United States
Environmental Protection
Agency
Office of Water
Washington DC
20460
EPA821-R-02-022
September 2002
vvEPA
Method 1600: Enterococci in
Water by Membrane Filtration
Using membrane-Enterococcus
Indoxyl-p-D-Glucoside Agar (mEI)
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U.S. Environmental Protection Agency
Office of Water (4303T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
EPA-821-R-02-022
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Acknowledgments
This method was developed under the direction of James W. Messer and Alfred P. Dufour of the U.S.
Environmental Protection Agency's (EPA) Human Exposure Research Division, National Exposure
Research Laboratory, Cincinnati, Ohio.
Disclaimer
The Engineering and Analysis Division, of the Office of Science and Technology, has reviewed and
approved this report for publication. The Office of Science and Technology directed, managed, and
reviewed the work of DynCorp in preparing this report. Neither the United States Government nor any of
its employees, contractors, or their employees make any warranty, expressed or implied, or assumes any
legal liability or responsibility for any third party's use of or the results of such use of any information,
apparatus, product, or process discussed in this report, or represents that its use by such party would not
infringe on privately owned rights. This document combines the information previously published in
Method 1600: Membrane Filter Test Method for Enter ococci in Water (EPA-821-R-97-004) (Reference
18.8) and Improved Enumeration Methods for the Recreational Water Quality Indicators: Enterococci
and Escherichia coll (EPA/821/R-97/004) (Reference 18.7). Mention of trade names or commercial
products does not constitute endorsement or recommendation for use.
Questions concerning this method or its application should be addressed to:
Robin K. Oshiro
Engineering and Analysis Division (4303T)
U.S. EPA Office of Water, Office of Science and Technology
1200 Pennsylvania Avenue, NW
Washington, DC 20460
oshiro.robin@epa.gov
202-566-1075
202-566-1053 (facsimile)
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Introduction
EPA has been increasingly concerned with the public health risks of infectious diseases caused by
microbial organisms in our nation's beaches. In response to this problem, EPA has established the
Beaches Environmental Assessment Closure and Health (BEACH) Program. This analytical method is
published for use in the BEACH Program.
In 1986, EPA issued a revision to its bacteriological ambient water quality criteria recommendations to
include new indicator bacteria, E. coll and enterococci, which provide a better correlation with
swimming-associated gastrointestinal illness than the previous criteria recommendations for fecal
coliform bacteria. These revised criteria are useful to public health officials because they enable
quantitative estimates of illness rates associated with swimming in polluted water.
This method is a revision of EPA's previous enterococci method, used since 1985 in ambient water
quality monitoring. It reduces analysis time to 24 hours and improves analytical quality. The method has
been validated in single- and multi-laboratory studies and has undergone peer review.
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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 1
5.0 Safety 2
6.0 Equipment and Supplies 2
7.0 Reagents and Standards 3
8.0 Sample Collection, Preservation, and Storage 5
9.0 Quality Control 6
10.0 Calibration and Standardization 6
11.0 Procedure 6
12.0 Data Analysis and Calculations 7
13.0 Method Performance 7
14.0 Reporting Results 7
15.0 Verification Procedure 7
16.0 Pollution Prevention 8
17.0 Waste Management 8
18.0 References 8
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Method 1600: Enterococci in Water by Membrane Filtration Using
membrane-Enterococcus lndoxyl-J3-D-Glucoside Agar (mEI)
September 2002
1.0 Scope and Application
1.1 This method describes a membrane filter (MF) procedure for the detection and enumeration of the
enterococci bacteria in water. Enterococci are commonly found in the feces of humans and other
warm-blooded animals. Although some strains are ubiquitous and not related to fecal pollution,
the presence of enterococci in water is an indication of fecal pollution and the possible presence
of enteric pathogens.
1.2 The enterococci test measures the bacteriological quality of recreational waters. Epidemiological
studies have led to the development of criteria which can be used to promulgate recreational
water standards based on the established relationship between health effects and water quality.
The significance of finding enterococci in recreational water samples is the direct relationship
between the density of enterococci in the water and swimming-associated gastroenteritis studies
of marine and fresh water bathing beaches (Reference 18.3, Reference 18.4).
1.3 The test for enterococci can be applied to potable, fresh, estuarine, marine, and shellfish growing
waters.
1.4 Since a wide range of sample volumes or dilutions can be analyzed by the MF technique, a wide
range of enterococci levels in water can be detected and enumerated.
2.0 Summary of Method
2.1 The MF method provides a direct count of bacteria in water based on the development of colonies
on the surface of the membrane filter (Reference 18.5). A water sample is filtered through the
membrane which retains the bacteria. Following filtration, the membrane containing the bacterial
cells is placed on a selective medium, mEI agar, and incubated for 24 h at 41°C. All colonies
(regardless of color) with a blue halo are recorded as enterococci colonies. Magnification and a
small fluorescent lamp are used for counting to give maximum visibility of colonies.
3.0 Definitions
3.1 In this method, enterococci are those bacteria which produce colonies with a blue halo after
incubation on mEI agar. Enterococci include Streptococcus faecalis, Streptococcus faecium,
Streptococcus avium, and their variants.
4.0 Interferences
4.1 Water samples containing colloidal or suspended particulate materials can clog the membrane
filter and prevent filtration, or cause spreading of bacterial colonies which could interfere with
identification of target colonies.
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Method 1600
5.0 Safety
5.1 The analyst/technician must know and observe the normal safety procedures required in a
microbiology laboratory while preparing, using, and disposing of cultures, reagents, and
materials, and while operating sterilization equipment.
5.2 Mouth-pipetting is prohibited.
6.0 Equipment and Supplies
6.1 Glass lens with magnification of 2-5x or stereoscopic microscope.
6.2 Lamp, with a cool, white fluorescent tube.
6.3 Hand tally or electronic counting device.
6.4 Pipet container, stainless steel, aluminum or borosilicate glass, for glass pipets.
6.5 Pipets, sterile, T.D. bacteriological or Mohr, glass or plastic, of appropriate volume.
6.6 Graduated cylinders, 100-1000 mL, covered with aluminum foil or kraft paper and sterile.
6.7 Membrane filtration units (filter base and funnel), glass, plastic or stainless steel, wrapped with
aluminum foil or kraft paper and sterilized.
6.8 Ultraviolet unit for sanitization of the filter funnel between filtrations (optional).
6.9 Line vacuum, electric vacuum pump, or aspirator for use as a vacuum source. In an emergency or
in the field, a hand pump or a syringe equipped with a check valve to prevent the return flow of
air, can be used.
6.10 Flask, filter, vacuum, usually 1 L, with appropriate tubing. A filter manifold to hold a number of
filter bases is optional.
6.11 Flask for safety trap placed between the filter flask and the vacuum source.
6.12 Forceps, straight or curved, with smooth tips to handle filters without damage.
6.13 Ethanol, methanol or isopropanol in a small, wide-mouth container, for flame-sterilizing forceps.
6.14 Burner, Bunsen or Fisher type, or electric incinerator unit for sterilizing loops and needles.
6.15 Thermometer, checked against a National Institute of Standards and Technology (NIST) certified
thermometer, or one that meets the requirements of NIST Monograph SP 250-23.
6.16 Petri dishes, sterile, plastic, 9 x 50 mm, with tight-fitting lids.
6.17 Bottles, milk dilution, borosilicate glass, screw-cap with neoprene liners, marked at 99 mL for
1:100 dilutions. Dilution bottles marked at 90 mL or tubes marked at 9 mL may be used for 1:10
dilutions.
6.18 Flasks, borosilicate glass, screw-cap, 250-2000 mL volume.
6.19 Membrane filters, sterile, white, grid marked, 47 mm diameter, with 0.45 ± 0.02 (im pore size.
6.20 Inoculation loops, at least 3 mm diameter, and needles, nichrome or platinum wire, 26 B & S
gauge, in suitable holders. Sterile disposable applicator sticks or plastic loops are alternatives to
inoculation loops.
6.21 Incubator maintained at 41 ± 0.5°C.
6.22 Waterbath maintained at 50°C for tempering agar.
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Method 1600
6.23 Test tubes, 20 x 150 mm, borosilicate glass or plastic.
6.24 Caps, aluminum or autoclavable plastic, for 20 mm diameter test tubes.
6.25 Test tubes, screw-cap, borosilicate glass, 16 x 125 mm or other appropriate size.
6.26 Whirl-Pak® bags.
7.0 Reagents and Standards
7.1 Purity of Reagents: Reagent grade chemicals shall be used in all tests. Unless otherwise
indicated, reagents shall conform to the specifications of the Committee on Analytical Reagents
of the American Chemical Society (Reference 18.9). The agar used in preparation of culture
media must be of microbiological grade.
7.2 Whenever possible, use commercial culture media as a means of quality control.
7.3 Purity of Water: Reagent water conforming to Specification Dl 193, reagent water conforming
Type II, Annual Book of ASTM Standards (Reference 18.1).
7.4 Buffered Dilution Water
7.4.1 Composition:
Sodium Dihydrogen Phosphate 0.58 g
Sodium Monohydrogen Phosphate 2.50 g
Sodium Chloride 8.50 g
Reagent-Grade Distilled Water 1.0 L
7.4.2 Preparation: Dissolve the ingredients in 1 L of reagent water in a flask and dispense in
appropriate amounts for dilutions in screw-cap bottles or culture tubes, and/or into
containers for use as rinse water. Autoclave after preparation at 121°C (15 Ib pressure)
for 15 min. Final pH should be 7.4 ± 0.2.
7.5 Phosphate Buffered Dilution Water (Reference 18.2)
7.5.1 Composition of Stock Phosphate Buffer Solution:
Phosphate dihydrogen phosphate 34.0 g
Reagent-Grade distilled water 500.0 mL
7.5.2 Preparation: Adjust the pH of the solution to 7.2 with 1 N NaOH, and bring the volume to
1 L with reagent-grade distilled water. Sterilize by filtration or autoclave at 121°C (15 Ib
pressure) for 15 min.
7.5.3 Preparation of Stock Magnesium Chloride Solution: Add 38 g anhydrous MgCl2 or 81.1
g MgCl2-6H2O to 1 L reagent-grade distilled water. Sterilize by filtration or autoclave at
121°C (15 Ib pressure) for 15 min.
7.5.4 Storage of Stock Solutions: After sterilization, store the stock solutions in the refrigerator
until used. Handle aseptically. If evidence of mold or other contamination appears, the
affected stock solution should be discarded and a fresh solution should be prepared.
7.5.5 Working Phosphate Buffered Dilution Water: Mix 1.25 mL of the stock phosphate buffer
and 5 mL of the MgCl2 stock per liter of reagent-grade distilled water. Dispense in
3 September 2002
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Method 1600
appropriate amounts for dilutions in screw-cap bottles or culture tubes, and/or into
containers for use as rinse water. Autoclave at 121°C (15 Ib pressure) for 15 min. Final
pH should be 7.0 ± 0.2.
7.6 mEI Agar
7.6.1 Composition of Basal Medium (mE Agar, Difco 0333)
Peptone 10.0 g
Sodium Chloride 15.0 g
Yeast Extract 30.0 g
Esculin 1.0 g
Actidione (Cycloheximide) 0.05 g
Sodium Azide 0.15g
Agar 15.0 g
Reagent-Grade Distilled Water 1.0 L
7.6.2 Preparation of mEI Medium: Add 71.2 g of dehydrated basal medium plus 0.75 grams of
indoxyl P-D glucoside to 1 L of reagent grade water in a flask and heat to boiling until
ingredients dissolve. Autoclave at 121°C (15 Ib pressure) for 15 min and cool in a 50°C
water bath.
7.6.3 Reagents Added After Sterilization: Mix 0.24 g nalidixic acid in 5 mL reagent-grade
sterile distilled water, add a few drops of 0. IN NaOH to dissolve; add to the mEI
medium. Add 0.02 g triphenyltetrazolium chloride separately to the mEI medium and
mix.
7.6.4 Alternately, the following solutions may be used:
7.6.4.1 Nalidixic acid: Add 0.48 g of nalidixic acid and 0.4 mL 10 N NaOH to 10 mL of
reagent-grade distilled water and mix. Filter-sterilize the solution, and add 5.2
mL per liter of medium.
7.6.4.2 Triphenyltetrazolium chloride (TTC): Add 0.1 g of TTC to 10 mL of reagent-
grade distilled water, and warm to dissolve. Filter-sterilize the solution, and add
2 mL per liter of medium.
7.6.5 Preparation of mEI Agar Plates: Pour the mEI agar into 9x50 mm petri dishes to a 4-5
mm depth (approximately 4-6 mL), and allow to solidify. Final pH of medium should be
7.1 ± 0.2. Store in a refrigerator.
7.7 Brain Heart Infusion Broth (BHIB) (Difco 0037, BD 4311059)
7.7.1 Composition:
Calf Brain Infusion 200.0 g
Beef Heart Infusion 25 0.0 g
Peptone lO.Og
Sodium Chloride 5.0 g
Disodium Phosphate 2.5 g
Dextrose 2.0 g
Reagent-Grade Distilled Water 1.0 L
7.7.2 Preparation: Dissolve 37 g of dehydrated brain heart infusion in 1 L of reagent grade
water. Dispense in 8-10 mL volumes in screw-cap tubes and autoclave at 121°C (15 Ib
pressure) for 15 min. If the medium is not used the same day as prepared and sterilized,
September 2002 4
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Method 1600
heat in boiling water bath for several min to remove absorbed oxygen, and cool quickly
without agitation, just prior to inoculation. The final pH should be 7.4 ± 0.2.
7.8 Brain Heart Infusion Broth (BHIB) with 6.5% NaC 1
7.8.1 Composition:
BHIB with 6.5% NaCl is the same as BHIB above, but with additional NaCl.
7.8.2 Preparation: Add 60.0 g NaCl per liter of medium. Since most commercially available
dehydrated media contain sodium chloride, this amount is subtracted from the 65 g per
liter required to make a final concentration of 6.5% NaCl.
7.9 Brain Heart Infusion Agar (BHIA) (Difco 0418, BD4311065)
7.9.1 Composition:
BHIA contains the same components as BHIB above with the addition of 15.0 g
of agar per L of BHIB.
7.9.2 Preparation: Suspend 52 g dehydrated BHIA in 1 L of reagent-grade distilled water. Heat
to boiling until the ingredients are dissolved. Dispense 10 mL of medium in screwcap
test tubes, and sterilize for 15 min at 121°C (15 Ib pressure). After sterilization, slant
until solid. Final pH should be 7.4 ± 0.2.
7.10 Bile Esculin Agar (BEA) (Difco 0879)
7.10.1 Composition:
Bacto Beef Extract 3.0 g
Bacto Peptone 5.0g
Bacto Oxgall 40.0 g
Bacto Esculin l.Og
Ferric Citrate 0.5 g
Bacto Agar 15.0g
Reagent-Grade Distilled Water 1.0 L
7.10.2 Preparation: Add 64.0 g dehydrated BEA to 1 L reagent-grade distilled water, and heat
to boiling to dissolve completely. Dispense 10-mL volumes in tubes for slants or larger
volumes into flasks for subsequent plating. Autoclave at 121°C (15 Ib pressure) for 15
min. Overheating may cause darkening of the medium. Cool in a 50°C waterbath, and
dispense into sterile petri dishes. Final pH should be 6.6 ± 0.2. Store in a refrigerator.
8.0 Sample Collection, Preservation, and Storage
8.1 Sampling procedures are described in detail in the USEPA microbiology methods manual,
Section II, A (Reference 18.2). Adherence to sample preservation procedures and holding time
limits is critical to the production of valid data. Samples shall not be analyzed if these conditions
are not met.
8.1.1 Storage Temperature and Handling Conditions
Ice or refrigerate bacteriological samples at a temperature of 1-4°C during transit to the
laboratory. Use insulated containers to assure proper maintenance of storage
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Method 1600
temperature. Take care that sample bottles are not totally immersed in water during
transit or storage.
8.1.2 Holding Time Limitations
Examine samples as soon as possible after collection. Do not hold samples longer than 6
h between collection and initiation of analyses.
9.0 Quality Control
9.1 See recommendations on quality control for microbiological analyses in the USEPA
microbiology methods manual, Part IV, C (Reference 18.2).
10.0 Calibration and Standardization
10.1 Check temperatures in incubators daily to ensure operation within stated limits.
10.2 Check thermometers at least annually against a NIST certified thermometer or one that meets the
requirements of NIST Monograph SP 250-23. Check mercury columns for breaks.
11.0 Procedure
11.1 Prepare the mEI agar as directed in 7.6.
11.2 Mark the petri dishes and report forms with sample identification and sample volumes.
11.3 Place a sterile membrane filter on the filter base, grid-side up and attach the funnel to the base so
that the membrane filter is now held between the funnel and the base.
11.4 Shake the sample bottle vigorously about 25 times to distribute the bacteria uniformly, and
measure the desired volume of sample or dilution into the funnel.
11.5 Select sample volumes based on previous knowledge of the pollution level, to produce 20-60
enterococci colonies on membranes. Sample volumes of 1-100 mL are normally tested at half log
intervals (e.g., 100, 30, 10, 3 mL).
11.6 Smaller sample size or sample dilutions can be used to minimize the interference of turbidity or
for high bacterial densities. Multiple volumes of the same sample or sample dilutions may be
filtered, and the results may be combined.
11.7 Filter the sample, and rinse the sides of the funnel at least twice with 20-30 mL of sterile buffered
rinse water. Turn off the vacuum and remove the funnel from the filter base.
11.8 Use sterile forceps to aseptically remove the membrane filter from the filter base, and roll it onto
the mEI agar to avoid the formation of bubbles between the membrane and the agar surface.
Reseat the membrane if bubbles occur. Run the forceps around the edge of the filter to be sure
that the filter is properly seated on the agar. Close the dish, invert, and incubate at 41 ± 0.5°C for
24 h.
11.9 After incubation, count and record colonies on those membrane filters containing, if practical,
20-60 colonies with a blue halo regardless of colony color as an enterococci. Use magnification
for counting and a small fluorescent lamp to give maximum visibility of colonies.
September 2002
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Method 1600
Figure 1. Enterococci on mEI Agar. Colonies with a blue halo are considered to be enterococci.
12.0 Data Analysis and Calculations
12.1 Use the following general rules to calculate the enterococci count per 100 ml of sample:
12.1.1 Select the membrane filter with an acceptable number of colonies (regardless of colony
color) with a blue halo (20-60). Calculate the number of enterococci per 100 mL
according to the following general formula:
Number of enterococci colonies
Enterococci/100 mL = X 100
Volume of sample filtered (mL)
12.1.2 See the USEPA microbiology methods manual, Part II, Section C, 3.5, for general
counting rules (Reference 18.2).
13.0 Method Performance
13.1 Specificity - The specificity of the medium used in this method is 6.0% false positive and 6.5%
false negative for various environmental water samples (Reference 18.6). The false positive rate
was calculated as the percent of colonies which reacted typically, but did not verify as members
of the enterococcus group. The false negative rate was calculated as the percent of all verified
enterococcus colonies not reacting typically.
13.2 Bias - The persistent positive or negative deviation of the results from the assumed or accepted
true value is not significant (Reference 18.6).
13.3 Precision - The precision among laboratories for marine water and surface water was 2.2% and
18.9% (Reference 18.6).
14.0 Reporting Results
14.1 There should be at least three volumes tested per sample. Report the results as enterococci per
100 mL of sample.
15.0 Verification Procedure
15.1 Colonies of any color having a blue halo after incubation on mEI agar can be verified as
enterococci. Verification of colonies may be required in evidence gathering and it is also
recommended as a means of quality control for the initial use of the test and for changes in
7 September 2002
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Method 1600
sample sites, lots of commercial media, or major ingredients in media compounded in the
laboratory. The verification procedure follows.
15.2 Using a sterile inoculating needle, transfer cells from the centers of at least 10 well-isolated
typical colonies into a BHIB tube and onto a BHIA slant. Incubate broth tubes for 24 h and slants
for48hat35±0.5°C.
15.3 After a 24 h incubation, transfer a loopful of material from each BHIB tube to BEA, BHIB and
BHIB with 6.5% NaCl.
15.3.1 Incubate the BEA and BHIB with 6.5% NaCl at 35 ± 0.5°C for 48 h.
15.3.2 Incubate the BHIB at 45 ± 0.5°C for 48 h.
15.4 Observe for growth.
15.5 After 48 h incubation, apply a Gram stain to growth from each BHIA slant.
15.6 Gram-positive cocci that grow and hydrolyze esculin on BEA (i.e., produce a black or brown
precipitate), and grow in BHIB at 45 ± 0.5°C and BHIB with 6.5% NaCl at 35 ± 0.5°C are
verified as enterococci.
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 It is the laboratory's responsibility to comply with all federal, state, and local regulations
governing waste management, particularly the biohazard and hazardous waste identification rules
and land disposal restrictions, and to protect 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.
17.2 Samples, reference materials, and equipment known or suspected to have viable enterococci
attached or contained must be sterilized prior to disposal.
17.3 Samples preserved with HC1 to pH <2 are hazardous and must be neutralized before being
disposed, or must be handled as hazardous waste.
17.4 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 NW, Washington, DC 20036.
18.0 References
18.1 Annual Book of ASTM Standards, Vol. 11.01, American Society for Testing and Materials,
Philadelphia, PA 19103.
September 2002
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Method 1600
18.2 Bordner, R., J.A. Winter and P.V. Scarpino (eds.), Microbiological Methods for Monitoring the
Environment, Water and Wastes, EPA-600/8-78-017. Office of Research and Development,
USEPA.
18.3 Cabelli, V. J., A. P. Dufour, M. A. Levin, L. J. McCabe, and P. W. Haberman, 1979. Relationship
of Microbial Indicators to Health Effects at Marine Bathing Beaches. Amer. Jour. Public Health.
69:690-696.
18.4 Dufour, A.P. 1984. Health Effects Criteria for Fresh Recreational Waters, EPA-600/1-84-004.
Office of Research and Development, USEPA.
18.5 Levin, M.A., J.R. Fischer and V.J. Cabelli. 1975. Membrane Filter Technique for Enumeration of
Enterococci in Marine Waters. Appl. Microbiol. 30:66-71.
18.6 Messer, J.W. and A.P. Dufour. 1998. A Rapid, Specific Membrane Filtration Procedure for
Enumeration of Enterococci in Recreational Water. Appl. Environ. Microbiol. 64:678-680.
18.7 Improved Enumeration Methods for the Recreational Water Quality Indicators: Enterococci and
Escherichiacoli. 2000. EPA/82 l/R-97/004. Office of Science and Technology, Washington
D.C.
18.8 Method 1600: Membrane Filter Test Method for Enterococci in Water. 1997. EPA-821-R-97-
004. Office of Water, Washington D.C.
18.9 Reagent Chemicals, American Chemical Society Specifications, American Chemical Society,
Washington, DC. For suggestions of the testing of reagents not listed by the American Chemical
Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, UK and the
United States Pharmacopeia.
September 2002
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