requirements (2.2). 

2.1 Ground Water Rule Summary 

The final GWR establishes a risk-targeted approach to target ground water systems that are susceptible to 

fecal contamination and then further target those systems that must take corrective action to protect public health. 

Key components of the GWR are: 

1. Sanitary surveys, 

2. Triggered source water monitoring, 

3. Corrective actions, and 

4. Compliance monitoring. 

Each of these components is discussed further below and Exhibit 2.1 provides a summary flowchart of the 

final GWR requirements. 

2.1.1 Sanitary Surveys 

The final GWR requires regular (every three years for CWSs and every five years for NCWSs) 

comprehensive sanitary surveys of 8 critical components: (1) source; (2) treatment; (3) distribution system; (4) 

finished water storage; (5) pumps, pump facilities, and controls; (6) monitoring and reporting, and data 

verification; (7) system management and operation; and (8) operator compliance with State requirements. The 

State may reduce the frequency of sanitary surveys for CWSs to at least once every five years if the water system 

has an outstanding performance record as determined by the State (e.g., no significant deficiencies documented in 

previous assessments and no history of total coliform MCL or monitoring violations under the Total Coliform Rule 

(TCR)) or the system maintains 4-log treatment of viruses using inactivation, removal, or State-approved 

combination of virus inactivation and removal. If a significant deficiency is identified, corrective action is required 

or a treatment technique violation is incurred. 

2.1.2 Source Water Monitoring 

In the final GWR, systems not achieving, or not performing compliance monitoring for, 4-log treatment of 

viruses (using inactivation, removal, or a State-approved combination of these technologies) must conduct 

triggered source water monitoring for the presence of at least one of the following fecal indicators: E. coli, 

enterococci, or somatic coliphage. The triggered monitoring requirements apply to systems that are notified that a 

TCR routine sample is total coliform-positive. Within 24 hours of receiving the total coliform-positive notice, 

GWSs must collect a source water sample and test it for the presence of a GWR fecal indicator. 
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If the State does not require corrective action (see Corrective Action section below) for the initial fecal 

indicator-positive source water sample immediately, the system must collect five additional source water samples 

within 24 hours of being notified of the initial fecal indicator-positive source water sample. The GWR requires 

systems to take corrective action if any of the five additional source water samples are fecal-indicator positive. 

The GWR provides States with the option to require systems to conduct assessment source water 

monitoring as needed and require systems to take corrective action for a fecal indicator-positive sample found 

during assessment monitoring. The purpose of this optional assessment source water monitoring requirement is to 

target source water monitoring to systems that the State determines are at risk for fecal contamination. 

2.1.3 Corrective Action 

The GWR requires that systems implement corrective action for; 

1) significant deficiencies, 

2) fecal-indicator positive samples if directed by the State after the initial fecal indicator-positive in 

triggered monitoring or for a fecal indicator-positive found during assessment monitoring, or 

3) a fecal indicator-positive sample in any of the five additional source water samples collected after the 

initial fecal indicator-positive source water sample during triggered monitoring. 

The system must implement at least one of the following corrective actions: correct all significant deficiencies; 

provide an alternate source of water; eliminate the source of contamination; or provide treatment that reliably 

achieves at least 4-log treatment of viruses. Furthermore, the system is required to notify the public served by the 

water system of any uncorrected significant deficiencies and/or source water contamination. (The State may also 

require notification of corrected significant deficiencies.) 

2.1.4 Compliance Monitoring 

Compliance monitoring requirements are the final defense against microbial contaminants provided by the 

final GWR. All GWSs that provide 4-log treatment of viruses, either as a corrective action or in lieu of GWR 

triggered source water monitoring, must conduct compliance monitoring to demonstrate continual treatment 

effectiveness. 
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Exhibit 2.1 Summary of System GWR Requirements 

All GWSs(1)

Initial and periodic sanitary surveys performed by the State

Community water systems (CWSs): every 3-5 years

Non-community water systems (NCWSs): every 5 years

Yes

Was

a sample fecal

indicatorpositive?

Complete or be in accordance 

with State-specified corrective 

action within 120 days of initial 

notification of contamination or 

significant deficiency

Conduct routine sampling 

under the Total Coliform Rule 

(TCR)

Implement State approved or specified corrective actions. 

Options include:

Eliminate source of contamination

Correct significant deficiency

Provide an alternate water source 

Provide treatment to achieve 4-log reduction of viruses

Continue State-required GWR 

compliance: sanitary surveys, 

triggered monitoring, TCR 

compliance, and 

assessment monitoring

No

Yes

Were any of the 5 

repeat samples 

positive?

No

Yes

Compliance monitoring – options include:

Serving 

>3,300 

people:

Continuously 

monitor 

residual 

disinfectant

Serving 

≤3,300 

people:

Monitor 

residual 

disinfectant 

daily via grab 

sample at 

peak flow

Chemical 

Disinfection

Alternative 

Treatment

Monitor the 

filtration 

process in 

accordance 

with Statespecified 

requirements

Membrane 

Filtration

Monitor the 

alternative 

treatment 

process in 

accordance 

with Statespecified 

requirements

No

Yes

No

Consult State 

within 30 days 

regarding appropriate 

corrective action, if 

necessary

Perform public notification 

and consult with the State 

within 24 hours.

Per State

direction, take 

corrective action or 

5 additional

samples

Was TCR

sample total 

coliform-positive?

Does system 

provide treatment 

≥4-logs(2)?

Did the 

State identify any 

significant 

deficiencies?(4)

Does the

State require 

assessment 

source water 

monitoring?

No

Was

a sample fecal

indicatorpositive?

Consult State within 30 days of notification regarding 

appropriate corrective action, if necessary

Yes

Does the State 

require corrective 

action?

Yes

No

Perform public notification 

and consult with the State 

within 24 hours.

Yes

Perform public notification 

and consult with the State 

within 24 hours

Yes

Conduct triggered 

source water 

monitoring(3)

No

No

Yes

Yes

(1) The GWR applies to all public water systems (PWSs) that use ground 

water, except public water systems that combine all of their ground water 

with surface water or with ground water under the direct influence of surface 

water prior to treatment. 

(2) Treatment using inactivation, removal, or State-approved combination to 

achieve a 4-log reduction of viruses before or at the first customer. Compliance 

monitoring required.

(3) If the State determines that the distribution system is deficient or causes total 

coliform-positive samples, the system may be exempted from triggered source 

water monitoring. 

(4) The State must provide the GWS with written notice describing any significant 

deficiencies within 30 days of identifying the significant deficiency. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2.2 Source Water Monitoring Methods Requirements 

Ground water systems conducting source water monitoring under the GWR must collect and analyze at 

least 100 mL of source water for one of three fecal indicators (E. coli, enterococci, or coliphage) using one of the 

following analytical methods: 

E. coli Methods: 

• Colilert (Standard Methods 9223 B) (APHA, 1998) 

• Colisure (Standard Methods 9223 B) (APHA, 1998) 

• Membrane Filter Method with MI Agar (EPA Method 1604) (USEPA 2002) 

• m-ColiBlue24 (Hach Company, Inc., Revision 2, 1999) 

• E*Colite Test (Charm Sciences, Inc., 1997) 

• EC-MUG (Standard Methods 9221 F) (APHA, 1998) 

• NA-MUG (Standard Methods 9222 G) (APHA, 1998) 

Enterococci Methods: 

• Multiple-Tube Technique - Azide Dextrose/BEA/BHI (Standard Methods 9230 B) (APHA, 1998) 

• Membrane Filter Technique with mE-EIA (Standard Methods 9230 C) (APHA, 1998) 

• Membrane Filter Technique with mEI Agar (EPA Method 1600) (USEPA 2006a) 

• Enterolert (Budnick, G.E. et al., 1996) 

Coliphage Methods: 

• Two-Step Enrichment Presence-Absence Procedure (EPA Method 1601) (USEPA 2001a) 

• Single Agar Layer Procedure (EPA Method 1602) (USEPA 2001b) 

Sample analysis must be initiated within 30 hours of sample collection for all analytical methods 

recognized by the GWR. Systems are encouraged but not required to hold samples below 10oC during transit. All 

analyses must be conducted by a laboratory certified by the State or EPA in accordance with specified analytical 

method requirements. Chapter 6 describes these methods in greater detail. 

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3. Basis for Ground Water Monitoring for Fecal Indicators 

Fecally contaminated ground water can be identified by monitoring for either pathogenic microorganisms 

or for non-pathogenic fecal indicator microorganisms whose presence suggests fecal contamination or a pathway 

for contamination and, therefore, the potential presence of pathogens. Monitoring for indicators is generally more 

practical than monitoring for actual pathogens. Only the more advanced water laboratories currently have the 

analytical capabilities to analyze water samples directly for pathogens. In addition, pathogen concentrations in 

water tend to be low, thereby requiring the analysis of larger sample volumes and increasing analytical costs; and 

many of the viruses associated with waterborne disease are either difficult or impossible to culture. For example, 

some viruses such as infectious norovirus and wild-type Hepatitis A virus are not culturable , while other viruses, 

such as enteroviruses, have variable, limited recovery and culturability. Some bacteria are also difficult to culture. 

Finally, laboratory analytical methods for fecal indicators are typically more widely available, more widely used, 

and significantly less expensive than methods for monitoring for individual enteric pathogens. 

Indicator data are important because illness can result from consuming ground water with fecal 

contamination in the absence of identified pathogens. EPA recognizes that any indicator organism may or may not 

co-occur with pathogens and that co-occurrence could be intermittent. 

The evaluation of fecal indicators for monitoring under the GWR is briefly discussed in Section 3.1, with 

more detail on the bacterial (E. coli and enterococci) and viral (coliphage) fecal indicators being provided in 

Sections 3.2 and 3.3, respectively. 

3.1 Indicators Evaluated 

Prior to proposal of the GWR, EPA carefully evaluated the existing scientific literature to identify the most 

appropriate indicators of fecal contamination in ground water. EPA considered a number of issues including, but 

not limited to, distribution, transport, and fate of fecal organisms in ground water (Pedley et al., 2006, DeBorde et 

al., 1998 and 1999). Detailed information on the scientific literature reviewed in support of fecal indicator 

selection for the rule can be found in the Occurrence and Monitoring Document for the Ground Water Rule 

(USEPA, 2006b). The organisms selected as the most reliable indicators of fecal contamination in ground water 

were E. coli, enterococci, and coliphage (male-specific and somatic). Other organisms that were considered for use 

as indicators are discussed in the bacterial (Section 3.2) and viral (Section 3.3) indicator sections below. For 

source water monitoring, the final GWR requires the use of E. coli, enterococci, or coliphage as fecal indicators in 

ground water based on the following: 

•	 E. coli, enterococci, and coliphage are closely associated with recent fecal contamination. 

•	 E. coli, enterococci, and coliphage are frequently found, sometimes in high concentrations, in sewage and 

septage. 

•	 E. coli, enterococci, or coliphage presence implies that other fecal pathogens (including enteric viruses) or 

a pathway for fecal pathogens could be present. 

•	 E. coli, enterococci, and coliphage are present in higher concentrations than other fecal pathogens 

(including enteric viruses), and are therefore easier to detect. 

•	 Approved analytical methods for E. coli, enterococci, and coliphage are simple, reliable, and inexpensive. 

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•	 The TCR allows for E. coli monitoring, therefore many laboratories are familiar with the methods used for 

the detection of E. coli. 

•	 Enterococci are recommended as indicators for fecally contaminated recreational waters and are 

commonly used as fecal indicators. 

3.2 Bacterial Indicators 

Bacteria that have been used as indicators of fecal contamination include the total coliform bacterial 

species, many of which are free-living in the environment, and fecal bacteria including E. coli. Fecal coliforms are 

coliform bacteria found in animal feces. Because total coliform bacteria are primarily free-living in the 

environment, when identified at the tap they are considered to be indicators of chlorine demand and distribution 

system contamination, as well as possible fecal contamination of source water. When total coliforms are identified 

in source ground water they may be indicators of surface or near-surface inflow to ground water as well as possible 

fecal contamination. However, they may not be representative of fecal contamination specifically, and therefore 

are not considered an appropriate fecal indicator for source water monitoring. Other bacteria that are used as 

indicators of fecal contamination include enterococci and Clostridium perfringens, a spore-forming anaerobic 

organism. Some indicator bacteria have specialized uses. For example, heterotrophic plate count (HPC) bacteria 

may be used to track treatment efficiency, and Bacillus is used as an indicator of surface or near-surface water 

inflow to ground water (Rice et al, 1996). Both HPC and total coliform bacteria are used to identify the presence 

of biofilm or other distribution system problems (Geldreich 1996, Carter et al. 2000). 

As indicated above, E. coli (Section 3.2.1) and enterococci (Section 3.2.2) were selected for use as 

bacterial indicators of fecal contamination. 

3.2.1 E. coli 

E. coli bacteria are a subgroup of the coliform group that can be found in high numbers in the intestines 

and feces of warm-blooded animals. E. coli is considered the most appropriate group of the coliform bacteria to 

indicate fecal contamination and the possible presence of enteric pathogens because it is generally believed that 

there are no significant non-fecal sources of E. coli, and E. coli generally does not grow extensively in the 

environment. However, exceptions have been reported in areas with little human impact and in warm, moist 

tropical/subtropical environments where E. coli has been shown to be part of the normal soil environment and grow 

in both soil and surface water (Hardina and Fujioka, 1991, Jimenez et al, 1989, and Rivera et al., 1988). This issue 

is discussed further in Section 4 in the context of selecting the most appropriate indicator for ground water 

monitoring. 

3.2.2 Enterococci 

Enterococci bacteria initially were a subgroup of fecal streptococci and consist of several species of 

bacteria in the genus Streptococcus. The current taxonomic approach separates enterococci into a separate genus 

(Hardie and Whiley, 1997). Enterococci are commonly found in relatively high numbers in the feces of humans 

and other warm-blooded animals. Although some strains are ubiquitous and not related to fecal contamination, the 

presence of enterococci in water is an indication of fecal contamination and the possible presence of enteric 

pathogens. Epidemiological studies conducted in fresh and marine waters have demonstrated there is a direct 

relationship between the density of enterococci and the risk of gastrointestinal illness associated with swimming 

(Cabelli, 1979). The risk would also be applicable to drinking water sources. 

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3.3 Viral Indicators: Coliphage 

Bacteriophages (“phages”) are viruses that infect bacteria. They can replicate only in a living host 

bacterial cell. While some phages are considered to be indicators of human enteric viruses (Curry 1999; Grabow 

2001), some are more frequently associated with fecal contamination than others. Feces-specific bacteriophages 

evaluated for potential use under the GWR included somatic coliphage, male-specific coliphage, and Bacteroides 

fragilis bacteriophages. Bacteriophages that infect and replicate in Bacteroides fragilis were determined not to be 

a useful fecal indicator because counts are usually low in United States waters. Coliphage are a group of 

bacteriophages that infect and replicate predominantly in E. coli and are considered to be indicators of human 

enteric viruses (Curry 1999; Grabow 2001). The two types of coliphage, somatic and male-specific, differ in the 

mechanism by which they infect host bacterial cells. 

•	 Somatic coliphage are viruses that infect host cells (E. coli) via receptors on the outer cell membrane. 

The majority of the somatic coliphage detected in water are host-specific, i.e., they can only replicate 

in E. coli. However, under certain conditions, closely related bacterial species may support the growth 

of somatic coliphage in water environments. 

•	 Male-specific coliphage (also referred to as FRNA coliphage) are viruses that infect host cells (E. coli) 

via the receptor sites on the F-pilus, a minute “fiber-like” structure produced by some bacteria for the 

exchange of genetic material. F-pili are only produced by actively growing bacteria under optimal 

conditions, usually at temperatures near 30°C or higher. This characteristic is important because in 

environmental waters, conditions rarely exist for the replication of male-specific coliphage. For all 

practical purposes, it is highly unlikely that male-specific coliphage can replicate in ground water 

environments (typically 8°C to 16°C). 

Conditions for coliphage replication typically exist in the gastrointestinal tract of humans and other warm 

blooded animals. Since human enteric viruses are released into the environment almost exclusively through human 

feces, coliphage reflect the potential origin and release of human viruses. Coliphage may be better indicators of 

fecal contamination than bacterial fecal indicators when viruses are the most likely pathogen of concern. The GWR 

specifically authorizes the use of coliphage as viral fecal indicators in ground water for the following reasons: 

•	 Coliphage more closely resemble human enteric viruses in shape, size, morphology, and composition 

(Grabow 2001). 

•	 Coliphage respond to water treatment and natural environments similarly to human enteric viruses. 

•	 Coliphage may be similar to the enteric viruses in transport efficiency through soil and aquifer materials 

due to similar size and shape. 

•	 Coliphage generally do not infect non-fecal bacteria and it is unlikely that they can reproduce in water 

environments due to strict conditions (e.g., temperature, log phase growth of their hosts) needed for 

replication (Grabow 2001). 

The process of selection by a State of the most appropriate source water monitoring indicator from those 

described above will take into consideration the chemical and physical elements of that state’s environment, as 

discussed in Chapter 4 of this document. 

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4.	 Determining the Appropriate Fecal Indicator for 

Source Water Monitoring 

A State’s selection of the most appropriate indicator(s) should consider factors such as aquifer type, 

historical water sampling data of the region, climate and other environmental factors. Based on available data, this 

chapter provides guidance regarding these factors, discussed in the following sections: Viral and Bacterial 

Transport (Section 4.2); Use of Multiple Fecal Indicators (Section 4.3); Tropical/Subtropical Environments 

(Section 4.4); Use of Historical Data (Section 4.5); and Coliphage Matrix Spike Sample Results (Section 4.6). 

4.1 Literature Overview 

As indicated in the USEPA Occurrence and Monitoring Document for the Final Ground Water Rule 

(USEPA, 2006b), EPA does not have a single preferred choice of indicator because no single indicator can 

definitively determine whether pathogens are present. With regard to occurrence, studies reviewed by the Agency 

observed both bacterial and viral indicators in various ground water systems surveyed. Some researchers observed 

that bacteria are more prevalent in ground water (e.g., Doherty, 1998; Francy et al., 2004; Femmer et al., 2000), 

while other researchers recovered viruses more frequently (e.g., Atherholt et al., 2003; Davis and Witt, 2000). 

In a comprehensive review of the current knowledge about distribution of pathogens in ground water and 

the factors that control their transport and attenuation, Pedley et al. (2006) provide information on both waterborne 

pathogens and indicator organisms, and describe the impact that system characteristics may have on the survival 

and migration of microorganisms in the subsurface. According to Pedley et al. (2006), while the current 

knowledge offers a number of guiding principles about the transport and attenuation of pathogens in ground water, 

the complex interaction of factors controlling the fate of pathogens is poorly understood and difficult to predict in 

some environments. The literature suggests that the properties of a ground water system that affect the transport 

and/or attenuation of microorganisms include but are not limited to: hydrogeologic conditions; flow mechanisms; 

light; temperature; pH; and soil properties, including moisture content, organic matter, iron content, nutrient 

content, and salt concentration. Additionally, characteristics of the microorganism, such as type, size, aggregation, 

microbial activity, heterogeneity of the population, predation and/or antagonism, and potential association of the 

microorganism with soil, will affect transport and attenuation (Pedley et al., 2006; Foppen and Schijven, 2006; 

USEPA, 2006b). 

While the available literature is variable and dependent on the ground water system, some factors may 

warrant the use of a specific indicator. Guidance provided in subsequent sections of this chapter is prescriptive to 

the extent that the available literature provides sufficient data to evaluate and make recommendations on a national 

level. Specific literature associated with these recommendations is cited below. For additional information on the 

current knowledge regarding pathogens in ground water, refer to Pedley et al. (2006) and to the Occurrence and 

Monitoring Document for the Final Ground Water Rule (USEPA, 2006b), which includes a review of the literature 

considered in development of the final GWR. 

4.2 Viral and Bacterial Transport 

Aquifers are broadly classified into two categories; porous and non-porous. In this document, non-porous 

aquifers (e.g., karst limestone, fractured igneous or metamorphic rock aquifers) as well as gravel aquifers are 

defined as sensitive aquifers. Non-porous aquifers are those that transmit ground water through large, well 

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connected openings such as fractures, solution enhanced fractures, conduits or caverns. The Source Assessment 

Guidance Manual provides additional explanation on what constitutes a sensitive aquifer and how best to identify 

one, and identifies pathways to obtained additional hydrogeologic information. 

Among the remaining (excluding gravel) porous aquifers (e.g., sand, or sand and gravel, aquifers), sand 

and gravel aquifers more efficiently transmit fecal contaminants than sand aquifers because average ground water 

velocity is higher. In general, for porous media aquifers the greater the grain size or grain size heterogenity, the 

more efficiently pathogenic microorganisms pass through the aquifer. 

All subsurface particles, including microbes, may be transported by flowing ground water. Particles may 

be removed from flow or be retarded. That is, they may permanently or temporarily become associated with the 

solid aquifer materials in either porous or non-porous aquifers. Microbial transport in porous media aquifers is an 

active research area and consensus is difficult in many issues in this field. It is generally agreed that microbe size is 

an important element in determining mobility in porous media, although many other factors, such as surface 

charge, may also have significant influence. Given the importance of microbe size, the significant (one-thousand 

fold) size difference between viruses (measured in nanometers) and bacteria (measured in micrometers) increases 

the likelihood that an infectious virus, rather than an infectious bacterium, will reach a GWS well in a porous 

aquifer. 

4.2.1 Sand Aquifers 

The thousand-fold size difference between viruses and bacteria may be particularly significant in sand 

aquifers for two reasons: 1) Viruses are less likely to be subject to removal or retardation at pore margins by 

straining, wedging, or micro-straining; and 2) viruses may be more likely to be excluded from the smaller pores 

where ground water velocities are slower. As a result of this pore-size exclusion (which is due indirectly to size 

because charge effects predominate for smaller particles), viruses may be favored over bacteria because the viruses 

remain in faster flowing ground water for longer periods. As the result of straining and pore-size exclusion, sand 

aquifers may facilitate virus transport as compared with bacterial transport. All other factors such as average 

ground water velocity being equal, this manual assumes that infectious viruses are more likely than infectious 

bacteria to be found in GWS well source water in sand aquifers because the viruses are smaller and thus more 

mobile in the subsurface. Some sand aquifers appear to more efficiently transmit viruses as compared with 

bacteria, thus, if targeted for assessment source water monitoring, sand aquifers should be monitored using 

coliphage rather than E. coli or enterococci. 

4.2.2 High Population Density Combined with On-site Wastewater Treatment Systems. 

Each aquifer has some risk that at any site, the natural attenuation capability may be overwhelmed by a 

combination of large wastewater discharge to the subsurface, high rate pumping and reduced recharge (and 

dilution). For example, areas with a high density population, using septic tanks and other on-site wastewater 

treatment systems discharge fecal contamination to the subsurface combined with restricted areal extent of an 

aquifer is an especially risky combination. This is because aquifer recharge by septage discharge in such 

environments is significant as compared to infiltrating precipitation. Some aquifers, such as barrier island or 

marine island aquifers, are capable of supplying only limited yield because over-pumping will result in seawater 

intrusion, permanently damaging the aquifer. Where population density is high and yield is limited, dilution and 

other natural attenuation processes are limited and fecal contamination is more likely. Barrier island aquifers are 

typically sand aquifers and, like all sand aquifers, may be more susceptible to viral rather than bacterial 

contamination. If targeted for additional monitoring, barrier island sand aquifers should be monitored using 

coliphage rather than E. coli or enterococci. 

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4.2.3 Other Aquifers 

In other aquifers, such as non-porous aquifers (e.g., fractured igneous or metamorphic rock aquifers) and 

gravel aquifers, average ground water velocities are exceptionally fast, and straining and pore-size exclusion are 

much less significant and bacteria and viruses are assumed to travel at equal rates. In general, straining and poresize exclusion effects are more significant in sand aquifers than in sand and gravel aquifers. In sand aquifers, 

ground water velocity is moderate because mean grain size is moderate. As ground water velocities increase 

because of increasing gravel content or increasing proximity to a pumping well, the differences between virus and 

bacterial transport efficiency become less important, and either a viral or bacterial indicator may be recommended. 

On the other hand, ground water velocities through the finest grained porous aquifers, such as shale and 

clay beds, are generally very slow. Because ground water velocity is very slow, these aquifers do not readily 

produce water in quantities sufficient to supply a PWS and thus are not considered further in this guidance. 

4.2.4 Non-Fecal Contamination and Proximity of Contamination to the Well 

Some microbial pathogens such as Legionella pneumophila (Costa et al., 2005; Riffard et al., 

2004), Helicobacter pylori, Naegleria fowlerii (Blair and Gerba, 2006), and perhaps Toxoplasma gondii 

(Sroka et al, 2006) are not associated with fecal contamination and, instead, may be resident members of 

aquifer ecosystems. For these microbes, transport from the surface or near surface is not an important risk 

element because the microbes can colonize the well gravel pack or the aquifer immediately surrounding the 

gravel pack. In these instances, the bacterial versus virus size difference and associated subsurface 

mobility differences become much less important. 

4.3 One Versus Two or More Fecal Indicators 

Although EPA’s Science Advisory Board (SAB) and the National Drinking Water Advisory Council 

(NDWAC) recommended that systems should monitor for coliphage and either E. coli or enterococci for source 

water monitoring, the available data did not provide for evaluation of such a measure on a national level. While 

coliphage data are available for many of the occurrence studies used to estimate national occurrence for E. coli, the 

methods used to measure coliphage were often based on high volume analysis and a variety of methods different 

than those specified under the final GWR. Thus, EPA could not determine whether the SAB/NDWAC proposal 

would provide additional effectiveness. Furthermore, EPA was concerned with the increase in sampling burden 

and cost relative to the additional number of fecally contaminated wells that would be identified using two 

indicators compared to the use of one indicator. Therefore, based on the data available, EPA requires all GWSs to 

monitor for a single fecal indicator under the final GWR, but encourages States to consider the use of multiple 

indicators where a net benefit seems likely. 

Pathogen and indicator occurrence in wells is intermittent. However, when indicators occur frequently in 

source water samples, then sampling for one fecal indicator is sufficient. When indicator occurrence is rare, then 

sampling for multiple fecal indicators may be more likely to recognize fecal contamination. For example, a well in 

Oregon (Lieberman et al, 2002, well number 31) was enteric virus positive in four of twelve monthly samples. It 

was also E. coli positive in six of twelve monthly samples. Thus, for this well E. coli sampling only is probably 

sufficient to indicate the fecal contamination hazard. In contrast, a well in North Carolina (Lieberman et al, 2002 

well number 99) was enteric virus positive in one of twelve monthly samples. None of the twelve E. coli samples 

were positive but one of the twelve enterococci and one of the twelve male-specific coliphage samples were 

positive. In this site with infrequent pathogen and indicator occurrence, assaying for multiple indicators will 

increase the likelihood that the well is identified as fecally contaminated. 

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4.4 Tropical/Subtropical Environments 

The GWR-approved bacterial indicators of fecal contamination, E. coli and enterococci, may not be 

reliable for assessing fecal contamination in tropical environments. There is a growing consensus among 

researchers that: (1) soil, sediments, water, and plants may be indigenous sources of E. coli and enterococci in 

tropical waters; (2) fecal indicators can multiply and persist in soil, sediment and water in some tropical and 

subtropical environments; and (3) tropical environments change the relationship between the presence of the 

indicators and potential health effects. 

The current literature suggests that E. coli and enterococci can be found in soil and surface waters of 

tropical environments in the absence of warm-blooded animals and, therefore, are endemic in tropical ecosystems 

and not simply indicators of fecal origin (Hardina and Fujioka, 1991; Rivera et al., 1988). In a study of Hawaiian 

freshwater streams, Hardina and Fujioka (1991) observed that E. coli was capable of replicating in stream water 

samples and that both E. coli and enterococci were present in soil samples free of fecal contamination. In 

subtropical environments, such as Florida, E. coli has been shown to grow in soil with high moisture content, 

(Solo-Gabrielle et al., 2000). As a result, positive bacterial indicator results for wells in these environments may 

not be due to fecal contamination. 

There are also non-fecal species of enterococci, such as Enterococcus casseliflavus that may grow in 

environmental settings (Niemi et al., 1993). Environmental proliferation of non-fecal enterococci further 

complicates the assessment of fecal contamination when detecting enterococci. Research is still needed to better 

define the parameters such as soil, nutrients, moisture, temperature, time of year, and latitudes that may promote 

natural proliferation of fecal indicator bacteria in the soil. 

Based on the literature, it appears that the presence of E. coli or enterococci from a ground water source in 

a tropical or subtropical ecosystem may not always be indicative of fecal contamination. As a result, selection of 

coliphage as an indicator in these environments merits consideration. However, as noted in Section 4.2 above, 

some microbial pathogens (e.g., Legionella pneumophila, Helicobacter pylori) are not associated with fecal 

contamination, and instead may be resident members of aquifer ecosystems. As such, detection of endemic, nonfecal E. coli and enterococci would not necessarily confirm that water from that source is safe. 

4.5 Use of Historical Source Water Monitoring Data 

Some GWSs may have historical source water data for total coliforms, E. coli, enterococci, or coliphage. 

Although historical data is unlikely to be useful in determining whether an indicator is appropriate, it may help 

determine that an indicator is not appropriate. With regard to fecal indicator selection, the following considerations 

may be useful regarding historical source water data: 

•	 If a GWS has a history of negative source water results for one of the GWR-approved indicators (E. 

coli, enterococci, or coliphage), the State may consider requiring that an alternate fecal indicator be 

selected for GWR monitoring. 

•	 A GWS may have a history of total coliform-negative source water samples, indicating that E. coli (a 

subset of total coliforms) is most likely not present at detectable levels in the source water. Since the 

GWS is unlikely to detect E. coli under GWR monitoring, another indicator may be more appropriate. 

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• A GWS may have a history of total coliform-positive source water samples that are not positive for E. 

coli, where data indicate that although coliform bacteria are present at detectable levels at the sampling 

location, E. coli are not present, or are present at levels below detection. Because the GWS is unlikely 

to detect E. coli under GWR monitoring, another indicator may be more appropriate. 

 
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5. Collecting and Shipping Ground Water Samples 

The GWR requires systems to collect source water samples and analyze for the presence of GWR fecal 

indicator(s) through triggered, and in some cases assessment, monitoring. This section provides an overview of 

recommendations for sample location and monitoring frequency (Section 5.1), sample containers and volume 

(Section 5.2), shipping regulations and documentation (Section 5.3), holding time (Section 5.4), and holding 

temperature and temperature monitoring (Section 5.5). Detailed sample collection protocols and sample packing 

and shipping procedures are provided in Appendix B for E. coli and enterococci samples, and Appendix C for 

coliphage samples. 

5.1 Sample Collection Location and Monitoring Frequency 

As indicated in Section 2.2.5, ground water samples used for triggered or assessment monitoring must be 

collected at a location prior to any treatment of the ground water source, unless the State approves a sampling 

location after treatment. If the system’s configuration does not allow for sampling at the well itself, the system may 

collect a sample at a State-approved location, if the sample is representative of the water quality of that well. 

With State approval, a system that uses more than one source of ground water may conduct source water 

monitoring at a representative ground water source or a subset of sources. Sample collection location and 

monitoring frequency information specific to triggered and assessment source water monitoring are detailed in 

Sections 5.1.1 and 5.1.2, respectively. 

5.1.1 Triggered Source Water Monitoring 

If a routine sample collected in accordance with §141.21(a) (TCR) is total coliform-positive a GWS that 

does not provide 4-log treatment of virus, as determined by the State, for its ground water source(s) must conduct 

triggered source water monitoring within 24 hours of receiving notification. A GWS is not required to comply 

with triggered source water monitoring if the cause of the total coliform-positive sample directly relates to the 

distribution system, as determined by the State. The GWS must collect at least one ground water source sample 

from each ground water source in use at the time the total coliform-positive sample was collected and test for E. 

coli, enterococci, or coliphage. The State may extend the 24-hour limit for triggered source water monitoring on a 

case-by-case basis, if the State determines that the system cannot collect the ground water source sample within 24 

hours due to circumstances beyond its control. If the State approves the use of E. coli as a fecal indicator for 

triggered source water monitoring, GWSs serving 1,000 people or fewer may use a TCR repeat sample collected 

from a ground water source to simultaneously meet the requirements of the TCR and satisfy the GWR’s triggered 

source water monitoring requirements for that ground water source only. 

 

If any initial triggered source water sample is fecal indicator-positive, the system must collect five 

additional water samples within 24 hours at the site, unless the State requires immediate corrective action to 

address contamination at that site. 

Consecutive and wholesale systems must comply with triggered source water monitoring provisions for 

their own sources. A consecutive GWS with a total coliform-positive sample must notify the wholesale system(s) 

within 24 hours of being notified of the test result. The wholesale system must, within 24 hours of notification, 

conduct triggered source water monitoring by collecting a sample from its ground water source(s) and analyzing for 

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5.1.2 Assessment Source Water Monitoring 

The GWR provides States with the option to require systems to conduct assessment source water 

monitoring at any time and require systems to take corrective action based on the results of these analyses. 

Assessment source water monitoring is not a requirement of the GWR but a recommended tool for States when 

targeting high-risk GWSs. States may identify high risk GWSs and require assessment source water monitoring 

based on information from hydrogeologic sensitivity assessments (HSAs), triggered monitoring results, or historical 

data from the system(s). 

If assessment monitoring is required, EPA recommends that States require collection of a minimum of 12 

ground water source samples that represent each month the system provides ground water to the public. Collection 

of samples from each well is also recommended, unless the system obtains written State approval to conduct 

monitoring at one or more wells within the GWS that are representative of multiple wells used by that system. 

5.2 Sample Containers and Volume 

Samples should be collected in sterile, plastic or glass containers with a leak-proof lid. The GWR requires 

GWSs conducting source water monitoring to analyze at least a 100-mL sample volume. However, EPA 

recommends that the GWS collect and ship more than 100-mL of sample to ensure that a minimum of 100 mL is 

available for analysis. The capacity of sample containers should be sufficient to allow at least a 1-inch headspace 

to facilitate mixing of the sample by shaking prior to analysis. Sample volume and container size recommendations 

are provided below. 

•	 E. coli and Enterococci Samples. The GWS should collect at least 120 mL of sample to ensure 

sufficient volume for sample analysis is available in the event of spillage at the laboratory. The 

capacity of sample containers should be at least 150 mL to allow at least a 1-inch headspace to 

facilitate mixing of the sample by shaking prior to analysis. 

•	 Coliphage Samples. If Method 1601 is used for coliphage sample analyses, either 100-mL or 1-L 

sample volumes may be analyzed (Method 1602 only accommodates 100-mL volumes). While the 

minimum sample volume requirement for the GWR is 100 mL, systems may wish to collect and 

analyze a 1-L sample volume to increase the sensitivity of the Method 1601 analysis. 

For 100-mL sample analyses, the GWS should collect 250 mL of sample for each of the two 

coliphage types to allow for sample re-analysis, if necessary. 

The capacity of sample containers should allow at least a 1-inch headspace to facilitate mixing of the 

sample by shaking prior to analysis. Alternatively, samples for male-specific and somatic sample 

analyses can be collected in separate containers. 

For 1-L sample analyses, the GWS should collect 2.5 L of sample for each of the 

two coliphage types to allow for sample re-analysis, if necessary. 

The capacity of sample containers should allow at least a 1-inch headspace to facilitate mixing of the 

sample by shaking prior to analysis. Alternatively, samples for male-specific and somatic sample 

analyses can be collected in separate containers. 

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Note: While samples may still be analyzed if spillage occurs at the laboratory, if spillage or leakage 

occurs during shipment, there is an opportunity for sample contamination to occur and the sample 

should not be analyzed. 

5.3 Shipping Regulations and Documentation 

Unless the sample is known or suspected to contain infectious agents (e.g., during an outbreak), samples 

should be shipped as noninfectious and should not be marked as infectious. U.S. Department of Transportation 

(DOT) regulations (49 CFR 172) prohibit interstate shipment of more than 4 L of solution known to contain 

infectious materials. State regulations may contain similar regulations for intrastate commerce. If an outbreak is 

suspected, ship less than 4 L per shipment. 

Sample Tracking Information. The GWS should record the following information on the sample 

collection form: 

• Name of system (public water system site identification number, if available) 

• Sample identification (number) 

• Sample site location 

• Sample type (e.g., triggered monitoring, assessment monitoring) 

• Date and time of collection 

• Analysis requested 

• Name of sampler 

• Any remarks 

Sample Container Information. The sample container should indicate the following: 

• Sample number 

• Name of system (public water system site identification number, if available) 

• Date and time of collection 

• Sample collection location 

• Analysis requested 

Chain-of-Custody. Sample collectors and laboratories should follow applicable State regulations 

pertaining to chain-of-custody (COC). Appendix A of the Manual for the Certification of Laboratories Analyzing 

Drinking Water (USEPA, 2005) provides detailed guidance on COC procedures, including the following: 

• Sample collection, handling, and identification 

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•	 Transfer of custody and shipment 

•	 Laboratory sample control procedures 

•	 Sample ID tag examples 

•	 Example COC record 

5.4 Holding Time 

The analytical holding time is defined as the time between sample collection and the start of sample 

analysis. During the time between sample collection and analysis, it is possible for stressed bacteria and/or bacterial 

viruses to suffer die-off or further injury due to adverse conditions during transit or the presence of substances in 

the water which can be toxic (e.g., heavy metals). Additionally, the presence of nutrients in the sample may lead to 

growth of background bacterial populations which could interfere with the analysis. As a result of these concerns, 

while the GWR requires that fecal indicator samples be analyzed within a 30-hour hold time period, EPA highly 

recommends that samples be analyzed as soon as possible after collection. 

5.5 Holding Temperature and Temperature Monitoring 

While not required, EPA strongly recommends that GWR monitoring samples that are not analyzed 

immediately following sample collection be chilled below 10°C to reduce biological activity and preserve the 

condition of ground water samples between collection and analysis. Samples for all analyses should remain above 

freezing at all times. Samples that arrive frozen should not be analyzed. Several options are available that mitigate 

the risk of sample contamination while providing an indication of sample temperature upon receipt at the 

laboratory and, in some cases, during shipment: 

•	 Temperature sample. Using this option, the GWS would fill a small, inexpensive sample bottle with 

water and pack this “temperature sample” next to the source water monitoring sample. The 

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

source water monitoring sample. Temperature sample bottles are not appropriate for use with bulk 

samples (i.e., 1-L samples) because of the potential affect that the difference in sample volume may 

have in temperature equilibration in the sample cooler. Example product: Cole Parmer catalog 

number U-06252-20. 

•	 Minimum/maximum thermometer. A minimum/maximum thermometer not only provides the current 

temperature of the shipping cooler, which is read upon receipt at the laboratory, but indicates the 

minimum and maximum temperatures that the sample experienced during shipment. Ideally, if the 

minimum/maximum thermometer is water-immersible, it should be placed in a temperature sample in 

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

coolant. This additional information may be used to determine whether the sample froze during 

shipment or exceeded the maximum temperature, even if the sample is received at an acceptable 

temperature. Example product: Cole Parmer catalog number U-08107-30. 

•	 iButton. A Thermocron7 iButton is a small, waterproof device that contains a computer chip to 

record temperature at different time intervals. The information is then downloaded from the iButton 

onto a computer. The iButton should be placed in a temperature sample in the cooler, rather than 

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placed directly in the cooler, where it may be affected by close contact with the coolant. Information 

on Thermocron7 iButtons is available from http://www.maxim-ic.com/products/ibutton/. 

•	 Stick-on temperature strips. A stick-on temperature strip may be applied to the outside of the sample 

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

other options, but still mitigates the risk of sample contamination while providing an indication of 

sample temperature upon receipt at the laboratory. Example product: Cole Parmer catalog number U90316-00. 

•	 Infrared thermometers. An infrared thermometer may be used to measure the temperature of the 

surface of the sample container upon receipt at the laboratory. The thermometer is pointed at the 

sample, and the temperature is measured without coming in contact with the sample volume. 

Example product: Cole Parmer catalog number EW-39641-04. 

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

ensure accurate measurements. See the U.S. EPA Manual for the Certification of Laboratories Analyzing 

Drinking Water (USEPA, 2005) for more information. 

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6. Understanding Ground Water Rule Fecal Indicator Methods 

Section 6.1 describes the formats available for the analysis of E. coli, enterococci, and coliphage. Sections 

6.2, 6.3, and 6.4 provide descriptions of the analytical methods approved for use under the GWR for the analysis of 

E. coli, enterococci, and coliphage, respectively. 

6.1 Selection of Method Format 

Three formats, presence/absence (Section 6.1.1), most probable number (Section 6.1.2), and direct plating 

(Section 6.1.3) are approved for use in the analysis of E. coli, enterococci, and coliphage samples under the Ground 

Water Rule (GWR). The type of format selected may be influenced by water sample type, quality, and character 

(e.g., organism density, turbidity). 

6.1.1 Presence/Absence 

A presence/absence procedure may be performed using a single vessel. Depending on the method and 

analyte (e.g., E. coli, coliphage), medium is added to the sample and thoroughly mixed. The mixture is incubated 

for the method-specified time at the method-specified temperature. Positive tubes/bottles are then 

confirmed/verified according to method-specific protocols. 

6.1.2 Most Probable Number (MPN) 

Multiple-Well 

A multiple-well procedure may be performed with sterilized disposable packets. The commercially 

available Quanti-Tray® or Quanti-Tray®/2000 multiple-well tests use Colilert® or Colisure® media to detect the 

presence of E. coli and EnterolertTM to detect the presence of enterococci. In these tests, the medium is added to a 

100-mL sample, mixed thoroughly, and poured into the tray. A tray sealer separates the sample into 51 wells 

(Quanti-Tray®) or 97 wells (Quanti-Tray®/2000) and seals the package which is subsequently incubated according 

to method-specific requirements. A single positive well constitutes a positive sample under the GWR. If 

enumeration is desired, MPN tables provided by the manufacturer can be used to estimate the number of bacteria 

per 100 mL of sample. 

Multiple-Tube 

In multiple-tube tests, serial dilutions may be used to obtain estimates of bacterial density over a range of 

concentrations, with replicate tubes analyzed at each ten-fold dilution/volume. The multiple-tube methodology is 

useful for detecting organisms in samples containing heavy particulate matter, toxic compounds (e.g., metals), or 

injured or stressed organisms. Generally, for nonpotable water samples, 5 replicate tubes at a minimum of 3 

dilutions/volumes (for a total of 15 tubes) are used. However, since the GWR requires 100 mL of sample to be 

analyzed, 10 tubes containing 10 mL of sample per tube should be analyzed. Tubes are incubated and positive 

results are reported and confirmed. Positive results are determined under specified conditions (e.g., production of 

acid and/or gas using multiple-tube fermentation tests, color change or fluorescence using enzyme substrate tests). 

A single positive tube constitutes a positive result for GWR compliance monitoring requirements. 
GWR Source Water Monitoring Methods 6-2 Revised March 2008 

Guidance Manual 

6.1.3 Direct Plating 

Membrane Filtration 

Membrane filtration (MF) is a direct-plating method in which sample dilutions/volumes are filtered 

through one or more 0.45-μm membrane filters that are subsequently transferred to Petri plates containing selective 

primary isolation agar or an absorbent pad saturated with selective broth. A second substrate medium is used in 

two-step MF procedures to confirm and/or differentiate the target organisms. The total sample volume to be 

analyzed may be distributed among multiple filters and diluted as necessary based on the anticipated water sample 

type, quality, and character (e.g., organism density, turbidity). Target colonies are detected by observing the 

presence of colonies that meet a specific morphology, color, or fluorescence under specified conditions. 

Membrane filtration results can be subject to interferences caused by water samples with high turbidity, toxic 

compounds, or large numbers of non-coliform (background) bacteria, and organisms damaged by chlorine or toxic 

compounds. 

Pour Plate (Coliphage Only) 

Pour plate is a direct-plating method in which 100 mL of sample is added to 100 mL of molten agar, mixed 

and poured into Petri plates. Following incubation plates are examined for plaques (circular lysis zones). A single 

plaque forming unit (PFU) constitutes a positive result for the GWR compliance monitoring requirements. 

6.2 E. coli Methods Approved for Ground Water Rule Monitoring 

The E. coli methods approved for use under the GWR are specified in Exhibit 6.1 and discussed in 

Sections 6.2.1 - 6.2.7, below. Exhibit 6.1 lists approved methods for GWR monitoring as of the date of this 

guidance document. Additional methods may have been approved for GWR monitoring since this date. 

Additional information, including additional approved analytical methods for GWR monitoring, can be 

found at the following URL: http://www.epa.gov/safewater/methods/methods.html. 

6.2.1 Colilert (Standard Methods 9223) 

Colilert® simultaneously detects total coliforms and E. coli in water. Commercially prepared media 

formulations are available in packets for presence-absence and multiple-well procedures, and disposable tubes for 

the multiple-tube procedure. The use of commercially prepared media is required for quality assurance and 

uniformity. Incubate the sample at 35.0°C±0.5°C for 24 hours.. If the response is unclear after the specified 

incubation period, the sample is incubated for up to an additional 4 hours at 35.0°C±0.5°C. After the appropriate 

incubation period, compare each bottle/tube/well to the reference color “comparator” provided by the 

manufacturer. A yellow color greater or equal to the comparator indicates the presence of total coliforms in the 

sample, and the bottle/tube/well is then checked for fluorescence under long-wavelength UV light (365-nm). The 

presence of fluorescence greater than or equal to the comparator is a positive result for E. coli. The concentration in 

MPN/100 mL, although not required under the GWR, can then be calculated based on the number of positive tubes 

or wells using MPN tables provided by the manufacturer 
GWR Source Water Monitoring Methods 6-3 Revised March 2008 

Guidance Manual 

Exhibit 6.1: E. coli Methods Approved for Use under the Ground Water Rule 

Media

Method 

Reference

Description of 

Positive Result Section

Colilert®

SM1 9223 Yellow, fluorescent 6.2.1

Colisure®

SM1 9223 Red/magenta, fluorescent 6.2.2

E*Colite — Blue/green, fluorescent 6.2.3

(LTB/P-A ) EC-MUG

(SM1 9221B/ 

SM1 9221D) 

SM

1

 9221F

(Growth and the presence 

of acid and/or gas in 

LTB/P-A), fluorescence in 

EC-MUG 6.2.4

(mEndo or 

LES Endo ) 

NA-MUG

(SM1 9222B/C 

) 

SM

1

 9222G

(Pink to red colonies with 

metallic (golden-green) 

sheen); fluorescence after 

transfer to NA-MUG 6.2.5

MI Medium

EPA Method 

1604 Blue colonies 6.2.6

m-ColiBlue24® — Blue colonies 6.2.7

1

Standard Methods for the Examination of Water and Wastewater, 20th edition. 

6.2.2 Colisure®

 (Standard Methods 9223) 

Colisure® simultaneously detects total coliforms and E. coli in water. Similar to the Colilert®

 method, 

Colisure® can be used in presence/absence, multiple-tube, or multiple-well procedures. The reagent is added to the 

sample and incubated at 35.0°C±0.5°C for 24 hours. In this method, coliform bacteria are those bacteria which 

produce a red or magenta color and E. coli also produce a fluorescent signal under a 6-watt, 365nm UV light after 

incubation 35±0.5°C for 24 hours. If after 24 hours the sample is a pink to orange color, incubate for an additional 

4 hours, to determine the appropriate result. Samples may be incubated for a maximum of 48 hours if necessary. A 

red/magenta color greater or equal to the comparator indicates the presence of total coliforms in the sample, and the 

presence of fluorescence greater than or equal to the comparator is a positive result for E. coli. The concentration 

in MPN/100 mL, although not required under the GWR, can then be calculated based on the number of positive 

tubes or wells using MPN tables provided by the manufacturer. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6.2.3 E*Colite 

The E*Colite test simultaneously detects total coliforms and E. coli. The E*Colite test involves a 

dehydrated medium to which a 100-mL water sample is added. The test consists of a sterile burst-a-seal bag 

divided into three compartments. A 100-mL water sample is added to the bag and sealed. Then the water sample 

is pushed through the burst-a-seal into the medium, and the two are mixed thoroughly. The bag is then incubated 

for 28 hours at 35.0°C±0.5°C. The bag may first be placed in a 35.0°C±0.5°C water bath for 10 minutes to bring 

the sample up to incubation temperature quickly. After incubation, the bag is observed for the presence of a 

blue/green color, which indicates a total coliform-positive sample. If the blue/green sample does not fluoresce after 

28 hours, the sample should be incubated an additional 20 hours for a maximum of 48 hours and re-checked for 

fluorescence. If a blue/green sample is also fluorescent under an ultraviolet light (366 nm), the sample is E. colipositive. 

6.2.4 EC-MUG (Standard Methods 9221B/9221D� 9221F) 

Positive total coliform tests from the presumptive medium (SM 9221B/9221D) are inoculated into EC 

broth supplemented with 4-methylumbelliferyl-β-D-glucuronide (MUG)EC-MUG, a differential medium used for 

the detection of E. coli. Growth from the presumptive total coliform test is transferred using a sterile loop or 

applicator stick to tubes containing EC-MUG broth. Inoculated tubes are incubated at 44.5°C±0.2°C for 24±2 

hours in a water bath. All tubes exhibiting growth are examined for bright blue fluorescence under longwavelength UV light (366-nm). If the sample exhibits growth and fluorescence, the sample is positive for E. coli. 

6.2.5 NA-MUG (Standard Methods 9222B/9222C � 9222G) 

Following initial isolation of total coliforms on mEndo or LES-Endo media, the filter is transferred to 

nutrient agar containing 4-methylumbelliferyl-β-D-glucuronide (NA-MUG) and incubated for 4 hours at 

35.0°C±0.5°C. Presumptive colonies (pink to red colonies with a metallic (golden-green) sheen) from mEndo that 

fluoresce under a long-wavelength UV light (366-nm) on NA-MUG are considered E. coli positive. 

6.2.6 MI Medium (EPA Method 1604) 

The MI medium method is a single-step membrane filtration procedure used to simultaneously detect total 

coliforms and E. coli (USEPA, 2002). In this method, a water sample is filtered through a 0.45-μm membrane 

filter, the filter is placed on an MI agar or broth saturated pad, and the medium is incubated at 35.0°C±0.5°C for 24 

hours. E. coli colonies exhibit a blue color. The plates can also be observed under long-wavelength UV light 

(366-nm) for the presence of total coliform species that fluoresce. Because the blue color from the breakdown of 

IBDG can mask fluorescence, non-fluorescent blue colonies are included in the total coliform count. Blue colonies 

regardless of fluorescence are considered E. coli. 

6.2.7 m-ColiBlue24® 

The m-ColiBlue24® method is a single-step membrane filtration procedure that simultaneously detects total 

coliforms and E. coli. A water sample is filtered through a 0.45-μm membrane filter, and the filter is transferred to 

a plate containing an absorbent pad saturated with m-ColiBlue24® broth. The filter is incubated at 35.0°C±0.5°C 

for 24 hours and examined for colony growth (Hach Co., 1999). Total coliforms are indicated by red colonies. The 

presence of E. coli is indicated by blue colonies. 

GWR Source Water Monitoring Methods 6-4 Revised March 2008 

Guidance Manual 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6.3 Enterococci Methods Approved for Ground Water Rule Monitoring 

The enterococci methods approved for use under the GWR are specified in Exhibit 6.2 and discussed below, 

in Sections 6.3.1 - 6.3.4. 

6.3.1 Multiple-Tube Technique (Standard Methods 9230B) 

The Azide Dextrose/BEA/BHI protocol for detecting enterococci in water employs a three-step procedure 

(presumptive fecal streptococcus, confirmed fecal streptococcus, and enterococcus) to determine the presence of 

enterococci bacteria (APHA, 1998). In the presumptive phase, azide dextrose broth is inoculated incubated at 35.0 

+ 0.5°C. Growth within 48±3 hours constitutes a positive presumptive reaction for fecal streptococci. 

Note: Although Standard Methods 9230B indicated the use of Pfizer selective enterococcus (PSE) agar for 

confirmation, it is no longer commercially available. Bile esculin agar (BEA) is an accepted alternative plating 

medium for confirmation testing. 

After enrichment during the presumptive phase, positive azide dextrose tubes are subjected to a fecal 

streptococci confirmation step. A portion of growth from each positive azide dextrose tube is streaked onto bile 

esculin agar (BEA). Inverted plates are incubated at 35.0°C±0.5°C for 24±2 hours and observed for the presence 

of brownish-black colonies with a brown halo. Such colonies are confirmed as fecal streptococci. Target colonies 

from the BEA medium are transferred to a tube of brain heart infusion (BHI) broth and incubated at 45°C±0.5°C 

for 48 hours. Simultaneously, target colonies from the BEA are transferred to BHI broth containing 6.5% NaCl 

and incubated at 35.0°C±0.5°C for 48 hours. Growth at 45.0°C in BHI broth and in BHI broth containing 6.5% 

NaCl at 35.0°C is indicative of enterococci. 

GWR Source Water Monitoring Methods 6-5 Revised March 2008 

Guidance Manual 
GWR Source Water Monitoring Methods 6-6 Revised March 2008 

Guidance Manual 

 Exhibit 6.2 Enterococci Methods Approved for Use under the GWR 

Media

Method 

Reference Description of Positive Results Section

Azide Dextrose / 

BEA / BHI SM1

 9230B

Growth at 45EC in BHI and growth 

in BHI with 6.5% NaCl at 35EC 6.3.1

mE-EIA SM1 9230C 

Pink to red colonies that form 

black or reddish-brown precipitate

on underside of filter 6.3.2 

mEI

EPA 

Method 

1600 All colonies with a blue halo 6.3.3

EnterolertTM

Budnick, 

G.E. et al., 

1996 

Presence of blue-white 

fluorescence 6.3.4

1

Standard Methods for the Examination of Water and Wastewater 20th edition. 

6.3.2 mE-EIA (Standard Methods 9230C) 

The mE-EIA agar method is a two-step membrane filtration procedure that detects enterococci based on the 

development of colonies on the surface of a filter when placed on a selective medium. In this method, a water 

sample is filtered through a 0.45-μm membrane filter and the filter is placed on mE agar. After the plate is 

incubated at 41°C±0.5°C for 48±3 hours, the filter is transferred to an esculin iron agar (EIA) plate and incubated at 

41°C±0.5°C for 20-30 minutes. After incubation, all pink to red colonies on mE agar that form a black or reddishbrown precipitate on the underside of the filter when placed on EIA are considered enterococci. Plates should be 

examined upside down to determine the presence of enterococci colonies. 

6.3.3 mEI (EPA Method 1600) 

The mEI agar method is a single-step membrane filtration procedure that detects enterococci in water 

samples (USEPA, 2006a). In this method, a water sample is filtered through a 0.45-μm membrane filter. The filter 

is placed on mEI agar and incubated at 41°C±0.5°C for 24 hours. Following incubation, all colonies with a blue 

halo, regardless of colony color, are considered enterococci. 

6.3.4 EnterolertTM (Budnick, G.E. et al., 1996) 

EnterolertTM is a commercially available enzyme-substrate test for the determination of enterococci in 

water (Budnick, G.E et al, 1996). In this method, the sample is mixed with the EnterolertTM medium and incubated 

for 24 hours at 41°C±0.5°C. After incubation, the presence of blue fluorescence is a positive result for enterococci. 

 If enumeration is desired, the concentration in MPN/100 mL can be calculated from the number of positive tubes 

or wells using MPN tables provided by the manufacturer. 
GWR Source Water Monitoring Methods 6-7 Revised March 2008 

Guidance Manual 

6.4 Coliphage Methods Approved for Ground Water Rule Monitoring 

The coliphage methods approved for use under the GWR are specified in Exhibit 6.3 and discussed below, 

in Sections 6.4.1 - 6.4.2. 

 Exhibit 6.3 Coliphage Methods Approved for Use under the GWR 

Media

Method 

Reference

Description of 

Positive Result Section

Two-Step Enrichment EPA Method 1601

Presence of plaques 

(circular lysis zones) 6.4.1

Single Agar Layer EPA Method 1602

Presence of plaques 

(circular lysis zones) 6.4.2

6.4.1 Two-Step Enrichment (Method 1601) 

The two-step enrichment procedure detects the presence or absence of somatic or male-specific coliphage 

based on the formation of plaques after enrichment. A 100-mL or 1-L ground water sample is supplemented with 

MgCl2 (magnesium chloride), log-phase host bacteria (E. coli Famp for male-specific coliphage or E. coli CN-13 for 

somatic coliphage), and tryptic soy broth (TSB) in an enrichment step. After overnight incubation, samples are 

“spotted” onto a lawn of host bacteria specific for each type of coliphage, incubated, and examined for plaque 

forming units, which indicate the presence of coliphage. 

6.4.2 Single Agar Layer (SAL) (Method 1602) 

The single agar layer procedure is a single-step procedure to detect somatic or male-specific coliphage. If 

desired, this method can be used to provide a direct count of somatic or male-specific coliphage. A 100-mL 

ground water sample is assayed by adding MgCl2 (magnesium chloride), log-phase host bacteria (E. coli Famp for 

male-specific coliphage or E. coli CN-13 for somatic coliphage), and 100 mL of double-strength molten tryptic soy 

agar to the sample. The sample is thoroughly mixed and the total volume is poured into 5 to 10 plates (dependent 

on plate size). After overnight incubation, plaques are counted and summed for all plates from a single sample. 

The quantity of coliphage in a sample can be expressed as plaque forming units (PFU)/100 mL, if enumeration is 

desired.
GWR Source Water Monitoring Methods 7-1 Revised March 2008 

Guidance Manual 

7. Evaluating Fecal Indicator Data

Data on sample measurements, sample processing times, and sample results are recorded at the laboratory when 

fecal indicator samples are processed and analyzed by the laboratory. Although only final results (e.g., presence/absence, 

CFU/100 mL, MPN/100 mL, PFU/100 mL) may be reported by the laboratory to the GWS, the “primary” data elements 

recorded by the laboratory will likely need to be consulted if questions on the data arise. This chapter provides an overview 

of the data recording and reporting processes and provides guidance on how to review and interpret the data. This 

information will be useful in the event that a GWS requests that a sample result be invalidated. 

7.1 Data Recording at the Laboratory 

The laboratories performing fecal indicator analyses under the Ground Water Rule (GWR) should record the 

following general types of information: 

• Sample identification information 

• The incubation start/read times and temperatures for each method to verify that method requirements were met 

• The name of the analyst performing the sample analysis 

• Primary measurements performed by the laboratory during sample analysis to determine the 

presence/absence/concentration of the fecal indicator 

• QC analysis results (e.g., positive/negative controls, blanks, OPR, etc.) 

Example bench sheets for each of the methods approved for use under the GWR (Section 6) can be found in 

Appendix D - Appendix F. 

7.1.1 Sample Identification Information 

Sample identification information is used to track the sample through sample collection, analysis, and data 

reporting. At a minimum, the laboratory should record the GWS PWSID, sample collection date, sample collection time, 

sample collection location, sample ID, and the fecal indicator(s) (e.g., enterococci , E. coli, somatic coliphage, malespecific coliphage) being analyzed. 

7.1.2 Primary Data 

The laboratory records all primary measurements necessary to determine the presence or absence of the GWR fecal 

indicator(s). Primary measurements may include the sample volume analyzed and the results of preliminary steps for the 

determination of the fecal indicator. If the GWS chooses to determine the concentration of the fecal indicator (rather than 

simply the presence or absence of the indicator), primary measurements may also include the number of positive tubes or 

wells for each volume analyzed (multiple-tube and multiple-well methods), the number of colony-forming units (membrane 

filtration methods), or the number of plaque forming units (Method 1602). 
GWR Source Water Monitoring Methods 7-2 Revised March 2008 

Guidance Manual 

7.1.3 Sample Results 

The final result of fecal indicator analyses will be reported as “present” or “absent” per volume analyzed unless the 

GWS elects to perform quantitative analyses. If the GWS requests the laboratory to determine the concentration of the 

fecal indicator in the sample, the final result will be reported as CFU/100 mL (for membrane filtration methods), MPN/100 

mL (for multiple-tube and multiple-well methods), or PFU/100 mL (for EPA Method 1602). 

7.2 Data Archiving 

GWR source water monitoring data must be maintained by the GWS for a period of five years. The requirements of 

The NELAC Institute (TNI) (formerly known as the National Environmental Laboratory Accreditation Conference 

(NELAC)) require also require data to be maintained by the laboratory for a period of five years. 

7.3 Evaluating Fecal Indicator Data 

If questions arise regarding the validity of E. coli or enterococci results submitted by the GWS, the information 

recorded by the laboratory on the data reporting forms should be carefully evaluated. Sections 8.3.1 through 8.3.3 provide 

guidance on how to review the data reporting forms and verify the accuracy. 

7.3.1 Data Completeness Check 

The first step in evaluating hard copy sample results for a GWR monitoring sample is to verify that the information 

below is included. If information is missing, incomplete, or incorrect, the State should request that the GWS contact the 

laboratory to request the missing information. 

• Sample result summary sheet. This form should include the following information: 

- Sample identification information 

- Sample result 

- Laboratory QC checklist or other verification from the laboratory that all QC requirements were met 

• Sample collection form. This form should have been completed by the GWS at the time of sample collection, 

indicating when and where the sample was collected. 

• Method bench sheet. This form should have been completed by the laboratory with primary sample processing 

and analysis data associated with the monitoring sample 

• Laboratory comments. If the laboratory encountered problems with the sample (e.g., receipt, processing, or 

analysis), they should be documented with the sample results; any of these issues may be associated with a 

GWS’s request to invalidate data. Possible issues include the following: 

- Sample arrived at the laboratory in unacceptable condition (i.e., leaking). 

- Sample holding time was exceeded. 

- Sample was frozen. 

- Sample holding temperature was not within acceptable range. 

- Unacceptable blank sample result. 

- Unacceptable positive or negative control result. 
GWR Source Water Monitoring Methods 7-3 Revised March 2008 

Guidance Manual 

- Media sterility checks were not acceptable. 

- Method incubation times or temperatures were not within acceptable range. 

- Membrane filtration: Too much sediment on the filter. 

- Membrane filtration: Confluent growth of non-target organism. 

- Membrane filtration: Pre- or post- filtration series sterility check not acceptable (e.g., contamination with 

E. coli or enterococci). 

- Quanti-Tray® was damaged or leaked. 

- Sample was not distributed to all wells in Quanti-Tray®. 

- Positive presumptive tubes prepared for multiple-tube analyses were not transferred into the appropriate 

confirmatory medium. 

- All rows of tubes prepared for multiple-tube analyses were not inoculated. 

Any of the above data qualifiers may result in the sample being considered invalid for GWR monitoring 

requirements. 

7.3.2 Evaluation of Data Against Method Quality Control Requirements 

The following items may be reviewed to verify that the laboratory analyzed the GWR E. coli or enterococci sample 

within the analytical controls specified by the method: 

• Sample condition upon receipt. If the sample was shipped to the laboratory, the completed sample collection 

form will reflect that the sample was received in acceptable condition (e.g., not leaking, etc.), and was not 

frozen. The temperature of the sample upon receipt should be noted. Systems are encouraged, but not required, 

to maintain the sample at <10°C during transport. If the sample is >10°C upon arrival, the sample result may be 

affected.

• QC samples associated with source water samples. Verification can be made that the required QC samples 

were run with the field sample(s). The frequency of analysis of quality control samples, including method 

blanks and positive and negative controls, depends on the method-specified requirements and the requirements 

in the Manual for the Certification of Laboratories Analyzing Drinking Water (USEPA, 2005). 

• Holding time. The sample collection date and time on the data collection form and the date and time of the 

initiation of sample analysis, recorded by the laboratory on the method bench sheet, can be used to verify that the 

laboratory began sample analysis within 30 hours of sample collection. 

• Incubation times and temperatures. The dates, times, and temperatures for the incubation initiation and 

completion of all method steps (recorded by the laboratory on the method bench sheet) can be used to verify 

consistency with the incubation times and temperatures.. 
GWR Source Water Monitoring Methods 8-1 Revised March 2008 

Guidance Manual 

8. Frequently Asked Questions 

8.1 Collecting and Shipping Ground Water Samples 

Does the sampling procedure differ for bacterial and viral indicators? 

Separate guidance for the collection of bacterial and viral indicators is provided in Section 5.2, as well as 

Appendix B (for bacterial indicators) and Appendix C (for viral indicators). The primary difference in the sample 

collection procedures is the flexibility to collect larger sample volumes for the analysis of coliphage using EPA 

Method 1601. 

If the GWS is within driving distance of a certified laboratory, is the system required to monitor the 

temperature of the samples in the same manner as samples shipped overnight? 

Under the GWR, systems are encouraged, but not required, to hold samples at <10°C regardless of whether 

samples are shipped overnight or delivered to a certified laboratory within driving distance. Samples for all 

analyses should remain above freezing at all times and samples that arrive frozen should not be analyzed. Please 

refer to Section 5.5 for detailed guidance on holding temperature and temperature monitoring. 

Can a ground water system analyze > 100 mL for bacterial indicators? 

Yes, more than 100 mL of sample may be analyzed. It should be noted that larger sample volumes may 

increase the analytical sensitivity of the method. However, certain conditions (e.g., high turbidity) may prohibit the 

analysis of larger sample volumes. 

8.2 Understanding Ground Water Rule Fecal Indicator Analyses 

If the method format allows for enumeration, can the GWS report the numerical value or do they have 

to report presence/absence? 

Unless otherwise specified by the State, the GWS should report the results to the State as “present” or 

“absent” regardless of method format. 

If the GWS initially selects membrane filtration for the analysis for E. coli or enterococci, can they 

switch to another format? 

Yes. Generally E. coli and enterococci samples should be analyzed using the same method during source 

water monitoring. However, if it is necessary to switch methods, Exhibits 6.1 and 6.2 lists methods approved for 

E. coli and enterococci, respectively. Additional methods may have been approved for GWR monitoring since this 

date. Additional information, including additional approved analytical methods for GWR monitoring, can be found 

at the following URL: http://www.epa.gov/safewater/methods/methods.html. 
GWR Source Water Monitoring Methods 8-2 Revised March 2008 

Guidance Manual 

Which methods are better for the analyses of the bacterial indicators? 

All bacterial methods approved under the GWR are considered comparable for the evaluation of the target 

analyte (E. coli or enterococci). 

Can a GWS use the methods in SM 9224 to analyze for coliphage? 

No. SM 9224 has not been approved for use under the GWR. The only methods approved for coliphage 

monitoring under the GWR are EPA Method 1601 and EPA Method 1602. See Section 6.4 of this manual for a 

detailed discussion of the methods approved for coliphage monitoring. Section 6.4 discusses approved methods for 

GWR monitoring as of the date of this guidance document. Additional methods may have been approved for GWR 

monitoring since this date. Additional information, including additional approved analytical methods for GWR 

monitoring, can be found at the following URL: http://www.epa.gov/safewater/methods/methods.html. 

Will the certification program be expanded to include enterococci and coliphage analyses? 

The Drinking Water Certification Manual (5th Edition) has been revised to include GWR-approved 

enterococci and coliphage methods. In addition, EPA’s training course “Laboratory Certification Officer’s Training 

Course” is providing auditors with training that will allow them to audit laboratories using both enterococci and 

coliphage methods. 

Are the methods recently approved for use under the Total Coliform Rule (e.g., Readycult, Chromocult) 

approved for E. coli monitoring under the GWR? 

No. Exhibit 6.1 lists approved methods for GWR monitoring as of the date of this guidance document. 

Additional methods may have been approved for GWR monitoring since this date. Additional information, 

including additional approved analytical methods for GWR monitoring, can be found at the following URL: 

http://www.epa.gov/safewater/methods/methods.html. 

8.3 Evaluating Fecal Indicator Data 

Is it up to the GWS to validate the data/final results submitted by the laboratory? 

Yes. The GWS should evaluate/validate the data submitted by the laboratory prior to submitting the 

results to the State. Data review and interpretation guidance are provided in Chapter 8 of this manual. In the event 

the GWS requests that a sample be considered invalid, the State should request the primary data from the GWS for 

evaluation. 

Can the State invalidate a positive source water sample result? 

Yes. The State may invalidate a positive source water sample if a laboratory establishes that improper 

sample analyses caused the positive result or if the State has substantial grounds to believe that a positive result was 

attributable to a circumstance or condition that did not reflect source water quality. The State must document this 

in writing. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

9. References
 

APHA. (American Public Health Association). 1998. Standard Methods for the Examination of Water and 

Wastewater (20th ed.). 1015 Fifteenth Street, NW., Washington, DC 20005. 

Atherholt, T., E. Feerst, B. Hovendon, J. Kwak, J. and D. Rosen. 2003. “Evaluation of indicators of fecal 

contamination in groundwater.” Journal of the American Water Works Association. 95(10):119-131. 

Blair, B.A. and C.P. Gerba, 2006. Occurrence of Naegleria fowleri in well water in Arizona. Unpublished report, 

University of Arizona, National Science Foundation Water Quality Center. 

Budnick, G.E., R.T. Howard and D.R. Mayo. 1996. Evaluation of enterolert for enumeration of 

enterococci in recreational waters. Appl Environ Microbiol. 62:3881-3884. 

Cabelli, V. J., A. P. Dufour, M. A. Levin, L. J. McCabe, and P. W. Haberman, 1979. Relationship of 

Microbial Indicators to Health Effects at Marine Bathing Beaches. American Journal of Public Health. 

69:690-696. 

Carter, J.T., Rice E.W., Buchburger, S.G., and Lee, Y. 2000. “Relationships between levels of heterotrophic 

bacteria and water quality parameters in a drinking water distribution system.” Water Research, 

34(5):1495-1502. 

Charm Sciences, Inc. 1997. Description of the E*Colite [reg] Test, ``Presence/Absence for Coliforms and 

E. Coli in Water,'' is available from Charm Sciences, Inc., 36 Franklin Street, Malden, MA 02148-4120. 

Costa, J., I. Tiago, M.S. da Costa, A. Verissimo. 2005. Presence and persistence of Legionella spp. in groundwater. 

Appl. Env. Micro. 71(3):663-671. 

Curry, D.S. 1999. “Final report of the septic siting project.” New York City Department of Environmental 

Protection, 210 pp. 

Davis, J.V. and Witt, III, E.C. 2000. “Microbiological and chemical quality of ground water used as a source of 

public supply in southern Missouri- phase I, May 1997-March 1998.” Water -Resources Investigations 

Report 00-4038, 77 pp., USGS, US DOI, Rolla, Missouri. 

DeBorde, D.C. 1998. “Sample collection data and analytical results document for conducting male-specific 

coliphage sampling of septic tanks, phase I.” Unpublished reported submitted by Montana Headwaters, 

Inc. to SAIC, Dec. 18, 1998. p. 24. 

DeBorde, D.C. 1999. “Sample collection data and analytical results document for conducting male-specific 

coliphage sampling of septic tanks, phase II.” Unpublished reported submitted by Montana Headwaters, 

Inc. to SAIC, Sept. 30, 1999. p. 12. 

Doherty, K. 1998. “Status of the New England ground water viral study.” Proceedings, American Water Works 

Association Annual Meeting, Dallas, Texas, June 23, 1998. American Water Works Association, Denver. 

Femmer, S. 2000. “Microbiological and chemical quality of ground water used as a source of public supply in 

southern Missouri - phase II, April-July, 1998.” Water-Resources Investigations Report 00-4260, US DOI, 

USGS, Rolla Missouri. 62 pp. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Foppen, J.W.A. and J.F. Schijven. 2006. “Evaluation of data from the literature on the transport and 

survival of Escherichia coli and thermotolerant coliforms in aquifers under saturated conditions.” 

Water Research: 40 (2006) pp. 401 – 426. 

Francy, D.S., R.N. Bushon, J. Stopar, E.J. Luzano, and G.S. Fout. 2004. “Environmental factors and chemical 

and microbiological water-quality constituents related to the presence of enteric viruses in ground water 

from small public water supplies in Southeastern Michigan.” USGS Scientific Investigations Report. 

54:3004-5219. 

Geldreich, E.E. 1996. Microbial Quality of Water Supply in Distribution Systems. Lewis Publishers, Boca Raton, 

Florida, 504 pp. 

Grabow, W. 2001. “Bacteriophages: Update on application as models for viruses in water.” Water SA. 

27(2):251-268. 

Hach Company, Inc. Revision 2, 1999. Description of m-ColiBlue24® Method, Total Coliforms and E. coli, is 

available from Hach Company, 100 Dayton Ave., Ames, IA 50010. Table IA, Note 18. 

Hardina, C.M. and R.S. Fujioka, 1991. “Soil: The environmental source of E. coli and enterococci in 

Hawaii’s streams.” Envir. Toxicol. Wat. Qual., 6:185. 

Hardie, J.M. and R.A. Whiley, 1997. “Classification and overview of the genera Streptococcus and 

Enterococcus.” Journal of Applied Microbiology Symposium Supplement, 83:1S – 11S. 

Jimenez, L. et al., Survival and activity of Salmonella typhimurium and Escherichia coli in tropical freshwater, 

Journal Applied Bacteriology, 1989 July, 67(1):61-9. 

Lieberman, R.J., L.C. Shadix, B.S. Newport, M.W.N. Frebis, S.E. Moyer, R.S. Safferman, R.E. Stetler, D. Lye, 

G.S. Fout, and D. Dahling. 2002. Microbial Monitoring of Vulnerable Public Ground Water Supplies. 

EPA/AWWARF. 

Niemi, R.M., Niemela, S.I., Bamford, D.H., Hantula, J., Hyvarinen, T., Forsten, T., and Raateland, A. 1993. 

“Presumptive fecal streptococci in environmental samples characterized by one-dimensional sodium 

dodecyl sulfate-polyacrylamide gel electrophoresis.” Appl Environ Microbiol. 59(7):2190-2196. 

Pedley. S., Yates, M., Schijven, J.F., West, J., Howard, G., and Barrett, M. 2006. “Pathogens: health relevance, 

transport, and attenuation.” In: Protecting groundwater for health: managing the quality of drinking water 

sources. O. Schmoll, G. Howard, J. Chilton, and I. Chorus, eds. IWA Publishing, London. 

Rice, E.W., K.R. Fox, R.J. Miltner, D.A. Lytle and C.H. Johnson (1996) Evaluating plant performance with 

endospores, Journal American Water Works Association, 88:122-130. 

Riffard, S., S. Springthorpe, L. Filion and S.A. Sattar. 2004. Occurrence of Legionella in Groundwater. American 

Water Works Association Research Foundation Report 90985F, Denver, Colorado, 164 pp. 

Rivera, S.C., Hazen, T.C., and Toranzos, G.A. 1988. “Isolation of fecal coliforms from pristine sites in a 

tropical rain forest.” Appl Environ Microbiol. 54(2):513-517. 

Solo-Gabriele, H.M., Wolfert, M.A., Desmarais, T.R., and Palmer, C.J. 2000. “Sources of Escherichia 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

coli in a coastal subtropical environment.” Appl Environ Microbiol. 66(1):230-237. 

Sroka, J., A. Wojcik-Fatla and J. Dutkiewicz. 2006. "Occurrence of Toxoplasma gondii in water from wells located 

on farms." Ann. Agric. Envron. Med. 13:169-175. 

U.S. Environmental Protection Agency. 2001a. Method 1601: Male-specific (F+) and Somatic Coliphage	 in 

Water by Two-step Enrichment Procedure. Office of Water, U.S. Environmental Protection Agency, 

Washington, DC 20460. EPA 821-R-01-030. 

U.S. Environmental Protection Agency. 2001b. Method 1602: Male-Specific (F+) and Somatic Coliphage	 in 

Water by Single Agar Layer (SAL) Procedure. Office of Water, U.S. Environmental Protection Agency, 

Washington, DC 20460. EPA 821-R-01-029. 

U.S. Environmental Protection Agency. 2002. Method 1604: Total Coliforms and Escherichia coli in Water by 

Membrane Filtration Using a Simultaneous Detection Technique (MI Medium). Office of Water, U.S. 

Environmental Protection Agency, Washington, DC 20460. EPA 821-R-02-024. 

U.S. Environmental Protection Agency. 2005. Manual for the Certification of Laboratories Analyzing Drinking 

Water: Criteria and Procedures Quality Assurance [Fifth Edition]. Office of Water, U.S. Environmental 

Protection Agency, Washington, DC 20460. EPA 815-R-05-004. 

U.S. Environmental Protection Agency. 2006a. Method 1600: Enterococci in Water by Membrane Filtration Using 

membrane-Enterococcus Indoxyl-B-D-Glucoside Agar (mEI). Office of Water, U.S. Environmental 

Protection Agency, Washington, DC 20460. EPA 821-R-06-009. 

U.S. Environmental Protection Agency. 2006b. Occurrence and Monitoring Document for the Final Ground Water 

Rule. Office of Water, U.S. Environmental Protection Agency, Washington, DC 20460. EPA-815-R-06­

012. 
 

 

Appendix A
 

Glossary
 

Revised March 2008 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Glossary
 

Analytical holding time - The time between the collection of the sample and the start of analysis.
 

Aquifer - A geologic formation, a group of formations, or a part of a formation that is water bearing. A geological
 

formation or structure that stores or transmits water, or both, such as to wells and springs.
 

Coliphage - Viruses that infect E. coli in Methods 1601 and 1602.
 

Community Water System (CWS) - A public water system that serves at least 15 service connections used by
 

year-round residents or regularly serves at least 25 year-round residents.
 

DAL - Double agar layer plate used to enumerate coliphage in Methods 1601 and 1602.
 

E. coli - A bacteria of the fecal coliform group that can be used as an indicator of fecal contamination in water.
 

Enteric pathogens - Pathogens that infect the gastrointestinal tract.
 

Enterococci - A bacteria in the fecal streptococci group that can be used as an indicator of fecal contamination in
 

water.
 

Fecal indicators - Organisms that can be used as indicators of fecal contamination of water.
 

Fractured bedrock aquifers - Under the Ground Water Rule all igneous and metamorphic aquifers.
 

Gravel aquifer - Unconsolidated water-bearing deposits of well-sorted pebbles, cobbles, and boulders.
 

Ground Water System (GWS) - For the purposes of the GWR and this guidance document, a GWS is defined as
 

any public water system that uses ground water as a source unless that ground water source is combined with 

surface water, or ground water under direct influence of surface water, prior to treatment under subpart H 

(“Filtration and Disinfection”). 

Hydrogeologic sensitivity assessment (HSA) - A determination of whether a ground water system obtains water 

from a hydrogeologically sensitive setting. 

Igneous aquifer - An aquifer consisting of rock formed by the cooling and solidification of magma.
 

Karst aquifer - An aquifer with geologic terrain within which flowing ground water has dissolved significant
 

portions of the area=s soluble (usually carbonate) rocks.
 

Lysis zone - Typically a circular zone of clearing indicating a sample is positive for coliphages in Method 1601.
 

Male-specific coliphage - Viruses (bacteriophages) that infect coliform bacteria only via the F-pilus.
 

Matrix spikes (MS) - A sample prepared by adding a known quantity of organisms to a specified amount of
 

sample matrix for which an independent estimate of target analyte concentration is available. A matrix spike is 

used to determine the effect of the matrix on a method=s recovery 

GWR Source Water Monitoring Methods A-1 Revised March 2008 

Guidance Manual 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Method blank - An aliquot of reagent water that is treated exactly as a sample, including exposure to all 

glassware, equipment, solvents, and procedures that are used with samples. The method blank is used to determine 

if analytes or interferences are present in the laboratory environment, the reagents, or the apparatus. 

Membrane filtration - Direct plating of a sample for detection and estimation of coliform densities. 

Metamorphic aquifer - Aquifer formed from rock that has been transformed under extreme pressure or 

heat from sedimentary, igneous, or other older metamorphic rock. 

Most probable number (MPN) format - A method format uses an index of the number of coliform bacteria that, 

more probably than any other number, would give the results shown by laboratory examination. It is not an actual 

enumeration. 

Multiple-well format - A method format that uses the number of positive wells to determine a result using an 

MPN index. 

National Environmental Laboratory Accreditation Conference (NELAC) - A cooperative association of States 

and Federal Agencies, formed to establish and promote mutually acceptable performance standards for the 

operation of environmental laboratories. 

Negative control culture - A culture that, when analyzed exactly like a field sample, will produce a negative result 

for a given type of media. 

Non-community water system (NCWS) - A public water system that is not a community water system. A noncommunity water system is either a transient non-community water systems or a non-transient non-community 

water system. 

Ongoing demonstration of capability (ODC) - Reagent water samples spiked with known quantities of analytes 

and analyzed exactly like a field sample. The purpose of this test is to assure that the results produced by the 

laboratory remain within limits specified in this method. 

Ongoing precision and recovery (OPR) - A reagent water sample method blank spiked with known quantities of 

analytes. The OPR is analyzed exactly like a sample. Its purpose is to assure that the results produced by the 

laboratory remain within the limits specified within this method for precision and recovery. 

Plaque - Circular zones of clearing (typically 1 to 10 mm in diameter) in lawn of host bacteria in DAL or SAL 

plates after incubation (Method 1601 and 1602) or SAL (Method 1601). 

Plaque forming units (PFU) - The units for reporting the concentration of coliphage in Method 1602. 

Positive control - For Method 1601 and 1602 a reagent water sample spiked with sewage filtrate or pure coliphage 

stock culture that is used to assess the stock coliphage suspensions, host bacterial cultures, and growth media are 

performing properly. 

Primacy - Primary enforcement responsibility-primary responsibility for administration and enforcement of 

primary drinking water regulations and related requirements applicable to public water systems within a State. 

Presence/absence format - A qualitative method format for detection of a microorganism where the result 

indicates whether or not the microorganism is present in the sample. The presence/absence format method will not 

GWR Source Water Monitoring Methods A-2 Revised March 2008 

Guidance Manual 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

give the numbers of a microorganism present. 

Quality assurance (QA) - The system of management controls that cover planning, implementation, and review of 

data collection activities and data use. 

Quality control (QC) - The technical functions that include all scientific precautions, such as calibrations, 

controls, and duplications, that are needed to acquire data of a known and adequate quality. 

Quantitative format - A method format for detection of a microorganism where the result indicates the 

concentration of microorganism in the sample. 

SAL - Single agar layer plate used to enumerate coliphage in Method 1601. 

Sanitary survey - An on-site review of the water source, facilities, equipment, operation, and maintenance of the 

public water system for the purpose of evaluating the adequacy of such source, facilities, equipment, operation, and 

maintenance for producing and distributing safe drinking water. 

Somatic coliphage - Those coliphage that infect host cells via the outer cell membrane but do not infect host cells 

via the F-pilus. 

Total Coliform Rule (TCR) - EPA rule that sets monitoring and compliance requirements for coliform bacteria in 

all PWSs. 

Turbidity - Unit of measurement quantifying the degree to which light traveling through a water column is 

scattered by the suspended organic (including algae) and inorganic particles. 

GWR Source Water Monitoring Methods A-3 Revised March 2008 

Guidance Manual 
 

 

 

 

Appendix B
 

Procedure for Collecting Ground Water Samples for
 

E. coli and Enterococci Analyses 

Revised March 2008 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Procedure for Collecting Ground Water Samples for 

E. coli and Enterococci Analyses 

1.0	 Materials 

1.1	 Check to make sure the following materials are available prior to collecting sample(s): 

$ Several pairs of new, powder-free latex gloves (Lab Safety Supply, cat. number 16285XL, or 

equivalent) 

$ 250 mL, Sterile glass or plastic containers with a leak-proof lids (Nalgene 2105-0008 or 

equivalent) 

$ Labels 

1.2	 The following additional materials will be necessary if the sample will be shipped off-site for analysis: 

$ Sample collection form 

$ Plastic bags (1 gallon) (Ziplock, or equivalent) 

$ Cooler, approximately 9-quart (Coleman, cat. number, 6209-703, or equivalent) 

$ Two large plastic trash bags 

$ One 8-lb. bag of ice or Gel ice packs (VWR, cat. number, 15715-105, or equivalent) 

$ Strapping tape 

$ Two, self-adhesive plastic airbill sleeves 

$ Airbill for shipment 

$ Duct tape 

2.0	 Collecting the Sample 

2.1	 If the sample will be analyzed on-site, record the sample number, sample location, samplers name, 

observations, and sampling date and time in a sampling log book. 

If the sample will be shipped off-site, record the following information on the sample collection form: 

• Name of system (e.g., Public Water System Identification number) 

• Sample site location 

• Sample type (assessment, triggered) 

• Sampler=s name 

• Sample number 

• Date of sample collection 

• Time of sample collection 

• Analysis requested 

2.2	 Water taps used for sampling should be free of aerators, strainers, hose attachments, mixing type faucets, 

and purification devices. The service line should be cleared before sampling by maintaining a steady water 

flow for at least two minutes (until the water changes temperature). 

2.3	 Adjust the flow of water out of the tap or hose so the water will not splash out when it is collected. 

GWR Source Water Monitoring Methods B-1 Revised March 2008 

Guidance Manual 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2.4	 Using aseptic technique (i.e., sanitize tap, do not touch the inside of the sample container), fill each of the 

E. coli and enterococci sample containers, leaving at least 1 inch of head space. Do not expose leave a 

container open for any longer than necessary. Record the system name, sampler=s name, sample number, 

sample type, date and time of sample collection, sample location, and analysis requested on the sample 

container. 

2.5	 Immediately following sample collection, tighten the sample container lid. If the sample will be shipped 

off-site for analysis, and will not be shipped for several hours, place the sample container upright in a 

refrigerator to maintain the sample at a temperature of <10EC prior to shipment. If a refrigerator is not 

available, wrap the sample with insulation such as bubble wrap or paper towels (to prevent freezing), place 

the sample in a ziplock bag, and place the bag containing the sample in the shipping cooler with wet ice or 

ice packs. Replace with fresh ice or ice packs immediately prior to shipment. 

3.0	 Packing the Sample (Applicable to Samples Shipped Off-Site for Analysis) 

3.1	 Insert two large plastic trash bags into the shipping cooler to create a double liner. Immediately before 

packing the cooler, disperse 6 pounds of ice into 3 to 4 plastic, ziplock bags. Gel packs or blue ice may be 

used in lieu of wet ice, as long as the sample is maintained at the appropriate temperature range. Seal the 

ziplock bags, expelling as much air as possible, and secure top with tape. 

Note: Shipping companies may delay sample shipments if leakage occurs. Double liners and 

ziplock bags around ice will help prevent leakage and delays. 

3.2	 Place the bag containing the samples into the shipping container. Place the ice or ice packs around, but not 

directly on, the sample bag to help prevent freezing. Seal each liner bag by twisting top of bag and tying in 

a knot. 

3.3	 Peel the backing off one of the plastic airbill sleeves and attach the sleeve to the inside of the cooler lid. 

Sign and date the sample collection form and fold the completed sample collection form and place it inside 

the plastic sleeve. 

3.4	 Close the cooler lid, seal the joints with duct tape, and secure the lid with strapping tape by taping the 

cooler at each end, perpendicular to the seal. 

Note: Be sure to seal the cooler joints as shipping companies may delay sample shipments if 

leakage occurs. 

3.5	 Peel the backing off of the second airbill sleeve and attach the sleeve to the outside of the cooler lid. 

Complete the shipping airbill with the laboratory address, billing information, sample weight, and shipping 

service. Remove the shipper=s copy of the airbill, and place the remainder of the airbill inside the plastic 

sleeve. 

4.0	 Shipping and Tracking 

4.1	 Contact the laboratory to notify them prior to sampling. This will allow the labs to ensure that they have 

the appropriate media ready for the samples. 

4.2 Ship samples on the day of collection and use a reliable shipping service for priority overnight delivery
 

GWR Source Water Monitoring Methods B-2 Revised March 2008 

Guidance Manual 
 

 

 

 

 

 

 

4.3	 Contact the laboratory to notify them of the sample shipment. Request that the laboratory contact you the 

next day if the sample is not received. 

4.4	 Using the airbill number on the shipper=s copy of the airbill, track the sample shipment using the shipping 

company=s web page or by contacting the shipping company over the phone. 

4.5	 If problems are encountered with the shipment, communicate with the shipping company to resolve, and 

update the laboratory regarding the status of the shipment. 

GWR Source Water Monitoring Methods B-3 Revised March 2008 

Guidance Manual 
 

 

 

 

Appendix C
 

Procedure for Collecting Ground Water Samples for
 

Somatic and/or Male-Specific Coliphage Analyses
 

Revised March 2008 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Procedure for Collecting Ground Water Samples for
 

Somatic and/or Male-Specific Coliphage Analyses
 

1.0	 Materials 

1.1	 Check to make sure the following materials are available prior to collecting sample: 

$ Several pairs of new, powder-free latex gloves (Lab Safety Supply, cat. number 16285XL, or 

equivalent) 

$	 Sterile glass or plastic containers with leak proof lids 

o 250 mL (Nalgene 2105-0008 or equivalent) for 100 mL samples 

o 4 L (Nalgene 2121-0010 or equivalent) for 1 L samples
 

$ Labels
 

1.2	 The following additional materials will be necessary if the sample will be shipped off-site for analysis: 

$	 Sample collection form 

$	 Plastic bags (1 gallon) (Ziplock, or equivalent) 

$	 For 100 mL samples: cooler, approximately 9-quart (Coleman, cat. number, 6209-703, or 

equivalent) 

$	 For 1 L samples: cooler, approximately 28-quart (Coleman, cat. number, 5277-718, or equivalent) 

$	 Two large plastic trash bags 

$	 One 8-lb. bag of ice or Gel ice packs (VWR, cat. number, 15715-105, or equivalent) 

$	 Strapping tape 

$	 Two, self-adhesive plastic airbill sleeves 

$	 Airbill for shipment 

$	 Duct tape 

2.0	 Collecting the Sample 

2.1	 If the sample will be analyzed on-site, record the sample number, sample location, samplers name, 

observations, and sampling date and time in a sampling log book. 

If the sample will be shipped off-site, record the following information on the sample collection form: 

•	 Name of system (e.g., Public Water System Identification number) 

•	 Sample site location 

•	 Sample type (assessment, triggered) 

•	 Sampler=s name 

•	 Sample number 

•	 Date of sample collection 

•	 Time of sample collection 

•	 Analysis requested 

2.2	 Water taps used for sampling should be free of aerators, strainers, hose attachments, mixing type faucets, 

and purification devices. The service line should be cleared before sampling by maintaining a steady water 

GWR Source Water Monitoring Methods C-1 Revised March 2008 

Guidance Manual 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

flow for at least two minutes (until the water changes temperature). Note: Sample bottles should not be 

pre-rinsed with sample when collecting somatic and/or male-specific coliphage samples. 

2.3	 Adjust the flow of water out of the tap or hose so the water will not splash out when it is collected. 

2.4	 Using aseptic techniques (i.e., sanitize tap, do not touch the inside of the sample container, etc.), fill each 

of the somatic and/or male-specific coliphage sample containers, leaving at least 1 inch of head space. Do 

not leave a container open for any longer than necessary. Record the system name, sampler=s name, 

sample number, sample type, date and time of sample collection, sample location, and analysis requested 

on the sample container. 

2.5	 Immediately following sample collection, tighten the sample container lid. If the sample will be shipped 

off-site for analysis, and will not be shipped for several hours, place the sample container upright in a 

refrigerator to maintain the sample at a temperature of < 8EC prior to shipment. If a refrigerator is not 

available, wrap the sample with insulation such as bubble wrap or paper towels (to prevent freezing), place 

the sample in a ziplock bag, and place the bag containing the sample in the shipping cooler with wet ice or 

ice packs. Replace with fresh ice or ice packs immediately prior to shipment. 

3.	 Packing the Sample (Applicable to Samples Shipped Off-Site for Analysis) 

3.1	 Insert two large plastic trash bags into the shipping cooler to create a double liner. Immediately before 

packing the cooler, disperse 6 pounds of ice into 3 to 4 plastic, ziplock bags. Gel packs or blue ice may be 

used in lieu of wet ice, as long as the sample is maintained at the appropriate temperature range. Seal the 

ziplock bags, expelling as much air as possible, and secure top with tape. 

Note: Shipping companies may delay sample shipments if leakage occurs. Double liners and 

ziplock bags around ice will help prevent leakage and delays. 

3.2	 Place the bag containing the samples into the shipping container. Place the ice or ice packs around, but not 

directly on, the sample bag to help prevent freezing. Seal each liner bag by twisting top of bag and tying in 

a knot. 

3.3	 Peel the backing off one of the plastic airbill sleeves and attach the sleeve to the inside of the cooler lid. 

Sign and date the sample collection form. Fold the completed sample collection form and place it inside 

the plastic sleeve. 

3.4	 Close the cooler lid, seal the joints with duct tape, and secure the lid with strapping tape by taping the 

cooler at each end, perpendicular to the seal. 

Note: Be sure to seal the cooler joints as shipping companies may delay sample shipments if 

leakage occurs. 

3.5	 Peel the backing off of the second airbill sleeve and attach the sleeve to the outside of the cooler lid. 

Complete the shipping airbill with the laboratory address, billing information, sample weight, and shipping 

service. Remove the shipper=s copy of the airbill, and place the remainder of the airbill inside the plastic 

sleeve. 

GWR Source Water Monitoring Methods C-2 Revised March 2008 

Guidance Manual 
 

 

 

 

 

 

 

 

 

 

 

 

4.0	 Shipping and Tracking 

4.1	 Contact the laboratory to notify them prior to sampling. This will allow the labs to ensure that they have 

the appropriate media and, in the case of coliphage, host organisms ready for the samples. 

4.2	 Ship samples on the day of collection and use a reliable shipping service for priority overnight delivery. 

4.3	 Contact the laboratory to notify them of the sample shipment. Request that the laboratory contact you the 

next day if the sample is not received. 

4.4	 Using the airbill number on the shipper=s copy of the airbill, track the sample shipment using the shipping 

company=s web page or by contacting the shipping company over the phone. 

4.5	 If problems are encountered with the shipment, communicate with the shipping company to resolve, and 

update the laboratory regarding the status of the shipment. 

GWR Source Water Monitoring Methods C-3 Revised March 2008 

Guidance Manual 
 

 

 

 

 

Appendix D 

E. coli Method Bench Sheets 

Note: Forms provided in this appendix are presented as guidance and are NOT required for 

compliance with GWR requirements. 

Revised March 2008 
Ground Water Rule Source Water Monitoring 

Presence/Absence: 

E. coli (Colilert® and Colisure) 

Presence/Absence (SM 9223) 

Laboratory: ____________________ Sample collection date: ___________________ Sample collection time: ___________________ 

PWS ID: Sampling point: 

Please circle the method used for analysis: 

Colilert Colisure 

Incubation start

Date 

Time 

Temperature (o

C) 

Incubation end 

Date 

Time 

Temperature (o

C) 

Additional incubation start 

Date 

Time 

Temperature (o

C) 

Additional incubation end 

Date 

Time 

Temperature (o

C) 

PRESENCE/ABSENCE RESULTS 

Analyst initials Volume analyzed (mL) 

Total coliforms 

"+" = present 

"-" = absent 

E. coli 

"+" = present 

"-" = absent 

Comments 

GWR Source Water Monitoring Methods Guidance Manual D - 1 Revised March 2008 
Ground Water Rule Source Water Monitoring
 

Multiple-Tube:
 

E. coli (Colilert® and Colisure)
 

Most Probable Number (SM 9223)
 

Laboratory: ____________________ Sample collection date: ___________________ Sample collection time: ___________________ 

PWS ID: Sampling point: 

Please circle the method used for analysis: 

Colilert Colisure 

Incubation start 

Date 

Time 

Temperature (o

C) 

Incubation end 

Date 

Time 

Temperature (o

C) 

Additional incubation start 

Date 

Time 

Temperature (o

C) 

Additional incubation end 

Date 

Time 

Temperature (o

C) 

Analyst 

initials 

Start 

Temp. 

Read 

Temp. 

Tube 1 

(10 mL) 

Tube 2 

(10 mL) 

Tube 3 

(10 mL) 

Tube 4 

(10 mL) 

Tube 5 

(10 mL) 

Tube 6 

(10 mL) 

Tube 7 

(10 mL) 

Tube 8 

(10 mL) 

Tube 9 

(10 mL) 

Tube 10 

(10 mL) 

Total coliforms/ 

E. coli 

Note: Complete either the presence/absence results table or the quantitative results table based on the type of 

analysis requested by the PWS. 

PRESENCE/ABSENCE RESULTS 

"+" = present 

"-" = absent 

Total coliforms E. coli 

QUANTITATIVE RESULTS 

Total coliforms E. coli 

Number of positive 

tubes 

Organisms per 100 mL 

Comments 

GWR Source Water Monitoring Methods Guidance Manual D - 2 Revised March 2008 
Ground Water Rule Source Water Monitoring 

Multiple-Well (51 Wells): 

E. coli (Colilert® and Colisure) 

Quanti-Tray 51 (SM 9223) 

Laboratory: __________________ Sample collection date: __________________ Sample collection time: __________________ 

PWS ID: Sampling point: 

Please circle the method used for analysis: 

Colilert Colisure 

Incubation start 

Date 

Time 

Temperature (o

C) 

Incubation end 

Date 

Time 

Temperature (o

C) 

Additional incubation start 

Date 

Time 

Temperature (o

C) 

Additional incubation end 

Date 

Time 

Temperature (o

C) 

Note: Complete either the presence/absence results table or the quantitative results table based on the type of 

analysis requested by the PWS. 

PRESENCE/ABSENCE RESULTS 

Analyst initials Volume analyzed (mL) 

Total coliforms 

"+" = present 

"-" = absent 

E. coli 

"+" = present 

"-" = absent 

QUANTITATIVE RESULTS 

Analyst initials Volume analyzed (mL) 

Total coliforms E. coli 

Number of positive 

wells 

Total coliforms 

per 100 mL 

Number of 

positive wells E. coli per 100 mL 

Comments 

GWR Source Water Monitoring Methods Guidance Manual D - 3 Revised March 2008 
Ground Water Rule Source Water Monitoring 

Multiple-Well (97 Wells): 

E. coli (Colilert® and Colisure) 

Quanti-Tray 2000 (SM 9223) 

Laboratory: __________________ Sample collection date: __________________ Sample collection time: __________________ 

PWS ID: Sampling point: 

Please circle the method used for analysis: 

Colilert Colisure 

Incubation start 

Date 

Time 

Temperature (o

C) 

Incubation end 

Date 

Time 

Temperature (o

C) 

Additional incubation start 

Date 

Time 

Temperature (o

C) 

Additional incubation end 

Date 

Time 

Temperature (o

C) 

Note: Complete either the presence/absence results table or the quantitative results table based on the type of 

analysis requested by the PWS. 

PRESENCE/ABSENCE RESULTS 

Analyst initials Volume analyzed (mL) 

Total coliforms 

"+" = present 

"-" = absent 

E. coli 

"+" = present 

"-" = absent 

QUANTITATIVE RESULTS 

Analyst initials

 Volume 

analyzed 

(mL) 

Total Coliforms E. coli 

Number of large 

positive wells 

Number of small 

positive wells 

Total coliforms per 

100 mL 

Number of large 

positive wells 

Number of small 

positive wells E. coli per 100 mL 

Comments 

GWR Source Water Monitoring Methods Guidance Manual D - 4	 Revised March 2008 
Ground Water Rule Source Water Monitoring 

Presence/Absence: E. coli (E*Colite) 

Laboratory: __________________ Sample collection date: __________________ Sample collection time: __________________ 

PWS ID: Sampling point: 

Incubation start 

Date 

Time 

Temperature (o

C) 

Incubation end 

Date 

Time 

Temperature (o

C) 

Note: Complete the presence/absence results table based on the type of analysis requested by the PWS. 

PRESENCE/ABSENCE RESULTS 

Analyst initials Volume analyzed (mL) 

Total coliforms 

"+" = present 

"-" = absent 

E. coli 

"+" = present 

"-" = absent 

Comments 

GWR Source Water Monitoring Methods Guidance Manual D - 5 Revised March 2008 
Ground Water Rule Source Water Monitoring 

Multiple-Tube Fermentation: E. coli (LTB/EC-MUG) 

(SM 9221B/9221F) 

Laboratory: ____________________ Sample collection date: ___________________ Sample collection time: ___________________ 

PWS ID: Sampling point: 

LTB incubation start LTB 24 hour incubation read LTB 48 hour incubation read 

Date 

Time 

Temperature (o

C) 

EC-MUG incubation 24 hour read (from 24 hour LTB) 

Date 

Time 

Temperature (o

C) 

EC-MUG incubation 24 hour read (from 48 hour LTB) 

Date 

Time 

Temperature (o

C) 

LTB 

LTB read Analyst 

initials 

Start 

Temp. 

Read 

Temp. 

Tube 1 

(10 mL) 

Tube 2 

(10 mL) 

Tube 3 

(10 mL) 

Tube 4 

(10 mL) 

Tube 5 

(10 mL) 

Tube 6 

(10 mL) 

Tube 7 

(10 mL) 

Tube 8 

(10 mL) 

Tube 9 

(10 mL) 

Tube 10 

(10 mL) 

24 hr LTB/ 

48 hr LTB 

EC-MUG 

EC-MUG read Analyst 

initials 

Start 

Temp. 

Read 

Temp. 

Tube 1 

(10 mL) 

Tube 2 

(10 mL) 

Tube 3 

(10 mL) 

Tube 4 

(10 mL) 

Tube 5 

(10 mL) 

Tube 6 

(10 mL) 

Tube 7 

(10 mL) 

Tube 8 

(10 mL) 

Tube 9 

(10 mL) 

Tube 10 

(10 mL) 

from 24 hr LTB 

from 48 hr LTB 

Note: Complete either the presence/absence results table or the quantitative results table based on the type of analysis 

requested by the PWS. 

PRESENCE/ABSENCE RESULTS 

E. coli 

"+" = present 

"-" = absent 

QUANTITATIVE RESULTS 

Number of positive tubes E. coli per 100 mL 

Comments 

GWR Source Water Monitoring Methods Guidance Manual D - 6 Revised March 2008 
Ground Water Rule Source Water Monitoring
 

Membrane Filtration: E. coli (mEndo or LES-Endo/NA-MUG)
 

(SM 9222B to 9222G)
 

Laboratory: __________________ Sample collection date: __________________ Sample collection time: __________________ 

PWS ID: Sampling point: 

mEndo or LES-Endo incubation start 

Date 

Time 

Temperature (o

C) 

mEndo or LES-Endo incubation end 

Date 

Time 

Temperature (o

C) 

NA-MUG incubation start 

Date 

Time 

Temperature (o

C) 

NA-MUG incubation end 

Date 

Time 

Temperature (o

C) 

Note: Complete either the presence/absence results table or the quantitative results table based on the type of analysis 

requested by the PWS. 

PRESENCE/ABSENCE RESULTS 

Analyst initials Volume filtered 

Total coliforms 

"+" = present 

"-" = absent 

E. coli 

"+" = present 

"-" = absent 

QUANTITATIVE RESULTS 

Analyst initials Volume filtered 

Total coliforms E. coli 

Number of 

colonies 

Total coliforms 

per 100 mL Number of colonies E. coli per 100 mL 

Comments 

GWR Source Water Monitoring Methods Guidance Manual D - 7 Revised March 2008 
Ground Water Rule Source Water Monitoring 

Membrane Filtration: E. coli (MI Medium) 

(EPA Method 1604) 

Laboratory: __________________ Sample collection date: __________________ Sample collection time: __________________ 

PWS ID: Sampling point: 

MI Medium incubation start 

Date 

Time 

Temperature (o

C) 

MI Medium incubation end 

Date 

Time 

Temperature (o

C) 

Note: Complete either the presence/absence results table or the quantitative results table based on the type of 

analysis requested by the PWS. 

PRESENCE/ABSENCE RESULTS 

Analyst initials Volume filtered 

Total coliforms 

"+" = present 

"-" = absent 

E. coli 

"+" = present 

"-" = absent 

QUANTITATIVE RESULTS 

Analyst initials Volume filtered 

Total coliforms E. coli 

Number of 

colonies 

Total coliforms 

per 100 mL Number of colonies E. coli 

per 100 mL 

Comments 

GWR Source Water Monitoring Methods Guidance Manual D - 8 Revised March 2008 
Ground Water Rule Source Water Monitoring 

Membrane Filtration: E. coli (m-ColiBlue24®) 

(m-ColiBlue24) 

Laboratory: __________________ Sample collection date: __________________ Sample collection time: __________________ 

PWS ID: Sampling point: 

m-ColiBlue24 incubation start 

Date 

Time 

Temperature (o

C) 

m-ColiBlue24 incubation end 

Date 

Time 

Temperature (o

C) 

Note: Complete either the presence/absence results table or the quantitative results table based on the type of 

analysis requested by the PWS. 

PRESENCE/ABSENCE RESULTS 

Analyst initials Volume filtered 

Total coliforms 

"+" = present 

"-" = absent 

E. coli 

"+" = present 

"-" = absent 

QUANTITATIVE RESULTS 

Analyst initials Volume filtered 

Total coliforms E. coli 

Number of 

colonies 

Total coliforms 

per 100 mL Number of colonies E. coli per 100 mL 

Comments 

GWR Source Water Monitoring Methods Guidance Manual D - 9 Revised March 2008 
 

 

 

 

 

Appendix E
 

Enterococci Method Bench Sheets
 

Note: Forms provided in this appendix are presented as guidance and are NOT required for
 

compliance with GWR requirements.
 

Revised March 2008 
Ground Water Rule Source Water Monitoring
 

Multiple-Tube: Enterococcus (Azide Dextrose/BEA/BHI/BHI-6.5% NaCl)
 

(SM 9230B)
 

Laboratory: _____________________ Sample collection date: _____________________ Sample collection time: _____________________ 

PWS ID: Sampling point: 

ADB incubation start ADB 24 hour incubation read ADB 48 hour incubation read 

Date 

Time 

24 hour BEA read (from 24 hour ADB) 

Date 

Time 

Temperature (o

C) 

24 hour BEA read (from 48 hour ADB) 

Date 

Time 

Temperature (o

C) 

48 hour BHI (at 45o

C) read (from 24 hour BEA) 

Date 

Time 

Temperature (o

C) 

48 hour BHI-6.5% NaCl read (from 24 hour BEA) 

Date 

Time 

Temperature (o

C) 

ADB 

ADB read Analyst 

initials 

Start 

Temp. 

Read 

Temp. 

Tube 1 

(10 mL) 

Tube 2 

(10 mL) 

Tube 3 

(10 mL) 

Tube 4 

(10 mL) 

Tube 5 

(10 mL) 

Tube 6 

(10 mL) 

Tube 7 

(10 mL) 

Tube 8 

(10 mL) 

Tube 9 

(10 mL) 

Tube 10 

(10 mL) 

24 hr ADB/ 

48 hr ADB 

BEA Plates 

BEA read Analyst 

initials 

Start 

Temp. 

Read 

Temp. 

Plate 1 

(10 mL) 

Plate 2 

(10 mL) 

Plate 3 

(10 mL) 

Plate 4 

(10 mL) 

Plate 5 

(10 mL) 

Plate 6 

(10 mL) 

Plate 7 

(10 mL) 

Plate 8 

(10 mL) 

Plate 9 

(10 mL) 

Plate 10 

(10 mL) 

from 24 hr ADB/ 

from 48 hr ADB 

BHI/BHI-6.5% NaCl READ 

BHI/ 

BHI-6.5% NaCl Analyst 

initials 

Start 

Temp. Read 

Temp. 

Tube 1 

(10 mL) 

Tube 2 

(10 mL) 

Tube 3 

(10 mL) 

Tube 4 

(10 mL) 

Tube 5 

(10 mL) 

Tube 6 

(10 mL) 

Tube 7 

(10 mL) 

Tube 8 

(10 mL) 

Tube 9 

(10 mL) 

Tube 10 

(10 mL) 

BHI (at 45o

C)/ 

BHI-6.5% NaCl 

Note: Complete either the presence/absence results table or the quantitative results table based on the type of analysis requested 

by the PWS. 

PRESENCE/ABSENCE RESULTS 

Enterococci 

"+" = present "-" = absent 

QUANTITATIVE RESULTS 

Number of positive tubes Enterococci per 100 mL 

Comments 

GWR Source Water Monitoring Methods Guidance Manual E-1 Revised March 2008 
Ground Water Rule Source Water Monitoring 

Membrane Filtration: Enterococcus (mE-EIA) 

(SM 9230C) 

Laboratory: _________________ Sample collection date: _________________ Sample collection time: ______________ 

PWS ID: Sampling point: 

mE incubation start 

Date 

Time 

Temperature (o

C) 

mE incubation end 

Date 

Time 

Temperature (o

C) 

EIA incubation start 

Date 

Time 

Temperature (o

C) 

EIA incubation end 

Date 

Time 

Temperature (o

C) 

Note: Complete either the presence/absence results table or the quantitative results table based on the type of 

analysis requested by the PWS. 

PRESENCE/ABSENCE RESULTS 

Analyst initials Volume filtered 

Enterococcus 

"+" = present 

"-" = absent 

QUANTITATIVE RESULTS 

Analyst initials Volume filtered Number of colonies Enterococci 

per 100 mL 

Comments 

GWR Source Water Monitoring Methods Guidance Manual E-2 Revised March 2008 
Ground Water Rule Source Water Monitoring 

Membrane Filtration: Enterococcus (mEI) 

(EPA Method 1600) 

Laboratory: _________________ Sample collection date: _________________ Sample collection time: ______________ 

PWS ID: Sampling point: 

mEI incubation start 

Date 

Time 

Temperature (oC) 

mEI incubation end 

Date 

Time 

Temperature (o

C) 

Note: Complete either the presence/absence results table or the quantitative results table based on the type of 

analysis requested by the PWS. 

PRESENCE/ABSENCE RESULTS 

Analyst initials Volume filtered 

Enterococcus 

"+" = present 

"-" = absent 

QUANTITATIVE RESULTS 

Analyst initials Volume filtered Number of colonies Enterococci 

per 100 mL 

Comments 

GWR Source Water Monitoring Methods Guidance Manual E-3 Revised March 2008 
Ground Water Rule Source Water Monitoring
 

Multiple-Tube:
 

Enterococcus (EnterolertTM)
 

Most Probable Number
 

Laboratory: _________________ Sample collection date: _________________ Sample collection time: ______________ 

PWS ID: Sampling point: 

Incubation start 

Date 

Time 

Temperature (o

C) 

Incubation end 

Date 

Time 

Temperature (o

C) 

Note: Complete the most probable number results table based on the type of analysis requested by the PWS. 

MOST PROBABLE NUMBER RESULTS 

Analyst initials Tube 1 

(10 mL) 

Tube 2 

(10 mL) 

Tube 3 

(10 mL) 

Tube 4 

(10 mL) 

Tube 5 

(10 mL) 

Tube 6 

(10 mL) 

Tube 7 

(10 mL) 

Tube 8 

(10 mL) 

Tube 9 

(10 mL) 

Tube 10 

(10 mL) 

Enterococci 

QUANTITATIVE RESULTS 

Number of positive tubes Enterococci per 100 mL 

Comments 

GWR Source Water Monitoring Methods Guidance Manual E-4 Revised March 2008 
 

 

 

 

 

Appendix F
 

Coliphage Method Bench Sheets
 

Note: Forms provided in this appendix are presented as guidance and are NOT required for
 

compliance with GWR requirements.
 

Revised March 2008 
Method 1601: Two-Step Enrichment, Presence/Absence 

Please complete one report form for each spot plate 

Section 1: General Information 

Laboratory name: Analyst(s): Phage type (circle one): Male-specific Somatic Volume analyzed (circle one): 100 mL 1 L 

PWS ID: Sample collection: Batch ID: Spot plate ID: 

Section 2: Sample Analysis Indicate "+" = presence "-" = absence 

Sample ID Sample collection date Sample collection time Enrichment start date Enrichment start time Enrichment start temp Enrichment end date Enrichment end time Enrichm

te

Positive Control Method Blank 

Spot plate Spot plate Start date: End date: Start time: End time: Start temp: End temp: Analysts initials: Analysts initials: 

Section 3: Comments 

• PFU = • V = 

GWR Source Water Monitoring Methods Guidance Manual F-1 
Method 1602: Single Agar Layer (SAL), Quantitative 

Section 1: General Information 

Laboratory name: Analyst(s): PWS ID: Sample collection point: Coliphage type (circle one): Male-specific Somatic Batch ID: 

Section 2: Sample Analysis 

Start date:Start time:Start temp:Analysts initials: 

End date: End time: End temp: Analysts initials: 

Sample ID Sample collection date Sample collection time No. PFU per Plate 1 No. PFU per Plate 2 No. PFU per Plate 3 No. PFU per Plate 4 

Section 3: Comments

GWR Source Water Monitoring Methods Guidance Manual F-2 
Section 1: General

Laboratory name:____________________ Filtrate collection date:___________________ Filtrate collection time:_Start date: Dilution Factor Inoculation volume Number of Plaques 

1 0.5 mL

Start time: 1 0.5 mLAnalyst initials: 0.1 0.5 mL

0.1 0.5 mL

End date: 0.01 0.5 mL

0.01 0.5 mL

End time:

0.001 0.5 mL

Analyst initials: 0.001 0.5 mLSection 3: Comments*Undiluted spiking suspension PFU / mL = (PFU1 + PFU2... PFUn)/(V1 + V2.... Vn)Where: 

• PFU = number of plaque forming units from plates of all countable sample dilutions 

(dilutions with 1 or more PFU per plate, excluding dilutions with all TNTC or all zeros)

• V = volume of undiluted sample in all plates with countable plaques

• n = number of useable counts

0.001 dilution

Filtrate Concentration

UndilutedUndiluted

0.1 dilution

Coliphage Enumeration by the Double Agar Layer Procedure (DAL, Section 11

0.01 dilution

0.001 dilution

Section 2: Coliphage enumeration 

0.1 dilution

0.01 dilution

GWR Source Water Monitoring Methods Guidance Manual F-3 
 

 

 

 

 

 

 

Appendix G
 

Ground Water Rule Source Water Monitoring
 

Quality Control Checklist for Presence Absence and Most Probable Number
 

E. coli or Enterococci Sample Results
 

Note: Forms provided in this appendix are presented as guidance and are NOT required for 

compliance with GWR requirements. 

Revised March 2008 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Ground Water Rule Source Water Monitoring
 

Quality Control Checklist for Presence Absence and Most Probable Number
 

E. coli or Enterococci Sample Results
 

No. 

Quality Control 

(QC) Procedure 

Sample Condition 

Description U

1 

Sample condition 

upon receipt 

Holding Time 

The sample was not frozen or leaking upon receipt 

2 Holding time 

General Quality Control 

The sample was analyzed within 30 hours of sample collection 

3 

Dilution/rinse water 

sterility check 

The dilution/rinse water sterility check was acceptable (target organism 

or potentially interfering materials were not found in the sterility check) 

4 Media sterility check The media sterility check was acceptable (target organism or potentially 

interfering materials were not found in the sterility check) 

5 

Positive/negative 

controls The positive/negative controls were acceptable 

6 

Media storage 

requirements The media storage requirements were not exceeded 

7 

Autoclave sterilization 

verification Autoclave sterilization verifications were acceptable 

8 

Incubator/waterbath 

temperature checks 

Incubator/waterbath temperatures were measured and recorded 2 times 

per day, 4 hours apart, and were within the temperature ranges specified 

in the method 

9 

Refrigerator/freezer 

temperature checks 

Refrigerator/freezer temperatures were measured and recorded once per 

day and were within acceptable temperature ranges 

10 

Sample processing 

equipment sterility 

checks 

Sample processing equipment sterility checks were acceptable (target 

organism or potentially interfering materials were not found in the sterility 

check) 

GWR Source Water Monitoring Methods G-1 Revised March 2008 

Guidance Manual 
GWR Source Water Monitoring Methods G-2 Revised March 2008 

Guidance Manual 

Presence Absence and Most Probable Number Specific QC

11 

Incubation time and 

temperature 

The following incubation time and temperature requirements were not 

exceeded: 

E. coli

Colilert 24 - 28 hours at 35.0EC"0.5EC 

Colisure 24 hours (up to 48 hours) at 35.0EC"0.5EC 

LTB 24"2 or 48"3 hours at 35.0EC"0.5EC 

EC-MUG 24"2 hours at 44.5EC"0.2EC 

E*Colite 28 hours (up to 48 hours) at 35.0EC"0.5EC 

Enterococci

Enterolert 24 hours at 41.0EC " 0.5EC 

ADB 24"2 or 48"3 hours 35.0EC"0.5EC 

BEA 24"2 hours at 35.0EC"0.5EC 

BHI 48 hours at 45.0EC"0.5EC 

BHI - 6.5% NaCl 48 hours at 35.0EC"0.5EC 

12 Preparation blank The preparation blank was not contaminated with the target organism 

13 

Verification of positive 

results 

The verifications, performed in accordance with method-specific 

requirements, were acceptable 

Note: Please see Sections 6.5.1 and 6.5.3 for detailed descriptions of quality control procedures for E. coli 

and enterococci methods. 
 

 

 

 

 

 

 

Appendix H
 

Ground Water Rule Source Water Monitoring
 

Quality Control Checklist for Membrane Filtration
 

E. coli or Enterococci Sample Results
 

Note: Forms provided in this appendix are presented as guidance and are NOT required for 

compliance with GWR requirements. 

Revised March 2008 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Ground Water Rule Source Water Monitoring
 

Quality Control Checklist for Membrane Filtration
 

E. coli or Enterococci Sample Results
 

No. 

Quality Control 

(QC) Procedure Description U

Sample Condition 

1 

Sample condition 

upon receipt The sample was not frozen or leaking upon receipt 

Holding Time 

2 Holding time The sample was analyzed within 30 hours of sample collection 

General Quality Control 

3 

Dilution/rinse water 

sterility check 

The dilution/rinse water sterility check was acceptable (target organism or 

potentially interfering materials were not found in the sterility check) 

4 Media sterility check The media sterility check was acceptable (target organism or potentially interfering materials were not found in the sterility check) 

5 

Positive/negative 

controls The positive/negative controls were acceptable 

6 

Media storage 

requirements The media storage requirements were not exceeded 

7 

Autoclave 

sterilization 

verification 

Autoclave sterilization verifications were acceptable 

8 

Incubator/waterbath 

temperature checks 

Incubator/waterbath temperatures were measured and recorded 2 times per 

day, 4 hours apart and were within the temperature ranges specified in the 

method 

9 

Refrigerator/freezer 

temperature checks 

Refrigerator/freezer temperatures were measured and recorded once per day 

and were within acceptable temperature ranges 

10 

Sample processing 

equipment sterility 

checks 

Sample processing equipment sterility checks were acceptable (no E. coli / 

enterococci or potentially interfering materials were found in the sterility 

checks) 

Membrane Filtration Specific QC 

11 

Incubation time and 

temperature 

The following incubation time and temperature requirements were not 

exceeded: 

E. coli 

mEndo6NA-MUG 24"2 hours at 35.0EC"0.5EC 4 hours at 35.0EC"0.5EC 

LES-Endo6NA-MUG 24"2 hours at 35.0EC"0.5EC 4 hours at 35.0EC"0.5EC 

MI Medium 24 hours at 35.0EC"0.5EC 

m-ColiBlue24 24 hours at 35.0EC"0.5EC 

Enterococci 

mE6EIA 48 " 3 hours at 41.0EC"0.5EC6

20-30 minutes at 41.0EC"0.5EC 

mEI 24"2 hours at 35.0EC"0.5EC 

GWR Source Water Monitoring Methods H-1 Revised March 2008 

Guidance Manual 
 

 

 

 

 

 

 

 

 

 

 

Membrane Filtration Specific QC (continued) 

12 

Filtration unit 

sterility check 

The filtration unit sterility check was acceptable (target organism or potentially 

interfering materials were not found in the sterility check) 

13 Preparation blank The preparation blank was not contaminated with the target organism 

14 Colony verification The verifications, performed in accordance with method-specific 

requirements, were acceptable 

Note: Please see Sections 6.5.1 and 6.5.2 for detailed descriptions of quality control procedures for E. coli and enterococci 

methods. 

GWR Source Water Monitoring Methods H-2 Revised March 2008 

Guidance Manual 
 

 

 

 

 

 

 

Appendix I
 

Ground Water Rule Source Water Monitoring
 

Quality Control Checklist for Method 1601: Two-Step Enrichment
 

Coliphage Sample Results
 

Note: Forms provided in this appendix are presented as guidance and are NOT required for 

compliance with GWR requirements. 

Revised March 2008 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Ground Water Rule Source Water Monitoring Quality Control Checklist for
 

Method 1601: Two-Step Enrichment Coliphage Sample Results
 

No. 

Quality Control 

(QC) Procedure Description U

Sample Condition 

1 

Sample condition 

upon receipt The sample was not frozen or leaking upon receipt 

Holding Time 

2 Holding time The sample was analyzed within 48 hours of sample collection 

General Quality Control 

3 

Media sterility checks The media sterility checks were acceptable (no coliphage or any 

potentially interfering organisms were found in the sterility check) for all 

media 

4 

Media storage 

requirements Media storage time and temperature requirements were met 

Coliphage Specific QC 

5 

Matrix spikes Matrix spike sample was spiked at the appropriate level for volume 

analyzed (100 mL or 1 L) and the results were acceptable (at least 1 of 3 

were positive) 

6 

Ongoing 

demonstration of 

capability (ODC) 

The ODC sample was spiked at the appropriate level for volume analyzed 

(100 mL or 1 L) and the results were acceptable (at least 1 of 3 were 

positive) 

7 

Incubation time and 

temperature 

The following incubation time and temperature requirements were met: 

10X TSB 16 - 24 hours at 36.0EC"1.0EC 

TSA (DAL plates) 16 - 24 hours at 36.0EC"1.0EC 

TSA (spot plates) 16 - 24 hours at 36.0EC"1.0EC 

8 Positive controls The positive controls were acceptable 

9 

Method blank The method blank was acceptable (no coliphage or any potentially 

interfering organisms were found in the sterility check) 

10 Spot plates A positive control and method blank were analyzed for each spot plate 

used for field samples 

11 

ODC and MS 

analyses ODC and MS analyses were conducted at the method-specified frequency 

Verification Specific QC 

12 Spot plates A positive control and method blank were analyzed for each spot plate 

13 

Incubation time and 

temperature 

The following incubation time and temperature requirements were met: 

TSA (spot plates) 16 - 24 hours at 36.0EC"1.0EC 

Note: Please see Sections 6.5.1 and 6.5.3 for detailed descriptions of quality control procedures for Method 1601. 

GWR Source Water Monitoring Methods I-1 Revised March 2008 

Guidance Manual 
 

 

 

 

 

 

 

Appendix J
 

Ground Water Rule Source Water Monitoring
 

Quality Control Checklist for Method 1602: Single Agar Layer (SAL)
 

Coliphage Sample Results
 

Note: Forms provided in this appendix are presented as guidance and are NOT required for 

compliance with GWR requirements. 

Revised March 2008 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Ground Water Rule Source Water Monitoring
 

Quality Control Checklist for Method 1602: Single Agar Layer (SAL)
 

Coliphage Sample Results
 

No. 

Quality Control 

(QC) Procedure Description U

Sample Condition 

1 

Sample condition 

upon receipt The sample was not frozen or leaking upon receipt 

Holding Time 

2 Holding time The sample was analyzed within 48 hours of sample collection 

General Quality Control 

3 Media sterility checks The media sterility checks were acceptable (no coliphage or any 

potentially interfering organisms found in the sterility check) for all media 

4 

Media storage 

requirements Media storage time and temperature requirements were met 

Coliphage Specific QC 

5 Matrix spikes Matrix spike was spiked at the appropriate level (80 PFU) and the results 

were acceptable (range for male-specific and somatic) 

6 

Ongoing precision 

and recovery (OPR) 

The OPR sample was spiked at the appropriate level (80 PFU) and the 

results were acceptable (range for male-specific and somatic) 

7 

Incubation time and 

temperature 

The following incubation time and temperature requirements were met: 

TSA (DAL plates) 16 - 24 hours at 36.0EC"1.0EC 

TSA (SAL plates) 16 - 24 hours at 36.0EC"1.0EC 

8 

Method blank The method blank was acceptable (no coliphage or any potentially 

interfering organisms were found in the sterility check) 

9 Positive controls The positive controls were acceptable 

Verification Specific QC 

10 Spot plates A positive control and method blank were analyzed for each spot plate 

11 

Incubation time and 

temperature 

The following incubation time and temperature requirements were met: 

TSA (spot plates) 16 - 24 hours at 36.0EC"1.0EC 

Note: Please see Section 6.5.1 and 6.5.4 for detailed descriptions of quality control procedures for Method 1602 

GWR Source Water Monitoring Methods J-1 Revised March 2008 

Guidance Manual