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.
April 2001
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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.
April 2001
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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
April 2001 10
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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
11 April 2001
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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.
13 April 2001
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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
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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
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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
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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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