United States Office of Water EPA 821-R-00-009
Environmental Protection Washington, D.C. 20460 April 2000
Agency
Method 1601: Male-specific (F+) and
Somatic Coliphage in Water by Two-step
Enrichment Procedure
April 2000 - Draft
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Acknowledgments
This method was prepared under the direction of William A. Telliard of the Engineering and Analysis Division
within the U.S. Environmental Protection Agency's (EPA) Office of Water. The EPA technical lead was Paul
Berger, of the Standards and Risk Management Division within the Office of Water. This document was
prepared under EPA Contract No. 68-C-98-139 by DynCorp Information & Enterprise Technology, Inc.
The contributions of the following persons and organizations to the development of this method are gratefully
acknowledged:
Sobsey, Mark, Ming Jing Wu, and Greg Lovelace, University of North Carolina, Department of
Environmental Sciences and Engineering, CB#7400, McGavran-Greenberg Building, Chapel Hill, NC
27599
Hsu, Fu-Chih, and Jim Larkin, Environmental Health Laboratories, 110 South Hill Street, South Bend, IN
46617
Chambers, Yildiz, City of San Diego Marine Microbiology Laboratory, 5530 Kiowa Drive, La Mesa, CA
91942
Cliver, Dean, Tadesse Mariam, and Mulugeta Tamene, University of California Davis, Department of Health
and Reproduction, School of Veterinary Medicine, Davis, CA 95616-8743
Danielson, Richard, BioVir Laboratory, 685 Stone Road Unit # 6, Benicia, CA 94510
Fujioka, Roger and Geeta Rijal, University of Hawaii, Water Resources Center, Holmes Hall 283, 2540 Dole
Street, Honolulu, ffl 96822
Karim, Mohammad and Dale Young, American Water Works System Research Laboratory, 1115 South
Illinois Street, Belleville, IL 62220-3731
Margolin, Aaron and Nicola Ballester, University of New Hampshire, Department of Microbiology, Biological
Sciences Building, Rudman Hall Room 285, Durham, NH 03824
Pillai, Suresh and Elisa Camacho, Texas A & M University, Department of Poultry Science, Kleberg Center
Room 418D, College Station, TX 77843
Williams, Fred and Ron Stetler U.S. Environmental Protection Agency, 26 West Martin Luther King Drive,
Cincinnati, OH, 45268
Yates, Marylynn, Omid Bakhtar, and Andre Salazar, University of California Riverside, Department of
Environmental Sciences, 2217 Geology, Riverside, CA 92521-0424
Disclaimer
This method is in final draft form. The method has been validated through a 10-laboratory round-robin. The
Agency welcomes comments on its technical merit. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
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Introduction
Coliphage presence in groundwater is an indication of fecal contamination. Method 1601 is a performance-
based method for detecting the presence of male-specific (F") and somatic coliphage in groundwater and other
waters. 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. The
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.
Note: Although Method 1601 has been validated through a 10-laboratory round-robin validation study,
the QC acceptance criteria and frequency of QC samples to be required in the method have not
been finalized. Final QC criteria and frequency will be specified in the next draft of the method.
This method is for use in the Environmental Protection Agency's (EPA's) data gathering and monitoring
programs under the Safe Drinking Water Act and the Clean Water Act.
Questions concerning this method or its application should be addressed to:
William A. Telliard
Engineering and Analysis Division (4303)
U.S. Environmental Protection Agency
401 M Street, SW
Washington, D.C. 20460
(202) 260-7120
Requests for additional copies of this publication should be directed to:
Water Resource Center
Mail Code RC-4100
401 M Street, SW
Washington, D.C. 20460
(202) 260-7786 or (202) 260-2814
Note: Prior to finalization of this method, each step of this method must be performed as written. After the
method is finalized, this method will be performance-based. At that point, the laboratory is
permitted to modify or omit any steps or procedure, provided that all performance requirements set
forth in the validated method are met. The laboratory may not omit any quality control analyses.
The terms "shall, " "must," and "may not" indicate steps and procedures required for producing
reliable results. The terms "should" and "may" indicate optional steps that may be modified or
omitted if the laboratory can demonstrate that the modified method produces results equivalent or
superior to results produced by this method.
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Table of Contents
1.0 Scope and Application 1
2.0 Summary of Method 1
3.0 Definitions 2
4.0 Interferences 2
5.0 Safety 2
6.0 Equipment and Supplies 3
7.0 Reagents and Standards 5
8.0 Sample Collection, Preservation, and Storage 8
9.0 Quality Control 9
10.0 Calibration and Standardization 12
11.0 Enumeration of Coliphage Stock Suspension 13
12.0 Preparation of Log-Phase Host Bacterial Cultures 17
13.0 Two-step Enrichment Procedure 17
14.0 Data Analysis and Calculations 20
15.0 Method Performance 22
16.0 Pollution Prevention 22
17.0 Waste Management 22
18.0 References 22
19.0 Tables, Diagrams, Flowcharts, and Validation Data 23
20.0 Glossary 23
IV
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Method 1601: Male-specific (F+) and Somatic Coliphage in Water by
Two-Step Enrichment Procedure
April 2000 Draft
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 groundwater 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 groundwater is affected by fecal contamination.
Note: Although this method may be used for water matrices other than groundwater, it has only been
validated for use in groundwater.
1.2 This method is designed to meet the monitoring requirements of the U.S. Environmental Protection
Agency (EPA). It is based on procedures developed for the determination of coliphage in water in the
Supplement to the 20* Edition of Standard Methods for the Examination of Water and Wastewater
(Reference 18.1).
1.3 This method is not intended for use in biosolids samples or as a test for microorganisms other than
coliphage. This method may be used hi groundwater and other water matrices where coliphage is
suspected to be present.
1.4 Each laboratory and analyst that uses this method must first demonstrate the ability to generate
acceptable results using the procedures in Section 9.0.
1.5 Any modification of the method beyond those expressly permitted is subject to the application and
approval of alternate test procedures under 40 CFR parts 136.4 and 136.5, and/or 141.27.
2.0 Summary of Method
2.1 Method 1601 describes a qualitative two-step enrichment procedure. A 100-mL or 1-L groundwater
sample is supplemented with MgCl2 (magnesium chloride), log-phase host bacteria (E. coli F^ for F+
coliphage and E. coli CN-13 for somatic coliphage), and tryptic soy broth (TSB) as an enrichment
step for coliphage. After an overnight incubation, samples are "spotted" onto a lawn of host bacteria
specific for that type of coliphage, incubated, and examined for circular lysis zones, which indicates
the presence of coliphage.
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3.0 Definitions
3.1 Coliphages are viruses (bacteriophages) that infect coliform bacteria and are indicators of fecal
contamination. This method is capable of detecting two types of coliphages: male-specific (F1") and
somatic.
3.2 F-factor is the fertility factor in certain strains of E. coli. It is a plasmid that, when present, codes for
pilus formation. The pilus allows for transfer of nucleic acid from one bacterium to another.
3.3 Male-specific coliphages (F*) are RNA or DNA viruses that infect via the F-pilus of male strains of
E. coli.
3.4 Somatic coliphages are DNA bacteriophages that infect host cells via the outer cell membrane.
3.5 Definitions for other terms used in this method are given in the glossary in Section 20.3.
4.0 Interferences
4.1 During the 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 in Section 13.2.11.
5.0 Safety
Caution: The biohazards and the risk of infection by pathogens associated with handling raw sewage
are high in this method. Use good laboratory practices when working with potentially
harmful samples.
5.1 Method 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.
5.2 The laboratory is responsible for maintaining a current awareness file of Occupational Safety and
Health Administration (OSHA) regulations regarding the safe handling of the chemicals specified in
this method. A reference file of material safety data sheets should be made available to all personnel
involved in these analyses. Additional information on laboratory safety can be found in Section 17.0.
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5.3 Samples may contain high concentrations of biohazardous agents and must be handled with gloves.
Any positive reference materials also must be handled with gloves in an appropriate laboratory hood.
The analyst must never place gloves near the face after exposure to media known or suspected to
contain pathogenic microorganisms. Laboratory personnel must change gloves after handling raw
sewage or any other items which may carry pathogenic microorganisms.
5.4 Mouth pipetting is prohibited.
6.0 Equipment and Supplies
Note: Brand names, suppliers, and part numbers are for illustrative purposes only. No
endorsement is implied. Equivalent performance may be achieved using apparatus and
materials other than those specified here, but demonstration of equivalent performance that
meets the requirements of this method is the responsibility of the laboratory.
6.1 Equipment for collection and transport of samples
6.1.1 Bottles for collection of water—Sterile, wide-mouth, polypropylene, bottles or carboys with
screw caps
6.1.2 Ice chest—Igloo, Coleman, styrofoam box or equivalent
6.1.3 Ice
6.1.3.1 Wet ice—purchased locally, or
6.1.3.2 Ice packs—Blue Ice, UTek cat. no. 429, or equivalent, frozen for use
6.1.4 Bubble wrap
6.2 Equipment and supplies for growth of microorganisms
6.2.1 Sterile dilution tubes with screw caps—Reusable or disposable, 16 x 150 mm, or 16 x 100
mm
6.2.2 Test tube rack—Size to accommodate tubes specified in Section 6.2.1
6.2.3 Glass or plastic, plugged, sterile serological pipettes—To deliver (TD), of appropriate
volume(s) (Falcon, Kimble, or equivalent)
6.2.4 Pipet bulbs, automatic pipetter—Pipet-Aid or equivalent
6.2.5 Inoculation loops—Nichrome or platinum wire, disposable, sterile plastic loops, or wooden
applicator, at least 3 mm in diameter or 10 uL volume (VWR, Fisher, DIFCO, or equivalent)
6.2.6 Micropipettors, adjustable—10- to 200-uL, and 100- to 1000-uL, with appropriate aerosol
resistant tips, Gilson, Eppendorf, or equivalent
6.2.7 Burner—Alcohol, Bunsen, Fisher, or equivalent
6.2.8 Sterile disposable petri dishes—100-mm-diameter dishes (Falcon #1029) or 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
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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, Coming, Nalgene, Kimble or equivalent
6.2.13 Stir bar—Fisher cat. no. 14-511-93, or equivalent
6.2.14 Stir plate—Fisher cat. no. 14-493-120S, or equivalent
6.2.15 Water bath capable of maintaining 36°C ± 1.0°C and 45°C to 48°C—Precision, VWR
Scientific, or equivalent
6.2.16 Sterilization filtration equipment—Millex type for syringe or larger Millipore type, sterile,
0.22-um pore size
6.2.17 Sterile, cotton-tipped applicators
6.2.18 Latex gloves for handling samples, supplies, and equipment—Microflex, San Francisco, CA,
stock no. UL-315-L, or equivalent
6.2.19 pH meter—Beckman, Coming, 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 V*" diameter cuvettes (Model #
4015) or 13 mm x 100 mm cuvettes
6.2.22 Cuvettes—1-cm light path, Beckman, Bausch and Lomb, or equivalent
6.2.23 Shaker flasks—Fluted Erlenmeyer, 125-mL with slip cap or sterile plug, Fisher (09-552-33
10-140-6, 10-041-5A) or equivalent
6.2.24 Shaker incubator—Capable of 36°C ± 1.0°C and 100 to 150 rpm, New Brunswick,
PsychoTherm, Innova, or equivalent or an ordinary shaker in an incubator
6.3 Supplies for collection and filtration of raw sewage for spiking (if not using stock coliphage, Sections
7.1.10 and 7.1.11)
6.3.1 Disposable filter disks—25-mm-diameter, 0.45-um pore size, sterile, low protein binding
(Gelman Acrodisc HT Tuffryn, No. 4184, cellulose acetate Corning No. 21053-25, or
equivalent)
6.3.2 Syringe—Sterile, disposable, 5-, 10-, or 20-mL
6.3.3 Polypropylene dilution tubes—Sterile, 10- to 20-mL, Falcon or equivalent
6.3.4 Sterile glass or polypropylene 250-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
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7.0 Reagents and Standards
7.1 General reagents
7.1.1 Reagent water—Conforming to Specification D 1193, Annual Book of ASTM Standards
(Reference 18.7).
7.1.2 E. coli CN-13 (somatic coliphage host)—Nalidixic acid-resistant mutant of E. coli C;
originated by Pierre Payment, Institute Armand Frappier, University of Quebec, Montreal,
Canada, frozen stock. ATCC#700609.
7.1.3 E. coli F^ — E. coli HS(pFamp)R (male-specific coliphage host)—originated by Victor
Cabelli, formerly of the Department of Microbiology, University of Rhode Island, Kingston,
RI, USA, frozen stock.
7.1.4 Preparation of frozen stock cultures—The laboratory shall obtain reference host bacterial
cultures (Sections 7.1.2 and 7.1.3) and use these to establish pure frozen stock cultures that
are maintained for the laboratory. Stocks are used as inoculum for log-phase host bacterial
cultures.
7.1.4.1 Establish pure frozen stock cultures by streaking host bacteria onto tryptic soy agar
with appropriate antibiotic (Section 7.2.1) to attain isolated colonies.
7.1.4.2 Incubate overnight, pick an individual colony, and grow to log phase (Section 12)
in tryptic soy broth with appropriate antibiotic (Sections 7.1.7.2 and 7.1.7.3).
7.1.4.3 Harvest broth by mixing sterile glycerol and broth with log-phase host bacteria in a
ratio of 1:4 in a freezer vial (Example: 200 uL sterile glycerol plus 800 uL log-
phase E. coli).
7.1.4.4 Label with E. coli strain and date of harvest.
7.1.4.5 Freeze stock cultures at -70°C, if possible. Cultures can be frozen -20°C if the
laboratory does not have the capability to freeze samples at -70°C).
7.1.5 100X nalidixic acid (Sigma N4382)—Dissolve 1 g of nalidixic acid sodium salt in 100 mL
reagent water. Filter through a sterile, 0.22-um-pore-size membrane filter assembly. Dispense
5 mL per freezer vial and store frozen at -20°C for up to one year. Thaw at room temperature
or rapidly in a 36°C ± 1.0°C water bath. Mix well prior to use.
7.1.6 100X ampicillin/streptomycin—Dissolve 0.15 g of ampicillin sodium salt (Sigma A9518) and
0.15 g streptomycin sulfate (Sigma S6501) in 100 mL of reagent water. Filter through a
sterile 0.22-um-pore-size membrane filter assembly. Dispense 5 mL per freezer vial and store
frozen at -20°C for up to one year. Thaw prior to use at room temperature or rapidly in a
36°C ± 1.0°C water bath.
Note: 100X nalidixic acid and 100X ampicillin/streptomycin are always added to medium after the
medium has been autoclaved.
7.1.7 Tryptic (or trypticase) soy broth (DIFCO 0370-15-5, or equivalent) (TSB)
7.1.7.1 TSB—Follow procedure as specified on bottle of media. If dehydrated medium is
not available, prepare the media by adding 17.0 g of tryptone, 3.0 g of soytone,
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2.5 g of dextrose, 5.0 g of sodium chloride, and 2.5 g of dipotassium phosphate to
1L of reagent water and heat to dissolve. Adjust pH to 7.3 with 1.0 N hydrochloric
acid or 1.0 N sodium hydroxide, if necessary. Autoclave at 121°C and 15 psi for
15 minutes. Check pH again after autoclaving by aseptically removing an aliquot of
medium. Adjust pH as necessary.
Note: To determine pH after autoclaving, aseptically remove an aliquot of medium and measure pH.
Discard aliquot after checking pH, to ensure that the medium is not contaminated.
7.1.7.2 TSB with nalidixic acid (for growth of E. coli CN-13)—Aseptically add 10 mL of
100X nalidixic acid (Section 7.1.5) to 1 L of autoclaved, cooled TSB (Section
7.1.7.1) and mix.
7.1.7.3 TSB with streptomycin/ampicillin (for growth off. coli f,^)—Aseptically add 10
mL of 100X streptomycin/ampicillin sulfate (Section 7.1.6) to 1 L of autoclaved,
cooled TSB (Section 7.1.7.1) and mix.
7.1.7.4 10X Tryptic soy broth—Dissolve 300 g TSB per liter of reagent water. Autoclave
for 15 minutes at 121°C and 15 psi. Be careful to remove broth as soon as possible
from the autoclave to prevent scorching. Store at 4°C ± 1°C until use.
7.1.8 10% (w/v) Sodium thiosulfate—Add 10 g sodium thiosulfate (Na2S2O3) per 90 mL reagent
water. Mix until dissolved. Bring to 100 mL and autoclave for 15 minutes at 121°C and 15
psi.
7.1.9 Magnesium chloride SOX (4M)—To 814 g of MgCl2-6H2O, add 300 mL reagent grade water.
Stir to dissolve. Bring to a final volume of 1 L, and mix thoroughly. Autoclave for 15 minutes
at 121°Cand 15 psi.
7.1.10 MS2 stock coliphage (ATCC# 15597-B1)
7.1.11 (|>X174 stock coliphage (ATCC#13706-B1)
7.1.12 Glycerol—Sigma#G6279 or equivalent. Autoclave for 15 minutes at 121°C and 15 psi.
Remove promptly to avoid scorching. Store at room temperature.
7.2 Double agar layer reagents
7.2.1 Tryptic soy agar or bottom agar (1.5%)—Prior to autoclaving the TSB in Section 7.1.7.1,
add 15 g of agar per liter of TSB. While stirring, heat to dissolve agar. Autoclave for 15
minutes at 121°C and 15 psi. Cool to 48°C ± 1.0°C and mix molten medium well for even
distribution.
7.2.1.1 For growth of somatic coliphages using E. coli CN-13 as host bacteria, add 10 mL
of 100X nalidixic acid (Section 7.1.5) per liter of autoclaved TSB. Swirl flask to
mix well and aseptically dispense 17 - 18 mL per 100-mm plate. Allow to solidify
with lids off in a biohazard hood for several minutes prior to use. Replace lids and
store inverted at 4°C ± 1°C for up to 2 weeks.
7.2.1.2 For growth of male-specific (F*) coliphages using E. coli F^p as host bacteria,
add 10 mL 100X ampicillin/streptomycin sulfate (Section 7.1.6) per liter of
autoclaved TSB. Swirl flask to mix well and aseptically dispense 17-18 mL per
100-mm plate. Allow to solidify with lids off in a biohazard hood for several
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minutes prior to use. Replace lids and store inverted at 4°C ± 1 °C for up to 2
weeks.
7.2.2 Top (soft) agar tubes (0.7%)—Prior to autoclaving the TSB in Section 7.1.7.1, add 7 g of
agar per liter of TSB. While stirring, heat to dissolve agar. Autoclave for 15 minutes at
121°C and 15 psi. Cool to 48°C ± 1.0°C.
7.2.2.1 Top agar with nalidixic acid (for growth of E. coli CN-13)—To 1 L of autoclaved
TSB (soft agar) (7.2.2), add 10 mL of 100X nalidixic acid. Dispense 5 mL per
sterile 10-mL tube, label, and keep at 45°C to 48°C until use. Tubes must be used
the day they are prepared.
7.2.2.2 Top agar tubes with ampicillin/streptomycin (for growth of E. coli F^)—To 1 L
of autoclaved TSB (soft agar) (Section 7.2.2), add 10 mL of 100X
ampicillin/streptomycin. Dispense 5 mL per sterile 10-mL tube, label, and keep at
45°C to 48°C until use. Tubes must be used the day they are prepared.
Note: When using top agar, add bacteria and sample to agar tubes immediately before plating to ensure
viability of both bacterial host and coliphage.
7.3 Two-step enrichment spot plate preparation
7.3.1 Log-phase host bacteria must be prepared in advance (Section 12.0). Dissolve 3 g TSB
(Section 7.1.7.1) 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.
7.3.2 Add 2 mL of log-phase host bacterium prepared from Section 12.0 and 1 mL 100X antibiotic
(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. 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, and creating the 1-cm grid on a circular
plastic dish and attaching to the bottom exterior of the plate with cellophane tape.
Note: Condensation may accumulate at the edges of stored plates and may drip over the agar surface if
tilted, ruining the spot pattern.
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8.0 Sample Collection, Preservation, and Storage
8.1 Samples are collected in plastic bottles or carboys and shipped to the laboratory for analysis. Samples
must be shipped at 2°C to 8°C. Samples must be stored at 4°C ± 1°C. Do not freeze.
8.2 Sample collection
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.
Note: Wet ice, Blue Ice® or similar products must be used to maintain a temperature of2°C to 8°C
during shipment. The samples must be protected from freezing.
Note: Unless the sample is known or suspected to contain infectious agents (e.g., during an outbreak),
samples should be shipped as noninfectious and should not be marked as infectious. U.S.
Department of Transportation (DOT) regulations (49 CFR 172) prohibit interstate shipment of
more than 4 L of solution known to contain infectious materials. State regulations may contain
similar regulations for intrastate commerce. If an outbreak is suspected, ship less than 4 Lot a
time.
8.3 The sampling team must maintain a log book with the following information for each sample:
8.3.1 Facility name and location
8.3.2 Date and time of collection
8.3.3 Name of analytical facility, contact, and phone number
8.3.4 Sample number
8.3.5 Sample location
8.4 The sample container must indicate the following:
8.4.1 Sample number
8.4.2 Date and time of collection
8.4.3 Sample collection location
8.5 Holding times. The following are maximum holding times beyond which the sample cannot be
retained.
8.5.1 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.
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8.6 Dechlorination procedure (if necessary)—For 1-L of water, add 0.5-mL 10% sodium thiosulfate.
9.0 Quality Control
9.1 Each laboratory that uses this method is required to operate a formal quality assurance (QA) program.
The minimum QA requirements for this program consist of an initial demonstration of laboratory
capability through performance of the initial precision and recovery (IPR) test (Section 9.3), analysis
of spiked samples to evaluate and document data quality, and analysis of standards and blanks as tests
of continued performance. Laboratory performance is compared to established performance criteria to
determine if the results of analyses meet the performance criteria of the method. Specific quality
control (QC) requirements for Method 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 18.4).
9.2 General QC requirements
Note: Although Method 1601 has been validated through a 10-laboratory round-robin validation study,
the QC acceptance criteria and frequency ofQC samples to be required in the method have not
been finalized. Final QC criteria and frequency will be specified in the next revision of the method.
9.2.1 The laboratory shall demonstrate the ability to generate acceptable performance with this
method by performing an IPR test. The procedure for performing the IPR is described in
Section 9.3.
9.2.2 The laboratory shall analyze method blanks to demonstrate freedom from contamination. The
procedures and criteria for analysis of a method blank are described in Section 9.4. The
laboratory shall analyze one method blank per spot plate for the two-step enrichment
procedure (Section 13).
9.2.3 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 analyze [number and frequency to be determined] positive controls for the two-step
enrichment procedure (Section 13).
9.2.4 The laboratory shall spike [number to be determined] separate sample aliquots from the
same groundwater source to monitor method performance. This matrix spike (MS) test is
described in Section 9.6. The laboratory shall analyze [number to be determined] MS
samples (Section 9.6) when samples are first received from a groundwater source for which
the laboratory has never before analyzed samples. The MS analysis is performed on
[number to be determined] additional sample aliquots collected from the groundwater
source at the same time as the routine field sample. If the laboratory routinely analyzes
samples from one or more groundwater sources, one MS analysis of [number to be
determined] samples must be performed per [number to be determined] field samples. For
example, when a laboratory receives the first sample from a source, the laboratory must
obtain [number to be determined] aliquots of this sample to be used for the MS. When the
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laboratory receives the [number to be determined] sample from this site, [number to be
determined] separate aliquots of this [number to be determined] sample must be collected
and spiked.
9.2.5 The laboratory shall, on an ongoing basis, demonstrate through analysis of the ongoing
precision and recovery (OPR) samples that the analytical system is in control (Section 9.7).
The laboratory shall analyze one OPR sample for each analytical batch [analytical batch to
be defined].
9.2.6 The laboratory shall maintain records to define the quality of data that are generated.
Development of accuracy statements is described in Sections 9.6 and 9.7.
9.2.7 The laboratory shall test media sterility by subjecting a representative portion of each media
batch to incubation at 35°C to 37°C for 48 to 72 hours and observe for indications of growth.
With respect to media, a batch is defined as 1 tube out of 50 in each lot or one tube, if the lot
contains less than 50 tubes.
9.2.8 The laboratory should participate in available interlaboratory performance studies conducted
by local, state, and federal agencies or commercial organizations. Review results to correct
unsatisfactory performance and record corrective actions.
9.2.9 A log book containing reagent and material lot numbers should be maintained along with
samples analyzed using each of the lots.
9.2.10 The laboratory shall maintain a record of the date and results of all QC samples described in
Section 9.2. A record of sterility check, OPR, and MS sample results must be maintained.
9.3 Initial precision and recovery (IPR)—The IPR test is performed to demonstrate acceptable
performance with the method.
Note: IPR tests must be accompanied by analysis of a method blank (Section 9.4).
9.3.1 A total of [number to be determined] spiked reagent water samples ([number to be
determined] samples spiked with each coliphage type) are required for the IPR test, as is a
method blank (Section 9.4) for each coliphage type.
9.3.2 IPR samples will be spiked in bulk, as follows: for each coliphage type (somatic and F*),
spike a [number to be determined]-mL "bulk" volume of reagent water with appropriate
antibiotics (for the 100-mL sample size) or a [number to be determined]'-L volume of reagent
water with appropriate antibiotics (for the 1-L sample size) with [number to be determined]
PFU (this will result in a spike of [number to be determined] PFU/sample). See Section 11
for enumeration of coliphage stock and Section 14.2 for spiking volume calculations.
9.3.3 Mix the spiked reagent water by swirling the container. For each coliphage type, dispense
100-mL aliquots (for the [number to be determined]-mL spiked reagent water) or 1-L
aliquots (for the [number to be determined]-^ spiked reagent water) into [number to be
determined] individual containers.
9.3.4 Analyze the [number to be determined] spiked samples ([number to be determined] for each
phage type resulting in [number to be determined] samples total) using the two-step
enrichment procedure (Section 13). At least [number to be determined] spiked samples, for
April 2000 Draft 10
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each coliphage type, must be positive for system performance to be acceptable. If system
performance is unacceptable, identify and correct the problem and repeat the IPR test.
9.4 Method blank (performed at the frequency specified in Section 9.2.2)
9.4.1 Prepare and analyze a reagent water sample containing no coliphage using the same
procedure as used for analysis of the field samples.
9.4.2 If coliphage, or any potentially interfering organisms are found in the blank, analysis of
additional samples must be halted until the source of contamination is eliminated, and a blank
shows no evidence of contamination. Any sample in a batch associated with a contaminated
blank must be recollected if holding time limits have been violated. Samples from a batch that
proves to have no coliphage in its blank may be reported along with that batch's sample data.
9.5 Positive controls (performed at the frequency specified in Section 9.2.3)
9.5.1 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 F+) at a concentration of approximately 20 PFU / 100 mL. If
spiking with MS2 (F*) or phi-X 174 (somatic), spike each flask with one coliphage type
(somatic or F+) at a concentration of approximately 60 PFU /100 mL. See Section 11 for
enumeration of stock/filtrate and Section 14.2 for spike volume calculations. Add appropriate
host bacteria (Section 12), growth media (with appropriate antibiotics), and other reagents to
each flask according to Section 13. Label each flask with the coliphage type and "+" or
"positive."
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
F+) at a concentration of approximately 20 PFU / L. If spiking with MS2 (F+) or
phi-X 174 (somatic), spike each flask with one coliphage type (somatic or F+) at a
concentration of approximately 60 PFU / L. See Section 11 for enumeration of stock/filtrate
and Section 14.2 for spike volume calculations. Add appropriate host bacteria (Section 12),
growth media (with appropriate antibiotics), and other reagents to each flask according to
Section 13. Label each flask with the coliphage type and "+" or "positive ."
9.6 Matrix spike—The laboratory shall spike and analyze [number to be determined] separate field
sample aliquots to determine the effect of the matrix on the method's coliphage recovery. The MS
shall be analyzed according to the frequency in Section 9.2.4.
9.6.1 Analyze an unspiked field sample according to the procedures specified in Section 13. To
[number to be determined] 100-mL or 1-L aliquots of the same field sample, add [number to
be determined] PFU of each type of stock coliphage (see Section 11 for enumeration).
9.6.2 Analyze these spiked samples according to the procedure in Section 13.
9.6.3 If [number to be determined] spiked samples are not positive, method performance is
unacceptable for that sample. If the results for the OPR sample and positive control sample
associated with this batch of samples are acceptable, a matrix interference may be causing the
poor recovery. If the results for the OPR and the positive control sample are not within their
control limits, the laboratory is not in control. The problem must be identified and corrected,
and the matrix spike and associated field samples reanalyzed.
11 April 2000 Draft
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9.7 Ongoing precision and recovery ([OPR]; laboratory control sample) (performed at the frequency
specified in Section 9.2.5)
9.7.1 Spike [number to be determined] reagent water samples with enumerated coliphage stock
suspension (Section 11), [number to be determined] samples for each coliphage type
(somatic and F+). 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.
9.7.2 Spike [number to be determined] coliphage into each sample (see Section 11 for enumeration
of coliphage stock and Section 14.2 for spiking volume calculations).
9.7.3 Analyze the [number to be determined] spiked samples using the two-step enrichment
procedure (Section 13). Coliphage must be detected in [number to be determined] samples. If
coliphage is not detected in [number to be determined] samples, method performance is
unacceptable. Identify and correct the problem and perform another OPR test before
continuing with the analysis of field samples.
9.8 The laboratory should periodically analyze an external QC sample, such as a performance evaluation
when available. The laboratory also should periodically participate in interlaboratory comparison
studies using the method.
9.9 The specifications contained in this method can be met if the analytical system is maintained under
control. The standards used for initial (Section 9.3) and ongoing (Section 9.7) precision and recovery
should be identical, so that the most precise results will be obtained.
10.0 Calibration and Standardization
10.1 Check temperatures in water baths, refrigerators, and -20°C freezers daily to ensure operation within
stated limits of method and record daily measurements in a log book.
10.2 Check temperatures in incubators twice daily, at least 4 hours apart, to ensure operation within stated
limits of method and record measurements in log book.
10.3 Check thermometers at least annually against an NIST-certified thermometer or one that meets the
requirements of MIST Monograph SP 250-23. Check mercury columns for breaks.
10.4 Calibrate pH meter prior to use, using standards of pH 4.0, 7.0, and 10.0. To calibrate, use the two
standards that are nearest to the desired pH.
10.5 Calibrate balances annually using ASTM-certified Class 2 reference weights.
10.6 Calibrate spectrophotometer prior to each use, following method described in owner's manual. Use
sterile TSB without antibiotics as the blank.
10.7 Laboratories must adhere to all applicable quality control requirements set forth in Reference Section
18.6.
April 2000 Draft 12
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11.0 Enumeration of Coliphage Stock Suspension
11.1 The double agar layer (DAL) procedure is used to enumerate stock suspensions of somatic and F+
coliphage for use in spiking quality control samples.
11.2 Prepare log-phase bacterial hosts from stock cultures incubated overnight. Inoculum from an
overnight bacterial host culture will reach log-phase more rapidly than inoculum from frozen stock.
11.2.1 Preparation of overnight stock cultures
11.2.1.1 Dispense 25 mL of tryptic soy broth (TSB) with nalidixic acid (Section
7.1.7.2) into a sterile 125-mL shaker flask. Label the flask. For proper
growth conditions, each flask should always contain 25 to 30 mL of medium.
11.2.1.2 Inoculate the flask with a loopful of E. coli CN-13 from the frozen stock
culture (Section 7.1.4). Label the flask.
11.2.1.3 Repeat Sections 11.2.1.1 and 11.2.1.2 using TSB with streptomycin and
ampicillin as the medium (Section 7.1.7.3) and E. coli f^ as the bacterial
host.
11.2.1.4 Place a sterile slip cap or plug on the shaker flasks and secure in shaker.
11.2.1.5 Incubate at 36°C ± 1.0°C and set shaker to 100 to 150 rpm overnight (18 to
20 hours).
11.2.1.6 Chill on wet ice or at 4°C ± 1 °C until ready for use.
11.2.2 Preparation of log-phase bacterial host cultures—See Section 12
11.3 Preparation of filtrate from raw sewage. This filtrate will be used as a QC spiking suspension. If
coliphage stock is available, skip this section and proceed to Section 11.4.
11.3.1 Collect approximately 100 mL of raw sewage in a 250-mL collection bottle.
11.3.2 Transport to the laboratory on ice.
11.3.3 Allow the raw sewage to settle at 4°C ± 1°C for 1 to 3 hours. This will make the filtration
process easier.
11.3.4 Remove a sterile, 20-mL syringe from its package, aseptically remove plunger from barrel,
and attach a filter disk to the syringe barrel.
11.3.5 Pipet 10 to 15 mL of supernatant from settled sewage into the syringe barrel.
11.3.6 Hold the assembly over a 15-mL polypropylene tube with screw-cap or snap-cap, insert the
plunger into the syringe barrel, and push the sewage through the filter into the sterile tube.
If filter clogs, change it as necessary and continue to filter sewage until at least 10 mL of
filtered sewage is obtained in the 15-mL polypropylene tube (filtration may require use of
numerous filters).
11.3.7 Cap the tube, label with source, date, and initials, and store the filtrate at 4°C ± 1 °C until
ready to assay. The filtrate should not be stored more than 24 hours, unless re-titered.
13 April 2000 Draft
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Note: Coliphage liter may decrease over time, therefore sewage filtrate should be assayed immediately for
total coliphage and used within 24 hours of preparation.
11.4 Dilution of coliphage stock or sewage filtrate (Section 11.3)—A minimum of four different
volumes/dilutions are used for DAL enumeration of the stock/filtrate:
• Undiluted
0.1
0.01
0.001
Additional dilutions may be necessary. TSB without antibiotics (Section 7.1.7.1) is used as the diluent
and as the method blank.
11.4.1 Aseptically add 9.0 mL of TSB without antibiotics (Section 7.1.7.1) into each of four (or
more) sterile dilution tubes (Section 6.2.1). Label them as "0.1," "0.01," "0.001," "method
blank," etc.
11.4.2 Add 1.0 mL of the stock/filtrate to the tube of TSB labeled "0.1." Cap the tube and vortex
for 5 seconds on a medium-high setting (if available) or until well-mixed.
11.4.3 Add 1.0 mL of the well-mixed 0.1 dilution to the tube of TSB labeled "0.01". Cap the tube
and vortex for 5 seconds on a medium-high setting (if available) or until well-mixed.
11.4.4 Add 1.0 mL of the well-mixed 0.01 dilution to the tube of TSB labeled "0.001." Cap the
tube and vortex for 5 seconds on a medium-high setting (if available) or until well-mixed.
11.4.5 Continue as necessary for subsequent dilutions.
11.4.6 Add 1.0 mL of TSB without antibiotics to the tube labeled "method blank". Cap the tube
and vortex for 5 seconds on a medium-high setting (if available) or until well-mixed.
11.5 Coliphage stock suspension enumeration procedure—In this procedure, a tube of molten top agar with
added host bacteria is inoculated with coliphage stock and will be poured into a bottom agar plate.
Four dilutions of stock/filtrate will be analyzed in duplicate for each coliphage type. As a result, nine
double-agar plates will be required for each coliphage type: two plates per dilution (undiluted, 0.1,
0.01, and 0.001) and one method blank plate.
11.5.1 Agar preparation
11.5.1.1 Place top agar tubes in a 45°C to 48°C water bath. The top agar should
remain molten in the water bath until ready for use. Four tubes are needed for
each volume of stock/filtrate (two for each bacterial host). Be sure to include
an additional two top agar tubes for the method blanks (a method blank is
required for each bacterial host). Half of the top agar tubes should contain
nalidixic acid (Section 7.2.2.1) for growth of E. coli CN-13; the other half
should contain ampicillin/streptomycin (Section 7.2.2.2) for growth of E. coli
"ampp
11.5.1.2 Disinfect a workspace near the water bath.
April 2000 Draft 14
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11.5.1.3 Assemble bottom agar plates and label or code so that the following
information is identifiable:
Dilution of stock filtrate or method blank
Bacterial host (E. coli CN-13 or E. coli F^
• Coliphage type (somatic for the E. coli CN-13 bacterial host or
for the E. coli F^ bacterial host)
Date
• Time
Note: The following plate-preparation steps are critical. Do not add bacterial host and stock/filtrate until
ready to plate.
11.5.2 Preparation of plates for enumeration of somatic coliphage (see Table 1 for inoculation
scheme)
11.5.2.1 With the top agar tube still in the water bath, aseptically inoculate a top agar
tube containing nalidixic acid with 30 to 100 uL of log-phase E. coli CN-13.
11.5.2.2 Immediately add 500 uL of undiluted stock/filtrate.
11.5.2.3 Mix the inoculum by rolling the tube briefly in palm of hand.
11.5.2.4 Pour contents into one of the two bottom agar plates marked "undiluted, E.
coli CN-13, somatic."
11.5.2.5 Duplicate analysis—Repeat Sections 11.5.2.1 through 11.5.2.4 for the
duplicate.
11.5.2.6 Repeat Sections 11.5.2.1 through 11.5.2.5 for each dilution volume.
11.5.3 Preparation of plates for enumeration of ¥+ coliphage—Repeat Section 11.5.2 using agar
containing ampicillin/streptomycin and log-phase E. coli F^. (see Table 1 for inoculation
scheme)
11.5.4 Preparation of somatic coliphage method blank (see Table 1 for inoculation scheme)
11.5.4.1 With the top agar tube still in the water bath, aseptically inoculate a top agar
tube containing nalidixic acid with 30 to 100 uL of log-phase E. coli CN-13.
11.5.4.2 Immediately add 500 uL of TSB from the "method blank" dilution tube.
11.5.4.3 Mix the inoculum by rolling the tube briefly in palm of hand.
11.5.4.4 Pour contents into a bottom agar plate marked "blank, E. coli CN-13,
somatic."
11.5.5 Preparation of the F^ coliphage method blank—Repeat Section 11.5.4 using agar containing
ampicillin/streptomycin and log-phase E. coli Fmnf (see Table 1 for inoculation scheme)
11.5.6 Retain undiluted stock/filtrate for use in preparing new dilutions for positive control and
OPR samples.
15 April 2000 Draft
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11.5.7 After the top agar hardens, cover, invert the plates and incubate for 16 to 24 hours at 36°C
± 1.0°C.
11.5.8 Circular zones of clearing (typically 1 to 10 mm in diameter) in lawn of host bacteria after
16 to 24 hours are plaques. Count the number of plaques on each plate.
11.5.9 Proceed to Section 14.1 and calculate the PFU / mL for each coliphage.
11.5.10 Use the enumerated somatic and F* stocks to spike the two-step enrichment IPR, OPR, MS,
and positive control samples as described in Section 9.
Tablet
Inoculation Scheme for Enumeration of Stock or Sewage Filtrate
c
-S
5. =
0)0
<0
a
|2|
TSA w/Nalidixic
Acid
TSA w/Ampicillin
and
Streptomycin sulfate
Add
30 - 100 ML of
E. co/iCN-13
per tube
Add
30 • 100 |iL of
£. co// Fmf
per tube
Coliphage Source (stock or sewage filtrate)
Undiluted
500
ML
i
U
u
500
ML
I
U
u
0.1 Dilution
inTSB
500
ML
1
U
u
500
ML
i
U
u
0.01 Dilution
inTSB
500
ML
i
U
u
500
ML
i
U
u
0.001 Dilution
inTSB
500
ML
1
U
u
500
ML
I
U
u
Method
Blank
500
ML
1
U
u
*Host bacteria are added first, then coliphage or TSB (for the blanks). Additional dilutions may be
necessary.
April 2000 Draft
16
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12.0 Preparation of Log-Phase Host Bacterial Cultures
12.1 Inoculate a shaker flask containing 25 mL of TSB containing nalidixic acid (Section 7.1.7.2) with 0.1
to 1.0 mL of E. coli CN-13. For proper growth conditions, each culture flask of host bacteria should
contain 25 to 30 mL of medium. As a result, several flasks of host bacteria may have to be prepared
(this depends on the number of samples and controls being run each day). Each 100-mL sample will
require a 0.5-mL inoculum of log-phase host bacteria and each 1-L sample will require a 5-mL
inoculum.
12.2 Repeat Section 12.1 using TSB with streptomycin and ampicillin (Section 7.1.7.3) as the medium and
E. coli F^ as the bacterial host.
12.3 After inoculation, place a sterile slip-cap or plug on the shaker flasks and secure in shaker incubator.
12.4 Incubate at 36°C ± 1.0°C and 100 to 150 rpm for approximately 4 hours or until cultures are visibly
turbid, indicating log-phase growth.
12.5 Aseptically remove 1 mL of culture from flask, dispense into a cuvette (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.
12.6 Chill on wet ice or at 4°C ± 1°C to slow replication until ready for use. The suspension may be stored
up to 48 hours. However, the best results occur when cultures are used immediately (within 6 hours).
12.7 Store remaining bacterial host culture at 4°C ± 1°C overnight to inoculate flasks for the preparation
of new log-phase bacterial hosts.
13.0 Two-step Enrichment Procedure
13.1 Enrichment procedure
13.1.1 100-mL samples
13.1.1.1 Dispense a 100-mL sample aliquot into each of two sterile, 100-mL or 125-
mL bottles. 100 mL of sample is required for each log-phase bacterial host
(E. coli CN-13 and E. coli F^). (If 100-mL bottles are used, be sure that
there is ample head space for the addition of reagents.)
13.1.1.2 Label bottle with the following information
Sample number
Bacterial host (E. coli CN-13 or E. coli F^)
Date
• Start time
• Sample volume
13.1.1.3 Record this information on a report form.
13.1.1.4 Add 1.25 mL of SOX MgCl2'6H2O (Section 7.1.9) to each groundwater
sample aliquot.
13.1.1.5 Add 0.5 mL of log-phase E. coli CN-13 (Section 12) to one sample aliquot
for each sample.
17 April2000 Draft
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13.1.1.6 Add 0.5 mL of log-phase E. coli F^p (Section 12) to the other sample aliquot
for each sample.
13.1.1.7 Add 5 mL of 10X TSB (Section 7.1.7.4) to all aliquots. This gives a final
concentration of 0.5X TSB.
13.1.1.8 To those bottles with E. coli CN-13 as host, add 1 mL of 100X nalidixic acid
(Section 7.1.5).
13.1.1.9 To those bottles with E. coli F^ as host, add 1 mL of 100X
ampicillin/streptomycin solution (Section 7.1.6).
13.1.1.10 Cap and invert each bottle five times to mix.
13.1.1.11 Prepare method blanks and positive controls as specified in Section 9.4 and
9.5.
13.1.1.12 Incubate the bottles for 16 to 24 hours at 36°C ± 1.0°C with no further
mixing. Proceed to Section 13.2.
13.1.2 1-L samples
13.1.2.1 Dispense a 1-L sample aliquot into each of two sterile, 1-L or 1.5-L bottles.
1 L of sample is required for each log-phase bacterial host (E. coli CN-13
and E. coli F^). (If 1-L bottles are used, be sure that there is ample head
space for the addition of reagents.)
13.1.2.2 Label bottle with the following information
• Sample number
Bacterial host (E. coli CN-13 or E. coli F^)
Date
• Time
• Sample volume
13.1.2.3 Record this information on a report form.
13.1.2.4 Add 12.5 mL of SOX MgCl2«6H2O (Section 7.1.9) to each groundwater
sample aliquot.
13.1.2.5 Add 5 mL of log-phase E. coli CN-13 (Section 12) to one sample aliquot for
each sample.
13.1.2.6 Add 5 mL of log-phase E. coli F^ (Section 12) to the other sample aliquot
for each sample.
13.1.2.7 Add 50 mL of 10X TSB (Section 7.1.7.4) to all aliquots. This gives a final
concentration of 0.5X TSB.
13.1.2.8 To those bottles with E. coli CN-13 as host, add 10 mL of 100X nalidixic
acid (Section 7.1.5).
13.1.2.9 To those bottles with E. coli F^ as host, add 10 mL of 100X
ampicillin/streptomycin solution (Section 7.1.6).
13.1.2.10 Cap and invert each bottle 5 times to mix.
13.1.2.11 Prepare method blanks and positive controls as specified in Section 9.4 and
9.5.
13.1.2.12 Incubate the bottles for 16 to 24 hours at 36°C ± 1.0°C with no further
mixing. Proceed to Section 13.2.
April 2000 Draft 18
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13.2 Spot-plate procedure
13.2.1 Prepare spot plates according to Section 7.3. 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.
13.2.2 After the overnight enrichment of 16 to 24 hours, mix the sample by inverting the bottle.
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.
13.2.3 Spot 10 uL 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.
13.2.4 Record the grid number and corresponding sample number into a log book.
13.2.5 Spot 10 uL of enriched sample from the bottle containing E. coli F^ host bacteria onto the
gridded spot plate that was prepared using E. coli F^p. If the analyst is extremely careful,
one 100-mm plate can be inoculated with up to 20 spots.
13.2.6 Record the grid number and corresponding sample number on the report form.
13.2.7 Spot the method blank and positive control samples as specified in Section 9.4 and 9.5.
13.2.8 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.
13.2.9 After inocula absorption, cover, invert the plate and incubate at 36°C ± 1.0°C for 16 to 24
hours.
13.2.10 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
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. This is also
scored a positive result; the bacterial colonies are from phage-resistant mutants.
13.2.11 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.
19 April 2000 Draft
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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.
13.2.11.1 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-um 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.
13.2.11.2 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.
13.2.11.3 Spot 10-uL volumes of filtered or centrifuged enrichment onto pre-poured
lawns of host bacteria as described in Section 13.2.
14.0 Data Analysis and Calculations
14.1 Calculation for the DAL procedure (Section 11)
14.1.1 Compute PFU / mL of filtered sewage using DAL plates that yield plaque counts within the
desired range of 20 to 300 PFU per plate for F+ coliphage and 20 to 100 PFU per plate for
somatic coliphage. There may be occasions when the total number of plaques on a plate will
be above or below the ideal range. If the plaques are not discrete, results should be recorded
as "too numerous to count" (TNTC).
14.1.2 The inoculation volume, as described in Section 11, is 500 uL (0.5 mL). Refer to Table 2
for dilution factors.
Table 2. Dilution factors for concentration of filtered sewage.
Sewage Concentration
Undiluted
0.1 Dilution
0.01 Dilution
0.001 Dilution
Dilution Factor
1
0.1
0.01
0.001
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14.1.3 Use the following equation to calculate PFU / mL of coliphage stock or filtered sewage for
each DAL plate:
number of plaques counted
PFU/mL = —
(inoculation volume in mL)(dilution factor)
14.1.4 If multiple plates are within the desired range, then PFU / mL of stock/filtrate should be
calculated for each of those plates and the average taken.
14.2 Calculation for preparing IPR, OPR, and positive control spikes
14.2.1 The stock/filtrate enumerated in Section 11 will be used as the spiking suspension. Use a
dilution of stock/filtrate that will result in a spike volume between 0.1 and 3.0 mL.
14.2.2 Use the following equation to determine the spiking volume:
(T/V)(B)
s=
(C)
where,
S = Spike volume (mL)
T = Target number of coliphage per sample (PFU)
V = Sample volume (mL)
B = Bulk sample volume (mL) (only necessary when multiple samples are spiked in
bulk for QC purposes)
C = Concentration of coliphage in the dilution to be used for spiking (PFU / mL)
14.2.3 For example, if:
T) A spike dose of 2 PFU per sample is needed
V) 100-mL samples will be spiked
B) A total of four samples will be spiked at the same time
(bulk sample volume = 400 mL)
C) The 10'2 dilution contains 6 PFU / mL
The equation would be solved as follows:
(2PFU/100mL)(400mL)
1.3 mL =
(6PFU/mL)
As a result, 1.3 mL of the 10"2 dilution would be spiked into the 400 mL bulk sample. The
400 mL bulk sample would be mixed and four, 100-mL aliquots dispensed. Each 100-mL
sample should contain approximately 2 PFU.
21 April 2000 Draft
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15.0 Method Performance
Note: Although Method 1601 has been validated through a IQ-laboratory round-robin validation study,
the QC acceptance criteria and frequency ofQC samples to be required in the method have not
been finalized. Final QC criteria and frequency will be specified in the next draft of the method.
15.1 Precision and accuracy statements will be placed within this document when they have been
established.
16.0 Pollution Prevention
16.1 The solutions and reagents used in this method pose little threat to the environment when recycled and
managed properly.
16.2 Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize the
volume of expired materials to be disposed.
17.0 Waste Management
17.1 The laboratory is responsible for complying with all Federal, State, and local regulations governing
waste management, particularly hazardous waste identification rules and land disposal restrictions,
and for protecting the air, water, and land by minimizing and controlling all releases from fume hoods
and bench operations. Compliance with all sewage discharge permits and regulations is also required.
An overview of requirements can be found in Environmental Management Guide for Small
Laboratories (EPA 233-B-98-001).
17.2 Samples, reference materials, and equipment known or suspected to have bacteriophage attached or
contained must be sterilized prior to disposal.
17.3 For further information on waste management, consult The Waste Management Manual for
Laboratory Personnel and Less Is Better: Laboratory Chemical Management for Waste Reduction,
both available from the American Chemical Society's Department of Government Relations and
Science Policy, 1155 16th Street N.W., Washington B.C. 20036.
18.0 References
18.1 American Public Health Association, American Water Works Association, Water Environment
Federation. Washington, D.C. Joint Task Group for Section 9224, 1997. Detection of Coliphages .
For Standard Methods for the Examination of Water and Waste Water 20th Edition Supplement, (draft
version - December 1997)
April 2000 Draft 22
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18.2 American Public Health Association, American Water Works Association, and Water Environment
Federation. 1995. Standard Methods for Water and Wastewater. 19th Edition. Sections 9020, 9030,
9040, 9050, and 9221.
18.3 U.S. Environmental Protection Agency, 1998. Method 1691: Municipal Biosolids Sampling
Guidance. Draft, September 1998. Office of Water, Washington, DC.
18.4 Bordner, R., J.A. Winter, and P.V. Scarpino (eds.). 1978. Microbiological Methods for Monitoring
the Environment, Water and Wastes. EPA-600/8-78-017. Office of Research and Development.
USEPA.
18.5 ICR Microbial Laboratory Manual, EPA/600/R-95/178, National Exposure Research Laboratory,
Office of Research and Development, U.S. Environmental Protection Agency, 26 Martin Luther King
Drive, Cincinnati, OH 45268.
18.6 Manual for the Certification of Laboratories Analyzing Drinking Water, EPA 815-B-97-001, Office
of Ground Water and Drinking Water, Technical Support Center, U.S. Environmental Protection
Agency, 26 Martin Luther King Drive, Cincinnati, OH 45268.
18.7 Annual Book of ASTM Standards. Vol. 11.01. American Society for Testing and Materials.
Philadelphia, PA 19103.
19.0 Tables, Diagrams, Flowcharts, and Validation Data
[To be added]
20.0 Glossary
These definitions and purposes are specific to this method but have been conformed to common usage
as much as possible.
20.1 Symbols
°C degrees Celsius
u micro
# number
% percent
20.2 Alphabetical characters and acronyms
ASTM American Society for Testing and Materials
CFR Code of Federal Regulations
DAL double agar layer method
DOT Department of Transportation
g gram
HC1 hydrochloric acid
IPR initial precision and recovery
KH2HPO4 potassium phosphate
L liter
M molar
mg milligram
MgCl2*6H2O magnesium chloride hexahydrate
mL milliliter
23 April 2000 Draft
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mm millimeter
MPN most probable number
MS matrix spike
NaOH sodium hydroxide
Na2S2O3 sodium thiosulfate
NIST National Institute of Standards and Technology
nm nanometer
OD optical density
OPR ongoing precision and recovery
OSHA Occupational Safety and Health Administration
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"
20.3 Additional definitions
Accuracy—A measure of the degree of conformity of a single test result generated by a specific
procedure to the assumed or accepted true value and includes both precision and bias.
Analyte—The organism tested for by this method. The analyte in this method is coliphage.
Analytical batch—[analytical batch to be defined].
Bias—the persistent positive or negative deviation of the average value of a test method from the
assumed or accepted true value.
Coliphage—Viruses that infect fecal coliforms, particularly E. coli.
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. coli CN-13, and
E. coli F^ (E. coli HS(pFamp)R).
Initial precision and recovery (IPR)—The IPR test is performed to establish the ability to
demonstrate control over the analytical system and to generate acceptable precision and recovery.
Male-specific coliphage—Viruses (bacteriophages) that infect coliform bacteria only via the
F-pilus and are indicators of fecal contamination.
April 2000 Draft 24
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May—This action, activity, or procedural step is neither required nor prohibited.
May not—This action, activity, or procedural step is prohibited.
Must—This action, activity, or procedural step is required.
Method blank —An aliquot of reagent water that is treated exactly as a sample and carried through all
portions of the procedure until determined to be negative or positive. The method blank is used to
determine if the sample has become contaminated by the introduction of a foreign microorganism
through poor technique.
Ongoing precision and recovery (OPR)—A reagent water sample spiked with known quantities of
analytes. The OPR is analyzed exactly like a sample. Its purpose is to assure that the results produced
by the laboratory remain within limits specified in this method fore precision and recovery.
Precision—The degree of agreement of repeated measurements of the same property, expressed in
terms of dispersion of test results about the arithmetical mean. Results are obtained by repetitive
testing of a homogeneous sample under specified conditions. The precision of a test method is
expressed quantitatively as the standard deviation computed from the results of a series of controlled
determinations.
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 number of virus present.
Relative Standard Deviation (RSD)—The standard deviation times 100 divided by the mean.
Reagent water—Water conforming to Specification D 1193, Annual Book of ASTM Standards
(Reference 18.7).
Should—This action, activity, or procedural step is suggested but not required.
Somatic coliphage—Those coliphage that infect host cells via the outer cell membrane but do not
infect host cells via the F-pilus.
25 April 2000 Draft
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