&EPA
    United States
    Environmental Protection
    Agency
Ground Water Rule Source Water
Monitoring Methods Guidance
Office of Water (4607M)
EPA815-R-07-019
Revised March 2008
                       Prepared/or:
                U.S. Environmental Protection Agency
               Office of Ground Water and Drinking Water
                   1200 Pennsylvania Ave, N.W.
                    Washington, B.C. 20460

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                                             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   Enterolert™ (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
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                                   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, immuno-
compromised) 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 unconnected 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
                                                              Initial and periodic sanitary surveys performed by the State
                                                             i Community water systems (CWSs): every 3-5 years
                                                             • Non-community water systems (NCWSs): every 5 years
                      Conduct routine sampling
                    under the Total Coliform Rule
                              (TCR)
       Did the   \
   State identify any
      significant
 \ deficiencies?'41 -^
                                                                Consult State within 30 days of notification regarding
                                                                    appropriate corrective action, if necessary
                                                              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
                                                                         Complete or be in accordance
                                                                         with State-specified corrective
                                                                         action within 120 days of initial
                                                                         notification of contamination or
                                                                             significant deficiency
                                                                       /Continue State-required GWR y
                                                                      /  compliance: sanitary surveys, V*
                                                                          triggered monitoring, TCR
                                                                              compliance, and
                                                                            assessment monitoring
                              Compliance monitoring - options include:
                                                                                            /  Per State
                                                                                              direction, take
                                                                                            corrective action or
                                                                                               5 additional
                                                                                                samples   /
                    Monitor the
                    alternative
                    treatment
                    process in
                    accordance
                    with State-
                     specified
                   requirements
Serving
<3,300
people:
Monitor
residual
disinfectant
daily via grab
sample at
peak flow










Serving
> 3,300
people:
Continuously
monitor
residual
disinfectant











Monitor the
filtration
process in
accordance
with State-
specified
requirements

     (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.
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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 2001 a)
       •   Single Agar Layer Procedure (EPA Method 1602) (USEPA 200Ib)

       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 10°C 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 pore-
size 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 Legionellapneumophila (Costa et al., 2005; Riffard et al.,
2004), Helicobacter pylori, Naegleriafowlerii (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., Legionellapneumophila, Helicobacter pylori) are not associated with fecal
contamination, and instead may be resident members of aquifer ecosystems. As such, detection of endemic, non-
fecal 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
the presence of a GWR fecal indicator.
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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 Farmer 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 Farmer catalog number  U-08107-30.

         •  iButton. A Thermocron® 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 Thermocron® 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 Farmer catalog number U-
            90316-00.

          •  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 Farmer 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 Enterolert™ 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.
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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-um 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
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    Exhibit 6.1:  E. coli Methods Approved for Use under the Ground Water Rule
Media
Colilert®
Colisure®
E*Colite
(LTB/P-A -*) EC-MUG
(mEndo or
LES Endo -»)
NA-MUG
Ml Medium
m-ColiBlue24®
Method
Reference
SM1 9223
SM1 9223
—
(SM1 922 1B/
SM1 92210^)
SM1 9221 F
(SM1 9222B/C
SM1 9222G
EPA Method
1604
—
Description of
Positive Result
Yellow, fluorescent
Red/magenta, fluorescent
Blue/green, fluorescent
(Growth and the presence
of acid and/or gas in
LTB/P-A), fluorescence in
EC-MUG
(Pink to red colonies with
metallic (golden-green)
sheen); fluorescence after
transfer to NA-MUG
Blue colonies
Blue colonies
Section
6.2.1
6.2.2
6.2.3
6.2.4
6.2.5
6.2.6
6.2.7
      1 Standard Methods for the Examination of Water and Wastewater, 20 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.
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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. coli-
positive.

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

       Positive total coliform tests from the presumptive medium (SM 922 IB/922 ID) are inoculated into EC
broth supplemented with 4-methylumbelliferyl-p-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 long-
wavelength 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-p-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-um 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-um 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.
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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.
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        Exhibit 6.2  Enterococci Methods Approved for Use under the GWR
Media
Azide Dextrose /
BEA/BHI
mE-EIA
mEI
Enterolert™
Method
Reference
SM1 9230B
SM1 9230C
EPA
Method
1600
Budnick,
G.E. etal.,
-IQQfi
Description of Positive Results
Growth at 45°C in BHI and growth
in BHI with 6.5% NaCI at 35°C
Pink to red colonies that form
black or reddish-brown precipitate
on underside of filter
All colonies with a blue halo
Presence of blue-white
fluorescence
Section
6.3.1
6.3.2
6.3.3
6.3.4
      'Standard Methods for the Examination of Water and Wastewater 2(fh 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-um 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 reddish-
brown 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-um 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   Enterolert™ (Budnick, G.E. et al., 1996)

       Enterolert™ 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 Enterolert™ 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.
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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
Two-Step Enrichment
Single Agar Layer
Method
Reference
EPA Method 1601
EPA Method 1602
Description of
Positive Result
Presence of plaques
(circular lysis zones)
Presence of plaques
(circular lysis zones)
Section
6.4.1
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.
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                                  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, male-
specific 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).
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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.

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           -   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..
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                                    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 mLfor 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.
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        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.
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                                           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, Maiden, 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, 210pp.

Davis, J.V. and Witt, III, E.G. 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.

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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, EJ. 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. <2wal., 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: IS - US.

Jimenez, L. et al., Survival and activity of Salmonella typhimurium and Escherichia coli in tropical freshwater,
        Journal Applied Bacteriology, 1989 July, 67(l):61-9.

Lieberman, R.J., E.G. 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(l):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.

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  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-l                        Revised March 2008
Guidance Manual

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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 non-
community 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

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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

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                  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-l                       Revised March 2008
Guidance Manual

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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 < 10 ° C 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

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                  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-l                       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 < 8 °C 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

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                                 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

-------
          Laboratory: _
                                    Ground Water Rule Source Water Monitoring
                                                 Presence/Absence:
                                           £. co// (Colilert® and Colisure)
                                            Presence/Absence (SM 9223)
Sample collection date: _
Sample collection time:
PWS ID:
                                      Sampling point:

Please circle the method used for analysis:
Colilert Colisure

Incubation start
Date
Time
Temperature (°C)







Incubation end
Date
Time
Temperature (°C)




Additional incubation start
Date
Time
Temperature (°C)







Additional incubation end
Date
Time
Temperature (°C)



PRESENCE/ABSENCE RESULTS
Analyst initials

Volume analyzed (mL)

Total conforms
"+" = present
"-" = absent

£. co/i
"+" = present
"-" = absent

Comments
GWR Source Water Monitoring Methods Guidance Manual
                                                        D-l
                                                                                                        RevisedMarch 2008

-------
                               Ground Water Rule Source Water Monitoring
                                               Multiple-Tube:
                                       £. co// (Colilert® and Colisure)
                                     Most Probable Number (SM 9223)
          Laboratory:
  PWS ID:
Sample collection date:


Sampling point:
Sample collection time:

Please circle the method used for analysis:
Colilert Colisure

Incubation start
Date
Time
Temperature (°C)







Incubation end
Date
Time
Temperature (°C)




Additional incubation start
Date
Time
Temperature (°C)



Additional incubation end
Date
Time
Temperature (°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
"+" = present
"-" = absent
Total conforms

£. co/i

QUANTITATIVE RESULTS

Number of positive
tubes
Organisms per 100 ml
Total conforms


£. co/i


  Comments
GWR Source Water Monitoring Methods Guidance Manual
                                                                                                        Revised March 2008

-------
                                Ground Water Rule Source Water Monitoring
                                           Multiple-Well (51 Wells):
                                        E. co// (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 (°C)




Additional incubation start
Date
Time
Temperature (°C)



Incubation end
Date
Time
Temperature (°C)




Additional incubation end
Date
Time
Temperature (°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 conforms
"+" = present
"-" = absent

E. co/i
"+" = present
"-" = absent

QUANTITATIVE RESULTS
Analyst initials



Volume analyzed (mL)



Total conforms
Number of positive
wells



Total coliforms
per 100 mL



£. co/i
Number of
positive wells



£. co// 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. co// (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 (°C)








Incubation end
Date
Time
Temperature (°C)

Additional incubation start
Date
Time
Temperature (°C)







Additional incubation end
Date
Time
Temperature (°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 initial

s Volume analyzed (mL)

Total coliforms E. coll
"+" = present "+" = present
"-" = absent "-" = absent

QUANTITATIVE RESULTS
Analyst initials



Volume
analyzed
(mL)



Total Coliforms
Number of large
positive wells



Number of small
positive wells



Total coliforms per
100mL



£ co//
Number of large
positive wells



Number of small
positive wells



£. co// 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 (°C)







Incubation end
Date
Time
Temperature (°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 conforms
"+" = present
"-" = absent

£ 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: £. coli (LTB/EC-MUG)
                                             (SM9221B/9221F)
          Laboratory:
Sample collection date:
Sample collection time:
 PWS ID:
                                     Sampling point:

Date
Time
Temperature (°C)
LTB Incubation start LTB 24 hour Incubation read LTB 48 hour Incubation read









EC-MUG Incubation 24 hour read (from 24 hour LTB)
Date
Time
Temperature (°C)



EC-MUG Incubation 24 hour read (from 48 hour LTB)
Date
Time
Temperature (°C)



                                                    LTB
                                                   EC-MUG
EC- MUG read
from 24 hr LTB
from 48 hr LTB
Analyst
initials


Start
Temp.


Read
Temp.


Tube 1
(10mL)


Tube 2
(10mL)


TubeS
(10mL)


Tube 4
(10mL)


TubeS
(10mL)


Tube6
(10mL)


Tube 7
(10mL)


TubeS
(10mL)


Tube 9
(10 mL)


Tube 10
(10mL)


 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 1 00 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 (°C)







mEndo or LES-Endo incubation end
Date
Time
Temperature (°C)




NA-MUG incubation start
Date
Time
Temperature (°C)







NA-MUG incubation end
Date
Time
Temperature (°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 conforms
"+" = present
"-" = absent



E. coli
"+" = present
"-" = absent



QUANTITATIVE RESULTS
Analyst initials



Volume filtered



Total conforms
Number of
colonies



Total conforms
per 100 mL



E. coli
Number of colonies



E. coli
per 100 mL



 Comments
GWR Source Water Monitoring Methods Guidance Manual
                                                     D-:
                                                                                                 Revised March 2008

-------
                              Ground Water Rule Source Water Monitoring
                                Membrane Filtration: E. coli (Ml Medium)
                                           (EPA Method 1604)
      Laboratory:.
Sample collection date:.
Sample collection time:
PWS ID:
                                    Sampling point:

Ml Medium incubation start
Date
Time
Temperature (°C)







Ml Medium incubation end
Date
Time
Temperature (°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 conforms
"+" = present
"-" = absent



E. coli
"+" = present
"-" = absent



QUANTITATIVE RESULTS
Analyst initials



Volume filtered



Total conforms
Number of
colonies



Total conforms
per 100 mL



E. coli
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-CoMBIue24)
      Laboratory:.
Sample collection date:.
Sample collection time:
PWS ID:
                                   Sampling point:

m-ColiBlue24 incubation start
Date
Time
Temperature (°C)







m-ColiBlue24 incubation end
Date
Time
Temperature (°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 conforms
"+" = present
"-" = absent



E. coli
"+" = present
"-" = absent



QUANTITATIVE RESULTS
Analyst initials



Volume filtered



Total conforms
Number of
colonies



Total conforms
per 100 mL



E. coli
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% NaCI)
                                                  (SM 9230B)
        Laboratory:.
Sample collection date: _
Sample collection time:.
PWS ID:
                                   Sampling point:

Date
Time
ADB incubation start ADB 24 hour incubation read



24 hour BEA read (from 24 hour ADB)
Date
Time
Temperature (°C)






ADB 48 hour incubation read



24 hour BEA read (from 48 hour ADB)
Date
Time
Temperature (°C)



48 hour BHI (at 45°C) read (from 24 hour BEA)
Date
Time
Temperature (°C)



48 hour BHI-6.5% NaCI read (from 24 hour BEA)
Date
Time
Temperature (°C)



                                                      ADB
                                                   BEA Plates
                                              BHI/BHI-6.5% NaCI READ
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-l
                                                                                                          RevisedMarch 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 (°C)







mE incubation end
Date
Time
Temperature (°C)




EIA incubation start
Date
Time
Temperature (°C)







EIA incubation end
Date
Time
Temperature (°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 (°C)







mEI incubation end
Date
Time
Temperature (°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
                                                                                          RevisedMarch 2008

-------
                            Ground Water Rule Source Water Monitoring
                                           Multiple-Tube:
                                    Enterococcus (Enterolert™)
                                       Most Probable Number
        Laboratory:
Sample collection date:
Sample collection time:
PWS ID:
                             Sampling point:

Incubation start
Date
Time
Temperature (°C)







Incubation end
Date
Time
Temperature (°C)



Note: Complete the most probable number results table based on the type of analysis requested by the PWS.
                               MOST PROBABLE NUMBER RESULTS

Enterococci
Analyst initials

Tube 1
(10 ml)

Tube 2
(10 ml)

TubeS
(10 ml)

Tube 4
(10 ml)

TubeS
(10 ml)

Tube 6
(10 ml)

Tube?
(10 ml)

TubeS
(10 ml)

Tube 9
(10 ml)

Tube 10
(10 ml)

QUANTITATIVE RESULTS
Number of positive tubes

Enterococci per 100 ml_

Comments
   GWR Source Water Monitoring Methods Guidance Manual
                                                 E-4
                                                                                     RevisedMarch 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:
Phage type (circle one): Male-specific Somatic
PWS ID:
Batch ID:
Analyst(s):
Volume analyzed (circle one): 100mL 1L
Sample collection:
Spot plate ID:
Section 2: Sample Analysis
Indicate "+" = presence "-" = absence
Sample ID


Positive Control
Method Blank
Sample collection
date




Sample collection
time




Enrichment start
date




Enrichment start
time





Spot plate
Start date:
Start time:
Start temp:
Analysts initials:





Enrichment start
temp




Enrichment end
date




Enrichment end
time




Enrichment end
temp




Results





Spot plate
End date:
End time:
End temp:
Analysts initials:





Section 3: Comments
 PFU =

   V =
  GWR Source Water Monitoring Methods Guidance Manual
                                                                                                                                                                       Revised March 2008

-------
                                            Method 1602: Single Agar Layer (SAL), Quantitative
Section 1: General Information
Laboratory name:
PWS ID:
Coliphage type (circle one): Male-specific Somatic
Analyst(s):
Sample collection point:
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
No. PFU per
  Plate 5
Total PFU per
100 mL sample
Section 3: Comments
   GWR Source Water Monitoring Methods Guidance Manual
                                  F-2
                                                                                           RevisedMarch 2008

-------
                     Coliphage Enumeration by the Double Agar Layer Procedure (DAL, Section 11.0)
Section 1: General
Laboratory name:_
Filtrate collection date:
Filtrate collection time:
Section 2: Coliphage enumeration
Start date:
Start time:
Analyst initials:
Filtrate Concentration
Undiluted
Undiluted
Dilution Factor
1
1
Inoculation volume Number of Plaques
0.5ml
0.5ml


PFU/ml



0.1 dilution
0.1 dilution
0.1
0.1
0.5ml
0.5ml





End date:
End time:
Analyst initials:
0.01 dilution
0.01 dilution
0.01
0.01
0.5ml
0.5ml





0.001 dilution
0.001 dilution
0.001
0.001
0.5ml
0.5ml





Section 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
GWR Source Water Monitoring Methods Guidance Manual
                    F-3
                           Revised March 2008

-------
                               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
Description
•
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
4
5
6
7
8
9
10
Dilution/rinse water
sterility check
Media sterility check
Positive/negative
controls
Media storage
requirements
Autoclave sterilization
verification
Incubator/waterbath
temperature checks
Refrigerator/freezer
temperature checks
Sample processing
equipment sterility
checks
The dilution/rinse water sterility check was acceptable (target organism
or potentially interfering materials were not found in the sterility check)
The media sterility check was acceptable (target organism or potentially
interfering materials were not found in the sterility check)
The positive/negative controls were acceptable
The media storage requirements were not exceeded
Autoclave sterilization verifications were acceptable
Incubator/waterbath temperatures were measured and recorded 2 times
per day, 4 hours apart, and were within the temperature ranges specified
in the method
Refrigerator/freezer temperatures were measured and recorded once per
day and were within acceptable temperature ranges
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
Guidance Manual
G-l
Revised March 2008

-------
Presence Absence and Most Probable Number Specific QC
11
12
13
Incubation time and
temperature
Preparation blank
Verification of positive
results
The following incubation time and temperature requirements were not
exceeded:
£. co//
Colilert 24-28 hours at 35. 0°C±0.5°C
Colisure 24 hours (up to 48 hours) at 35.0°C±0.5°C
LIB 24±2 or 48±3 hours at 35.0°C±0.5°C
EC-MUG 24±2 hours at 44.5°C±0.2°C
E*Colite 28 hours (up to 48 hours) at 35.0°C±0.5°C
Enterococci
Enterolert 24 hours at 41.0°C ± 0.5°C
ADB 24±2 or48±3 hours 35.0°C±0.5°C
BEA 24±2 hours at 35.0°C±0.5°C
BHI 48 hours at 45.0°C±0.5°C
BHI - 6.5% NaCI 48 hours at 35.0°C±0.5°C
The preparation blank was not contaminated with the target organism
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.
GWR Source Water Monitoring Methods
Guidance Manual
G-2
Revised March 2008

-------
                                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
•
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
4
5
6
7
8
9
10
Dilution/rinse water
sterility check
Media sterility check
Positive/negative
controls
Media storage
requirements
Autoclave
sterilization
verification
Incubator/waterbath
temperature checks
Refrigerator/freezer
temperature checks
Sample processing
equipment sterility
checks
The dilution/rinse water sterility check was acceptable (target organism or
potentially interfering materials were not found in the sterility check)
The media sterility check was acceptable (target organism or potentially
interfering materials were not found in the sterility check)
The positive/negative controls were acceptable
The media storage requirements were not exceeded
Autoclave sterilization verifications were acceptable
Incubator/waterbath temperatures were measured and recorded 2 times per
day, 4 hours apart and were within the temperature ranges specified in the
method
Refrigerator/freezer temperatures were measured and recorded once per day
and were within acceptable temperature ranges
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
mEndo-NA-MUG 24±2 hours at 35.0°C±0.5°C 4 hours at 35.0°C±0.5°C
LES-Endo-NA-MUG 24±2 hours at 35.0°C±0.5°C 4 hours at 35.0°C±0.5°C
Ml Medium 24 hours at 35.0°C±0.5°C
m-ColiBlue24 24 hours at 35.0°C±0.5°C
Enterococci
mE-EIA 48 ± 3 hours at 41.0°C±0.5°C-
20-30 minutes at 41 .0°C±0.5°C
mEI 24±2 hours at 35.0°C±0.5°C

GWR Source Water Monitoring Methods
Guidance Manual
H-l
Revised March 2008

-------
Membrane Filtration Specific QC (continued)
12
13
14
Filtration unit
sterility check
Preparation blank
Colony verification
The filtration unit sterility check was acceptable (target organism or potentially
interfering materials were not found in the sterility check)
The preparation blank was not contaminated with the target organism
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
Guidance Manual
H-2
Revised March 2008

-------
                                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
•
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
4
Media sterility checks
Media storage
requirements
The media sterility checks were acceptable (no coliphage or any
potentially interfering organisms were found in the sterility check) for all
media
Media storage time and temperature requirements were met


Coliphage Specific QC
5
6
7
8
9
10
11
Matrix spikes
Ongoing
demonstration of
capability (ODC)
Incubation time and
temperature
Positive controls
Method blank
Spot plates
ODC and MS
analyses
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)
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)
The following incubation time and temperature requirements were met:
10XTSB 16-24hoursat36.0°C±1.0°C
TSA (DAL plates) 16-24 hours at 36.0°C±1 .0°C
TSA (spot plates) 16-24 hours at 36.0°C±1.0°C
The positive controls were acceptable
The method blank was acceptable (no coliphage or any potentially
interfering organisms were found in the sterility check)
A positive control and method blank were analyzed for each spot plate
used for field samples
ODC and MS analyses were conducted at the method-specified frequency







Verification Specific QC
12
13
Spot plates
Incubation time and
temperature
A positive control and method blank were analyzed for each spot plate
The following incubation time and temperature requirements were met:
TSA (spot plates) 16-24 hours at 36.0°C±1.0°C


    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
Guidance Manual
1-1
Revised March 2008

-------
                                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
•
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
4
Media sterility checks
Media storage
requirements
The media sterility checks were acceptable (no coliphage or any
potentially interfering organisms found in the sterility check) for all media
Media storage time and temperature requirements were met


Coliphage Specific QC
5
6
7
8
9
Matrix spikes
Ongoing precision
and recovery (OPR)
Incubation time and
temperature
Method blank
Positive controls
Matrix spike was spiked at the appropriate level (80 PFU) and the results
were acceptable (range for male-specific and somatic)
The OPR sample was spiked at the appropriate level (80 PFU) and the
results were acceptable (range for male-specific and somatic)
The following incubation time and temperature requirements were met:
TSA (DAL plates) 1 6 - 24 hours at 36.0°C±1 .0°C
TSA (SAL plates) 1 6 - 24 hours at 36.0°C±1 .0°C
The method blank was acceptable (no coliphage or any potentially
interfering organisms were found in the sterility check)
The positive controls were acceptable





Verification Specific QC
10
11
Spot plates
Incubation time and
temperature
A positive control and method blank were analyzed for each spot plate
The following incubation time and temperature requirements were met:
TSA (spot plates) 1 6 - 24 hours at 36.0°C± 1 .0°C


   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
Guidance Manual
J-l
Revised March 2008

-------