United States         Office of Water          EPA 821 -R-00-010
Environmental Protection    Washington, D.C. 20460      April 2000
Agency



Method 1602: Male-specific (F+) and


Somatic Coliphage in Water by Single


Agar Layer (SAL) Procedure



April 2000  -  Draft

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                                   Acknowledgments

This method was prepared under the direction of William A. Telliard of the Engineering and Analysis Division
within the U.S. Environmental Protection Agency's (EPA) Office of Water. The EPA technical lead was Paul
Berger, of the Standards and Risk Management Division within the Office of Water. This document was
prepared under EPA Contract No. 68-C-98-139  by DynCorp Information & Enterprise Technology, Inc.


The contributions of the following persons and organizations to the development of this method are gratefully
acknowledged:

Sobsey, Mark, Ming Jing Wu, and Greg Lovelace, University of North Carolina, Department of
       Environmental Sciences and Engineering, CB#7400, McGavran-Greenberg Building, Chapel Hill, NC
       27599

Hsu, Fu-Chih, and Jim Larkin, Environmental Health Laboratories, 110 South Hill Street, South Bend, IN
       46617

Chambers, Yildiz, City of San Diego Marine Microbiology Laboratory, 5530 Kiowa Drive, La Mesa, CA
       91942

Cliver, Dean, Tadesse Mariam, and Mulugeta Tamene, University of California Davis, Department of Health
       and Reproduction, School of Veterinary Medicine, Davis, CA 95616-8743

Danielson, Richard, BioVir Laboratory, 685 Stone Road Unit # 6, Benicia, CA 94510

Fujioka, Roger and Geeta Rijal, University of Hawaii, Water Resources Center, Holmes Hall 283, 2540 Dole
       Street, Honolulu, HI 96822

Karim, Mohammad and Dale Young, American Water Works System Research Laboratory, 1115 South
       Illinois Street, Belleville, IL 62220-3731

Margolin, Aaron and Nicola Ballester, University of New Hampshire, Department of Microbiology, Biological
       Sciences Building, Rudman Hall Room 285, Durham, NH 03824

Pillai, Suresh and Elisa Camacho, Texas A & M University, Department of Poultry Science, Kleberg Center
       Room 418D, College Station, TX 77843

Williams, Fred and Ron Stetler U.S. Environmental Protection Agency, 26 West Martin Luther King Drive,
       Cincinnati, OH, 45268

Yates, Marylynn, Omid Bakhtar, and Andre Salazar, University of California Riverside, Department of
       Environmental Sciences, 2217 Geology, Riverside, CA 92521-0424


                                          Disclaimer


This method is in final draft form. The method has been validated through a 10-laboratory round-robin. The
Agency welcomes comments on its technical merit. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.

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                                         Introduction

Coliphage presence in groundwater is an indication of fecal contamination. Method 1602 is a performance-
based method for enumerating male-specific (F+) and somatic coliphage in groundwater and other waters. The
single agar layer procedure requires the addition of host bacteria, magnesium chloride, and double-strength
molten agar medium to the sample, followed by pouring the mixture into plates using the total volume. All
plates from a single sample are examined for plaque formation (zones of bacterial host lawn clearing). The
quantity of coliphage in a sample is expressed as plaque forming units (PFU) /100 mL.
Note:  Although Method 1602 has been validated through a 10-laboratory round-robin validation study,
       the QC acceptance criteria and frequency of QC samples to be required in the method have not
       been finalized. Final QC criteria and frequency will be specified in the next draft of the method.
This method is for use in the Environmental Protection Agency's (EPA's) data gathering and monitoring
programs under the Safe Drinking Water Act and the Clean Water Act.

Questions concerning this method or its application should be addressed to:

William A. Telliard
Engineering and Analysis Division (4303)
U.S. Environmental Protection Agency
401 M Street, SW
Washington, D.C. 20460
(202)260-7120

Requests for additional copies of this publication should be directed to:

Water Resource Center
Mail Code RC-4100
401 M Street, SW
Washington, D.C. 20460
(202) 260-7786 or (202) 260-2814
Note: Prior to finalization of this method, each step of this method must be performed as written. After the
       method is finalized, this method will be performance-based. At that point, the laboratory is
       permitted to modify or omit any steps or procedure, provided that all performance requirements set
       forth in the validated method are met. The laboratory may not omit any quality control analyses.
       The terms "shall," "must, " and "may not" indicate steps and procedures required for producing
       reliable results. The terms "should" and "may" indicate optional steps that may be modified or
       omitted if the laboratory can demonstrate that the modified method produces results equivalent or
       superior to results produced by this method.

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                                    Table of Contents







1.0    Scope and Application	  1




2.0    Summary of Method	  1




3.0    Definitions 	  2




4.0    Interferences	  2




5.0    Safety	  2




6.0    Equipment and Supplies  	  3




7.0    Reagents and Standards	  5




8.0    Sample Collection, Preservation, and Storage	  7




9.0    Quality Control	  8




10.0   Calibration and Standardization	  12




11.0   Enumeration of Coliphage Stock Suspension	  12




12.0   Preparation of Log-Phase Host Bacterial Cultures	  16




13.0   Single Agar Layer (SAL) Procedure 	  17




14.0   Data Analysis and Calculations	  19




15.0   Method Performance	  21




16.0   Pollution Prevention  	  21




17.0   Waste Management	  21




18.0   References	  22



19.0   Tables, Diagrams, Flowcharts, and Validation Data	  22




20.0   Glossary	  22
                                               IV

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    Method 1602: Male-specific (F+) and Somatic Coliphage in Water by
                        Single Agar Layer (SAL) Procedure

                                     April 2000 Draft
1.0   Scope and Application

1.1    The single agar layer (SAL) procedure detects and enumerates male-specific (F+) and somatic
       coliphages in groundwater and other waters. This method is intended to help determine if groundwater
       is affected by fecal contamination.
Note:  Although this method may be used for water matrices other than groundwater, it has only been
       validated for use in groundwater.
1.2    This method is designed to meet the monitoring requirements of the U.S. Environmental Protection
       Agency (EPA). It is based on procedures developed for the determination of coliphage in water in the
       Supplement to the 20th Edition of Standard Methods for the Examination of Water and Wastewater
       (Reference 18.1).
1.3    This method is not intended for use in biosolids samples or as a test for microorganisms other than
       coliphage. This method may be used in groundwater and other water matrices where coliphage is
       suspected to be present.
1.4    Each laboratory and analyst that uses this method must first demonstrate the ability to generate
       acceptable results using the procedures in Section 9.0.
1.5    Any modification of the method beyond those expressly permitted is subject to the application and
       approval of alternate test procedures under 40 CFR parts 136.4 and 136.5, and/or 141.27.


2.0   Summary of Method

2.1    Method 1602 describes the single agar layer (SAL) procedure. A 100-mL groundwater sample is
       assayed by adding MgCl2 (magnesium chloride), host bacteria (E. coli F^p for F+ coliphage and 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 an overnight incubation, circular lysis zones (plaques) are counted and summed
       for all plates from a single sample. The quantity of coliphage in a sample is expressed as plaque
       forming units (PFU) / 100 mL. For quality control purposes, both a coliphage-positive reagent water
       sample (OPR) and a negative reagent water sample (method blank) are analyzed for each type of
       coliphage with each sample batch.

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3.0    Definitions

3.1     Coliphages are viruses (bacteriophages) that infect coliform bacteria and are indicators of fecal
        contamination. This method is capable of detecting two types of coliphages: male-specific (F*) and
        somatic.
3.2     F-factor is the fertility factor in certain strains ofE. coli. It is a plasmid that, when present, codes for
        pilus formation. The pilus allows for transfer of nucleic acid from one bacterium to another.
3.3     Male-specific coliphages (F*) are RNA or DNA viruses that infect via the F-pilus of male strains of
        E. coli.
3.4     Somatic coliphages are DNA bacteriophages that infect host cells via the outer cell membrane.
3.5     Definitions for other terms used in this method are given in the glossary in Section 20.3.
4.0    Interferences

4.1     During the single agar layer procedure the sample and host bacteria should not remain in contact with
        each other for more than 10 minutes prior to plating and after plating the agar must harden within 10
        minutes. Increased contact time or agar hardening time may result in replication of phages such that
        the initial phage concentration is overestimated. The entire plating procedure from combining sample
        with host to hardening of single-agar layer plates should not exceed 20 minutes.
5.0    Safety


Caution:      The biohazards and the risk of infection by pathogens associated with handling raw sewage
               are high in this method. Use good laboratory practices when working with potentially
               harmful samples.
5.1     Method 1602 does not purport to address all of the safety problems associated with its use. It is the
        responsibility of the laboratory to establish appropriate safety and health practices prior to use of this
        method. The analyst/technician must know and observe the safety procedures required in a laboratory
        that handles biohazardous material while preparing, using, and disposing of cultures, reagents, and
        materials. The analyst/technician must use proper safety procedures while operating sterilization
        equipment. Equipment and supplies that have come into contact with biohazardous material or are
        suspected of containing biohazardous material must be sterilized prior to disposal or re-use. Field and
        laboratory staff collecting and analyzing environmental samples are under some risk of exposure  to
        pathogenic microorganisms. Staff should apply safety procedures used for handling pathogens to all
        samples.
5.2     The laboratory is responsible for maintaining a current awareness file of Occupational Safety and
        Health Administration (OSHA) regulations regarding the safe handling of the chemicals specified in
        this method.  A reference file of material safety data sheets should be made available to all personnel
        involved in these analyses. Additional information on laboratory safety can be found in Section 17.0.

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5.3    Samples may contain high concentrations of biohazardous agents and must be handled with gloves.
       Any positive reference materials also must be handled with gloves in an appropriate laboratory hood.
       The analyst/technician must never place gloves near the face after exposure to media known or
       suspected to contain pathogenic microorganisms. Laboratory personnel must change gloves after
       handling raw sewage or any other items which may carry pathogenic microorganisms.
5.4    Mouth pipetting is prohibited.
6.0   Equipment and Supplies
       Note:  Brand names, suppliers, and part numbers are for illustrative purposes only. No
               endorsement is implied. Equivalent performance may be achieved using apparatus and
               materials other than those specified here, but demonstration of equivalent performance that
               meets the requirements of this method is the responsibility of the laboratory.
6.1    Equipment for collection and transport of samples
       6.1.1   Bottles for collection of water—Sterile, wide-mouth, polypropylene, 4-L (or smaller) bottles
               or carboys with screw caps
       6.1.2   Ice chest—Igloo, Coleman, styrofoam box or equivalent
       6.1.3   Ice
               6.1.3.1  Wet ice—purchased locally, or
               6.1.3.2  Ice packs—Blue Ice, UTek cat. no. 429, or equivalent, frozen for use
       6.1.4   Bubble wrap
6.2    Equipment and supplies for growth of microorganisms
       6.2.1   Sterile dilution tubes with screw caps—Reusable or disposable, 16 * 150 mm, or 16 * 100
               mm
       6.2.2   Test tube rack—Size to accommodate tubes specified in Section 6.2.1
       6.2.3   Glass or plastic, plugged,  sterile serological pipettes—To deliver (TD), of appropriate
               volume(s) (Falcon, Kimble, or equivalent)
       6.2.4   Pipet bulbs, automatic pipetter—Pipet-Aid or equivalent
       6.2.5   Inoculation loops—Nichrome or platinum wire, disposable, sterile plastic loops, or wooden
               applicator, at least 3 mm in diameter or 10 uL volume (VWR, Fisher, DIFCO, or equivalent)
       6.2.6   Micropipettors, adjustable—10- to 200-uL, and 100- to 1000-uL, with appropriate aerosol
               resistant tips, Gilson, Eppendorf, or equivalent
       6.2.7   Burner—Alcohol, Bunsen, Fisher, or equivalent
       6.2.8   Sterile disposable petri dishes—100-mm  -diameter dishes (Falcon # 1029) or 150-mm-dishes
               (Falcon #1058) or equivalent
       6.2.9   Incubator capable of maintaining 36°C ± 1.0 °C for growth of microorganisms

                                                 3                               April 2000 Draft

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       6.2.10  Beakers—2- and 4-L, sterile, polypropylene, glass, or polycarbonate
       6.2.11  Polypropylene, glass, or polycarbonate bottles—Wide-mouth, 100-mL or 1-L, square or
               round, autoclavable with screw cap
       6.2.12  Erlenmeyer flasks—1-L and 2-L, sterile, Corning, Nalgene, Kimble or equivalent
       6.2.13  Stir bar—Fisher cat. no. 14-511-93, or equivalent
       6.2.14  Stirplate-Fishercat.no.  14-493-120S, or equivalent
       6.2.15  Water bath capable of maintaining 36°C ± 1.0°C and 45°C to 48°C —Precision, VWR
               Scientific, or equivalent
       6.2.16  Sterilization filtration equipment—Millex type for syringe or larger Millipore type, sterile,
               0.22-um pore size
       6.2.17  Sterile, cotton-tipped applicators
       6.2.18  Latex gloves for handling  samples, supplies, and equipment—Microfiex, San Francisco, CA,
               stock no. UL-315-L, or equivalent
       6.2.19  pH meter—Beckman, Corning, or equivalent
       6.2.20  Vortex mixer—Vortex Genie, or equivalent
       6.2.21  Spectrophotometer or colorimeter (with wavelengths in visible range)—Spectronic 20,
               Spectrum Instruments, Inc., or equivalent, with cell holder for Vi" diameter cuvettes (Model #
               4015) or 13 mm x 100 mm cuvettes
       6.2.22  Cuvettes—1-cm light path, Beckman, Bausch and Lomb, or equivalent
       6.2.23  Shaker flasks—Fluted Erlenmeyer, 125-mL with slip cap or sterile plug, Fisher (09-552-33
               10-140-6, 10-041-5A) or equivalent or equivalent
       6.2.24  Shaker incubator—Capable of 36°C ± 1.0 °C and 100 to 150 rpm, New Brunswick,
               PsychoTherm, Innova, or equivalent or an ordinary shaker in an incubator
6.3    Supplies for collection and filtration of raw sewage for spiking (if not using stock coliphage, Sections
       7.1.10 and 7.1.11)
       6.3.1   Disposable filter disks—25-mm-diameter, 0.45-um pore size,  sterile, low protein binding
               (Gelman Acrodisc HT Tuffryn, No. 4184, cellulose acetate Corning No. 21053-25, or
               equivalent)
       6.3.2   Syringe—Sterile, disposable, 5-, 10-, or 20-mL
       6.3.3   Polypropylene dilution tubes—Sterile, 10- to 20-mL, Falcon or equivalent
       6.3.4   Sterile glass or polypropylene 250-rnL bottles for collection of raw sewage
6.4    Miscellaneous lab ware and supplies
       6.4.1   Lint-free tissues-—KimWipes or equivalent
       6.4.2   Weigh boats
       6.4.3   Graduated cylinders—Sterile, polypropylene or glass, 100-mL, 250-mL, and 1-L
       6.4.4   Autoclave
       6.4.5   Thermometers—Range of 0°C to 100°C
       6.4.6   Balance—Capable of weighing to 0.1 mg for samples having  a mass up to 200 g
       6.4.7   Freezer vials—Sterile, 5-mL screw cap, Nunc or equivalent

April 2000 Draft                                  4

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7.0    Reagents and Standards

7.1     General reagents
        7.1.1   Reagent water—Conforming to Specification D 1193, Annual Book of ASTM Standards
               (Reference 18.7).
        7.1.2   E. coli CN-13 (somatic coliphage host)—Nalidixic acid-resistant mutant of E. coli C;
               originated by Pierre Payment, Institute Armand Frappier, University of Quebec, Montreal,
               Canada, frozen stock. ATCC#700609.
        7.1.3   E. coli Fjmp — E. coli HS(pFamp)R (male-specific coliphage host)—originated by Victor
               Cabelli, formerly of the Department of Microbiology, University of Rhode Island, Kingston,
               RI, USA, frozen stock.
        7.1.4   Preparation of frozen stock cultures—The laboratory shall obtain reference host bacterial
               cultures (Sections 7.1.2 and 7.1.3) and use these to establish pure frozen stock cultures that
               are maintained for the laboratory. Stocks are used as inoculum for log-phase host bacterial
               cultures.
               7.1.4.1   Establish pure frozen stock cultures by streaking host bacteria onto tryptic soy agar
                        (Section 7.2.1) with appropriate antibiotic to attain isolated colonies.
               7.1.4.2  Incubate overnight, pick an individual colony, and grow to log phase (Section 12)
                        in tryptic soy broth with appropriate antibiotic (Sections 7.1.7.2 and 7.1.7.3).
               7.1.4.3  Harvest broth by mixing sterile glycerol and broth with log-phase host bacteria in a
                        ratio of 1:4 in a freezer vial (Example: 200 uL sterile glycerol plus 800 uL log-
                        phase E. coli).
               7.1.4.4  Label with E. coli strain and date of harvest.
               7.1.4.5  Freeze stock cultures at -70°C, if possible. Cultures can be frozen -20°C if the
                        laboratory does not have the capability to freeze samples at -70°C).
        7.1.5   100X nalidixic acid (Sigma N4382)—Dissolve 1 g of nalidixic acid sodium salt in 100 mL
               reagent water. Filter  through a sterile, 0.22-um-pore-size membrane filter assembly. Dispense
               5 mL per freezer vial and store frozen at -20°C for up to one year. Thaw at room temperature
               or rapidly in a 36°C  ±  1.0 °C water bath. Mix well prior to use.
        7.1.6   100X ampicillin/streptomycin—Dissolve 0.15 g of ampicillin sodium salt (Sigma A9518) and
               0.15  g streptomycin  sulfate (Sigma S6501) in  100 mL of reagent water. Filter through a
               sterile 0.22-um-pore-size membrane filter assembly. Dispense 5 mL per freezer vial and store
               frozen at -20°C for up to one year. Thaw prior to use at room temperature or rapidly in a
               36°C±1.0°C water bath.
Note:  100X nalidixic acid and 100X ampicillin/streptomycin are always added to medium after the
        medium has been autoclaved.
        7.1.7   Tryptic (or trypticase) soy broth (DIFCO 0370-15-5, or equivalent) (TSB)
               7.1.7.1   TSB—Follow procedure as specified on bottle of media. If dehydrated medium is
                        not available, prepare the media by adding 17.0 g of tryptone, 3.0 g of soytone,

                                                 5 .                               April 2000 Draft

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                        2.5 g of dextrose, 5.0 g of sodium chloride, and 2.5 g of dipotassium phosphate to
                        1L of reagent water and heat to dissolve. Adjust pH to 7.3 with 1.0 N hydrochloric
                        acid or 1.0 N sodium hydroxide, if necessary. Autoclave at 121°C and 15 psi for
                        15 minutes. Check pH again after autoclaving by aseptically removing an aliquot of
                        medium. Adjust pH as necessary.
Note:  To determine pH after autoclaving, aseptically remove an aliquot of medium and measure pH.
        Discard aliquot after checking pH, to ensure that the medium is not contaminated.
               7.1.7.2  TSB with nalidixic acid (for growth of E. coli CN-13)—Aseptically add 10 mL of
                        100X nalidixic acid (Section 7.1.5) to 1 L of autoclaved, cooled TSB (Section
                        7.1.7.1) and mix.
               7.1.7.3  TSB with streptomycin/ampicillin (for growth of E. coli F^)—Aseptically add 10
                        mL of 100X streptomycin/ampicillin sulfate (Section 7.1.6) to 1 L of autoclaved,
                        cooled TSB (Section 7.1.7.1) and mix.
               7.1.7.4  10X Tryptic soy broth—Dissolve 300 g TSB per liter of reagent water. Autoclave
                        for  15 minutes at 121°C and 15 psi. Be careful to remove broth as soon as possible
                        from the autoclave to prevent scorching. Store at 4°C ± 1°C until use.
        7.1.8   10% (w/v) Sodium thiosulfate—Add 10 g sodium thiosulfate (Na2S2O3) per 90 mL reagent
               water. Mix until dissolved. Bring to 100 mL and autoclave for 15 minutes at 121°C and 15
               psi.
        7.1.9   Magnesium chloride SOX (4M)—To 814  g of MgCl2-6H2O, add 300 mL reagent grade water.
               Stir to dissolve. Bring to a final volume of 1 L, and mix thoroughly. Autoclave for 15 minutes
               atl21°Cand!5psi.
        7.1.10 MS2 stock coliphage (ATCC# 15597-B1)
        7.1.11 X174 stock coliphage (ATCC#13706-B1)
        7.1.12 Glycerol—Sigma#G6279 or equivalent. Autoclave for 15 minutes at 121 °C and 15 psi.
               Remove promptly to avoid scorching. Store at room temperature.
7.2     Double agar layer reagents
        7.2.1   Tryptic soy agar or bottom agar (1.5%)—Prior to autoclaving the TSB in Section 7.1.7.1,
               add 15 g of agar per liter of TSB. While stirring, heat to dissolve agar. Autoclave for 15
               minutes at 121°C and 15 psi. Cool to 48°C ± 1.0 °C and mix molten medium well for even
               distribution.
               7.2.1.1  For growth of somatic coliphage using E. coli CN-13 as host bacteria, add 10 mL
                        of 100X nalidixic acid (Section 7.1.5) per liter of autoclaved TSA. Swirl flask to
                        mix well and aseptically dispense 17-18 mL per 100-mm plate. Allow to solidify
                        with lids off in a biohazard hood for several minutes prio'r to use. Replace lids and
                        store inverted at 4°C ± 1°C for up to 2 weeks.
               7.2.1.2  For growth of male-specific (F*) coliphage using E. coli F^ as host bacteria,
                        add 10 mL 100X ampicillin/streptomycin sulfate (Section 7.1.6) per liter of
                        autoclaved TSA. Swirl flask to mix well and aseptically dispense 17-18 mL per
                        100-mm plate.  Allow to solidify with lids off in a biohazard hood for several

April 2000 Draft                                  6

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                       minutes prior to use. Replace lids and store inverted at 4°C ± 1°C for up to 2
                       weeks.
       7.2.2  Top (soft) agar tubes (0.7%)—Prior to autoclaving the TSB in Section 7.1.7.1, add 7 g of
              agar per liter of TSB. While stirring, heat to dissolve agar. Autoclave for 15 minutes at
              121°C and 15 psi. Cool to 48°C ± 1.0 °C.
              7.2.2.1   Top agar with nalidixic acid (for growth of E. coli CM-13)—To 1 L of autoclaved
                       TSA (soft agar) (Section 7.2.2), add 10 mL of 100X nalidixic acid. Dispense 5 mL
                       per sterile 10-mL tube, label, and keep at 45°C to 48°C until use. Tubes must be
                       used the day they are prepared.
              7.2.2.2  Top agar tubes with ampicillin/streptomycin (for growth of E.  coli F^p)—To 1 L
                       of autoclaved TSA (soft agar) (Section 7.2.2), add 10 mL of 100X
                       ampicillin/streptomycin. Dispense 5 mL per sterile 10-mL tube, label, and keep at
                       45°C to 48°C until use. Tubes must be used the day they are prepared.
Note:  When using top agar, add bacteria and sample to agar tubes immediately before plating to ensure
       viability of both bacterial host and coliphage.
7.3    Single agar layer
       7.3.1  2X tryptic soy agar for single agar layer (SAL) method (2X TSA)—To make double-strength
              (2X) TSA, double all components of TSB from Section 7.1.7.1 (except reagent water) and
              add 18 g of agar per liter. Heat to dissolve while stirring. Autoclave for 15 minutes at 121°C
              and 15 psi. Cool to 48°C± 1.0 °C. Medium may become darker after autoclaving but this
              should not impact media performance.
              7.3.1.1   2X TSA with nalidixic acid (for growth of E. coli CN-13)—To 1 L of autoclaved
                        2X TSA, add 20 mL of 100X nalidixic acid and mix well. Keep molten at 45°C to
                        48°C in water bath until use. Agar must be used on the day of preparation.
              7.3.1.2   2X TSA with ampicillin/streptomycin (for growth of E. coli F^)—To 1 L of
                        autoclaved 2X TSA, add 20 mL of 100X ampicillin/streptomycin and mix well.
                        Keep molten at 45°C to 48°C in water bath until use. Agar must be used on the day
                        of preparation.
8.0   Sample Collection, Preservation, and Storage

8.1    Samples are collected in plastic bottles or carboys and shipped to the laboratory for analysis. Samples
       must be shipped at 2°C to 8°C. Samples must be stored at 4°C ± 1°C. Do not freeze.
8.2    Sample collection: Collect 250 mL of sample for each of the two coliphage types to allow for sample
       re-analysis, if necessary.
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Note:   Wet ice, Blue Ice® or similar products must be used to maintain a temperature of2°C to 8°C
        during shipment. The samples must be protected from freezing.
Note:  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 at a
        time.
8.3    The sampling team must maintain a log book with the following information for each sample:
       8.3.1   Facility name and location
       8.3.2   Date and time of collection
       8.3.3   Name of analytical facility, contact, and phone number
       8.3.4   Sample number
       8.3.5   Sample location
8.4    The sample container must indicate the following:
       8.4.1   Sample number
       8.4.2   Date and time of collection
       8.4.3   Sample collection location
8.5    Holding times. The following are maximum holding times beyond which the sample cannot be
       retained.
       8.5.1   Single agar layer procedure—Between collection of sample and beginning of analysis: 48
               hours
       8.5.2   Raw sewage sample—Between collection of sewage sample and analysis: 24 hours, unless re-
               titered and titer has not decreased by more than 50%. If liter has not decreased by more than
               50%, the sample can be stored for up to 72 hours.
8.6    Dechlorination procedure (if necessary)—For 1-L of water, add 0.5-mL 10% sodium thiosulfate.


9.0   Quality Control

9.1    Each laboratory that uses this method is required to operate a formal quality assurance (QA)
       program. The minimum QA requirements for this program consist of an initial demonstration of
       laboratory capability through performance of the initial precision and recovery (IPR) test (Section
       9.3), analysis of spiked samples to evaluate and document data quality, and analysis of standards and
       blanks as tests of continued performance. Laboratory performance is compared to established
       performance criteria to determine if the results of analyses meet the performance criteria of the
       method. Specific quality control (QC) requirements for Method 1602 are provided below. General

April 2000 Draft                                  8

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        recommendations on QA and QC for facilities, personnel, laboratory equipment, instrumentation, and
        supplies used in microbiological analyses are provided in the USEPA Microbiology Methods Manual,
        Part IV, C (Reference 18.4).
9.2     General QC requirements
Note: Although Method 1602 has been validated through a 10-laboratory round-robin validation study,
       the QC acceptance criteria and frequency of QC samples to be required in the method have not
       been finalized. Final QC criteria and frequency will be specified in the next revision of the method.
        9.2.1   The laboratory shall demonstrate the ability to generate acceptable performance with this
               method by performing an IPR test. The procedure for performing the IPR is described in
               Section 9.3.
        9.2.2   The laboratory shall analyze method blanks to demonstrate freedom from contamination. The
               procedures and criteria for analysis of a method blank are described in Section 9.4. The
               laboratory shall analyze one method blank for each analytical batch [analytical batch to be
               defined].
        9.2.3   The laboratory shall spike a separate sample aliquot from the same groundwater source to
               monitor method performance. This matrix spike (MS) test is described in Section 9.5. The
               laboratory shall analyze one MS sample (Section 9.5) when samples are first received from a
               groundwater source for which the laboratory has never before analyzed samples. The MS
               analysis is performed on an additional (second) sample aliquot collected from the groundwater
               source at the same time as the routine field sample. If the laboratory routinely  analyzes
               samples from one or more groundwater sources, one MS analysis  must be performed per
               [number to be determined] field samples. For example, when a laboratory receives the first
               sample from a source, the laboratory must obtain a second aliquot of this sample to be used
               for the MS. When the laboratory receives the [number to be determined] sample from this
               site, a separate aliquot of this [number to be determined] sample  must be collected and
               spiked.
        9.2.4   The laboratory shall, on an ongoing basis, demonstrate through analysis of the ongoing
               precision and recovery (OPR) samples that the analytical system is in control (Section 9.6).
               The laboratory shall analyze one OPR sample for each analytical batch [analytical batch to
               be defined].
        9.2.5   The laboratory shall maintain records to define the quality of data that are generated.
               Development of accuracy statements is described in Sections 9.5 and 9.6
        9.2.6   The laboratory shall test media sterility by subjecting a representative portion of each media
               batch to incubation at 36°C ± 1.0 °C for 48 to 72 hours and observe for indications of
               growth. With respect to media, a batch is defined as 1 tube out of 50 in each lot or one tube,
               if the lot contains less than 50 tubes.
        9.2.7   The laboratory should participate in available interlaboratory performance studies conducted
               by local, state, and federal agencies or commercial organizations.  Review results to correct
               unsatisfactory performance and record corrective actions.
        9.2.8   A log book containing reagent and material lot numbers should be maintained along with
               samples analyzed using each of the lots.

                                                 9                                April 2000 Draft

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       9.2.9   The laboratory shall maintain a record of the date and results of all QC samples described in
               Section 9.2. A record of sterility check, OPR, and MS sample results must be maintained.
9.3    Initial precision and recovery (IPR)—The IPR test is performed to demonstrate acceptable
       performance with the method.
Note: IPR tests must be accompanied by analysis of a method blank (Section 9.4).
       9.3.1   A total of [number to be determined] spiked reagent water samples ([number to be
               determined] samples spiked with each coliphage type) are required for the IPR test, as is a
               method blank (Section 9.4) for each coliphage type.
       9.3.2   For each coliphage type (somatic and F*), use an appropriate volume of the enumerated stock
               suspension prepared in Section 11, to spike a [number to be determined]-mL "bulk" reagent
               water sample with approximately 320 coliphage (resulting in 80 PFU / sample). Aliquot each
               sample into [number to be determined], 100-mL samples (see Section 11 for enumeration of
               coliphage stock and Section 14.2 for spiking volume calculations).
       9.3.3   Analyze the [number to be determined] spiked samples and two method blanks (one for each
               coliphage type) using the SAL procedure (Section 13).
       9.3.4   Compute  the percent recovery of coliphage in each sample using the following equation:
                                      TV
                        R = 100 X —

               where
               R = percent recovery
               N = number of coliphage detected (PFU / sample)
               T = number of coliphage spiked (PFU / sample)

       9.3.5   Using all  sample results from an IPR test, compute the average percent recovery (x) and the
               relative standard deviation of the recovery (RSDr). (See glossary for definition of RSDr.)
               Compare RSDr and x with the corresponding limits for IPR (Section 15). If RSDr and x meet
               the acceptance criteria, system performance is acceptable and analysis of blanks and samples
               may begin. If RSDr or x falls outside the range for recovery, system performance is
               unacceptable. In this event, identify and correct the problem and repeat the test.
9.4    Method blank (performed at the frequency specified in Section 9.2.2)
       9.4.1   Prepare and analyze a reagent water sample containing no coliphage using the same
               procedure as used for analysis  of the field samples.
       9.4.2   If coliphage, or any potentially interfering organisms are found in the blank, analysis of
               additional samples must be halted until the source of contamination is eliminated, and a blank
               shows no evidence of contamination. Any sample in a batch associated with a contaminated
               blank must be recollected if holding time limits have been violated. Samples from a batch that
               proves to have no  coliphage in its blank may be reported along with that batch's sample  data.
April 2000 Draft                                  10

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9.5    Matrix spike—The laboratory shall spike and analyze [number to be determined] separate field
       sample aliquot to determine the effect of the matrix on the method's coliphage recovery. The MS shall
       be analyzed according to the frequency in Section 9.2.3.
       9.5.1   Analyze an unspiked field sample according to the SAL quantitative procedure (Section 13).
               To a second aliquot of the same field sample, add a volume of stock coliphage equal to 80
               PFU /100 mL (see Section 11 for enumeration of coliphage stock and Section 14.2 for spike
               volume calculations).
       9.5.2   Compute the percent recovery (R) of coliphage using the following equation:

               R= 100x(Nsp-NusP)/T

               where
               R is the percent recovery
               Nsp is the number of coliphage detected in the spiked sample
               Nusp is the number of coliphage detected in the unspiked sample
               T is the number of coliphage spiked

       9.5.3   Compare the coliphage recovery with the corresponding limits in Section 15. If the recovery
               for coliphage falls outside its limit, method performance is unacceptable for that sample. If
               the results for the OPR sample associated with this batch of samples are within their
               respective control limits, a matrix interference may be causing poor recovery. If the results for
               the OPR are  not within their control limits, the laboratory is not in control. The problem must
               be identified and corrected. The matrix spike and associated field sample should be
               reanalyzed.
9.6    Ongoing precision and recovery ([OPR]; laboratory control sample) (performed at the frequency
       specified in Section 9.2.4)
       9.6.1   Spike [number to be determined] 100-mL reagent water samples with enumerated coliphage
               stock suspension (Section 11), [number to be determined] sample for each coliphage type
               (somatic and F+) will be spiked.
       9.6.2   Spike 80 coliphage into each sample (see Section 11 for enumeration of coliphage stock and
               Section 14.2 for spiking volume calculations).
       9.6.3   Analyze the [number to be determined] spiked samples using the SAL procedure (Section
               13).
       9.6.4   Compute the percent recovery of coliphage in each OPR sample using the following equation:
                           W
               R= 100 X  —

               where
               R = percent recovery
               N = the number of coliphage detected
               T = the number of coliphage spiked
                                                11                               April 2000 Draft

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       9.6.5  Compare R with the corresponding limits for ongoing precision and recovery in Section 15. If
              R meets the acceptance criteria, system performance is acceptable and analysis of samples
              may begin. If R falls outside the range for recovery, system performance is unacceptable. In
              this event, correct the problem and repeat the test.
       9.6.6  As part of the QA program for the laboratory, method precision for samples should be
              assessed and records retained. After the analysis of five OPR samples for which the reagent
              water spike recovery of coliphage is determined, the laboratory should compute the average
              percent recovery (R) and the standard deviation of the percent recovery (sr). Express the
              precision assessment as a percent recovery interval from R - 2 sr to R + 2 sr for each
              coliphage type. For example, if R = 80% and sr = 30%, the accuracy interval is expressed as
              20% to 140%. The assessment should be updated on a regular basis (e.g., after each 5 to 10
              new accuracy measurements).
9.7    The laboratory should periodically analyze an external QC sample, such as a performance evaluation
       when available. The laboratory also should periodically participate in interlaboratory comparison
       studies using the method.
9.8    The specifications contained in this method can be met if the analytical system is  maintained under
       control. The standards used for initial (Section 9.3) and ongoing (Section 9.6) precision and recovery
       should be identical, so that the most precise results will be obtained.
10.0  Calibration and Standardization

10.1   Check temperatures in water baths, refrigerators, and -20°C freezers daily to ensure operation within
       stated limits of method and record daily measurements in a log book.
10.2   Check temperatures in incubators twice daily, at least 4 hours apart, to ensure operation within stated
       limits of method and record measurements in log book.
10.3   Check thermometers at least annually against an NIST-certified thermometer or one that meets the
       requirements of MIST Monograph SP 250-23. Check mercury columns for breaks.
10.4   Calibrate pH meter prior to use, using standards of pH 4.0, 7.0, and 10.0. To calibrate, use the two
       standards that are nearest to the desired pH.
10.5   Calibrate balances annually using ASTM-certified Class 2 reference weights.
10.6   Calibrate spectrophotometer prior to each use, following method described in owner's manual. Use
       sterile TSB without antibiotics as the blank.
10.7   Laboratories must adhere to all applicable quality control requirements set forth in Reference 18.6.
11.0   Enumeration of Coliphage Stock Suspension

11.1    The double agar layer (DAL) procedure is used to enumerate stock suspensions of somatic and F+
        coliphage for use in spiking quality control samples.
11.2    Prepare log-phase bacterial hosts from stock cultures incubated overnight. Inoculum from an
        overnight bacterial host culture will reach log-phase more rapidly than inoculum from frozen stock.
April 2000 Draft                                  12

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        11.2.1   Preparation of overnight stock cultures
                 11.2.1.1     Dispense 25 mL of tryptic soy broth (TSB) with nalidixic acid (Section
                              7.1.7.2) into a sterile 125-mL shaker flask. Label the flask. For proper
                              growth conditions, each flask should always contain 25 to 30 mL of medium.
                 11.2.1.2     Inoculate the flask with a loopful of E. coli CN-13 from the frozen stock
                              culture (Section 7.1.2). Label the flask.
                 11.2.1.3     Repeat Sections  11.2.1.1 and 11.2.1.2 using TSB with streptomycin and
                              ampicillin as the medium (Section 7.1.7.3) and E. coli F^ (Section 7.1.3) as
                              the bacterial host.
                 11.2.1.4     Place a sterile slip cap or plug on the shaker flasks and secure in shaker.
                 11.2.1.5     Incubateat 36°C ± 1.0 °C and set shaker to lOOto 150 rpm overnight (18 to
                              20 hours).
                 11.2.1.6     Chill on wet ice or at 4°C ± 1 °C until ready for use.
        11.2.2   Preparation of log-phase bacterial host cultures—See Section 12
11.3    Preparation of filtrate from raw sewage. This filtrate will be used as a QC spiking suspension. If
        coliphage stock is available, skip this section and proceed to Section 11.4.
        11.3.1   Collect approximately 100 mL of raw sewage in a 250-mL collection bottle.
        11.3.2   Transport to the laboratory on ice.
        11.3.3   Allow the raw sewage to settle at 4°C ± 1°C for 1 to 3 hours. This will make the filtration
                 process easier.
        11.3.4   Remove a sterile, 20-mL syringe from its package, aseptically remove plunger from barrel,
                 and attach a filter disk to the syringe barrel.
        11.3.5   Pipet 10 to  15 mL of supernatant from settled sewage into the syringe barrel.
        11.3.6   Hold the assembly  over a 15-mL polypropylene tube with screw-cap or snap-cap, insert the
                 plunger into the syringe barrel, and push the sewage through the filter into the sterile tube.
                 If filter clogs, change it as necessary and continue to filter sewage until at least 10 mL of
                 filtered sewage is obtained in the 15-mL polypropylene tube (filtration may require use of
                 numerous filters).
        11.3.7   Cap the tube, label with source, date, and initials, and store the filtrate at 4°C ± 1°C until
                 ready to assay. The filtrate should not be stored more than 24 hours, unless re-titered.
Note:  Coliphage liter may decrease over time, therefore sewage filtrate should be assayed immediately for
        total coliphage and used within 24 hours of preparation. Re-titer this sewage filtrate the day it is
        used for IPR, OPR, and MS samples.
                                                 13                                April 2000 Draft

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11.4   Dilution of coliphage stock or sewage filtrate (Section 11.3)—A minimum of four different
       volumes/dilutions are used for DAL enumeration of the stock/filtrate:
       •         Undiluted
                 0.1
                 0.01
                 0.001
       Additional dilutions may be necessary. TSB without antibiotics (Section 7.1.7.1) is used as the diluent
       and as the method blank.
       11.4.1   Aseptically add 9.0 mL of TSB without antibiotics (Section 7.1.7.1) into each of three (or
                 more) sterile dilution tubes (Section 6.2.1). Label them as "0.1," "0.01," "0.001," "method
                 blank," etc.
       11.4.2   Add 1.0 mL of the stock/filtrate to the tube of TSB labeled "0.1." Cap the tube and vortex
                 for 5 seconds on a medium-high setting (if available) or until well-mixed.
       11.4.3   Add 1.0 mL of the well-mixed 0.1 dilution to the tube of TSB labeled "0.01". Cap the tube
                 and vortex for 5 seconds on a medium-high setting (if available) or until well-mixed.
       11.4.4   Add 1.0 mL of the well-mixed 0.01 dilution to the tube of TSB labeled "0.001." Cap the
                 tube and  vortex for 5 seconds on a medium-high setting (if available) or until well-mixed.
       11.4.5   Continue as necessary for subsequent dilutions.
       11.4.6   Add 1.0 mL of TSB without antibiotics to a tube labeled "method blank". Cap the tube and
                 vortex for 5 seconds on a medium-high setting (if available) or until well-mixed.
11.5   Coliphage stock suspension enumeration procedure—In this procedure, a tube of molten top agar with
       added host bacteria is inoculated with coliphage stock and will be poured into a bottom agar plate.
       Four dilutions of stock/filtrate will be analyzed in duplicate for each coliphage type. As a result, nine
       double-agar plates will be required for each coliphage type: two plates per dilution (undiluted, 0.1,
       0.01, and 0.001) and one method blank plate.
       11.5.1   Agar preparation
                 11.5.1.1     Place top agar tubes in a 45°C to 48°C water bath. The top agar should
                              remain molten in the water bath until ready for use. Four tubes are needed for
                              each volume of stock/filtrate (two for each bacterial host). Be sure to include
                              additional two top agar tubes for the method blanks (a method blank is
                              required for each bacterial host). Half of the top agar tubes should contain
                              nalidixic acid (Section 7.2.2.1) for growth of E. coli CN-13; the other half
                              should contain ampicillin/streptomycin (Section 7.2.2.2) for growth of E. coli
                              "amp"
                 11.5.1.2    Disinfect a workspace near the water bath.
                 11.5.1.3    Assemble bottom agar plates and label or code so that the following
                              information is identifiable:
                              •       Dilution of stock filtrate or method blank
                              •       Bacterial host (E. coli CN-13 or E. coli F^p)
                              •       Coliphage type (somatic for the E. coli CN-13 bacterial host or F+
                                      for the E. coli F^ bacterial host)
                                      Date
                              •       Time
April 2000 Draft                                   14

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Note:  The following plate-preparation steps are critical. Do not add bacterial host and stock/filtrate until
        ready to plate.
        11.5.2   Preparation of plates for enumeration of somatic coliphage (see Table 1 for inoculation
                 scheme)
                 11.5.2.1     With the top agar tube still in the water bath, aseptically inoculate a top agar
                              tube containing nalidixic acid with 30 to  100 uL of log-phase E. coli CN-13.
                 11.5.2.2     Immediately add 500 uL of undiluted stock/filtrate.
                 11.5.2.3     Mix the inoculum by rolling the tube briefly in palm of hand.
                 11.5.2.4     Pour contents into one of the two bottom agar plates marked "undiluted, E.
                              coli CN-13, somatic."
                 11.5.2.5     Duplicate analysis—Repeat Sections 11.5.2.1 through 11.5.2.4 for the
                              duplicate.
                 11.5.2.6     Repeat Sections 11.5.2.1 through 11.5.2.5 for each dilution volume.
        11.5.3   Preparation of plates for enumeration of F+ coliphage—Repeat Section 11.5.2 using agar
                 containing ampicillin/streptomycin and log-phase E. coli F^. (see Table 1 for inoculation
                 scheme)
        11.5.4   Preparation of somatic coliphage method blank (see Table 1 for inoculation scheme)
                 11.5.4.1     With the top agar tube still in the water bath, aseptically inoculate a top agar
                              tube containing nalidixic acid with 30 to  100 uL of log-phase E. coli CN-13.
                 11.5.4.2     Immediately add 500 uL of TSB.
                 11.5.4.3     Mix the inoculum by rolling the tube briefly in palm of hand.
                 11.5.4.4     Pour contents into a bottom agar plate marked "blank, E. coli CN-13,
                              somatic."
        11.5.5   Preparation of the F+ coliphage method blank—Repeat Section 11.5.4 using agar containing
                 ampicillin/streptomycin and log-phase E. coli F^p (see Table 1 for inoculation scheme)
        11.5.6   Retain undiluted stock/filtrate for use in preparing new  dilutions for OPR and MS samples.
        11.5.7   After the top agar hardens, cover, invert the plates and incubate for 16 to 24 hours at 36°C
                 ± 1.0 °C.
        11.5.8   Circular zones of clearing (typically 1 to 10 mm in diameter) in lawn of host bacteria after
                 16 to 24 hours are plaques. Count the number of plaques on each plate.
        11.5.9   Proceed to Section 14.1 and calculate the PFU / mL for each coliphage.
                                                 15                                April 2000 Draft

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Top Agar
Tubes Contain

TSA w/Nalidixic
Acid

TSA w/Ampicillin
and
Streptomycin sulfate


Add
30 -100 pL of
£. CO//CN-13
per tube

Add
30 - 100 ML of
E.co//Famp
per tube
Coliphage Source (stock or sewage filtrate)
Undiluted
500
ML
1

U

U
500
ML
i

U

u
0.1 Dilution
inTSB
500
ML
i

U

u
500
ML
1

U

U
0.01 Dilution
inTSB
500
ML
i

U

u
500
ML
I

U

u
0.001 Dilution
inTSB
500
ML
1

U

u
500
ML
i

U

u

Method
Blank
500
ML
i

U

u
*Host bacteria are added first, then coliphage or TSB (for the blanks). Additional dilutions may be
necessary.
12.0  Preparation of Log-Phase Host Bacterial Cultures

12.1   Inoculate a shaker flask containing 25 mL of TSB containing nalidixic acid (Section 7.1.7.2) with 0.1
       to 1.0 mL of E. coli CN-13. For proper growth conditions, each culture flask of host bacteria should
       contain 25 to 30 mL of medium. As a result, several flasks of host bacteria may have to be prepared
       (this depends on the number of samples and controls being run each day). Each 100-mL sample will
       require a 0.5-mL inoculum of log-phase host bacteria.
12.2   Repeat Section 12.1 using TSB with streptomycin and ampicillin (Section 7.1.7.3) as the medium and
       E. coli F^p as the bacterial host.
12.3   After inoculation, place a sterile slip-cap or plug on the shaker flasks and secure in shaker incubator.
12.4   Incubate at 36°C ± 1.0 °C and 100 to  150 rpm for approximately 4 hours or until cultures are visibly
       turbid, indicating log-phase growth.
12.5   Aseptically remove 1 mL of culture from flask, dispense into a cuvette, and read absorbance at 520
       nm (Section 6.2.22). An absorbance reading between 0.1 and 0.5 optical density (OD) units  is an
       indication of log-phase growth. If proper OD has not been reached, place cultures back into  shaker
       incubator and take readings every 30 minutes until an OD of between 0.1 and 0.5  is reached.
April 2000 Draft
16

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12.5   Aseptically remove 1 mL of culture from flask, dispense into a cuvette, and read absorbance at 520
       nm (Section 6.2.22). An absorbance reading between 0.1 and 0.5 optical density (OD) units is an
       indication of log-phase growth. If proper OD has not been reached, place cultures back into shaker
       incubator and take readings every 30 minutes until an OD of between 0.1 and 0.5 is reached.
12.6   Chill on wet ice or at 4°C ± 1°C to slow replication until ready for use. The suspension may be stored
       up to 48 hours. However, the best results occur when cultures are used immediately (within 6 hours).
12.7   Store remaining bacterial host culture at 4°C ± 1°C overnight to inoculate flasks for the preparation
       of new log-phase bacterial hosts.
13.0  Single Agar Layer (SAL) Procedure

13.1   Refer to Section 12 for preparation of log-phase host bacterial cultures.
Note:  During the single agar layer procedure the sample and host bacteria should not remain in contact
       with each other for more than 10 minutes prior to plating and after plating the agar must harden
       within 10 minutes. Increased contact time or agar hardening time may result in replication of
       phages such that the initial phage concentration is overestimated. The entire plating procedure
       from combining sample with host to hardening of single-agar layer plates should not exceed 20
       minutes.
13.2   Preparation of SAL media
       13.2.1   Prepare 100 mL of 2X ISA with nalidixic acid for E. coli CN-13 as described in Section
                7.3.1.1.
       13.2.2   Add the 100 mL of 2X ISA with nalidixic acid to a 250-mL to 500-mL size Erlenmeyer
                flask and place in a 45°C to 48°C water bath to equilibrate. The liquid in the waterbath
                must come up to the level of the media and care must be taken to ensure that the flask does
                not tip-over in the waterbath.
       13.2.3   Prepare 100 mL of 2X TSA with ampicillin/streptomycin for E. coli F^p as described in
                Section 7.3.1.2.
       13.2.4   Add the 100 mL of 2X TSA with ampicillin/streptomycin to a 250-mL to 500-mL size
                Erlenmeyer flask and place in a 45°C to 48°C water bath to equilibrate.
       13.2.5   Keep the agar molten between 45°C and 48°C until use in the SAL assay.
       13.2.6   Prepare method blanks as specified in Section 9.4.
13.3   Disinfect a work space near the water baths.
13.4   Assemble and label plates with bacterial host (E. coli CN-13 or E. coli F^p), date, and time.
                                                17                              April 2000 Draft

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13.5   Addition of sample and host bacteria to SAL media
       13.5.1   Dispense two, 100-mL aliquots of sample into separate 250-mL to 500-mL size Erlenmeyer
                flasks. Also dispense 100 mL of reagent water into a 250-mL to 500-mL size Erlenmeyer
                flask (this will be used to determine sample temperature changes in the following steps and
                will be referred to as the "temperature flask").
       13.5.2   Add 0.5 mL of sterile 80X MgCl2-6H2O (Section 7.1.9) to each flask.
       13.5.3   Place the flasks into the 36 °C ± 1.0 °C water bath for 5 minutes or until sample water just
                reaches the water bath temperature.
Note:  All components should be warmed before assay to avoid solidification prior to pouring plates.
Note:  The following steps are critical. Temperature must be monitored closely to ensure that coliphages
       are not inactivated and also to ensure that the agar does not harden prematurely.
       13.5.4  Add 10 mL of log-phase E. coli CN-13 (Section 12) to one flask of sample water.
       13.5.5  Add 10 mL of log-phase E. coli Famp (Section 12) to the other flask of sample water.
       13.5.6  Add 10 mL of water to the temperature flask.
       13.5.7  Immediately transfer temperature flask and flasks containing sample and log-phase bacteria
                to the 45°C to 48°C water bath. The approximate temperature of the samples should be
                determined by monitoring the temperature of the water in the temperature flask. When
                water in the temperature flask reaches 43 °C ± 1.0°C, remove samples from the water bath
                and proceed to the next step immediately.
Note:  Samples should remain in contact.with host for a minimum of three minutes before plates are
       poured.
        13.5.8  Add the sample/^, coli CN-13 mixture to the 100 mL of 2X ISA containing nalidixic acid.
                13.5.8.1     Pour the contents into a series of petri dishes at 20 mL per
                             100-mm-diameter dish or 40 mL per 150-mm-diameter dish.
Note:  This procedure requires either five, 150-mm plates or ten, 100-mm plates per 100-mL sample.
        13.5.9  Combine the sample/^, coli  F^p mixture with the 100 mL of 2X TSA containing
                ampicillin/streptomycin.
April 2000 Draft                                 18

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                13.5.9.1     Pour the contents into a series of petri dishes at 20 mL per 100-mm diameter
                            dish or 40 mL per 150-mm dish.
Note:  Plates should be dry before they are inverted, as condensation drops on the agar surface may
       appear to be plaques.  When reading plates examine plaques closely.
       13.5.10  Allow the agar to harden, cover, invert, and incubate for 16 - 24 hours at 36°C ± 1.0°C.
       13.5.11  Circular zones of clearing (typically 1 to 10 mm in diameter) in lawn of host bacteria in
                SAL plates after 16-24 hours are considered to be plaques. Count all plaques per plate
                series and go to Section 14.2 for calculation of PFU /100 mL.
14.0  Data Analysis and Calculations

14.1   Calculation for the DAL procedure (Section 11)
       14.1.1   Compute PFU / mL of filtered sewage using DAL plates that yield plaque counts within the
                desired range of 20 to 300 PFU per plate for F+ coliphage and 20 to 100 PFU per plate for
                somatic coliphage. There may be occasions when the total number of plaques on a plate will
                be above or below the ideal range. If the plaques are not discrete, results should be recorded
                as "too numerous to count" (TNTC).
       14.1.2   The inoculation volume, as described in Section 11, is 500 uL (0.5 mL). Refer to Table 2
                for dilution factors.

                Table 2.   Dilution factors for concentration of filtered sewage.
Sewage Concentration
Undiluted
0.1 Dilution
0.01 Dilution
0.001 Dilution
Dilution Factor
1
0.1
0.01
0.001
       14.1.3   Use the following equation to calculate PFU / mL of coliphage stock or filtered sewage for
                each DAL plate:
                PFU/mL =
                                      number of plaques counted
                               (inoculation volume in mL)(dilution factor)
                                              19
April 2000 Draft

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       14.1.4   If multiple plates are within the desired range, then PFU / mL of filtered sewage should be
                calculated for each of those plates and the average taken.
14.2   Calculation for the SAL procedure (Section 13)
       14.2.1   The SAL method (SAL) is validated for use with 100 mL sample volumes. 100% of each
                sample should be plated.
       14.2.2   For each sample, count total number of plaques from all plates.
Note:  If the plaques are not discrete, results should be recorded as "too numerous to count" (TNTC). The
       remaining sample should be diluted, as appropriate, and re-analyzed.
       1 4.2.3   Total number of plaques per 100 mL sample = PFU / 100 mL.
1 4.3   Calculation for preparing IPR, MS, and OPR spikes
       14.3.1   The stock/filtrate enumerated in Section 1 1 will be used as the spiking suspension. Use a
                dilution of stock/filtrate that will result in a spike volume between 0. 1 and 3.0 mL.
       1 4.3.2   Use the following equation to determine the spiking volume:
                        S=
                               (Q
                       where,
                       S = Spike volume (mL)
                       T = Target number of coliphage per sample (PFU)
                       V = Sample volume (mL)
                       B = Bulk sample volume (mL) (only necessary when multiple samples are spiked in
                       bulk for QC purposes)
                       C = Concentration of coliphage in the dilution to be used for spiking (PFU / mL)

       1 4.3.3   For example, if:
                T) A spike dose of 2 PFU per sample is needed
                V) 100-mL samples will be spiked
                B) A total of four samples will be spiked at the same time
                (bulk sample volume = 400 mL)
                C) The 10'2 dilution contains 6 PFU / mL

                The equation would be solved as follows:

                           (2 PFU / 100mL)(400mL)
                 1.3 mL =
                                   (6PFU/mL)
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                As a result, 1.3 mL of the 10~2 dilution would be spiked into the 400 mL bulk sample. The
                400 mL bulk sample would be mixed and four, 100-mL aliquots dispensed. Each 100-mL
                sample should contain approximately 2 PFU.
15.0  Method Performance
Note:  Although Method 1602 has been validated through a 10-laboratory round-robin validation study,
       the QC acceptance criteria and frequency ofQC samples to be required in the method have not
       been finalized. Final QC criteria and frequency will be specified in the next revision of the method.
15.1   Precision and accuracy statements will be placed within this document when they have been
       established.
16.0  Pollution Prevention

16.1   The solutions and reagents used in this method pose little threat to the environment when recycled and
       managed properly.
16.2   Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize the
       volume of expired materials to be disposed.
17.0  Waste Management

17.1   The laboratory is responsible for complying with all Federal, State, and local regulations governing
       waste management, particularly hazardous waste identification rules and land disposal restrictions,
       and for protecting the air, water, and land by minimizing and controlling all releases from fume hoods
       and bench operations. Compliance with all sewage discharge permits and regulations is also required.
       An overview of requirements can be found in Environmental Management Guide for Small
       Laboratories (EPA 233-B-98-001).
17.2   Samples, reference materials, and equipment known or suspected to have bacteriophage attached or
       contained must be sterilized prior to disposal.
17.3   For further information on waste management, consult The Waste Management Manual for
       Laboratory Personnel and Less Is Better: Laboratory Chemical Management for Waste Reduction,
       both available from the American Chemical Society's Department of Government Relations and
       Science Policy, 1155 16th Street N.W., Washington D.C. 20036.
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18.0  References

18.1   American Public Health Association, American Water Works Association, Water Environment
       Federation. Washington, D.C. Joint Task Group for Section 9224, 1997. Detection of Coliphages .
       For Standard Methods for the Examination of Water and Waste Water 20th Edition Supplement, (draft
       version - December 1997)
18.2   American Public Health Association, American Water Works Association, and Water Environment
       Federation. 1995. Standard Methods for Water and Wastewater. 19th Edition. Sections 9020, 9030,
       9040, 9050, and 9221.
18.3   U.S. Environmental Protection Agency, 1998. Method 1691: Municipal Biosolids Sampling
       Guidance. Draft, September 1998. Office of Water, Washington, DC.
18.4   Bordner, R., J.A. Winter, and P.V. Scarpino (eds.). 1978. Microbiological Methods for Monitoring
       the Environment, Water and Wastes. EPA-600/8-78-017. Office of Research and Development.
       USEPA.
18.5   ICR Microbial Laboratory Manual, EPA/600/R-95/178, National Exposure Research Laboratory,
       Office of Research and Development, U.S. Environmental Protection Agency, 26 Martin Luther King
       Drive, Cincinnati, OH 45268.
18.6   Manual for the Certification of Laboratories Analyzing Drinking Water, EPA 815-B-97-001, Office
       of Ground Water and Drinking Water, Technical Support Center, U.S. Environmental Protection
       Agency, 26 Martin Luther King Drive, Cincinnati, OH 45268.
18.7   Annual Book ofASTM Standards. Vol. 11.01. American Society for Testing and Materials.
       Philadelphia, PA 19103.


19.0  Tables, Diagrams, Flowcharts, and Validation Data

[To be added]


20.0  Glossary

       These definitions and purposes are specific to this method but have been conformed to common usage
       as much as possible.
20.1   Symbols
              °C            degrees Celsius
              u             micro
              #             number
              %             percent

20.2   Alphabetical characters and acronyms
              ASTM        American Society for Testing and Materials
              CFR          Code  of Federal Regulations
              DAL          double agar layer method
              DOT          Department of Transportation
              g             gram

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              HC1           hydrochloric acid
              IPR           initial precision and recovery
              KH2HPO4     potassium phosphate
              L             liter
              M             molar
              mg            milligram
              MgCl2*6H2O   magnesium chloride hexahydrate
              mL            milliliter
              mm           millimeter
              MPN          most probable number
              MS            matrix spike
              NaOH         sodium hydroxide
              Na2S2O3       sodium thiosulfate
              NIST          National Institute of Standards and Technology
              nm            nanometer
              OD            optical density
              OPR          ongoing precision and recovery
              OSHA         Occupational Safety and Health Administration
              PFU          plaque forming unit
              psi            pounds per square inch
              QA            quality assurance
              QC            quality control
              rpm           revolutions per minute
              SAL          single agar layer method
              TNTC         too numerous to  count
              ISA          tryptic soy agar
              TSB          tryptic soy broth
              USEPA       United States Environmental Protection Agency
              X             "times"

20.3   Additional definitions

       Accuracy—A measure of the degree of conformity of a single test result generated by a specific
       procedure to the assumed or accepted true value and includes both precision and bias.

       Analyte—The organism tested for by this method. The analyte in this method is coliphage.

       Analytical batch—[analytical batch to be defined].

       Bias—the persistent positive or negative deviation of the average value of a test method from the
       assumed or accepted true value.

       Coliphage—Viruses that infect fecal coliforms, particularly E. coli.

       Host bacteria—Are those bacteria that allow the bacteriophage to penetrate and replicate within them,
       ultimately lysing, resulting in the release of the progeny bacteriophage. Host bacteria are  essential for
       virus replication. The hosts used in this method are: E. coli CN-13, and
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       E. coli F^ (E. coli HS(pFamp)R).

       Initial precision and recovery (IPR)—The IPR test is performed to establish the ability to
       demonstrate control over the analytical system and to generate acceptable precision and recovery.

       Male-specific coliphage—Viruses (bacteriophages) that infect coliform bacteria only via the
       F-pilus and are indicators of fecal contamination.

       May—This action, activity, or procedural step is neither required nor prohibited.

       May not—This action, activity, or procedural step is prohibited.

       Must—This action, activity, or procedural step is required.

       Method blank—An aliquot of reagent water that is treated exactly as a sample and carried through all
       portions of the procedure until determined to be negative or positive. The method blank is used to
       determine if the sample has become contaminated by the introduction of a foreign microorganism
       through poor technique.

       Ongoing precision and recovery—A reagent water sample method blank spiked with known quantities
       of analytes. The OPR is analyzed exactly like a  sample. Its purpose is to assure that the results
       produced by the laboratory remain within the limits specified within this method for precision and
       recovery.

       Precision—The degree of agreement of repeated measurements of the same property, expressed in
       terms of dispersion of test results about the arithmetical mean. Results are obtained by repetitive
       testing of a homogeneous sample under specified conditions. The precision of a test method is
       expressed quantitatively as the standard deviation computed from the results of a series of controlled
       determinations.

       Relative Standard Deviation (RSD)—The standard deviation times 100 divided by the mean.

       Reagent water—Water conforming to Specification D  1193, Annual Book of ASTM Standards
       (Reference 18.7).

       Should—This action, activity, or procedural step is suggested but not required.

       Somatic  coliphage—Those coliphage that infect host cells via the outer cell membrane but do not
       infect host cells via the F-pilus.
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