United States         Office of Water          EPA 821-R-01-030
          Environmental Protection     Washington, D.C. 20460       April 2001
          Agency



&EPA    Method 1601: Male-specific (F+) and


          Somatic Coliphage in Water by Two-step


          Enrichment Procedure
          April 2001

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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

Pope, Misty, Kevin Cornell, Ken Miller, Jason Kempton, and Jessica Pulz, DynCorp Information and
        Enterprise Technologies, 6101 Stevenson Avenue, Alexandria, VA 22304

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

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                                       Disclaimer



Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

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                                         Introduction

Coliphage presence in ground water is an indication of fecal contamination. Method 1601 (two-step
enrichment procedure) is a performance-based method for detecting the presence of male-specific (F+) and
somatic coliphage in ground water and other waters. Laboratories are permitted to modify or omit any steps or
procedure, with the exception of the coliphage stock enumeration procedure (Section 11.3), provided that all
performance requirements set forth in the validated method are met. The laboratory may not omit any quality
control analyses.

The two-step enrichment procedure requires enrichment of coliphage in a nutrient broth with host bacteria
followed by  spotting onto a lawn of host bacteria and assessing lysis zone formation in the lawn. This two-step
enrichment method was validated as a qualitative, presence-absence method, and Method 1601 was written
with this use in mind. Although the method potentially may be used as a quantitative assay of coliphage
concentrations in an MPN format, the two-step enrichment method has not been validated this way.
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
       U.S. EPA Office of Water
       Analytical Methods Staff
       1200 Pennsylvania NW
       Mail Code 4303
       Washington, DC 20460
       Email: telliard.william@epa.gov

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
                                                IV

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                                   Table of Contents






1.0    Scope and Application	 1




2.0    Summary of Method	 1




3.0    Definitions  	 1




4.0    Interferences	 2




5.0    Safety	 2




6.0    Equipment and Supplies  	 2




7.0    Reagents and Standards	 4




8.0    Sample Collection, Preservation, and Storage	 9




9.0    Quality Control	  10




10.0   Calibration and Standardization	  15




11.0   Enumeration of Coliphage QC Spiking Suspensions	  15




12.0   Two-step Enrichment Procedure for Sample Analysis	  18




13.0   Data Analysis and Calculations	  21




14.0   Method Performance	  22




15.0   Pollution Prevention  	  24




16.0   Waste Management	  24




17.0   References	  25




18.0   Flowcharts  	  26




19.0   Glossary	  31
                                             VI

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    Method 1601: Male-specific (F+) and Somatic Coliphage in Water by
                          Two-Step Enrichment Procedure

                                         April 2001
1.0   Scope and Application

1.1    The two-step enrichment procedure determines the presence or absence of male-specific (F+) and
       somatic coliphages in ground water and other waters. The two-step enrichment method was validated
       as a qualitative, presence-absence method, and Method 1601 was written with this use in mind. The
       two-step enrichment method potentially may be used as a quantitative assay of coliphage
       concentrations in an MPN format, however, the method has not been validated this way. This method
       is intended to help determine if ground water is affected by fecal contamination.
1.2    Although this method may be used for water matrices other than ground water, it has only been
       validated for use in ground water.
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 ground water 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 1601 describes a qualitative (presence/absence) two-step enrichment procedure for coliphage.
       A 100-mL or  1-L ground water sample is supplemented with MgCl2 (magnesium chloride), log-phase
       host bacteria (E. coll Famp for male-specific coliphage and E. coll CN-13 for somatic coliphage), and
       tryptic soy broth (TSB) as an enrichment step for coliphage. After an overnight incubation, samples
       are "spotted" onto a lawn of host bacteria specific for each type of coliphage, incubated, and
       examined for circular lysis zones, which indicate the presence of coliphages.
3.0   Definitions

3.1    Coliphages are viruses (bacteriophages) that infect E. coll 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. coll. 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. coll.
3.4    Somatic coliphages are DNA viruses 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 19.3.
                                                                                     April 2001

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Method 1601: Two-Step Enrichment Procedure
4.0    Interferences

4.1     During the enrichment phase of the two-step enrichment procedure, other bacteria in the water sample
        can grow and interfere with the spot-test confirmation step. Bacteria may grow over the lysis zone and
        obscure visualization, resulting in a false negative. Generally, when bacteria have overgrown lysis
        zones, they appear as raised colonies or a confluent growth of raised colonies. As a result, they are
        distinguishable from the surrounding lawn of host bacteria. If this problem is noted, follow the
        procedure described in Section 12.2.11.
5.0    Safety

5.1     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.
5.2     Method 1601 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 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 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.
5.3     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 16.0
        Waste Management.
5.4     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 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.
5.5     Mouth pipetting is prohibited.
6.0    Equipment and Supplies

Please 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 in this section, 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, bottles or carboys with
               screw caps
        6.1.2   Ice chest—Igloo, Coleman, styrofoam box or equivalent
April 2001

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                                                        Method 1601: Two-Step Enrichment Procedure
       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 x 150 mm, or
               16 x 100mm
       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, 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 yL volume (VWR, Fisher, DIFCO, or equivalent)
       6.2.6   Micropipettors, adjustable—10- to 200-(iL, and 100- to 1000-(iL, with appropriate aerosol
               resistant tips, Gibson, Eppendorf, or equivalent. Please note: To avoid cross-contamination,
               micropipettors should be wiped down with a 1 : 100 solution of household bleach followed
               by a 10% solution of sodium thiosulfate. Alternatively, disposable pipets (Serological,
               Pasteur, or equivalent) may be used.
       6.2.7   Burner—Alcohol, Bunsen, Fisher, or equivalent
       6.2.8   Sterile disposable petri dishes—100-mm-diameter dishes (Falcon #1029) or equivalent
       6.2.9   Incubator capable of maintaining 36°C ± 1.0°C for growth of microorganisms
       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, 125-mL,  1-L, or
               1.5-L 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 Stir plate—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-(jm pore size
       6.2.17 Sterile, cotton-tipped applicators
       6.2.18 Latex gloves for handling samples, supplies,  and equipment—Microflex, 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 1A" 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, 10-041-5 A) or equivalent

                                                 3                                        April 2001

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Method 1601: Two-Step Enrichment Procedure
       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 (Section 7.4.3)
       6.3.1   Disposable filter disks—25-mm-diameter, 0.45-(jm pore size, sterile, low protein binding
               (Gelman Acrodisc HT Tuffryn, No. 4184, cellulose acetate Corning No. 21053-25, or
               equivalent)
       6.3.2   Syringes—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-mL 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
       6.4.8   Light box—VWR 21475-460 or equivalent
7.0   Reagents and Standards
7.1    General reagents
       7.1.1  Reagent water—Should conform to Specification D 1193, Annual Book of ASTM Standards
              (Reference 17.5).
       7.1.2  10% (w/v) Sodium thiosulfate—Add 10 g sodium thiosulfate (Na2S2O3) per 90 mL reagent
              water. Mix until dissolved. Bring to a final volume of 100 mL and autoclave for 15 minutes at
              121°Cand 15 psi.
       7.1.3  Stock 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 at 121°C and 15 psi.
       7.1.4  Glycerol—Sigma #G6279 or  equivalent. Autoclave for 15 minutes at 121°C and 15 psi.
              Remove promptly to avoid scorching. Store at room temperature.
       7.1.5  Household bleach
       7.1.6  Ethanol—70% or greater

7.2    Antibiotic stocks— Antibiotics must always be added to medium after the medium has been
       autoclaved.
       7.2.1  Stock nalidixic acid  (Sigma N4382, or equivalent)—Please note: Nalidixic acid is
              considered toxic. Wear suitable protective clothing, gloves, and eye/face protection and use
              in a chemical fume hood.
              7.2.1.1   For growth of E. coli CN-13, the host bacteria for somatic coliphage.
              7.2.1.2   Dissolve 1 g of nalidixic acid sodium salt in  100 mL reagent water. Filter through a

April 2001                                      4

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                                                        Method 1601: Two-Step Enrichment Procedure
                        sterile, 0.22-(jm-pore-size membrane filter assembly. Dispense 5 mL per 5-mL
                        freezer vial, date 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.
       7.2.2   Stock ampicillin/streptomycin
               7.2.2.1   For growth of E. coll Famp, the host bacteria for male-specific coliphage.
               7.2.2.2   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-(jm-pore-size membrane filter assembly. Dispense 5 mL per 5-mL freezer
                        vial, date 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. Mix well prior to use.

7.3    Media
       7.3.1   Tryptic (or trypticase) soy broth (TSB)—(DIFCO 0370-15-5, or equivalent)
               7.3.1.1   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,
                        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. Discard aliquot after checking pH, to ensure
                        that the medium is not contaminated.
               7.3.1.2   TSB with nalidixic acid (for growth of E1. coli CN-13)—Aseptically add 10 mL of
                        stock nalidixic acid (Section 7.2.1) to 1 L of autoclaved, cooled (48°C ± 1.0°C)
                        TSB (Section 7.3.1.1)  and mix. Please note: Antibiotics must always be added to
                        medium after the medium has been autoclaved and cooled.
               7.3.1.3   TSB with streptomycin/ampicillin (for growth of E. coli Famp )—Aseptically add
                        10 mL of stock streptomycin/ampicillin sulfate  (Section 7.2.2) to 1 L of
                        autoclaved, cooled (48°C ± 1.0°C) TSB (Section 7.3.1.1) and mix. Please note:
                        Antibiotics must always be added to medium after the medium has been
                        autoclaved and cooled.
               7.3.1.4   10X Tryptic soy broth (TSB)—Add 300 g TSB per liter of reagent water and heat
                        to  dissolve. 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.
       7.3.2   1.5% tryptic soy agar (TSA)—To be used in streak plates (Section 7.5.2.1) and as bottom
               layer of agar (Section  11.3.1.3) during the double agar layer (DAL) coliphage stock QC
               sample spiking suspension enumeration procedure. Prior to autoclaving the  TSB prepared as
               described in  Section 7.3.1.1, 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 to ensure even  distribution.
               7.3.2.1   For growth of somatic  coliphages using E. coli  CN-13 as host bacteria, aseptically
                        add 10 mL of stock nalidixic acid (Section 7.2.1) per liter of autoclaved 1.5% TSA
                        (Section 7.3.2). Please note: Antibiotics must always be added to medium after
                        the medium has been autoclaved and cooled. Swirl flask until well mixed and
                        aseptically dispense  17 - 18 mL per 100-mm plate. Allow to solidify with lids off
                        in  a biohazard hood for several minutes prior to use.  If not used immediately,
                        replace lids and store inverted at 4°C ±  1°C for up to 2 weeks.


                                                 5                                       April 2001

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Method 1601: Two-Step Enrichment Procedure
               7.3.2.2  For growth of male-specific (F+) coliphages using E. coll Famp as host bacteria,
                        aseptically add 10 mL stock ampicillin/streptomycin sulfate (Section 7.2.2) per liter
                        of autoclaved 1.5% TSA (Section 7.3.2). Please note: Antibiotics must always be
                        added to medium after the medium has been autoclaved and cooled. Swirl flask
                        until well mixed and aseptically dispense 17-18 mL per 100-mm plate. Allow to
                        solidify with lids off in a biohazard hood for several minutes prior to use. If not
                        used immediately, replace lids and store  inverted at 4°C ± 1°C for up to 2 weeks.
       7.3.3   0.7% tryptic soy agar (TSA)—"Soft" agar for use as the top layer of agar (Section
               11.3.1.1) during the double agar layer (DAL) coliphage stock QC sample spiking suspension
               enumeration procedure. Prior to autoclaving the TSB in Section 7.3.1.1, add 7 g of agar per
               liter of TSB. While stirring, heat to dissolve agar. Autoclave for 15 minutes at 121°C and
               15psi.  Coolto48°C± 1.0°C.
               7.3.3.1  0.7% TSA top agar tubes with nalidixic acid (for growth of E. coli CN-13)—To
                        1 L of autoclaved 0.7% TSA (soft agar) (Section 7.3.3), aseptically add 10 mL of
                        stock nalidixic acid (Section 7.2.1). Please note: Antibiotics must always be
                        added to medium after the medium has been autoclaved and cooled. 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.
               7.3.3.2  0.7% TSA top agar tubes with ampicillin/streptomycin (for growth of E. coli
                        Famp)—To 1 L of autoclaved 0.7% TSA  (soft agar) (Section 7.3.3), aseptically add
                        10 mL of stock ampicillin/streptomycin (Section 7.2.2). Please note: Antibiotics
                        must always be added to medium after the medium has been autoclaved and
                        cooled. 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.
       7.3.4   Spot plates—To be used during the two step enrichment procedure (Section 12). Please
               note: Condensation may accumulate at the edges of stored spot plates and may drip over
               agar surface if tilted, ruining the spot pattern.  If the stored spot plates have condensation,
               incubate plates for approximately 10 minutes to reduce condensation prior to inoculation.
               7.3.4.1  Log-phase host bacteria must be prepared in advance (Section 7.5.4). Dissolve 3 g
                        TSB (Section 7.3.1.1) and 0.75 g bacteriological grade agar per 100 mL of reagent
                        grade water. Heat and mix to dissolve. Autoclave for 15 minutes at 121°C and
                        15 psi. Cool to 45°C  to 48°C in a water bath.
               7.3.4.2  Add 2 mL of log-phase host bacterium prepared as directed in Section 7.5.4 and
                        1 mL stock antibiotic (Section 7.2). Please note: Antibiotics must always be
                        added to medium after the medium has been autoclaved and cooled. Nalidixic
                        acid is used with E. coli CN-13,  and ampicillin/streptomycin is used with E. coli
                        Famp. Swirl to mix, and pour 20 mL per 100-mm diameter, sterile petri plate. Allow
                        to solidify. Label plates with name of host bacterium. Plates may be used that day
                        or stored at 4°C ± 1°C for up to four days before use. Divide the bottom of the
                        plate into a grid of 1-cm squares using a permanent marking pen. Number each
                        square for ease of reference. Other alternatives include: 1) gridded petri dishes, 2)
                        adhesive grids, or 3) creating the 1-cm grid on a circular plastic dish and attaching
                        to the bottom  exterior of the plate with cellophane tape.

7.4    Coliphage stock
       7.4.1   MS2 stock coliphage (ATCC#15597-B1)—Male-specific (F+) coliphage. Refer to
               http://www.atcc.org/SearchCatalogs/faqBacteriology.cfnrfqlO for initial preparation of stock. May
               be stored at 2°C to 8°C for up to 5 years.

April 2001                                        6

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                                                         Method 1601: Two-Step Enrichment Procedure
        7.4.2  phi-X 174 stock coliphage (ATCC#13706-B 1)—Somatic coliphage. Refer to
               http://www.atcc.org/SearchCatalogs/faqBacteriology.cfm#qlO for initial preparation of stock. May
               be stored at 2°C to 8°C for up to 5 years.
        7.4.3  Coliphage stock from sewage filtrate—This filtrate will be used as a spiking suspension for
               QC samples.
               7.4.3.1  Collect approximately 100 mL of raw sewage in a 250-mL collection bottle.
               7.4.3.2  Transport to the laboratory on ice. Analysis of raw sewage filtrate should begin
                        within 24 hours of collection.
               7.4.3.3  Allow the raw sewage to settle at 4°C ± 1°C for 1  to 3 hours. This will make the
                        filtration process easier.
               7.4.3.4  Remove a sterile, 20-mL syringe from its package, aseptically remove plunger from
                        barrel, and attach a filter disk to the syringe barrel.
               7.4.3.5  Pipet 10 to 15 mL of supernatant from settled sewage into the syringe barrel.
               7.4.3.6  Hold the assembly over a sterile  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).
               7.4.3.7  Cap the tube, label with source, date, and initials, and store the filtrate at
                        4°C ± 1°C until ready to assay. If filtrate is stored more than 24 hours, it must be
                        re-titered before use.

7.5     Host bacteria stock cultures
        7.5.1   Pure host bacteria cultures
               7.5.1.1  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.
               7.5.1.2  E. coli Famp— 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. ATCC#700891.
        7.5.2  Frozen host bacteria stock cultures—The laboratory shall obtain reference host bacterial
               cultures (Sections 7.5.1.1 and 7.5.1.2) and use these to establish pure frozen host stock
               cultures that are maintained by the laboratory. Frozen stocks are used as inoculum for
               overnight host bacteria stock cultures (Section 7.5.3).
               7.5.2.1  Establish pure frozen stock cultures by streaking host bacteria onto  1.5% TSA
                        plates with appropriate antibiotic (Section 7.3.2) to attain isolated colonies.
               7.5.2.2  Incubate inoculated plates overnight, pick an individual colony and inoculate into
                        tryptic soy broth with appropriate antibiotics (Sections 7.3.1.2 and 7.3.1.3), and
                        grow to log phase  (Section 7.5.4).
               7.5.2.3  Harvest broth by mixing  sterile glycerol and broth with log-phase host bacteria in a
                        ratio of 1:4 in a 5-mL freezer vial. Prepare log-phase host bacteria as described in
                        Section 7.5.4, below. (Example: 200 ^L sterile glycerol plus 800 ^L log-phase
                        E. coli).
               7.5.2.4  Label with E. coli  strain and date of harvest.
               7.5.2.5  Freeze host bacteria  stock cultures at -70°C, if possible. Cultures can be frozen at


                                                  7                                         April 2001

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Method 1601: Two-Step Enrichment Procedure
                         -20°C if the laboratory does not have the capability to freeze samples at -70°C.
               7.5.2.6  Host bacteria stored at -70°C may be retained for up to one year. If stored at
                        -20°C, the host bacteria may be retained for up to two months.
        7.5.3  Overnight host bacteria stock cultures—Inoculum from an overnight bacterial host culture
               will reach log-phase more rapidly than inoculum from frozen stock.
               7.5.3.1  Dispense 25 mL of tryptic soy  broth (TSB) with nalidixic acid (Section 7.3.1.2)
                        into a sterile 125-mL shaker flask. For proper growth conditions, each flask should
                        always contain 25 to 30 mL of medium.
               7.5.3.2  Inoculate the flask with a loopful of E. coll CN-13 from the frozen stock culture
                        (Section 7.5.2).
               7.5.3.3  Repeat Sections 7.5.3.1 and 7.5.3.2 using TSB with streptomycin/ampicillin as the
                        medium (Section 7.3.1.3) and E. coll F^p as the bacterial host.
               7.5.3.4  Place a sterile  slip cap or plug on the shaker flasks, label flasks, and secure in
                        shaker.
               7.5.3.5  Incubate at 36°C ± 1.0°C and set shaker to 100 to 150 rpm overnight (18 to 20
                        hours).
               7.5.3.6  Chill on wet ice or at 4°C ± 1°C until ready for use.
        7.5.4  Log-phase host bacteria stock cultures  (Section  18, Flow chart 1)
               7.5.4.1  To a 125-mL shaker flask containing 25 mL of TSB with nalidixic acid (Section
                        7.3.1.2) add 0.1 to 1.0 mL of overnight E. coll CN-13 host bacteria stock culture
                        (Section 7.5.3  or 7.5.4.7). 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 analyzed using the two-step
                        enrichment procedure  (Section 12) will require a 0.5-mL inoculum of log-phase
                        host bacteria.
               7.5.4.2  Repeat Section 7.5.4.1 using TSB with streptomycin/ampicillin (Section 7.3.1.3) as
                        the medium and E. coll Famp as  the bacterial host.
               7.5.4.3  After inoculation, place a sterile slip-cap or plug on the shaker flasks and secure in
                        shaker incubator.
               7.5.4.4  Incubate at 36°C ± 1.0°C and 100 to 150 rpm for approximately 4 hours or until
                        cultures are visibly turbid (cloudy), indicating log-phase growth.
               7.5.4.5  Aseptically remove 1 mL of culture from flask, dispense into a cuvette (Section
                        6.2.22), and read absorbance at 520 nm. 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.
               7.5.4.6  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).
               7.5.4.7  Store remaining bacterial host culture at  4°C ± 1°C overnight to inoculate flasks
                        for the preparation of  new log-phase bacterial hosts.
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                                                       Method 1601: Two-Step Enrichment Procedure
8.0   Sample Collection, Preservation, and Storage

Please 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.

8.1    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 using wet ice, Blue Ice®, or similar products to maintain temperature.
       Samples must be stored at 4°C ± 1°C. Do not freeze.
8.2    Sample  collection
       8.2.1   Two-step enrichment procedure using 100-mL samples: Collect 250 mL of sample for
               each of the two coliphage types to allow for sample re-analysis, if necessary.
       8.2.2   Two-step enrichment procedure using 1-L samples: Collect 2.5 L of sample for each of the
               two coliphage types to allow for sample re-analysis, if necessary.
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
       for testing.
       8.5.1   Two-step  enrichment 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 titer has not decreased by more
               than 50%, the sample can be stored for up to 72 hours.
8.6    Dechlorination procedure—Although this method was validated for use with unchlorinated ground
       water, it potentially can be used with chlorinated waters. If the sample has been chlorinated, add
       0.5-mL  of 10% sodium thiosulfate per  1-L of sample at time of sample collection.
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Method 1601: Two-Step Enrichment Procedure
9.0    Quality Control

9.1     Each laboratory that uses Method 1601 is required to operate a formal quality assurance (QA)
        program. The minimum QA requirements consist of an initial demonstration of laboratory capability
        through performance of the initial demonstration of capability (IDC) test (Section 9.3), ongoing
        analysis of spiked reagent water and field samples to evaluate and document data quality, and analysis
        of positive controls and method blanks as tests of continued acceptable performance. Spiked sample
        results are compared to acceptance criteria based on data generated during the interlaboratory
        validation of Method 1601 involving 10 laboratories and 10 raw ground water matrices. Specific
        quality control (QC) requirements for Method 1601 are provided below. General 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 17.3).
9.2     General QC requirements—Positive control samples may be spiked with enumerated sewage
        filtrate or pure cultures of MS2 (male-specific) or phi-X 174 (somatic) coliphage. All other spiked QC
        samples must be spiked with enumerated sewage filtrate or equivalent.
        9.2.1   Initial demonstration of capability (IDC). The laboratory shall demonstrate the ability to
               generate acceptable performance with this method by performing an IDC test before
               analyzing any field samples. The procedure for performing the IDC is described in Section
               9.3. IDC tests must be accompanied by a method blank (Section 9.2.2) for each coliphage
               type.
        9.2.2   Method blanks. The laboratory shall analyze method blanks (reagent water sample
               containing no coliphage) to demonstrate freedom from contamination. The procedures and
               criteria for analysis of a method blank are described in Section 9.4. At a  minimum, the
               laboratory shall analyze one method blank per spot plate (Section 13). In an effort to
               determine if cross-contamination is an issue, the method blank should be spotted onto the
               lawn of host bacteria immediately following the positive control spot.
        9.2.3   Positive controls. The laboratory shall analyze positive controls to ensure that stock
               coliphage suspensions, host bacterial cultures, and growth media are performing properly
               (Section 9.5). The laboratory shall inoculate one positive control spot per spot plate (Section
               13). If multiple spot plates are inoculated with samples on the same day, a single enriched
               positive control sample may be used to inoculate multiple spot plates on that day.
        9.2.4   Matrix spikes (MS).  The laboratory shall analyze  one set of MS samples for each coliphage
               type when samples are first received from a ground water source for which the laboratory has
               never before analyzed samples (Section 9.6). In addition, the laboratory shall analyze one set
               of MS samples on an  ongoing basis after every 20th field sample for each ground water
               source. For  example, when a laboratory receives the first sample from a  source, the
               laboratory must obtain additional aliquots of the field samples to be used for the MS test.
               When the laboratory receives the 20th field sample from this site, additional aliquots of this
               sample must be collected and spiked. MS samples should be collected at the same time as
               routine field samples.
        9.2.5   Ongoing demonstration of capability (ODC). The laboratory shall demonstrate that the
               analytical system is in control on an ongoing basis through analysis of ODC samples (Section
               9.7). The laboratory shall analyze one set of ODC samples after every 20 field and MS
               samples or one per week, whichever occurs more frequently.
        9.2.6   Method modification validation/equivalency demonstration requirements. Method 1601 is
               a performance-based method and the laboratory is permitted to modify certain method
               procedures to improve performance or lower the costs of measurements, provided that all

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                                                        Method 1601: Two-Step Enrichment Procedure
               quality control (QC) tests cited in Section 9.2.6 are performed and all QC acceptance criteria
               are met. The laboratory is not permitted to modify the double agar layer QC spiking
               suspension enumeration procedure (Section 11.3).
               9.2.6.1   Method modifications at a single  laboratory. Each time a modification is made to
                        this method for use in a single laboratory, the laboratory is required to validate the
                        modification according to Tier 1 of EPA's performance-based measurement system
                        (PBMS) (Table 4 and Reference 17.6) to demonstrate that the modification
                        produces results equivalent or superior to  results produced by this method as
                        written. Briefly, each time a modification  is made to this method, the laboratory is
                        required to demonstrate acceptable  modified method performance through the IDC
                        test (Section 9.3). IDC results must meet the QC acceptance criteria in Table  1 in
                        Section 9.3, and should be comparable to  previous results using the unmodified
                        procedure. Although not required, the laboratory also should perform an Expanded
                        MS test (Section 9.8) to demonstrate the performance of the modified method in at
                        least one  real-world matrix before analyzing field samples using the modified
                        method.
               9.2.6.2   Method modifications for nationwide approval. If the laboratory or a
                        manufacturer seeks EPA approval of a method modification for nationwide use, the
                        laboratory or manufacturer must validate the modification according to Tier 2 of
                        EPA's PBMS (Table 4 and Reference 17.6). Briefly, at least three laboratories
                        must perform IDC tests (Section 9.3) and Expanded MS tests (Section 9.8) using
                        the modified method, and all tests must meet the QC acceptance criteria specified in
                        Tables 1 and 3 in Sections 9.3 and  9.8. Upon nationwide approval, laboratories
                        electing to use the modified method still must demonstrate acceptable initial and
                        ongoing performance in their laboratory according to the requirements in Section
                        9.2.
       9.2.7   Media sterility checks. The laboratory shall test media sterility by incubating one unit (tube
               or plate) of each batch of medium at 36°C ±  1.0°C  for 48 to 72 hours and observing for
               growth. Also, if media is stored in the refrigerator after sterilization, the media must be stored
               overnight at room temperature and all media with growth discarded.
       9.2.8   Record maintenance. The laboratory shall maintain records to define the quality of data that
               are generated. 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, IDC, ODC, and MS sample
               results must be maintained. In addition, a log  book containing reagent and material lot
               numbers should be maintained along with samples analyzed using each of the lots.
       9.2.9   Performance studies. The laboratory should  periodically analyze external QC samples, such
               as performance evaluation (PE) samples, when  available. The laboratory should also
               participate in available interlaboratory performance  studies conducted by local, state, and
               federal agencies or commercial organizations. The laboratory should review results, correct
               unsatisfactory performance, and record corrective actions.
       9.2.10 The specifications contained in this method can be met if the analytical system is maintained
               under control.

9.3    Initial demonstration of capability (IDC)—The  IDC test is performed to demonstrate acceptable
       performance with the method as written prior to analysis of field samples  or to evaluate acceptable
       performance of a method modification. IDC test samples must be spiked with enumerated sewage
       filtrate or equivalent. IDC tests must be accompanied  by analysis of a method blank (Section 9.4).
       9.3.1   A total of 10 spiked reagent water samples per coliphage type (male-specific and somatic) and

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Method 1601: Two-Step Enrichment Procedure
               sample volume are required for the IDC test. 100-mL reagent water samples should be used if
               the laboratory will analyze 100-mL field samples by this procedure, while 1-L reagent water
               samples should be used if the laboratory will analyze 1-L field samples.
       9.3.2   IDC samples must be spiked in "bulk" for each coliphage type at concentrations as specified
               in Table 1. For example, for 1-L somatic IDC samples, spike 10-L of reagent water with 14
               PFU to achieve the target spike concentration of 1.4 PFU per sample. Please refer to Section
               11 for the coliphage spiking suspension enumeration procedure and Section 13.2 for spike
               volume calculations.

Table 1.	Initial demonstration of laboratory capability (IDC)
Coliphage
type
F+
F+
Somatic
Somatic
Sample
size
100-mL
1-L
100-mL
1-L
Target spike
concentration
(PFU per sample)
1.3
1.2
1.5
1.4
"Bulk" volume
to be spiked
1000 mL
10L
1000 mL
10L
Bulk spike
concentration
(PFU per bulk
volume)
13
12
15
14
Minimum
number of
positive
samples out of
10
5
3
5
5
       9.3.3   Mix the spiked reagent water by swirling the container. For each coliphage type and volume,
               dispense 10 aliquots into individual containers.
       9.3.4   Analyze the spiked IDC samples according to the two-step enrichment procedure (Section
               12). See Table  1 for the minimum number of samples that must be positive for each set of
               10 IDC samples. If system performance is unacceptable, identify and correct the problem and
               repeat the IDC 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 or QC 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 repeat
               of the method blank analysis shows no evidence of contamination. Any sample in a batch
               associated with a contaminated blank should be recollected (if holding times have  been
               violated) and reanalyzed. Samples from a batch that proves to have no coliphage  in its  blank
               may be reported.
9.5    Positive control
       9.5.1
             performed at the frequency specified in Section 9.2.3.
Positive controls for 100-mL samples—Add 100 mL of reagent water to each of two 250 to
500-mL sterile Erlenmeyer flasks. If spiking with sewage filtrate, spike each flask with one
coliphage type (somatic or male-specific) at a concentration of approximately 20 PFU /
100 mL. If spiking with MS2 (male-specific) or phi-X 174 (somatic), spike each flask with
one coliphage type at a concentration of approximately 60 PFU /100 mL. See Section 11 for
enumeration of stock/filtrate and Section 13.2 for spike volume calculations. Label each flask
with the coliphage type and "+" or "positive." Analyze positive control sample by the two-
step enrichment procedure as described in Section 12.
April 2001
                                  12

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                                                       Method 1601: Two-Step Enrichment Procedure
       9.5.2  Positive controls for 1-L samples—Add 1 L of sterile reagent water to each of two sterile,
              1-L bottles.  If spiking with sewage filtrate, spike each flask with one coliphage type (somatic
              or male-specific) at a concentration of approximately 20 PFU / L. If spiking with MS2 (male-
              specific) or phi-X 174 (somatic), spike each flask with one coliphage type at a concentration
              of approximately 60 PFU / L. See Section 11 for enumeration of stock/filtrate and Section
              13.2 for spike volume calculations. Label each flask with the coliphage type and "+" or
              "positive." Analyze positive control sample by the two-step enrichment procedure, as
              described  in Section 12.

9.6    Matrix spike (MS) analyses for ongoing assessment of method performance—The
       laboratory shall analyze MS samples according to the frequency in Section 9.2.4. The laboratory shall
       spike and analyze  field samples from each ground water source to assess method performance in each
       matrix. For each coliphage type and sample volume, at a minimum, one out of three MS samples must
       be positive. 100-mL MS samples must be analyzed if 100-mL field samples are being analyzed by this
       procedure, while 1-L MS samples must be analyzed  if 1-L field samples are being analyzed by this
       procedure. MS test samples must be  spiked with enumerated sewage filtrate or equivalent.
       9.6.1  The ongoing MS sample must be collected and analyzed at the same time as the unspiked field
              sample from the same source.
       9.6.2  MS samples must be spiked in "bulk" for each coliphage type and sample volume. Spike the
              bulk samples at concentrations according to Table 2. For example, for 1-L somatic MS
              samples, spike 3-L of ground water with 4.2 PFU of somatic coliphage to achieve a target
              spike concentration of 1.4 PFU per sample, based on stock coliphage enumeration. See
              Section 11 for enumeration of coliphage stock and Section  13.2 for spiking volume
              calculations.
       9.6.3  Analyze MS samples according to the two-step enrichment procedure (Section 12).
       9.6.4  One or more MS samples out of three must be positive for method performance to be
              considered acceptable for that ground water source.  If the matrix spike results are
              unacceptable and the ODC sample and positive control sample results associated with this
              batch of samples are acceptable, a matrix interference may be causing the poor results.

9.7    Ongoing demonstration of capability (ODC)—Performed at the frequency specified in Section
       9.2.5. To demonstrate that the laboratory is in control for each coliphage type, at a minimum, one out
       of three reagent water spiked samples must be positive. 100-mL reagent water samples should be used
       if 100-mL field samples are being analyzed by this procedure, while 1-L reagent water samples should
       be used if 1-L field samples are being analyzed by this procedure. ODC test samples must be spiked
       with enumerated sewage filtrate or equivalent.
       9.7.1  ODC samples must be spiked in "bulk" for each coliphage  type. Spike the bulk samples at
              concentrations according to Table 2. For example, for 1-L somatic ODC samples, spike 3-L
              of reagent water with 4.2 PFU of somatic coliphage to achieve a target spike concentration  of
              1.4 PFU per sample, based on stock coliphage enumeration. See Section  11 for enumeration
              of coliphage stock and Section 13.2 for spiking volume calculations.
       9.7.2  Mix the spiked reagent water by swirling the container and dispense three aliquots into
              individual containers.
       9.7.3  Analyze the spiked ODC samples according to Section  12.  One or more out of three samples
              must be positive for each coliphage type and sample volume being evaluated. If not, method
              performance is unacceptable. Identify and correct the problem and perform another ODC test
              before continuing with the analysis of field samples.
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Method 1601: Two-Step Enrichment Procedure
Table 2.       MS and ODC sample spiking requirements for ongoing evaluation of method
              performance
Coliphage
type
F+
F+
Somatic
Somatic
Sample
size
100-mL
1-L
100-mL
1-L
Target spike
concentration
(PFU per sample)
1.3
1.2
1.5
1.4
Number of samples
that must be spiked
( 1 must be positive)
3
3
3
3
"Bulk"
volume
to be spiked
300-mL
3-L
300-mL
3-L
Bulk spike
concentration
(PFU per bulk
volume)
3.9
3.6
4.5
4.2
9.8    Expanded MS test— If IDC and Expanded MS test performance is equal to or better than the criteria
       set forth in Tables 1 and 3, then the modified version of the method is acceptable. Expanded MS
       analyses for evaluation of modified method performance are as follows. Please note: the expanded
       MS test must be accompanied by an unspiked matrix sample. If the unspiked matrix sample tests
       positive, the Expanded MS test must be re-run.
       9.8.1  A total of 10 spiked field samples per coliphage type (male-specific and somatic) and volume
              are required. 100-mL matrix spike samples should be used if 100-mL field samples are being
              analyzed by this procedure, while 1-L matrix spike samples should be used if 1-L field
              samples are being analyzed by this procedure.
       9.8.2  Samples must be spiked in "bulk" for each coliphage type. Spike the bulk samples at
              concentrations according to Table 3. For example, for 1-L somatic IDC samples, spike 10-L
              of ground water with a target spike dose of 14 PFU of somatic coliphage. See Section 11 for
              enumeration of coliphage stock and Section 13.2 for spiking volume calculations.

Table 3.	Expanded MS test requirements for evaluation of method modification performance
Coliphage
type
F+
F+
Somatic
Somatic
Sample
size
100-mL
1-L
100-mL
1-L
Target spike
concentration (PFU
per sample)
1.3
1.2
1.5
1.4
"Bulk"
volume to be
spiked
1000-mL
10-L
1000-mL
10-L
Target bulk Spike
(PFU per bulk
volume)
13
12
15
14
Minimum number
of positive
samples out of 10
4
2
5
5
       9.8.3  Mix the spiked ground water by swirling the container. For each coliphage type and volume
              dispense 10 aliquots into individual containers.
       9.8.4  Analyze the spiked ground water samples according to the two-step enrichment procedure
              (Section 12). Table 3 specifies the minimum number of samples that must be positive for each
              set of 10 Expanded MS  samples.
April 2001
14

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                                                       Method 1601: Two-Step Enrichment Procedure
10.0  Calibration and Standardization

10.1   At a minimum, check temperatures in water baths, refrigerators, -20°C freezers, and -70°C freezers
       daily to ensure operation within stated limits of method and record daily measurements in a log book.
10.2   At a minimum, 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 NIST Monograph SP 250-23. The mercury column should not be separated.
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  17.4.
11.0  Enumeration of Coliphage QC Spiking Suspensions
11.1   The double agar layer (DAL) procedure is used to enumerate stock suspensions of somatic and male-
       specific coliphage for use in spiking quality control samples.
11.2   Dilution of coliphage stock (Section 7.4.1 or 7.4.2)  or sewage filtrate (Section 7.4.3)—A minimum of
       four different volumes/dilutions are necessary for the (DAL) enumeration of the coliphage stock or
       sewage filtrate (Section 18, Flow chart 2):
                Undiluted
                0.1
                0.01
                0.001
       Additional dilutions may be necessary. TSB without antibiotics (Section 7.3.1.1) is used as the diluent
       and as the method blank.
       11.2.1   Aseptically add 9.0 mL of TSB without antibiotics (Section 7.3.1.1) into each of four (or
                more) sterile dilution tubes (Section 6.2.1).  Label them as "0.1," "0.01," "0.001," "method
                blank," etc.
       11.2.2   Add 1.0 mL of the coliphage stock or sewage 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.
       11.2.3   Add 1.0 mL of the well-mixed 0.1 dilution to atube with 9 mL of TSB labeled "0.01". Cap
                the tube and vortex for 5  seconds on a medium-high setting (if available) or until well-
                mixed.
       11.2.4   Add 1.0 mL of the well-mixed 0.01 dilution to atube with 9 mL of TSB labeled "0.001."
                Cap the tube and vortex for 5 seconds on a medium-high setting (if available) or until well-
                mixed.
       11.2.5   Add 1.0 mL of TSB  without antibiotics (Section 7.3.1.1) to the tube labeled "method
                blank." Cap the tube and  vortex for 5 seconds on a medium-high setting (if available) or
                until well-mixed.
11.3   Coliphage spiking suspension enumeration by double  agar layer (DAL) procedure (Section 18, Flow
       chart 3)—In this procedure, a tube of molten 0.7%  TSA "top agar" with added host bacteria is
       inoculated with coliphage stock and will be poured into a 1.5% TSA "bottom agar" plate. Four


                                               15                                      April 2001

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Method 1601: Two-Step Enrichment Procedure
        dilutions of coliphage stock or sewage filtrate will be analyzed in duplicate for each coliphage type.
        As a result, nine double-agar layer 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. Please note: Laboratories are
        not permitted to modify or omit any aspects of the coliphage stock enumeration procedure (Section
        11.3). As a result, magnesium chloride or calcium chloride must not be added the sample or media.
        11.3.1   Agar preparation
                 11.3.1.1     Place 0.7% TSA top agar tubes with antibiotics (Section 7.3.3.1 and 7.3.3.2)
                              in a 45°C to 48°C water bath. The top agar should remain molten in the
                              water bath until ready for use. 18 tubes are necessary to enumerate four
                              dilution volumes in duplicate for each phage. The 18 tubes also includes an
                              additional method blank tube for each phage type. Nine of the top agar tubes
                              should contain nalidixic acid (Section 7.3.3.1) for growth of E.  coli CN-13;
                              the other nine should contain ampicillin/streptomycin (Section 7.3.3.2) for
                              growth of E. coli Famp.
                 11.3.1.2     As a precaution against contamination, disinfect a workspace near the water
                              bath with a 1 : 100 dilution of household bleach and allow to dry. If
                              workspace can be corroded by bleach, use an ethanol solution of 70% or
                              greater.
                 11.3.1.3     Assemble 1.5% TSA bottom agar plates (Section 7.3.2) and label so that the
                              following information is identifiable:
                              •       Dilution of stock filtrate or method blank
                                      Bacterial host (E. coli CN-13 or E. coli Famp)
                              •       Coliphage type (somatic for the E. coli CN-13  bacterial host or
                                      male-specific for the E. coli Famp bacterial host)
                                      Date
                              •       Time

Please note: The following steps are critical. To ensure viability of bacterial host and coliphage, do not add
bacterial host and coliphage spiking suspension until ready to plate.

        11.3.2   Preparation of plates for enumeration of somatic coliphage
                 11.3.2.1     With the top agar tube still in the water bath, aseptically inoculate a top agar
                              tube containing nalidixic acid with 100 (iL of log-phase E.  coli CN-13.
                 11.3.2.2     Immediately add 500 yL (0.5 mL) of undiluted coliphage stock or sewage
                              filtrate.
                 11.3.2.3     Mix the inoculum by rolling the tube briefly in palm of hand.
                 11.3.2.4     Pour contents into one of the two bottom agar plates marked "undiluted,
                              E. coli CN-13,  somatic."
                 11.3.2.5     Duplicate analysis—Repeat Sections 11.3.2.1 through  11.3.2.4 forthe
                              duplicate.
                 11.3.2.6     Repeat Sections 11.3.2.1 through 11.3.2.5 for each dilution volume.
        11.3.3   Preparation of plates for enumeration of male-specific (F+) coliphage—Repeat Section
                 11.3.2 using agar containing ampicillin/streptomycin and log-phase E. coli F^p
        11.3.4   Preparation of somatic coliphage method blank
                 11.3.4.1     With the top agar tube still in the water bath, aseptically inoculate a top agar
                              tube containing nalidixic acid with 100 (iL of log-phase E.  coli CN-13.
April 2001                                        16

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                                                Method 1601: Two-Step Enrichment Procedure
         11.3.4.2     Immediately add 500 yL (0.5 mL) of TSB from the "method blank" dilution
                      tube.
         11.3.4.3     Mix the inoculum by rolling the tube briefly in palm of hand.
         11.3.4.4     Pour contents into a bottom agar plate marked "method blank,
                      E. coll CN-13, somatic."
11.3.5   Preparation of the male-specific (F+) coliphage method blank—Repeat Section 11.3.4 using
         agar containing ampicillin/streptomycin and log-phase E. coll Famp
11.3.6   Store undiluted coliphage stock or sewage filtrate at 4°C ± 1.0°C for use in preparing new
         dilutions for positive controls, IDC, ODC, and MS samples.
11.3.7   After the top agar hardens, cover, invert the plates and incubate for 16 to 24 hours at
         36°C± 1.0°C.
11.3.8   Circular zones of clearing (typically  1 to 10 mm in diameter) in lawn of host bacteria after
         16 to 24 hours of incubation are plaques. Count the number of plaques on each plate.
         Please note: The use of a light box. (Section 6.4.8) to evaluate results is recommended.
11.3.9   Proceed to Section  13.1 and calculate the PFU / mL for each coliphage.
11.3.10  Use the enumerated somatic and male-specific stocks to spike the two-step enrichment IDC,
         ODC, MS, and positive control samples as described in Section 9.
                                         17                                      April 2001

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Method 1601: Two-Step Enrichment Procedure
12.0 Two-step Enrichment Procedure for Sample Analysis
12.1   Enrichment procedure—Instead of adding antibiotics to each individual sample as described in
       Sections 12.1.1.8, 12.1.1.9, 12.1.2.8, and 12.1.2.9 laboratories may add antibiotics directly to the
       1 OX TSB, AFTER the 1 OX TSB has been autoclaved and cooled to 48 °C ± 1.0 °C. Regardless of
       which technique is used, the laboratory's QC samples must be prepared and analyzed the same way
       that field samples are analyzed.
       12.1.1   100-mL samples (Section 18, Flow chart 4)
                12.1.1.1     Dispense a 100-mL sample aliquot into each of two sterile, 100-mL or
                            125-mL bottles. If 100-mL bottles are used, be sure that there is ample head
                            space for the addition of reagents.
                12.1.1.2    Label bottle with the following information
                                    Sample number
                            •       Bacterial host (E. coll CN-13 or E. coll Famp)
                                    Date
                            •       Start time
                                    Sample volume
                12.1.1.3    Record this information on a report form.
                12.1.1.4    Add 1.25 mL of stock magnesium chloride (Section 7.1.3) to each ground
                            water sample aliquot.
                12.1.1.5    Add 0.5 mL of log-phase E. coll CN-13 (Section 7.5.4) to one sample aliquot
                            for each sample.
                12.1.1.6    Add 0.5 mL of log-phase E. coll Famp (Section 7.5.4) to the other sample
                            aliquot for each sample.
                12.1.1.7    Add 5 mL of 10X TSB (Section 7.3.1.4) to all aliquots. This gives a final
                            concentration of 0.5X TSB.
                12.1.1.8    To those bottles with E. coll CN-13 as host, add 1 mL of stock nalidixic acid
                            (Section 7.2.1).
                12.1.1.9    To those bottles with E. coll F^p as host, add 1 mL of stock
                            ampicillin/streptomycin solution (Section 7.2.2).
                12.1.1.10   Cap and invert each bottle five times to mix.
                12.1.1.11   Prepare  method blanks and positive controls as specified in Sections 9.4 and
                            9.5.
                12.1.1.12   Incubate the bottles for 16 to 24 hours at 36°C ± 1.0°C with no further
                            mixing.  Proceed to Section 12.2.
       12.1.2   1-L samples (Section 18, Flow chart 5)
                12.1.2.1     Dispense a 1-L sample aliquot into each of two sterile,  1-L or 1.5-L bottles.
                            If 1-L bottles are used, be sure that there is ample head space for the addition
                            of reagents.
                12.1.2.2    Label bottle with the following information
                                    Sample number
                            •       Bacterial host (E. coll CN-13 or E. coll Famp)
                                    Date
                            •       Time
                                    Sample volume
                12.1.2.3    Record this information on a report form.

April 2001                                      18

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                                                        Method 1601: Two-Step Enrichment Procedure
                 12.1.2.4     Add 12.5 mL of stock magnesium chloride (Section 7.1.3) to each ground
                              water sample aliquot.
                 12.1.2.5     Add 5 mL of log-phase E. coll CN-13 (Section 7.5.4) to one sample aliquot
                              for each sample.
                 12.1.2.6     Add 5 mL of log-phase E. coll F^p (Section 7.5.4) to the other sample
                              aliquot for each sample.
                 12.1.2.7     Add 50 mL of 10X TSB (Section 7.3.1.4) to all aliquots. This gives a final
                              concentration of 0.5X TSB.
                 12.1.2.8     To those bottles with E. coli CN-13 as host, add 10 mLof stock nalidixic
                              acid (Section 7.2.1).
                 12.1.2.9     To those bottles with E. coli Famp as host, add lOmL of stock
                              ampicillin/streptomycin solution (Section 7.2.2).
                 12.1.2.10    Cap and invert each bottle 5  times to mix.
                 12.1.2.11    Prepare method blanks and positive controls as specified in Sections 9.4 and
                              9.5.
                 12.1.2.12    Incubate the bottles for 16 to 24 hours at 36°C ± 1.0°C with no further
                              mixing. Proceed to Section 12.2.
12.2   Spot-plate procedure (Section 18, Flow charts 4 and 5)
        12.2.1   Prepare spot plates according to Section 7.3.4. Once prepared,  spot plates may be used on
                 the same day or held at 4°C ± 1°C for up to 4 days prior to use.
        12.2.2   After the overnight enrichment of 16 to 24 hours, mix the sample by inverting the bottle 25
                 times or more.

Please note: If the positive control or method blank exhibits  an inappropriate  response or if there is a
problem with overlapping spots, it will be necessary to re-spot all associated samples. As a result, the
enriched samples may be stored at 4°C ± 1 °C for up to 48 hours.

        12.2.3   Spot 10 \jL of enriched sample from the bottle containing E. coli CN-13 host bacteria onto
                 the gridded spot plate that was prepared using E. coli CN-13. Record spot time on the
                 report form.
        12.2.4   Record the grid number and corresponding  sample number into a log book.
        12.2.5   Spot 10 (A of enriched sample from the bottle containing E. coli Famp host bacteria onto the
                 gridded spot plate that was prepared using E. coli Famp. If the analyst is extremely careful,
                 one 100-mm plate can be inoculated with up to 15 spots.
        12.2.6   Record the grid number and corresponding  sample number on the report form.
        12.2.7   Spot the method blank and positive control samples as specified in Section 9.4 and 9.5.
        12.2.8   Allow inocula to absorb into medium. This will take approximately 30 to 60 minutes. The
                 inocula must not be allowed to run across the plate.
        12.2.9   After inocula absorption, cover, invert, and incubate the plate at 36°C ± 1.0°C for 16 to 24
                 hours.
        12.2.10  Lysis zone formation (typically a circular zone of clearing) indicates a sample is positive
                 for coliphages. If the spot contains an intact lawn of bacteria  indistinguishable from the
                 background lawn of bacteria,  this indicates a negative result. However, other outcomes of
                 the spot assay are possible. A positive result also may appear as one or more small plaques
                 or areas of clearing of the host bacteria lawn within the spot, despite the presence of some

                                                 19                                       April 2001

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Method 1601: Two-Step Enrichment Procedure
                 portion of the host bacteria lawn within the spot. A positive result may also appear as a
                 zone of lysis containing small, discrete colonies of bacteria within the spot.  These bacterial
                 colonies are from phage-resistant mutants. Please note: The use of a light box (Section
                 6.4.8) to evaluate sample results is recommended.
        12.2.11  It is possible that the circular spot area contains confluent bacterial growth or a very large
                 number of bacterial colonies that are distinct from the background lawn of host bacteria.
                 This result could make it difficult or impossible to determine if lysis of host bacteria has
                 occurred. In this case, the bacteria must be removed from the enrichment culture material
                 before re-spotting onto a new pre-poured lawn of host bacteria in a spot plate. The
                 interfering bacteria are removed from the enrichment material by filtration or centrifugation.

Please note: A preliminary examination of spot plates for lysis zones may be completed after 6 hours of
incubation to determine if there will be bacterial interferences. Alternatively, the filtration or centrifugation
step (Section 12.2.11.1 or Section 12.2.11.2) may be performed before the initial spot.

                 12.2.11.1    To remove interfering bacteria by filtration, push a 0.5 to 1.0 mL volume of
                              the enrichment sample through a 25-mm-diameter, 0.45-(jm pore-size, sterile,
                              low-protein binding  filter (Section 6.3.1) using a 1 to 3 mL syringe, and
                              collect the filtrate in a sterile microcentrifuge tube.
                 12.2.11.2    To remove interfering bacteria by centrifugation, pipet a 0.5 to  1-mL volume
                              of the enrichment sample into a  1.8-mL capacity, sterile microcentrifuge
                              tube. Centrifuge at 5,000 to 10,000 X G for 10 minutes. Recover most of
                              the supernatant by aspirating with a micropipet. Place the recovered
                              supernatant in a  1.5-mL capacity, sterile microcentrifuge tube.
                 12.2.11.3    Spot 10-(iL volumes of filtered or centrifuged enrichment onto pre-poured
                              lawns of host bacteria as described in Section 12.2.
12.3   Spot plate coliphage confirmation procedure—Although not required, laboratories may use the spot
        plate procedure for confirmation of lysis zones if one or more such zones on a spot plate are
        questionable.
        12.3.1    Pick lysis zone with a sterile Pasteur (or other) pipette and transfer it to a tube with 0.5 mL
                 TSB (Section 7.3.1.1).
        12.3.2   Allow the inoculated broth to stand 5 minutes at room temperature.
        12.3.3   Cap the tube and vortex for 5 seconds on a medium-high setting (if available)  or until well-
                 mixed.
        12.3.4   Prepare spot plates according to Section 7.3.4. Once prepared, spot plates may be used on
                 the same day or held at 4°C ± 1°C for up to 4 days prior to use.
        12.3.5   Spot 10 microliters of inoculated broth to a spot plate with appropriate E. coll host, using
                 the same E. coll host on which the  phage was initially isolated. Record spot time.
        12.3.6   Spot the method blank and positive control samples as specified in Section 9.4 and 9.5.
        12.3.7   Allow inocula to absorb into medium. This will take approximately 30 to 60 minutes. The
                 inocula must not be allowed to run across the plate.
        12.3.8   After inocula absorption, cover, invert, and incubate the plate at 36°C ± 1.0°C for 16 to 24
                 hours.
        12.3.9   Lysis zone formation (typically a circular zone of clearing) indicates confirmation for
                 coliphages. If the spot contains an intact lawn of bacteria indistinguishable from the
                 background lawn of bacteria, this indicates a negative result. However, other outcomes of
                 the spot assay are possible. A positive confirmation also may appear as one or more small

April 2001                                        20

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                                                       Method 1601: Two-Step Enrichment Procedure
                plaques or areas of clearing of the host bacteria lawn within the spot, despite the presence
                of some portion of the host bacteria lawn within the spot.  A positive result may also appear
                as a zone of lysis containing small, discrete colonies of bacteria within the spot.  These
                bacterial colonies are from phage-resistant mutants. Please note: The use of a light box
                (Section 6.4.8) to evaluate sample results is recommended.
13.0  Data Analysis and Calculations

13.1   Calculation of QC sample spiking suspension concentrations from the double agar layer (DAL)
       enumeration procedure (Section 11)
       13.1.1   The number of plaque forming units (PFU) per mL in the coliphage spiking suspension will
                be calculated using DAL plates that yield plaque counts within the desired range of zero to
                300 PFU per plate for male-specific (F+) coliphage and zero to 100 PFU per plate for
                somatic coliphage. There may be occasions when the total number of plaques on a plate will
                be above the ideal range. If the count exceeds the upper range or if the plaques are not
                discrete, results should be recorded as "too numerous to count" (TNTC).
       13.1.2   For each coliphage type, sum the number of PFU from all dilutions with plaques (on either
                of the duplicate plates), excluding dilutions with all TNTC or all zeros. (See equation in
                Section 13.1.5)
       13.1.3   Sum the undiluted sample volumes used to inoculate all replicate plates at all dilutions
                having useable counts (as defined above). (See equation in Section 13.1.5)
       13.1.4   Divide the sum of the PFU by the sum of the undiluted sample volume to obtain PFU/ mL
                in the spiking suspension. (See equation in Section 13.1.5)
       13.1.5   The equation for Sections 13.1.1 through 13.1.4 is as follows:

                Undiluted spiking suspension PFU / mL = (PFU! + PFU2... PFUn)/(Vi + 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 (0)

                        V=    volume of undiluted sample in all plates with countable plaques

                        n =     number of useable counts

Example DAL data
Dilution
Undiluted
1 : 10
1 : 100
1 : 1,000
PFU / plate (for each duplicate
plate)
TNTC, TNTC
35, 37
0,3
0, 0
Volume of undiluted spiking suspension (mL)
0.5ml
0.05
0.005
0.0005
                                                21
April 2001

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Method 1601: Two-Step Enrichment Procedure
                Example: (35 + 37 + 0 + 3)/(0.05 + 0.05 + 0.005 + 0.005) = 75/0.11 = 682 PFU / mL

                In this example, the undiluted spiking suspension contains approximately 682 PFU per mL,
                the 1 : 10 dilution contains approximately 68.2 PFU per mL, the 1 :100 dilution contains
                approximately 6.82 PFU per mL, and the 1  : 1000 dilution contains approximately 0.682
                PFU per mL.

13.2   Calculation for preparing IDC, ODC, MS, and positive control spikes
       13.2.1 Use a dilution of the QC sample spiking suspension that will result in a bulk spike volume
              between 0.1 and 3.0 mL for the spike concentration specified in Section 9.
       13.2.2 Use the following equation to determine the spiking volume:

                      „    (TX.B)
                             (Q

                     where,

                     S =     Spike volume (mL)

                     T =     Target number of coliphage per sample (PFU)

                     B =    Number of samples that will be spiked (only necessary when multiple QC
                             samples are spiked in bulk)

                     C =     Concentration (PFU/mL) in the dilution to be  used for spiking

       13.2.3 For example, for 1-L, somatic IDC samples (Section 9.3.2):
              T) A spike dose of 1.4 PFU is needed per 1-L sample
              B) A total often, 1-L samples will be spiked at the same time
              C) The 1 :  100 dilution contains 6.82 PFU / mL

              The equation would be solved as follows:

                             (1.4PFU)(10)
                      O      \          / \,    /     r\ r\
                      S = (6.82 PFU /mL) = Z°

              As a result, approximately 2.1 mL of the 1 : 100 dilution would be spiked into the 10-L bulk
              sample. The 10-L mL bulk sample would be mixed and ten, 1-L aliquots dispensed. Each
              1-L sample should contain approximately 1.4 PFU.
14.0  Method Performance

14.1   The QC acceptance criteria listed in Tables 1, 2 and 3, are based on data generated through the
       interlaboratory validation of Method 1601 involving 10 laboratories and 10 raw ground water
       matrices. Detailed Method QC procedures applicable to these criteria are discussed in Section 9.


April 2001                                     22

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                                                     Method 1601: Two-Step Enrichment Procedure
Table 1.
Initial demonstration of laboratory capability (IDC)
Coliphage
type
F+
F+
Somatic
Somatic
Sample
size
100-mL
1-L
100-mL
1-L
Target spike
concentration
(PFU per sample)
1.3
1.2
1.5
1.4
"Bulk" volume
to be spiked
1000 mL
10L
1000 mL
10L
Bulk spike
concentration
(PFU per bulk
volume)
13
12
15
14
Minimum
number of
positive
samples out of
10
5
3
5
5
Table 2.       MS and ODC sample spiking requirements for ongoing evaluation of method
              performance
Coliphage
type
F+
F+
Somatic
Somatic
Sample
size
100-mL
1-L
100-mL
1-L
Target spike
concentration
(PFU per sample)
1.3
1.2
1.5
1.4
Number of samples
that must be spiked
(>1 must be positive)
3
3
3
3
"Bulk"
volume
to be spiked
300-mL
3-L
300-mL
3-L
Bulk spike
concentration
(PFU per bulk
volume)
3.9
3.6
4.5
4.2
Table 3.
Expanded MS test requirements for evaluation of method modification performance
Coliphage
type
F+
F+
Somatic
Somatic
Sample
size
100-mL
1-L
100-mL
1-L
Target spike
concentration (PFU
per sample)
1.3
1.2
1.5
1.4
"Bulk"
volume to be
spiked
1000-mL
10-L
1000-mL
10-L
Target bulk Spike
(PFU per bulk
volume)
13
12
15
14
Minimum number
of positive
samples out of 10
4
2
5
5
14.2   Method 1601 is a performance-based method and the laboratory is permitted to modify certain method
       procedures to improve performance or lower the costs of measurements, provided that all quality
       control (QC) tests cited in Section 9.2.6 are performed and all QC acceptance criteria are met. The
       laboratory is not permitted to modify the double agar layer QC spiking suspension enumeration
       procedure (Section 11.3).
       14.2.1 Method modifications at a single laboratory. Each time a modification is made to this
              method for use in a single laboratory, the laboratory is required to validate the modification
              according to Tier 1 of EPA's performance-based measurement system (PBMS) (Table 4 and
              Reference 17.6).
                                              23
                                                                        April 2001

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Method 1601: Two-Step Enrichment Procedure
        14.2.2 Method modifications for nationwide approval. If the laboratory or a manufacturer seeks
               EPA approval of a method modification for nationwide use, the laboratory or manufacturer
               must validate the modification according to Tier 2 of EPA's PBMS (Table 4 and Reference
               17.6). Please note: After a method modification is validated for nationwide use, each
               individual laboratory electing to use the modification still must demonstrate acceptable
               initial and on-going performance with the modified method through the analysis of method
               blanks, media sterility checks, positive controls, ODC samples, and MS samples.
Table 4.
Tier 1 and Tier 2 Validation/Equivalency Demonstration Requirements
Test
IDC
(Section 9.3)
Method blank
(Section 9.4)
Expanded MS
(Section 9.8)
Description
10 replicates of
spiked reagent water
Unspiked reagent
water
10 replicates of
spiked matrix water
Tier 1 modification'1'
Required. Must be accompanied by a
method blank.
Required
Recommended, but not required. Must be
accompanied by an unspiked field sample
collected at the same time as the MS
sample
Tier 2 modification'2'
Required per laboratory
Required per laboratory
Required per laboratory.
Each laboratory must
analyze a different water.
       Please note:
(1)     If a modification will be used only in one laboratory, these tests must be performed and the results must meet
       all of the QC acceptance criteria in the method (these tests also are required the first time a laboratory uses
       the validated version of the method). After the initial demonstration that the modification is equivalent to the
       procedure specified in this method, the laboratory must continue to demonstrate acceptable ongoing
       performance with the modified method through the  analysis of media sterility checks, method blanks, positive
       controls, ODC samples, and MS samples.
(2)     If nationwide approval of a modification is sought for one type of water matrix (such as ground water), a
       minimum of 3 laboratories must perform the tests and the results from each lab individually must meet all QC
       acceptance criteria in the method. If more than 3 laboratories are used in a study, a minimum of 75% of the
       laboratories must meet all QC acceptance criteria.
15.0   Pollution  Prevention

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

16.1   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).

16.2   Samples, reference materials, and equipment known or suspected to have bacteriophage attached or
       contained must be sterilized prior to disposal.
April 2001
                                  24

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                                                      Method 1601: Two-Step Enrichment Procedure
16.3   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.
17.0  References

17.1   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)
17.2   American Public Health Association, American Water Works Association, and Water Environment
       Federation. 1995. Standard Methods for Water and Wastewater.  20th Edition. Sections 9020, 9030,
       9040, 9050, and 9221.
17.3   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.
17.4   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.
17.5   Annual Book of ASTMStandards. Vol. 11.01. American Society  for Testing and Materials.
       Philadelphia, PA 19103.
17.6   USEPA. EPA Guide to Method Flexibility and Approval of EPA  Water Methods,
       EPA 821-D-96-004. Office of Water, Engineering and Analysis Division, Washington,  DC 20460
       (1996).
                                              25                                      April 2001

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Method 1601: Two-Step Enrichment Procedure
18.0   Flowcharts

We greatly appreciate Fred Williams (USEPA, Cincinnati, OH) for providing the original flow charts on
which all of the following flowcharts are based.

Flow chart 1.   Preparation of log-phase host bacteria stock cultures (Section 7.5.4)
                              Add 25-30 ml of TSB with antibiotics to each shaker flask
                                       W
                                      CN1 CN2 CN3          F1  F2  F3
                                   TSB w/naidixic for somatics   TSB w/amp & strep for maEe^peeifics
                                  Add 0.1 -1.0 ml of host bacteria to each flask
                                 c^t3   RRl
                                        CN1 CN2 CN3
                                   TSB w/naBdixic for somatics
             F1  F2  F3

       TSB wfamp & strep for maBe^pecifics
                                Sncubate in shaker at 36°C and 100-150 rpm for 4 hr
                                                                   36°C
                      Additional
                      30 minutes
                      incubation
                                   Aseptically transfer 1.0 ml to cuvette and read
                                             absorbance at 520 nm
                                    If OD 0.1-0
                                                                IFOD 0.1-0,5
                                            Chill on wet ice or at 4°C
                                                          Ji\Ji\i
                                         uyo>%^/L\LL
                              Additional
                              30 minutes
                              incubation
April 2001
26

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                                                        Method 1601: Two-Step Enrichment Procedure
Flow chart 2.   Dilution of coliphage QC spiking suspensions (Section 11.2)
       1
Add 10 ml of undiluted sewage filtrate
                       undiluted
               Add 9.0 m!_ TSB without antibiotics to each dilution tube
                         _    _    _     _.
                       undiluted   0.1   0.01   0.001
               Vortex undiluted tube 5 seconds. Transfer 1.0 ml from undiluted tube to 0.1 tube.
                          1,0 mL




                        n    m    r    rr
                       undilited   0.1   0.01    0.001
               Vortex 0.1 tube 5 seconds. Transfer 1.0 ml from 0.1 tube to 0.01 tube.
                               1,0ml.
                      undiluted   0.1   0.01    0.001
               Vortex 0.01 tube 5 seconds. Transfer 1.0 m!_ from 0.01 tube to 0.001 tube.
                                     IJtmL
                      undiluted   0.1   0.01    0.001
                                                 27
                                                                            April 2001

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Method 1601: Two-Step Enrichment Procedure
Flow chart 3.  Coliphage spiking suspension enumeration by double agar layer (DAL) procedure
             (Section 11.3)
1
Place eighteen 0.7% TSA top agar tubes with antibiotics in 46.5'C water bath
nine for somatic ^^ ""\^ nine for mate-specific
0.7%TSAvrfnaldiwc acid 0.7% TSAw/amp & strep




2
T _
JWwi ifHfL^-ftkjiu
Btnk u y u j u y ii\y y u yu [
UndiUed 0.1 0.01 0.001 0.001 0.01
IT
-|T rr II
fWlWH 46.5°C
J U U ^ Bbnk
0.1 Undilited
Label eighteen corresponding 1 .5% TSA bottom agar plates containing antibiotics
nine for somatic nine for male-specific
1 .5%TSA w/na jd xic acid 1 £% TSA w/amp & slrep



3


gj • • • • 3 Bbnk g55E
3 	 :3 OJ01
S=i:^ 	 g^ 0,1
J! 	 '.,]§ UndiUed
=3 Btont
For each TSA top agar tube in water bath complete steps A through D below:
A. Add 100 uL (0.1 mL) fog-phase B. Immediately, add 500 uL (05 mL) from

host bacteria to tube with £
appropriate coEiphage stock diEution tube
appropriate antibiotic (Flow chart 2)








A
CKM3 Famji
A A
F-1 F-1
P P

U U
0.7%TSA-mth naidixic acid 0.7%TSA ™tli amp/strap
GB GentEy mix tube in paEm Dn

^^T
Invert and incubate at 36°C for 16-24 hr
A
Vi
[I For method blanks add
500 uL (05 mL) TSB instead
y of coliphage stock dilution
0.7%TSAwilh antibiolic
Pour tube into 1 £% TSA bottom agar plate with
appropriate antibiotic and label
"m~

CN-1 3 and naidixic acid Fampand amp/strep
for somatic for male-epecific





5


OJX)1 grrr— ^g g >.'..•- :jg gZE
^^ ^J 1^
o.i g^1' '" i:]g gzw": '"3 §5?:
UndUed g5=g gLEE^ g=
Bbnk S^ESS
After incubation, count plaques, and
CN.13
©
^Eg gEE^Sg oall
^e^^ojoi
^^§ g1'"''"'''"1'"1'"'^ 0.1
^^§ ggjig^ UndiMed
g==g B|ank
record results
Famp
@
April 2001
28

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                                                               Method 1601: Two-Step Enrichment Procedure
Flow chart 4.  Two-step enrichment procedure for 100-mL samples (Sections 12.1.1 and 12.2)
                  1OO-mL samples
   •    for somatics (CN}//   \for male-specifics (F )
  100 mL              //          \              100 mL
  reagent	CN+  m  ,	"	»        ,	
     CN blank
                                              water
                                          F blank
               CN1 CN2 CN3
          Add 1.25 ml stock magnesium chloride
  CN+

CN blank (
              CN1 CN2 CN3
                                         F+

                                          F blank
            Add 0.5 ml           Add 0.5 ml
3          log-phase CN-13       log-phase Fam
                 1               _1_
                  CN.13
                   LULU
                CNl CN2 CN3
                  Add 5 ml 10XTSB
                                             F*

                                             I F blank
        CN+

      CN blank
                 CN-13
                  LULU
               CN1 CN2 CN3
                        LiJlULL
                        F1  F2  F3
                                    F+

                                     F blank
  Add 1 ml         Add 1 ml
stock nalidixic     stock amp/strep
  CN+

CN blank
                 CN-13
                  LULU
               CN1 CN2 CN3
               Add from sewage filtrate
        20 PFU somatics    20 PFU mate-specifics
    CN blank
    (OPFU)
           rnr
        I  CN-13
        LULULU
        CNl CN2 CN3      F1  F2  F3
                              LLf
                              F blank
                              (0 PFU)
              Invert 5 times to mix
                         m
                                                        8
                                                            Incubate 36°C for 16-24 hrs
                                                            c

                                                          CN blan
                                                                         -ULULU
                                                                         CN1 CN2 CN3
                                                                                     JLULL
                                                                                   F1  F2  F3
                                                                          •p  M in
                                                                          \-,    f
                                                                                rar
                                                                              LULL
                                                                              F1 F2


                                                          After incubation, invert 25 times to mix
                                                       10
                                                              Remove 10 ML of each bottle
                                                                 to grid on petri plate
                                                                                   Note: Interfering
                                                                                   bacteria may be
                                                                                   removed from
                                                                                   enriched sample by
                                                                                   filtration or
                                                                                   centrifugation.
                                                                                   Filtration: Push
                                                                                   1.0 mL of enriched
                                                                                   sample through a
                                                                                   0.45-4im fifter using
                                                                                   a1-to3-ml_
                                                                                   syringe.
                                                                                   Centrifugation:
                                                                                   Centrifuge 1.0 mL of
                                                                                   enriched sample at
                                                                                   5,000 to 10,000 XG
                                                                                   for 10 minutes.
                                                                    CN-13
                                                              11
                                                                  Allow to absorb for 30 min
                                                                        CN-13
                                                                       Snvert and incubate at 36
-------
Method 1601: Two-Step Enrichment Procedure
Flow chart 5.   Two-step enrichment procedure for 1 -L samples (Sections 12.1.2 and 12.2)
                       14. samples
1L
reagent	 CN+
                                $or male-specifics (F+)
                 CN1 CN2 CN3
                                 F1  F2  F3
             Add 12.5 mL stock magnesium chloride
                CN1 CN2 CN3
             Add 5.0 ml
           log-phase CN-13
                                 Add 5.0 ml
                                log-phase Fam|
                 CN1 CN2 CMS
                                    LULL
                                 F1 F2  F3
    _      Add 10 ml
    *S   stock nalidixic
                   Add 10 ml
                 stock amp/strep
                       1
       CN+

     CN blank \
                 CN-13
                 ULUL
                CN1 CN2 CN3
      Z*         Add from sewage filtrate
           20 PFU somatics    20 PFU male-specifics
    CN blank
    (0 PFU)
                               'amp
                               JLiJU
 CN-13
 ULUL
CN1 CN2 CN3


  Invert 5 times to mix
                                             (OPRJ)
                                                 8
                                                 CN*
                                                CN blank
                                                                      Incubate 36°C for 16-24 hrs
                                                                                     o
                                                                                      C
                                                                           CN-13
                                                                         QjLULlJ
                                                                         CN1 CN2 CN3
                                                                                                       Ft

                                                                                                        F bbnk
                                                                                          F,mp

                                                                                       F1  F2  F3


                                                                    After incubation, invert 25 times to mix
                                                            10
                                                           Remove 10pL of each bottle
                                                              to grid on petri plate
                                                                                              Note: Interfering
                                                                                              bacteria may be
                                                                                              removed from
                                                                                              enriched sample by
                                                                                              filtration or
                                                                                              centrifugation.
                                                                                              Filtration: Push
                                                                                              1.0 mL of enriched
                                                                                              sample through a
                                                                                              0.45"Um fifter using
                                                                                              a1-to3-ml_
                                                                                              syringe.
                                                                                              Centrifugation:
                                                                                              Centrifuge 1.0 mL of
                                                                                              enriched sample at
                                                                                              5,000 to 10,000 XG
                                                                                              for 10 minutes.
                                                                     CN-13
11
                                                                        Allow to absorb for 30 min
                                                                       CN-13
                                                                I  2.    Invert and incubate at 361: for 16-24 hr
                                                                               CN-13
                                                                      After incubation, examine for the presence
                                                                          of lysis zones and record results
                                                                              CN-13
April 2001
                                                    30

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                                                     Method 1601: Two-Step Enrichment Procedure
19.0  Glossary
       These definitions and purposes are specific to this method but have been conformed to common usage
       as much as possible.
19.1   Symbols
              °C
                            degrees Celsius
                            micro
                            number
                            percent
19.2   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
              HC1           hydrochloric acid
              IDC           initial demonstration of capability
              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
              ODC          ongoing demonstration of capability
              OSHA         Occupational Safety and Health Administration
              psi            pounds per square inch
              QA           quality assurance
              QC           quality control
              rpm           revolutions per minute
              TNTC         too numerous to count
              TSA          tryptic soy agar
              TSB          tryptic soy broth
              USEPA        United  States Environmental Protection Agency
              X             "times"
                                              31
                                                                                     April 2001

-------
Method 1601: Two-Step Enrichment Procedure
19.3   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.

       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 E. coll.

       Enrichment—In this method, enrichment is meant as the increase in number of bacteriophage through
       the addition to the growth medium of host bacteria allowing coliphage replication.

       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. coll CN-13, and
       E. coli Famp (E. coli HS(pFamp)R).

       Initial  demonstration of capability (IDC)—The IDC test is performed to establish the ability to
       demonstrate control over the analytical system and to demonstrate acceptable performance.

       Lysis zone—In this method, typically a circular zone of clearing indicating a sample is positive for
       coliphages.

       Male-specific coliphage—Viruses (bacteriophages) that infect coliform bacteria only via the
       F-pilus.

       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 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.

       Plaque—Circular zones of clearing (typically 1 to 10 mm in diameter) in lawn of host bacteria in
       DAL plates after incubation.

       Presence-absence—A qualitative method for detection of a microorganism where the result indicates
       whether or not the microorganism is present in the sample. The presence-absence method will not give
       the numbers of virus present.

       Reagent water—Water conforming to Specification D 1193, Annual Book of ASTM Standards
       (Reference 17.5) or specifications in Standard Methods 9020 B.4.d (Reference 17.2)

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


April 2001                                        32

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