f/ EPA
United States Office of Science and EPA/821 /R-97/004
Environmental Protection Technology March 2000
Agency Washington DC 20460
Improved Enumeration Methods
for the Recreational Water
Quality Indicators: Enterococci
and Eschenc\Y\a co\\
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Contents
ABSTRACT 1
1 INTRODUCTION 1
2 SAFETY PRECAUTIONS 3
3 SAMPLE COLLECTION, PRESERVATION,
AND STORAGE 3
3.1 Storage Temperature and Handling
Conditions 3
3.2 Holding Time Limitations 4
4 CALIBRATION AND STANDARDIZATION 4
5 PURITY OF REAGENTS 4
6 PURITY OF WATER 4
7 QUALITY CONTROL 4
8 METHOD PERFORMANCE
CHARACTERISTICS—DEFINITIONS 5
9 TEST METHODS FOR ENTEROCOCCI 5
9.1 Summary 5
9.2 Original Enterococci Method
(Method 1106.1) 6
9.2.1 Equipment and Supplies 6
9.2.2 Reagents and Media 8
9.2.2.1 Phosphate Buffered
Saline 8
9.2.2.2 Phosphate Buffered Dilution
Water 8
9.2.2.3mEAgar 9
9.2.2.4EsculinIronAgar 10
9.2.2.5 Brain Heart Infusion Broth .... 11
9.2.2.6 Brain Heart Infusion Broth
with 6.5% NaCl 11
9.2.2.7 Brain Heart Infusion Agar 11
9.2.2.8BileEsculinAgar 12
9.2.3 Original Enterococci Test Procedure ... 12
9.2.4 Calculation of Results 14
9.2.5 Reporting Results 14
9.2.6 Verification Procedure 14
9.2.7 Method Performance 15
9.3 Modified Enterococci Method
(Method 1600) 15
9.3.1 Equipment and Supplies 15
9.3.2 Reagents and Media 17
9.3.2.1 Phosphate Buffered Saline 17
-------
9.3.2.2 Phosphate Buffered Dilution
Water 18
9.3.2.3mEIAgar 18
9.3.2.4 Brain Heart Infusion Broth .... 19
9.3.2.5 Brain Heart Infusion Broth
with 6.5% NaCl 20
9.3.2.6 Brain Heart Infusion Agar 20
9.3.2.7 Bile Esculin Agar 20
9.3.3 Modified Enterococci Test Procedure .. 21
9.3.4 Calculation of Results 22
9.3.5 Reporting Results 22
9.3.6 Verification Procedure 22
9.3.7 Method Performance 23
10 TEST METHODS FOR E. COLI 23
10.1 Summary 23
10.2 Original E. coliMethod (Method 1103.1) 24
10.2.1 Equipment and Supplies 24
10.2.2 Reagents and Media 26
10.2.2.1 Phosphate Buffered Saline .. 26
10.2.2.2 Phosphate Buffered Dilution
Water 27
10.2.2.3 mTECAgar 28
10.2.2.4 Urea Substrate Medium 28
10.2.2.5 NutrientAgar 29
10.2.2.6 Tryptic Soy Broth;
Trypticase Soy Broth 30
10.2.2.7 Simmons Citrate Agar 30
10.2.2.8 Tryptone 1%;
Tryptophane Broth 30
10.2.2.9 EC Broth 31
10.2.2.10 Oxidase Reagent 31
10.2.2.11 Kovacs Indole Reagent 31
10.2.3 Original E. coliTest Procedure 31
10.2.4 Calculation of Results 33
10.2.5 Reporting Results 34
10.2.6 Verification Procedure 34
10.2.7 Method Performance 35
10.3 Modified E. ozflMethod 35
10.3.1 Equipment and Supplies 36
10.3.2 Reagents and Media 38
10.3.2.1 Phosphate Buffered
Saline 38
10.3.2.2 Phosphate Buffered
Dilution Water 38
10.3.2.3 Modified mTEC Agar 39
10.3.2.4 NutrientAgar 40
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10.3.2.5 Tryptic Soy Broth;
Trypticase Soy Broth 40
10.3.2.6 Simmons Citrate Agar 41
10.3.2.7 Tryptone 1%; Tryptophane
Broth 41
10.3.2.8 EC Broth 41
10.3.2.9 Oxidase Reagent 42
10.3.2.10 Kovacs Indole Reagent 42
10.3.3 Modified £ coA'Test Procedure 42
10.3.4 Calculation of Results 44
10.3.5 Reporting Results 44
10.3.6 Verification Procedure 44
10.3.7 Method Performance 45
REFERENCES 46
Disclaimer 47
Acknowledgments 48
PHOTOGRAPHS
1. Enterococci on mE Agar 13
2. Enterococci on Esculin Iron Agar (EIA) 14
3. Enterococci on mEI Agar 22
4. Urea Substrate Medium 29
5. Escherichia colicolonies on mTEC Agar 33
6. Escherichia coli colonies on an absorbent pad
saturated with Urea Substrate Medium 33
7. Escherichia coli colonies on modified mTEC Agar 43
III
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ABSTRACT
In 1986, the U.S. Environmental Protection Agency
(USEPA) recommended two new indicator organisms for recreational
water quality assessment. They were enterococci (for both marine and
fresh waters) and Escherichia coli (E. coR. for fresh waters only).
These organisms were chosen based on epidemioloncal studies conducted
o _r o
at various beaches in the United States that showed a strongpositive
correlation between the organisms and the occurrence of swimming-
associated gastroenteritis. The two new target organisms required the
use of media designed specifically for enumeration of them from
ambient waters. Since then, these two media (mE Agarfor
enterococci andmTEC Agarfor E. coli) have been improved, resulting
in faster and easier enumeration. The modified media are mEIAgar
for enterococci and modified mTEC Agarfor E. coli.
The purpose of this manual is to provide specific step-by-step
instructions for both the original and the revised test methods (mE,
mEI, mTEC, and modified mTEC) used for the new USEPA
recommended recreational water quality indicators. This manual is
intended to supplement the 1999 USEPA video entitled "Improved
Enumeration Media for the 'Recreational Water Quality Indicators
Enterococci and Escherichia coli." although it may be used without the
video.
1. INTRODUCTION
Epidemiological studies of marine and fresh water
bathing beaches have established a direct relationship
between the density of enterococci and E. coli in water and
the occurrence of swimming-associated gastroenteritis.
Recognition of this relationship has led to the development
of criteria that can be used to establish recreational water
standards (USEPA, 1986a).
In 1976, the U.S. Environmental Protection Agency
recommended fecal coliforms as indicators of recreational
water quality (USEPA, 1976). The guidelines were based on
a 1968 recommendation from the National Technical
Advisory Committee of the Department of the Interior to
the Federal Water Pollution Control Administration. Since
then, USEPA has conducted multi-site epidemiological
studies that found that enterococci have a much higher
correlation with swimming-associated gastroenteritis in both
\ntroduct\on T
-------
fresh and marine water environments than fecal coliforms. E.
coli was found to have a high correlation with gastroenteritis
in fresh water environments only (USEPA, 1986a).
In 1986, USEPA recommended that these two
indicators be used as bacterial water quality indicators to
monitor recreational waters (USEPA, 1986b). This change in
indicators was based on the development of two new media
(Dufour etai, 1981; Levin etai, 1975; USEPA, 1985) for
ambient water, namely, mE Agar for enterococci and mTEC
Agar for E. colt. Since then, these media have been improved,
allowing faster (24-hour) and easier (one-step) enumeration
of the target organisms. The improved media (Messer and
Dufour, 1998; USEPA, 1997) are mEI Agar for enterococci
and modified mTEC Agar for_E. coli. These media are
recommended for enumeration of the target organisms from
ambient waters and are not intended for enumeration from
other water sources, such as drinking water.
Four test methods for measuring bacteriological
densities in ambient waters are described in this manual: the
original and a revised method for detecting enterococci, and
the original and a revised method for detecting E. coli. All
four methods use a membrane filter procedure.
The original test method (Levin etai., 1975; USEPA,
1985) for enterococci was introduced in 1986 (USEPA,
1986b). It uses two media: a primary isolation medium, mE
Agar, and Esculin Iron Agar (EIA) for the confirmation of
colonies on the transferred filter. The revised method,
introduced in 1997 (USEPA, 1997), uses a single medium,
reduces analysis time from 48 hours to 24 hours, and
improves analytical quality. For the revised method (Messer
and Dufour, 1998; USEPA, 1997), the original mE Agar
medium was modified by reducing the concentration of
triphenyltetrazolium chloride and adding a chromogenic
cellobiose analogue, indoxyl-B-D-glucoside.
The mTEC method (Dufour etai., 1981; USEPA,
1985), originally developed in 1981, is a two-step method
utilizing the fermentation of lactose at 44.5°C to detect
thermotolerant coliforms. A second substrate medium
2 \ntroduct\on
-------
containing urea is used to distinguish urease-negativeE. colt
from other thermotolerant coliforms that can hydrolyze urea.
Many laboratories are reluctant to use the mTEC procedure
because of the need to transfer the membrane to a substrate
medium after incubation on the primary medium.
The modified mTEC method is a single-step method
that uses one medium and does not require the transfer of
the membrane filter to another substrate. The modified
medium contains a chromogen, 5-bromo-6-chloro-3-
indolyl-B-D-glucuronide, which is catabolized to glucuronic
acid and a red- or magenta-colored compound by E. mh
that produce the enzyme B-D-glucuronidase.
2 SAFETY PRECAUTIONS
The analyst/technician must know and observe safety
procedures required in a microbiology laboratory while
preparing, using, and disposing of cultures, reagents, and
materials and while operating sterilization equipment.
Mouth-pipetting is prohibited.
3 SAMPLE COLLECTION,
PRESERVATION, AND STORAGE
Sampling procedures are described in detail in the
USEPA microbiology methods manual (Bordner et ai, 1978,
Section II, A). Briefly, samples should be collected in sterile
containers and stored on ice until analyzed. Samples should
not be held longer than 6 h prior to analysis, and analyses
should be completed within 8 h after collection of the
samples (Bordner etal., 1978; CFR, 1999). Adherence to
sample preservation procedures and holding time limits is
critical to the production of valid results. Samples must not
be analyzed if these conditions are not met.
3.1 Storage Temperature and Handling
Conditions
Refrigerate bacteriological samples at a temperature
of 1^4-°C during transit to the laboratory. Use insulated
containers to ensure proper maintenance of storage tempera-
ture. Take care that sample bottles are not totally immersed in
water from melting ice during transit or storage.
\ntroduct\on 3-
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3.2 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 (Bordner et al., 1978; CFR, 1999).
4 CALIBRATION AND
STANDARDIZATION
Check temperatures in incubators daily to ensure
operation within recommended limits.
Check thermometers at least annually against a
National Institute of Standards and Technology (NIST)-
certified thermometer or one that meets the requirements
of NIST Monograph SP 250-23. Check mercury columns
for breaks (APHA, 1998).
5 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 (1981). For suggestions on
testing reagents not listed by the American Chemical
Society, see Rosin (1967) and U.S. Pharmacopeia (1974).
Agar used in the preparation of culture media must
be of microbiological grade. Whenever possible, use
commercial culture media as a means of quality control.
6 PURITY OF WATER
Reagent-grade distilled water should conform to
Specification Dl 193-91, Type II water, as specified by the
American Society for Testing and Materials (1993).
7 QUALITY CONTROL
See recommendations on quality control for microbio-
logical analyses in the USEPA microbiology methods manual,
Part IV, A-C (Bordner etal., 1978).
4 Calibration and Standardization
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8 METHOD PERFORMANCE
CHARACTERISTICS—DEFINITIONS
(APHA, 1998; ASTM, 1993)
Precision—The degree of agreement of repeated
measurements of the same sample, usually reported as the
standard deviation.
Bias—Consistent deviation of measured values from
the true value, caused by systematic errors in a procedure.
Specificity—The ability of a method to select and
distinguish the target bacteria from all others in the same
sample. The specificity of a method is usually reported as
the percentage of false-positive and false-negative results.
False-Positive Rate—The percentage of test results that
are read as positive when they are really negative.
False-Negative Rate—The percentage of test results that
are read as negative when they are really positive.
9 TEST METHODS FOR ENTEROCOCCI
9.1 Summary
Two test methods to detect and enumerate entero-
cocci in water are presented here: the original method and a
revised method, both using a membrane filter (MF) proce-
dure. The two methods provide direct counts of enterococci
in the water based on the number of colonies that develop
on the surface of a membrane filter.
In the original method (Levin etal., 1975; USEPA,
1985), a water sample is filtered through the membrane,
which retains the bacteria. Following filtration, the mem-
brane containing the bacteria is incubated on a selective
medium, mE Agar, for 48 h at 41±0.5°C. The filter is
transferred to EIA and incubated at 41±0.5°C for 20-30 min.
Pink to red enterococci colonies on mE Agar will develop a
black or reddish-brown precipitate on the underside of the
filter on EIA. These colonies are counted under a fluorescent
Method Performance Characteristics 5
-------
lamp and a glass lens (2-5x magnification), or a stereoscopic
microscope may be used.
In the modified method (Messer and Dufour, 1998;
USEPA, 1997), the membrane filter containing the bacterial
cells is placed on mEI Agar and incubated for 24 h at
41 ±0.5°C. All colonies with a blue halo, regardless of colony
color, are recorded as enterococci colonies. A stereoscopic
microscope provides maximum visibility of colonies when
counting. The revised method reduces analysis time from 48
h to 24 h and improves analytical quality.
9.2 Original Enterococci Method
(Method 1106.1)
9.2 A Equ\pment and SuppUes
» Stereoscopic microscope or glass lens (2-5x
magnification).
» Lamp with a cool, white fluorescent bulb and
diffuser.
» Hand tally or electronic counting device.
» Pipets, sterile, To Deliver (T.D.) bacteriological or
Mohr, glass or plastic, of appropriate volume.
» Graduated cylinders, 100—1000 mL, sterile,
covered with aluminum foil or kraft paper.
» Membrane filtration units (filter base and funnel),
sterile; glass, plastic, or stainless steel; wrapped
with aluminum foil or kraft paper to maintain
sterility.
» Ultraviolet unit for sanitizing the filter funnel
between filtrations (optional).
» Line vacuum, electric vacuum pump, or aspirator.
(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.)
» Filter flask, vacuum, usually 1 L, with appropriate
tubing. A filter manifold to hold a number of
filter bases is optional.
6 Qr\g\na\ \Lr\terococc\ Method
-------
Vacuum flask for safety trap, placed between the
filter flask and the vacuum source.
Forceps, straight or curved, with smooth tips to
handle filters without damage.
Ethanol, methanol, or isopropanol in a small, wide-
mouth container, for flame-sterilizing forceps.
Burner, Bunsen or Fisher type, or electric incinerator
unit for sterilizing loops and needles.
Thermometer, checked against a National Institute
of Standards and Technology (NIST)-certified
thermometer, or one traceable to a NIST
thermometer.
Petri dishes, sterile, plastic, 9x50 mm, with tight-
fitting lids.
Bottles, milk dilution, borosilicate glass, screwcap
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.
Flasks, borosilicate glass, screwcap, 250—2000 mL
volume.
Membrane filters, sterile, white, grid-marked,
47-mm diameter, with 0.45±0.02 (am pore size.
Inoculation loops, at least 3-mm diameter, and
needles, nichrome and platinum wire, 26 B&S
gauge, in suitable holders. Sterile disposable
applicator sticks or plastic loops are alternatives to
inoculation loops.
Incubator maintained at 41±0.5°C.
Waterbath maintained at 50°C for tempering agar.
Test tubes, 20x150 mm, borosilicate glass or
plastic.
Test tube caps, aluminum or autoclavable plastic,
for 20-mm diameter test tubes.
Test tubes, borosilicate glass, 16x125 mm or other
appropriate size, with screwcaps.
Whirl-Pak ® bags
Or\g\na\ \Lnterococc\ Method 7
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9.2.2 Reagents and W\ed\a
Preparation of the following reagents and media used
in the original enterococci test are described below:
» Phosphate buffered saline or phosphate buffered
dilution water
» rnE Agar
» Esculin Iron Agar (EIA)
» Brain Heart Infusion Broth (BHIB)
» Brain Heart Infusion Broth (BHIB) with 6.5%
NaCl
» Brain Heart Infusion Agar (BHIA)
» Bile Esculin Agar (BEA)
9.2.2.1 Phosphate Buffered Saline
Ingredients:
sodium dihydrogen phosphate 0.58 g
sodium monohydrogen phosphate 2.5 g
sodium chloride 8.5 g
reagent-grade distilled water 1.0 L
Preparation: Dissolve the ingredients in 1 L of reagent-
grade distilled water in a flask, and dispense in appropriate
amounts for dilutions in screwcap 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
7.4±0.2.
9.2.2.2 Phosphate Buffered Dilution Water
(APHA, 1998; Bordner eta\., 1978)
Stock phosphate buffer solution:
phosphate dihydrogen phosphate 34.0 g
reagent-grade distilled water 500 mL
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 nitration or autoclave at 121 °C (15 Ib
pressure) for 15 min.
8 Qr\g\na\ \Lnterococc\ Method
-------
Stock magnesium chloride solution: Add 38 g anhydrous
MgCl2or 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.
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.
Workingphosphate buffered dilution water. Mix 1.25 mL
of the stock phosphate buffer and 5 mL of the MgQ2 stock
per liter of reagent-grade distilled water. Dispense in
appropriate amounts for dilutions in screwcap 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.
9.2.2.3 mE Agar (Difco 0333-17)
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 0.15 g
agar 15.0 g
reagent-grade distilled water 1.0 L
«:Add 71.2 g dehydrated mE basal
medium to 1 L of reagent-grade distilled water in a flask,
and heat to boiling until the ingredients dissolve using a
magnetic stirrer. Autoclave at 121°C (15 Ib pressure) for 15
min, and cool in a 50°C waterbath.
Reagents added after sterilisation: Mix 0.24 gnalidixic acid
in 5 mL of reagent-grade distilled water, add 0.2 mL of 10 N
NaOH. Allow the mixture to dissolve, and add the mixture
Or\g\na\ \Lnterococc\ Method 9
-------
to the basal medium. Add 0.15 g triphenyltetrazolium
chloride to the basal medium and mix.
Alternately, the following solutions may be used:
(a) Nalidixic add: 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.
(b) Triphenyltetra%>lium chloride (TIC): Add 0.25 g of
TTC to 25 mL of reagent-grade distilled water, and warm to
dissolve. Filter-sterilize the solution, and add 15 mL per liter
of medium.
tare mE Afar: Pour the mE Agar into 9x50 mm
o o
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.
9.2.2.4 Esculin Iron Agar (EIA)
(Difco 0488-15-4)
Ingredients:
esculin 1.0 g
ferric citrate 0.5 g
agar 15.0 g
reagent-grade distilled water 1.0 L
baration: Add 16.5 g dehydrated EIA to 1 L of
reagent-grade distilled water in a flask, and heat to boiling
until the ingredients are dissolved. Autoclave the medium at
121°C (15 Ib pressure) for 15 min, and cool in a 50°C
waterbath. After cooling, pour the medium into 9x50 mm
petri dishes to a depth of 4-5 mm (approximately 4—6 mL),
and allow to solidify. Final pH should be 7.1 ±0.2. Store in a
refrigerator.
10 Or\g\na\ \Lnterococc\ Method
-------
9.2.2.5 Brain Heart Infusion Broth (BHIB)
(Difco 0037-17, BD 4311059)
Ingredients:
calf brain infusion 200.0 g
beef heart infusion 250.0 g
proteose peptone 10.0 g
sodium chloride 5.0 g
disodium phosphate 2.5 g
dextrose 2.0 g
reagent-grade distilled water 1.0 L
Preparation: Dissolve 37 g dehydrated BHIB in 1 L of
reagent-grade distilled water. Dispense in 10-niL volumes
in screwcap tubes, and autoclave at 121°C (15 Ib pressure)
for 15 min. Final pH should be 7.4±0.2.
9.2.2.6 Brain Heart Infusion Broth (BHIB)
with 6.5% NaCl
Ingredients:
BHIB with 6.5% NaCl is the same as BHIB broth above,
but with additional NaCl.
Preparation: Add 60.0 g NaCl per liter of medium.
Since most commercially available dehydrated media already
contain 5 g of sodium chloride, this amount is subtracted
from the 65 g per liter required to make a final concentration
of 6.5% NaCl.
9.2.2.7 Brain Heart Infusion Agar (BHIA)
(Difco 0418-17-7, BD 4311065)
Ingredients:
BHIA contains the same components as BHIB (See
above.) with the addition of 15.0 g agar per liter of BHIB.
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
Or\g\na\ \Lnterococc\ Method ,11
-------
pressure). After sterilization, slant until solid. Final pH
should be 7.4±0.2.
9.2.2.8 Bile Esculin Agar (BEA)
(Difco 0879-02-6)
Ingredients:
Bacto beef extract 3.0 g
Bacto peptone 5.0 g
Bacto oxgall 40.0 g
Bacto esculin 1.0 g
ferric citrate 0.5 g
Bacto agar 15.0 g
reagent-grade distilled water 1.0 L
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.
9.2.3 Or\9\na\ Enterococc\ Test Procedure
» Prepare the mE Agar as directed under "Reagents
and Media" above.
» Mark the petri dishes and report form with the
sample identification and volume.
» 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 held between the
funnel and the base.
» Shake the sample bottle vigorously at least 25
times to distribute the bacteria uniformly, and
measure the desired volume of sample or dilution
into the funnel.
» Select sample volumes based on previous
knowledge of the pollution level, to produce
20-60 enterococci colonies on the membranes.
Sample volumes of 1—100 mL are normally tested
at half-log intervals (e.g., 100, 30,10, 3 mL).
12 Or\g\na\ \Lnterococc\ Method
-------
Smaller sample sizes or sample dilutions can be
used to minimize the interference of turbidity or for
high bacterial densities. Multiple volumes of the
same sample or dilutions of sample may be filtered,
and the results maybe combined.
Filter the sample, and rinse the sides of the funnel
at least twice with 20—30 mL of sterile buffered
water. Turn off the vacuum, and remove the
funnel from the filter base.
Use sterile forceps to aseptically remove the
membrane filter from the filter base, and roll it
onto the mE Agar to avoid the formation of
bubbles between the membrane and the agar
surface. Reseat the filter 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 48 h.
(See photo 1.)
After incubation, transfer the membranes to El A
plates that have been warmed up at room temp-
erature for 20—30 min, and incubate at 41 +0.5°C for
an additional 20—30 min. (See photo 2.)
After the second incubation, count and record
colonies on those membrane filters containing, if
practical, 20-60 pink-to-red colonies with black or
reddish-brown precipitate on the underside of the
membrane. Use magnification for counting and a
small fluorescent lamp to give maximum visibility
of colonies.
Photo 1. Enterococci
on mE Agar. Colo-
nies that are pink to
dark red are consid-
ered to be presump-
tive enterococci.
Or\g\na\ \Lr\terococc\ Method 13
-------
9.2 A Ca\cu\at\on of ResuVls
Use the following general rules to calculate the entero-
cocciper 100 rnL of sample.
Select and count membranes ideally containing 20-60
pink-to-red colonies that form a black or reddish-brown
precipitate on the underside of the filter when placed on EIA.
Calculate the final value using the following formula:
100 (number of enteiococci colonies)
Knterococci/100 mL =
(volume of sample filtered, in mL)
See the USEPA microbiology methods manual, Part II,
Section C, 3.5, forgeneral counting rules (Bordner «"«/.,
1978).
9.2.5 Reporting Resu\ts
There should be at least three volumes tested per
sample. Report the results as enterococci per 100 mL of
sample.
9.2.6 V er\f \cat\on Procedure
Pink-to-red colonies on mE Agar that produce a black
or reddish-brown precipitate after incubation on EIA can be
verified as enterococci. Verification of colonies may be
required in evidence gathering. It is also recommended as a
means of quality control for the initial use of the test and for
changes in sample sites, lots of commercial media, or major
ingredients in media compounded in the laboratory. The
verification procedure follows.
Photo 2. Enterococci
on Esculin Iron
Agar (EIA). Colonies
that are pink to dark
red on mE Agar and
have a reddish
brown to black
precipitate on the
underside of the filter
when placed on EIA
are confirmed as
enterococci.
Or\g\na\ \Lr\terococc\ Method
-------
» 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 agar slants for
48hat35±0.5°C.
» After a 24-h incubation, transfer a loopful of
material from each BHIB tube to each of the
following media:
• BEA and incubate at 35±0.5°C for 48 h.
• BHIB and incubate at 45±0.5°C for 48 h.
• BHIB with 6.5% NaCl and incubate at 35±0.5°
for 48 h.
» Observe for growth on all media.
» After 48-h incubation, apply a Gram stain to
growth from each BHIA slant.
» 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.
9 .2.1 Method Performance (USEP Av, 1985)
The specificity for this medium, as reported for various
environmental water samples, was 10% false-positive and
11.7% false-negative results.
9.3 Modified Enterococci Method
(Method 1600)
9.3 A Equ\pment and SuppUes
» Stereoscopic microscope or glass lens (2-5x
magnification).
» Lamp with a cool, white fluorescent bulb and
diffuser.
» Hand tally or electronic counting device.
» Pipets, sterile, To Deliver (T.D.) bacteriological or
Mohr, glass or plastic, of appropriate volume.
» Graduated cylinders, 100—1000 mL, sterile,
W\od\f\ed \Lr\terococc\ Method .15
-------
covered with aluminum foil or kraft paper.
Membrane filtration units (filter base and funnel),
sterile; glass, plastic, or stainless steel; wrapped with
aluminum foil or kraft paper to maintain sterility.
Ultraviolet unit for sanitizing the filter funnel
between filtrations (optional).
Line vacuum, electric vacuum pump, or aspirator.
(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.)
Filter flask, vacuum, usually 1 L, with appropriate
tubing. A filter manifold to hold a number of
filter bases is optional.
Vacuum flask for safety trap, placed between the
filter flask and the vacuum source.
Forceps, straight or curved, with smooth tips to
handle filters without damage.
Ethanol, methanol, or isopropanol in a small,
wide-mouth container, for flame-sterilizing
forceps.
Burner, Bunsen or Fisher type, or electric
incinerator unit for sterilizing loops and needles.
Thermometer, checked against a National Institute
of Standards and Technology (NIST)-certified
thermometer, or one traceable to a NIST
thermometer.
Petri dishes, sterile, plastic, 9x50 mm, with tight-
fitting lids.
Bottles, milk dilution, borosilicate glass, screwcap
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.
Flasks, borosilicate glass, screwcap, 250-2000 mL
volume.
Membrane filters, sterile, white, grid-marked,
47-mm diameter, with 0.45+0.02 (am pore size.
Inoculation loops, at least 3-|om diameter, and
IE. Modified tnterococci Method
-------
needles, nichrome and platinum wire, 26 B&S
gauge, in suitable holders. Sterile disposable
applicator sticks or plastic loops are alternatives to
inoculation loops.
» Incubatormaintainedat41+0.5°C.
» Waterbath maintained at 50°C for tempering agar.
» Test tubes, 20x150 mm, borosilicate glass or plastic.
» Test tube caps, aluminum or autoclavable plastic, for
20-mm diameter test tubes.
» Test tubes, borosilicate glass, 16x125 mm or other
appropriate size, with screwcaps.
» Whirl-Pak®bags.
9.3.2 Reagents and W\ed\a
Preparation of the following reagents and media used
in the revised enterococci test are described below:
» Phosphate buffered saline or phosphate buffered
dilution water
» mEI Agar
» Brain Heart Infusion Broth (BHIB)
» Brain Heart Infusion Broth (BHIB) with
6.5% NaCl
» Brain Heart Infusion Agar (BHIA)
» Bile Esculin Agar (BEA)
9.3.2.1 Phosphate Buffered Saline
Ingredients:
sodium dihydrogen phosphate 0.58 g
sodium monohydrogen phosphate 2.5 g
sodium chloride 8.5 g
reagent-grade distilled water 1.0 L
Preparation: Dissolve ingredients in 1 L of reagent-
grade distilled water in a flask, and dispense in appropriate
amounts for dilutions in screwcap 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
7.4±0.2.
W\od\f\ed \Lnterococc\ Method ,17
-------
9.3.2.2 Phosphate Buffered Dilution Water
(APHA, 1998; Bordner ela\.f 1978)
Stock phosphate buffer solution:
phosphate dihydrogen phosphate 34.0 g
reagent-grade distilled water 500 mL
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.
Stock magnesium chloride solution: Add 38 g anhydrous
MgCl2or 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.
Storage of stock solutions: After sterilization, store the
stock solution 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.
Workingphosphate buffered dilution water: Mix 1.25 mL
of the stock phosphate buffer and 5 mL of the MgQ2 stock
per liter of reagent-grade distilled water. Dispense in
appropriate amounts for dilutions in screwcap 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.
9.3.2.3 mEI Agar
Ingredients of basal medium (mEAgar, Difco 0333-17):
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 0.15 g
agar 15.0 g
reagent-grade distilled water 1.0 L
11 Modified tnterococci Method
-------
baration of mEI medium: Add 71.2 g dehydrated mE
basal medium plus 0.75 g indoxyl-6-D-glucoside to 1 L of
reagent-grade distilled water in a flask, and heat to boiling
until the ingredients dissolve. Autoclave at 121 °C (15 Ib
pressure) for 15 min, and cool in a 50°C waterbath.
^agents added after sterilisation: Mix 0.24 g nalidixic
acid in 5 mL of reagent-grade distilled water; add a few
drops of 0.1 N NaOH to dissolve; add to the mEI medium.
Add 0.02 g triphenyltetrazolium chloride separately to the
mEI medium and mix.
Alternately, the following solutions may be used:
(a) Nalidixic add: 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.
(b) Triphenyltetra^pHum 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.
Preparation of mEI agarplates: 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.
9.3.2.4 Brain Heart Infusion Broth (BHIB)
(Difco 0037-17, BD 4311059)
Ingredients:
calf brain infusion 200.0 g
beef heart infusion 250.0 g
proteose peptone 10.0 g
sodium chloride 5.0 g
disodium phosphate 2.5 g
dextrose 2.0 g
reagent-grade distilled water 1.0 L
W\od\f\ed \Lnterococc\ Method ,19
-------
Preparation: Dissolve 37 gdehydrated BHIB in 1 L of
reagent-grade distilled water. Dispense in 10-mL volumes in
screwcap tubes, and autoclave at 121 °C (15 Ib pressure) for 15
min. Final pH should be 7.4+0.2.
9.3.2.5 Brain Heart Infusion Broth (BHIB)
with6.5%NaCl
Ingredients: BHIB with 6.5% NaCl is the same as
BHIB described above, but with additional NaCl.
baration: Add 60.0 g NaCl per liter of medium.
Since most commercially available dehydrated media already
contain 5 g of sodium chloride, this amount is deducted
from the 65 g per liter required to make a final concentra-
tion of 6.5% NaCl.
9.3.2.6 Brain Heart Infusion Agar (BHIA)
(Difco 0418-17-7, BD 4311065)
Ingredients: BHIA contains the same components as
BHIB (See above.) with the addition of 15.0 g agar per liter
of BHIB.
baration: 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.
9.3.2.7 Bile Esculin Agar (BEA)
(Difco 0879-02-6)
Ingredients:
Bacto beef extract 3.0 g
Bacto peptone 5.0 g
Bacto oxgall 40.0 g
Bacto esculin 1.0 g
ferric citrate 0.5 g
Bacto agar 15.0 g
reagent-grade distilled water 1.0 L
Preparation: Add 64.0 g dehydrated BEA to 1 L of
reagent-grade distilled water, and heat to boiling to dissolve
completely. Dispense in 10-mL volumes in tubes for slants or
20 Modified tnterococci Method
-------
larger volumes into flasks for subsequent plating. Autoclave
at 121 °C (15 Ib pressure) fbrlSmin. 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.
9.3.3 W\od\f \ed Enterococc\ Test Procedure
» Prepare the mEI Agar as directed above.
» Mark the petri dishes and report form with the
sample identification and volume.
» 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 held between the
funnel and the base.
» Shake the sample bottle vigorously at least 25
times to distribute the bacteria uniformly, and
measure the desired volume of sample or dilution
into the funnel.
» 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).
» Smaller sample sizes or sample dilutions can be
used to minimize the interference of turbidity or
for high bacterial densities. Multiple volumes of
the same sample or dilutions of sample may be
filtered, and the results may be combined.
» 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.
» 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. (See photo 3.)
W\od\f\ed \Lnterococc\ Method 21
-------
Photo 3. Entero-
cocci on mEI Agar.
Colonies with a
blue halo are
considered to be
enterococci.
» After incubation, count and record as enterococci any
colonies (regardless of color) with a blue halo on
the membranes, ideally containing 20-60 colonies.
Use magnification for counting and a small
fluorescent lamp to give maximum visibility of
colonies.
9.3.4 Ca\cu\at\on of ResuVls
To calculate the number of enterococci per 100 mL of
sample:
» Select and count as enterococci any colonies
(regardless of color) with a blue halo on the
membranes, ideally containing 20—60 colonies.
» Calculate the final value using the following
formula:
Enteiococci/100 mL =
100 (number of enteiococci colonies)
(volume of sample filtered, in mL)
See the USEPA microbiology methods manual, Part II,
SectionC,3.5,forgeneralcountingrules (Botdnetetal., 1978).
9.3.5 Reporting ResuVls
There should be at least three volumes tested per
sample. Report the results as enterococci per 100 mL of
sample.
9.3.6 V er\f \cation Procedure
Colonies with a blue halo, regardless of color, can be
verified as enterococci. Verification of colonies may be
W\od\f\ed \Lr\terococc\ Method
-------
required in evidence gathering. It is also recommended as a
means of quality control for the initial use of the test and for
changes in sample sites, lots of commercial media, or major
ingredients in media compounded in the laboratory. The
verification procedure follows.
» 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 agar slants for
48hat35±0.5°C.
» After a 24-h incubation, transfer a loopful of
material from each BHIB tube to each of the
following media:
• BEA and incubate at 35±0.5°C for 48 h.
• BHIB and incubate at 45±0.5°C for 48 h.
• BHIB with 6.5% NaCl and incubate at
35±0.5°C for 48 h.
» Observe for growth on all media.
» After 48-h incubation, apply a Gram stain to
growth from each BHIA slant.
» 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.
9 .3 .1 Method Performance (W\esser and Duf our ,
•\998-, USEPAv,
The false-positive and false-negative rates, reported for
various environmental water samples, were 6.0% and 6.5%,
respectively. The precision among laboratories for marine
water and surface water was 2.2% and 18.9%, respectively, and
the bias was not significant.
10 TEST METHODS FOR t.COU
10.1 Summary
Two test methods for the detection and enumeration
of Escherichia mh in water are presented here. The original
mTEC Agar enumeration method (Dufour et al., 1981) for
Test Methods fort. co\\ ,23
-------
E. coli was introduced by EPA in 1986 (USEPA, 1986b). The
revised method was developed in 1998 by the Agency. Both
the mTEC and modified mTEC Agar methods use the
membrane filter procedure. The two membrane filter
methods provide a direct count of E. mh in water based on
the development of colonies that grow on the surface of a
membrane filter.
In both methods, a water sample is filtered through
the membrane, which retains the bacteria. After filtration, the
membrane containing the bacteria is placed on a selective and
differential medium, either mTEC (Dufour etaL, 1981;
USEPA, 1985), or modified mTEC Agar, incubated at
35±0.5°C for 2 h to resuscitate the injured or stressed bacteria,
and then incubated at 44.5±0.2°C for 22 h. With the original
method, the filter is transferred from mTEC Agar to a filter
pad saturated with Urea Substrate Medium. After 15—20
min, yellow, yellow-green, or yellow-brown colonies on
mTEC are counted with the aid of a fluorescent lamp and a
glass lens (2—5x magnification) or stereoscopic microscope.
The modified method eliminates the transfer of the mem-
brane filter to another substrate. The target colonies on
modified mTEC Agar are red in color after the incubation
period.
10.2 Original E. co\\ Method (Method 1103.1)
"\ 0.2 A Equ\pment and SuppUes
» Glass lens, 2—5x magnification, or stereoscopic
microscope.
» Lamp with cool, white fluorescent bulb and diffuser.
» Hand tally or electronic counting device.
» Pipets, sterile, To Deliver (T.D.) bacteriological or
Mohr, glass or plastic, of appropriate volume.
» Graduated cylinders, 100-1000 mL, s terile, covered
with aluminum foil or kraft paper.
» Membrane filtration units (filter base and funnel),
sterile; glass, plastic, or stainless steel; wrapped
with aluminum foil or kraft paper to maintain
sterility.
» Ultraviolet unit for s anitizing the filter funnel
between filtrations (optional).
24 Original \L. co\\ Method
-------
Line vacuum, electric vacuum pump, or aspirator.
(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.)
Filter flask, vacuum, usually 1 L, with appropriate
tubing. A filter manifold to hold a number of
filter bases is optional.
Vacuum flask for safety trap, placed between the
filter flask and the vacuum source.
Forceps, straight or curved, with smooth tips to
handle filters without damage.
Ethanol, methanol, or isopropanol in a small,
wide-mouth container, for flame-sterilizing
forceps.
Burner, Bunsen or Fisher type, or electric
incinerator unit for sterilizing inoculation loops.
Thermometer, checked against a National Institute
of Standards and Technology (NIST)-certified
thermometer, or one traceable to a NIST
thermometer.
Petri dishes, sterile, plastic, 9x50 mm, with tight-
fitting lids; or 15x60 mm, glass or plastic, with
loose-fitting lids; or 15x100 mm.
Bottles, milk dilution, borosilicate glass, screwcap
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.
Flasks, borosilicate glass, screwcap, 250—2000 mL
volume.
Membrane filters, sterile, white, grid-marked,
47-mm diameter, with 0.45±0.02-|om pore size.
Absorbent pads, sterile, 47-mm diameter (usually
supplied with membrane filters).
Inoculation loops, at least 3-mm diameter, and
needles, nichrome and platinum wire, 26 B&S
gauge, in suitable holders. Disposable applicator
sticks or plastic loops are alternatives to inoculation
loops. Note: A platinum loop is required for the
cytochrome oxidase test in the verification
procedure.
Original \L. co\\ Method 25
-------
» Incubator maintained at 35±0.5°C, with
approximately 90% humidity if loose-lidded petri
dishes are used.
» Waterbath maintained at 44.5±0.2°C.
» Waterbath maintained at 50°C for tempering agar.
» Test tubes, 20x150 mm, borosilicate glass or plastic.
» Test tubes, 10x75 mm, borosilicate glass.
» Test tube caps, aluminum or autoclavable plastic, for
20-mm diameter test tubes.
» Test tubes, 16x125 mm or other appropriate size,
with screwcaps.
» Filter paper.
» Whirl-Pak® bags.
"\0.2..2. Reagents and W\ed\a
Preparation of the following reagents and media used
in the original E. coli test are described below:
» Phosphate buffered saline or phosphate buffered
dilution water
» mTEC Agar
» Urea Substrate Medium
» Nutrient Agar
» Tryptic Soy Broth or Trypticase Soy Broth
» Simmons Citrate Agar
» Tryptone 1% or Tryptophane Broth
» EC Broth
» Oxidase Reagent
» Kovacs Indole Reagent
10.2.2.1 Phosphate Buffered Saline
Ingredients:
sodium dihydrogen phosphate 0.58 g
sodium monohydrogen phosphate 2.5 g
sodium chloride 8.5 g
reagent-grade distilled water 1.0 L
26 Original \L. co\\ Method
-------
n: Dissolve the ingredients above in 1 L of
reagent-grade distilled water in a flask, and dispense in
appropriate amounts for dilutions in screwcap 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 7.4±0.2.
10.2.2.2 Phosphate Buffered Dilution Water
(APHA, 1998; Bordner ela\.f 1978)
Stock phosphate buffer solution:
phosphate dihydrogen phosphate 34.0 g
reagent-grade distilled water 500 mL
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.
Stock magnesium chloride solution: Add 38 g anhydrous
MgCl2or 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.
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.
Workingphosphate buffered dilution water: Mix 1.25 mL
of the stock phosphate buffer and 5 mL of the MgQ2 stock
per liter of reagent-grade distilled water. Dispense in
appropriate amounts for dilutions in screwcap 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.
Original \L. co\\ Method ,27
-------
10.2.2.3 mTEC Agar (Difco 0334-15-0)
Ingredients:
proteose peptone #3 5.0 g
yeast extract 3.0 g
lactose 10.0 g
NaQ 7.5 g
dipotassium phosphate 3.3 g
monopotassium phosphate 1.0 g
sodium lauryl sulfate 0.2 g
sodium desoxycholate 0.1 g
brom cresol purple 0.08 g
brom phenol red 0.08 g
agar 15-Og
reagent-grade distilled water 1.0 L
Preparation: Add 45.3 g dehydrated mTEC Agar to
1 L of reagent-grade distilled water in a flask, and heat to
boiling until the ingredients dissolve. Autoclave at 121°C (15
Ib pressure) for 15 min, and cool in a 50°C waterbath. Pour
the medium into each 9x50 mm culture dish to a
4-5 mm depth (approximately 4-6 mL), and allow to
solidify. Final pH should be 7.3±0.2. Store in a refrigerator.
10.2.2.4 Urea Substrate Medium
Ingredients:
urea 2.0 g
phenol red 0.01 g
reagent-grade distilled water 100.0 mL
Preparation: Add dry ingredients to 100 mL reagent-
grade distilled water in a flask. Stir to dissolve, and adjust to
pH 3^1 with 1 N HC1. The substrate solution should be a
straw-yellow color at this pH. (See photo 4.)
28 Original t. co\\ Method
-------
10.2.2.5
Nutrient Agar (Difco 0001 -17-0,
BD4311472)
peptone
beef extract
agar
reagent-grade distilled water
5.0 g
3.0 g
15.0 g
1.0 L
«: Add 23 g of dehydrated Nutrient Agar to 1
L of reagent-grade distilled water, and mix well. Heat to
boiling to dissolve the agar completely. Dispense in screwcap
tubes, and autoclave at 121 °C (15 Ib pressure) for 15 min.
Remove the tubes and slant. Final pH should be 6.8+0.2.
Photo 4. Urea
Substrate Medium.
After adjusting the pH
of the medium to 3-
4, the Urea Substrate
Medium should be
straw-yellow in color.
Original \L. co\\ Method
-------
10.2.2.6 Tryptic Soy Broth (Difco 0370-17);
Trypticase Soy Broth (BD 99071)
Ingredients:
tryptone or trypticase 17.0 g
soytone or phytone 3.0 g
sodium chloride 5.0 g
dextrose 2.5 g
dipotassium phosphate 2.5 g
reagent-grade distilled water 1.0 L
baration: Add 30 g of dehydrated Tryptic/Trypticase
Soy Broth to 1 L of reagent-grade distilled water. Warm the
broth, and mix gently to dissolve the medium completely.
Dispense in screwcap tubes, and autoclave at 121°C (15 Ib
pressure) for 15 min. Final pH should be 7.3+0.2.
10.2.2.7 Simmons Citrate Agar (BD 4311620,
Difco 0091-17-1)
magnesium sulfate 0.2 g
mono ammonium phosphate 1-Og
dipotassium phosphate 1.0 g
sodium citrate 2.0 g
sodium chloride 5.0 g
brom thymol blue 0.08 g
agar 15.0g
reagent-grade distilled water 1.0 L
Preparation: Add 24.2 g Simmons Citrate Agar to 1 L
of reagent-grade distilled water. Heat to boiling to dissolve
completely. Dispense into screwcap tubes, and autoclave at
121°C (15 Ib pressure) for 15 min. Cool the tubes and slant.
Final pH should be 6.8±0.2.
10.2.2.8 Tryptone 1% (Difco 0123-01);
Tryptophane Broth (BD 4321717 and
4321718)
Ingredients:
tryptone or trypticase peptone 10.0 g
reagent-grade distilled water 1.0 L
30 Original t. co\\ Method
-------
Add 10 g tryptone or trypticase peptone to
1 L of reagent-grade distilled water, and heat, mixing until
dissolved. Dispense in 5-mL volumes into tubes, and
autoclave at 121°C (15 Ib pressure) for 15 min. Final pH
should be 7.2±0.2.
10.2.2.9 EC Broth (Difco 0314-01-0,
BD4311187)
Ingredients:
tryptose or trypticase peptone 20.0 g
lactose 5.0 g
bile salts no. 3 or bile salts mixture 1.5 g
dipotassium phosphate 4.0 g
monopotassium phosphate 1.5 g
sodium chloride 5.0 g
reagent-grade distilled water 1.0 L
Preparation: Add 37 g dehydrated EC Broth to 1 L of
reagent-grade distilled water, and warm to dissolve com-
pletely. Dispense into fermentation tubes (20x150 mm tubes
containing inverted 10x75 mm vials). Autoclave at 121 °C (15
Ib pressure) for 15 min. Final pH should be 6.9+0.2.
10.2.2.10 Oxidase Reagent
Ingredients:
N, N, N', N'-tetramethyl-p-phenylenediamine
dihydrochloride, 1% aqueous solution
(1 g per 100 mL sterile reagent-grade distilled water).
10.2.2.11 Kovacs Indole Reagent
Ingredients:
p-dimethylaminobenzaldehyde 10.0 g
amyl or isoamyl alcohol 150.0 mL
concentrated (12 M) hydrochloric acid 50.0 mL
Preparation: Dissolve p-dimethylaminobenzaldehyde
in alcohol, slowly add hydrochloric acid, and mix.
"\0.2.3 Or\g\na\ E. co\\ Test Procedure
» Prepare mTEC Agar and Urea Substrate Medium
as directed above in the "Reagents and Media"
section.
Original \L. co\\ Method ,31
-------
Mark the petri dish and report form with the
sample identification and volume.
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 held between the
funnel and the base.
Shake the sample bottle vigorously at least 25
times to distribute the bacteria uniformly, and
measure the desired volume of sample or dilution
into the funnel.
Select sample volumes based on previous
knowledge of the pollution level, to produce
20—80 E. mh colonies on the membranes. Sample
volumes of 1-100 mL are normally tested at half-
log intervals (e.g., 100, 30,10, 3 mL).
Smaller sample sizes 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 maybe filtered,
and the results maybe combined.
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.
Use sterile forceps to aseptically remove the
membrane filter from the filter base, and roll it
onto the mTEC 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 35±0.5°C
for 2 h.
After a 2-h incubation at 35±0.5°C, transfer the
plate to a Whirl-Pak® bag, seal the bag, place the
bag with the plate inverted in a test-tube rack, and
put the rack in a 44.5±0.2°C waterbath for 22-24 h.
After 22-24 h, remove the plate from the waterbath.
Place an absorbent pad in a new petri dish or the lid
of the same petri dish, and saturate the pad with
Urea Substrate Medium. Aseptically transfer the
32 Original t. co\\ Method
-------
membranes from mTEC Agar to the absorbent
pads saturated with Urea Substrate Medium, and
allow to sit at room temperature for 15-20 min.
(See photo 5.)
» After incubation on the urea substrate at room
temperature, count and record the number of
yellow, yellow-green, or yellow-brown colonies on
the membrane filters, ideally containing 20-80
colonies. (See photo 6.)
"\ 0.2..4 Ca\cu\at\on of Resu\ts
Select the membrane filter with an acceptable number
of yellow, yellow-green, or yellow-brown colonies (20—80) on
the urea substrate, and calculate the number of-E. «>/z per 100
mL according to the following general formula:
Photo 5. Escher\ch\a
co\\ colonies on
mTEC Agar. Colo-
nies that are yellow,
yellow-green, or
yellow-brown are
E. coll,
Photo 6. EschencVYia
co\\ colonies on an
absorbent pad
saturated with Urea
Substrate Medium.
E. co\\ colonies
remain yellow,
yellow-green, or
yellow-brown when
the filter is placed on
the Urea Substrate
Medium, while
nontarget colonies
turn pink or purple.
Original \L. co\\ Method
-------
100 (number of E. mli colonies counted)
E. foli/WOmL= i ;
(volume of sample filtered, in mL)
See the USEPA microbiology methods manual, Part II,
Section C, 3.5, for general counting rules (Bordner «"«/.,
1978).
"\ 0.2.5 Reporting Resu\ts
There should be at least three volumes filtered per
sample. Report the results as E. mlipet 100 mL of sample.
"\ 0.2.6 V er\f \cat\on Procedure
Yellow, yellow-green, or yellow-brown colonies from
the urease test can be verified as E. mli. Verification of
colonies may be required in evidence gathering and is also
recommended as a means of quality control for the initial use
of the test and for changes in sample sites, lots of commer-
cial media, or major ingredients in media compounded in the
laboratory. The verification procedure follows.
» Using a sterile inoculation loop, transfer growth
from the centers of at least 10 well-isolated colonies
to Nutrient Agar plates or slants and to Trypticase
Soy Broth. Incubate the agar and broth cultures for
24hat35±0.5°C.
» After incubation, remove a loopful of growth from
the Nutrient Agar slant with a platinum loop, and
deposit it on the surface of a piece of filter paper
that has been saturated with freshly prepared
Cytochrome Oxidase Reagent. If the spot where
the bacteria were deposited turns deep purple within
15 seconds, the test is positive.
» Transfer growth from the Trypticase Soy Broth tube
to Simmons Citrate Agar, Tryptone Broth, and an
EC Broth fermentation tube.
• Incubate the Simmons Citrate Agar and Tryptone
Broth for 48 h at 35±0.5°C.
• Incubate the EC Broth at 44.5±0.2°C in a
waterbath for 24 h. The water level must be
above the level of the EC Broth in the tube.
• Add 0.5 mL of Kovacs Indole Reagent to the
48-h Tryptone Broth culture, and shake the tube
34 Original \L. co\\ Method
-------
gently. A positive test for indole is indicated by a
deep red color which develops in the alcohol layer
on top of the broth.
• E. colite EC gas-positive, indole-positive, and
oxidase-negative, and does not utilize citrate (i.e.,
the medium remains green).
» Alternately, commercially available multi-test
identification systems may be used to verify
colonies. Inoculate the colonies into an
identification system forEnterobacteriaceae that
includes lactose fermentation, O-nitrophenyl-B-D-
galactopyranoside (ONPG), and cytochrome oxidase
test reactions.
"\O.Z.l Method Performance (Dufour et a\., "\98V,
USEPAv, 1985)
Using multilaboratory testing, the precision of the
mTEC method was found to be fairly representative of what
would be expected from counts with a Poisson distribution.
The bias of the mTEC method has been reported to be —2%
of the true value. Because of the instability of microbial
populations in water, each laboratory analyzed its own
samples. Therefore, no full measure of recovery or bias was
possible. However, all laboratories analyzed a single surrogate
sample prepared from a freeze-dried culture of E. colt. The
mean count (X) and the overall standard deviation of the
counts (ST) (including the variability among laboratories for
this standardized-E. colt sample) were 31.6 colonies/
membrane and 7.61 colonies/membrane, respectively.
The false-positive rate reported for mTEC medium
averaged 9% for marine and fresh water samples. Less than
1% of the E. colt colonies observed gave a false-negative
reaction.
The upper counting limit (i.e., the number of colonies
above which unacceptable counting errors occur) for E. colt
on mTEC Agar has been reported as 80 colonies per filter.
10.3 Modified E. co\\ Method
The revised Escherichia colt method is a single-step
method that uses one medium, modified mTEC Agar, and
Modified \L. co\\ Method 35
-------
does not require the transfer of the membrane filter to
another medium or other substrate. The modified medium
contains a chromogen (5-bromo-6-chloro-3-indolyl-B-D-
glucuronide), which is catabolized to glucuronic acid and a
red- or magenta-colored compound by.E. coli that produce
the enzyme B-D-glucuronidase.
The apparatus and equipment, and sampling, filtration,
and verification procedures for this modified mTEC method
are identical to those of the original mTEC method.
"\ 0.3 A Equ\pment and SuppUes
» Glass lens, 2—5x magnification, or stereoscopic
microscope.
» Lamp with cool, white fluorescent bulb and
diffuser.
» Hand tally or electronic counting device.
» Pipets, sterile, To Deliver (T.D.) bacteriological or
Mohr, glass or plastic, of appropriate volume.
» Graduated cylinders, 100-1000 mL, s terile, covered
with aluminum foil or kraft paper.
» Membrane filtration units (filter base and funnel),
sterile, glass, plastic, or stainless steel, wrapped with
aluminum foil or kraft paper to maintain sterility.
» Ultraviolet unit for s anitizing the filter funnel
between nitrations (optional).
» Line vacuum, electric vacuum pump, or aspirator.
(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.)
» Filter flask, vacuum, usually 1 L, with appropriate
tubing. A filter manifold to hold a number of filter
bases is optional.
» Vacuum flask for safety trap, placed between the
filter flask and the vacuum source.
» Forceps, straight or curved, with smooth tips to
handle filters without damage.
» Ethanol, methanol, or isopropanol in a small,
wide-mouth container, for flame-sterilizing
forceps.
36 Modified t. co\\ Method
-------
Burner, Bunsen or Fisher type, or electric incinerator
unit for sterilizing inoculation loops.
Thermometer, checked against a National Institute
of Standards and Technology (NIST)-certified
thermometer, or one traceable to a NIST
thermometer.
Petri dishes, sterile, plastic, 9x50 mm, with tight-
fitting lids; or 15x60 mm, glass or plastic, with
loose-fitting lids; or 15x100 mm.
Bottles, milk dilution, borosilicate glass, screwcap
with neoprene liners, marked at 99 mL for 1:100
dilutions. Dilution bottles marked at 90 mL or
tubes marked at 9 mL maybe used for 1:10
dilutions.
Flasks, borosilicate glass, screwcap, 250-2000 mL
volume.
Membrane filters, sterile, white, grid-marked,
47-mm diameter, with 0.45+0.02 (am pore size.
Inoculation loops, at least 3-mm diameter, and
needles, nichrome and platinum wire, 26 B&S
gauge, in suitable holders. Disposable applicator
sticks or plastic loops are alternatives to
inoculation loops. Note: A platinum loop is
required for the cytochrome oxidase test in the
verification procedure.
Incubator maintained at 35±0.5°C, with
approximately 90% humidity if loose-lidded petri
dishes are used.
Waterbath maintained at 44.5±0.2°C.
Waterbath maintained at 50°C for tempering agar.
Test tubes, 20x150 mm, borosilicate glass or
plastic.
Test tubes, 10x75 mm, borosilicate glass.
Test tube caps, aluminum or autoclavable plastic,
for 20-mm diameter test tubes.
Test tubes, 16x125 mm or other appropriate size,
with screwcaps.
Filter paper.
Whirl-Pak® 1
Modified \L. co\\ Method 37
-------
"\0.3.2. Reagents and W\ed\a
Preparation of the following reagents and media used
in the revised E. mh test are presented below:
» Phosphate buffered saline or phosphate buffered
dilution water
» Modified mTEC Agar
» Nutrient Agar
» Tryptic Soy Broth or Trypticase Soy Broth
» Simmons Citrate Agar
» Tryptone 1% or Tryptophane Broth
» EC Broth
» Oxidase Reagent
» Kovacs Indole Reagent
10.3.2.1 Phosphate Buffered Saline
Ingredients:
sodium dihydrogen phosphate 0.58 g
sodium monohydrogen phosphate 2.5 g
sodium chloride 8.5 g
reagent-grade distilled water 1.0 L
Preparation: Dissolve the ingredients above in 1 L of
reagent-grade distilled water in a flask, and dispense in
appropriate amounts for dilutions in screwcap 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 7.4±0.2.
10.3.2.2 Phosphate Buffered Dilution Water
(APHA, 1998; Bordner ela\.f 1978)
Stock phosphate buffer solution:
phosphate dihydrogen phosphate 34.0 g
reagent-grade distilled water 500 mL
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.
38 Modified t. co\\ Method
-------
Stock magnesium chloride solution: Add 38 g anhydrous
MgCl2or 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.
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.
Workingphosphate buffered dilution water: Mix 1.25 mL
of the stock phosphate buffer and 5 mL of the MgQ2 stock
per liter of reagent-grade distilled water. Dispense in
appropriate amounts for dilutions in screwcap 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.
10.3.2.3 Modified mTEC Agar
Ingredients:
proteose peptone #3 5.0 g
yeast extract 3.0 g
lactose 10.0 g
Nad 7.5 g
dipotassium phosphate 3.3 g
monopotassium phosphate 1.0 g
sodium lauryl sulfate 0.2 g
sodium desoxycholate 0.1 g
chromogen (5-bromo-6-chloro-3-
indolyl-6-D-glucuronide) 0.5 g
agar 15.0 g
reagent-grade distilled water 1.0 L
•baration: Add 45.6 g dehydrated modified mTEC
medium to 1 L of reagent-grade distilled water in a flask,
and heat to boiling until the ingredients dissolve. Autoclave
at 121°C (15 Ib pressure) for 15 min, and cool in a 50°C
waterbath. Pour the medium into each 9x50 mm culture dish
Modified \L. co\\ Method 39
-------
to a 4-5 mm depth (approximately 4-6 mL), and allow to
solidify. Final pH should be 7.3+0.2. Store in a
refrigerator.
10.3.2.4 Nutrient Agar (Difco 0001 -17-0,
BD4311472)
Ingredients:
peptone 5.0 g
beef extract 3.0 g
agar 15.0 g
reagent-grade distilled water 1.0 L
Preparation: Add 23 g dehydrated Nutrient Agar to 1 L
of reagent-grade distilled water, and mix well. Heat to boil-
ing to dissolve the agar completely. Dispense in screwcap
tubes, and autoclave at 121°C (15 Ib pressure) for 15 min.
Remove the tubes and slant. Final pH should be 6.8+0.2.
10.3.2.5 Tryptic Soy Broth (Difco 0370-17);
Trypticase Soy Broth (BD 99071)
Ingredients:
tryptone or trypticase 17.0 g
soytone or phytone 3.0 g
sodium chloride 5.0 g
dextrose 2.5 g
dipotassium phosphate 2.5 g
reagent-grade distilled water 1.0 L
ttaration: Add 30 g dehydrated Tryptic/Trypticase
Soy Broth to 1 L of reagent-grade distilled water. Warm the
broth, and mix gently to dissolve the medium completely.
Dispense in screwcap tubes, and autoclave at 121°C (15 Ib
pressure) for 15 min. Final pH should be 7.3±0.2.
40 Modified \L. co\\ Method
-------
10.3.2.6 Simmons Citrate Agar (BD 4311620,
Difco 0091-17-1)
Ingredients:
magnesium sulfate 0.2 g
mono ammonium phosphate 1-Og
dipotassium phosphate 1.0 g
sodium citrate 2.0 g
sodium chloride 5.0 g
brom thymol blue 0.08 g
agar 15.0 g
reagent-grade distilled water 1.0 L
Preparation: Add 24.2 g Simmons Citrate Agar to 1 L of
reagent-grade distilled water. Heat to boiling to dissolve
completely. Dispense in screwcap tubes, and autoclave at
121°C (15 Ib pressure) for 15 min. Cool the tubes in a 50°C
waterbath and slant. Final pH should be 6.8+0.2.
10.3.2.7 Tryptone1% (Difco 0123-01);
Tryptophane Broth (BD 4321717 and
4321718)
Ingredients:
tryptone or trypticase peptone 10.0 g
reagent-grade distilled water 1.0 L
Preparation: Add 10 g tryptone or trypticase peptone
to 1 L of reagent-grade distilled water, and heat, mixing
until dissolved. Dispense in 5-mL volumes in tubes, and
autoclave at 121°C (15 Ib pressure) for 15 min. Final pH
should be 7.2±0.2.
10.3.2.8 EC Broth (Difco 0314-01-0,
BD4311187)
Ingredients:
tryptose or trypticase peptone 20.0 g
lactose 5.0 g
bile salts #3 or bile salts mixture 1.5 g
dipotassium phosphate 4.0 g
monopotassium phosphate 1.5 g
sodium chloride 5.0 g
reagent-grade distilled water 1.0 L
Modified \L. co\\ Method ,41
-------
Preparation: Add 37 g dehydrated EC medium to 1 L
of reagent-grade distilled water, and warm to dissolve
completely. Dispense into fermentation tubes (20x150 mm
tubes containing inverted 10x75 mm vials). Autoclave at
121°C (15 Ib pressure) for 15 min. Final pH should be
6.9±0.2.
10.3.2.9 Oxidase Reagent
Ingredients:
N, N, N', N'-tetramethyl-p-phenylenediamine
dihydrochloride, 1% aqueous solution (1 gper 100 mLsterile
reagent-grade distilled water).
10.3.2.10 Kovacs Indole Reagent
Ingredients:
p-dimethylaminobenzaldehyde 10 g
amyl or isoamyl alcohol 150 mL
concentrated (12 M) hydrochloric acid 50 mL
baration: Dissolve p-dimethylaminobenzaldehyde
in alcohol, slowly add hydrochloric acid, and mix.
"\0.3.3 W\od\f\ed E. co\\ Test Procedure
» Prepare the modified mTEC Agar as directed
above in the "Reagents and Media" section.
» Mark the petri dish and report form with sample
identification and volume.
» 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 held between the
funnel and the base.
» Shake the sample bottle vigorously at least 25
times to distribute the bacteria uniformly, and
measure the desired volume of sample or dilution
into the funnel.
» Select sample volumes based on previous
knowledge of the pollution level, to produce
20-80 E. coli colonies on the membranes. Sample
volumes of 1-100 mL are normally tested at half-
log intervals (e.g., 100, 30,10, 3 mL).
42 Modified t. co\\ Method
-------
Smaller sample sizes 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.
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.
Use sterile forceps to aseptically remove the
membrane filter from the filter base, and roll it
onto the modified mTEC Agar to avoid the
formation of bubbles between the membrane and
the agar surface. Reseat the filter 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 35±0.5°C
for 2 h.
After a 2-h incubation at 35±0.5°C, transfer the
plate to a Whirl-Pak® bag, seal the bag, place the
bag with the plate inverted in a test-tube rack, and
put the rack in a 44.5±0.2°C waterbath for
22-24 h.
After 22-24 h, remove the plate from the
waterbath, and count and record the number of
red or magenta colonies with the aid of an
illuminated lens with a 2-5x magnification or a
stereoscopic microscope. (See photo 7.)
Photo 7. EschencVYia
co\\ colonies on
modified mTEC Agar.
E. co\\ colonies are
red to magenta.
Modified \L. co\\ Method
-------
"\ 0.3.4 Ca\cu\at\on of ResuVls
Select the membrane filter with an acceptable number
of magenta or red colonies (20—80), and calculate the number
of E. mh per 100 mL according to the following general
formula:
100 (number of E. colt colonies counted)
E. coli/\QQ mL = ^ : ;
(volume of sample filtered, in mL)
See the USEPA microbiology methods manual, Part II,
Section C, 3.5 for general counting rules (Bordner etaL, 1978).
"\ 0.3.5 Reporting Resu\ts
There should be at least three volumes filtered per
sample. Report the results as E. coliptt 100 mL of sample.
"\ 0.3.6 V er\f \cat\on Procedure
Red or magenta colonies can be verified as E. coli.
Verification of colonies may be required in evidence
gathering and is also recommended as a means of quality
control for the initial use of the test and for changes in
sample sites, lots of commercial media, or major ingredients
in media compounded in the laboratory. The verification
procedure follows.
» Using a sterile inoculation loop, transfer growth
from the centers of at least 10 well-isolated typical
colonies to Nutrient Agar plates or slants and to
Trypticase Soy Broth. Incubate the agar and broth
cultures for 24 h at 35±0.5°C.
» After incubation, remove a loopful of growth
from the Nutrient Agar with a platinum loop and
deposit it on the surface of a piece of filter paper
that has been saturated with freshly prepared
Cytochrome Oxidase Reagent. If the spot where
the bacteria were deposited turns deep purple
within 15 seconds, the test is positive.
» Transfer growth from the Trypticase Soy Broth to
Simmons Citrate Agar, Tryptone Broth, and an EC
Broth fermentation tube.
• Incubate the Simmons Citrate Agar and
Tryptone Broth for 48 h at 35±0.5°C.
44 Modified \L. co\\ Method
-------
• Incubate the EC Broth at 44.5±0.2°C in a
waterbath for 24 h. The water level must be
above the level of the EC Broth in the tube.
• Add 0.5 mL of Kovacs Indole Reagent to the 48-
h Tryptone Broth culture, and shake the tube
gently. A positive test for indole is indicated by
a deep red color that develops in the alcohol
layer on top of the broth.
» E. foliis EC gas-positive, indole-positive, and
oxidase-negative, and does not utilize citrate (i.e.,
the medium remains green).
» Alternately, commercially available multi-test
identification systems may be used to verify
colonies. Inoculate the colonies into an
identification system for Entenbacteriaceae that
includes lactose fermentation, O-nitrophenyl-p-D-
galactopyranoside (ONPG), and cytochrome
oxidase test reactions.
"\ 0.3.1 Method Performance
The false-positive and false-negative rates, reported
for various environmental water samples, were <1% and
4%, respectively.
Modified \L. co\\ Method ,45
-------
REFERENCES
American Public Health Association (APHA), American
Water Works Association, Water Environment Federa-
tion. (1998). Standard methods for the examination of
water and wastewater (20th ed). Clesceri LS, Greenberg
AE, Eaton AD, eds. Washington, DC: American Public
Health Association.
American Society for Testing and Materials (ASTM). (1993).
Standard specification for reagent water. In: 1993 Annual
book of standards. Section 11: water and environmental
technology. Vol 11.01: water (1), D 1193-91. Philadelphia,
PA: American Society for Testing and Materials.
Bordner R, Winter J, Scarpino P. (1978). Microbiological
methods for monitoring the environment: water and
wastes. Environmental Monitoring and Support
Laboratory, Office of Research and Development, U.S.
Environmental Protection Agency, Cincinnati, OH.
EPA-600/8-78/017.
Code of Federal Regulations (CFR). (1999). Protection of
environment. Code of Federal Regulations, Title 40, Part
136.3. pp. 27-29.
Committee on Analytical Reagents of the American Chemical
Society. (1981). Reagent chemicals: American Chemical
Society specifications (6th ed). Washington DC: Ameri-
can Chemical Society.
Dufour AP, Strickland ER, Cabeffi VJ. (1981). Membrane filter
method for enumerating Escherichia colt. Appl Environ
Microbiol41:1152-1158.
Levin MA, Fischer JR, Cabelli VJ. (1975). Membrane filter
technique for enumeration of enterococci in marine
waters. Appl Microbiol 30:66-71.
Messer JW, Dufour AP. (1998). A rapid, specific membrane
filtration procedure for enumeration of enterococci in
recreational water. Appl Environ Microbiol 64:678-680.
46 References
-------
Rosin J. (1967). Reagent chemicals and standards. Princeton,
NJ: D. Van Nostrand.
United States Pharmacopeia Convention. (1974). United
States pharmacopeia (19th ed). Rockville, MD: United
States Pharmacopeia Convention, Inc.
U.S. Environmental Protection Agency (USEPA). (1976).
Fecal coliform bacteria. In: Quality criteria for water
("The Red Book"). Office of Water and Hazardous
Materials, Washington, DC. pp. 42-50. Available from:
National Technical Information Service, Springfield, VA,
PB93-184620.
USEPA. (1985). Test methods for Escherichia mli and
enterococci in water by the membrane filter procedure.
Environmental Monitoring and Support Laboratory,
Cincinnati, OH. EPA-600/4-85/076.
USEPA. (1986a). Ambient water quality criteria for bacteria-
1986. Office of Water Regulations and Standards, Criteria
and Standards Division, Washington, DC. EPA-440/5-
84/002.
USEPA. (1986b). Bacteriological ambient water quality criteria;
availability. Federal Register 51(45):8012-8016.
USEPA. (1997). Method 1600: membrane filter test methods
for enterococci in water. Office of Water, Washington,
DC. EPA-821/R-97/004.
Disclaimer
This manual has been reviewed by the USEPA Office
of Water and approved for publication. Mention of trade
names or commercial products does not constitute endorse-
ment or recommendation for use.
D\sc\a\mer 47
-------
Acknowledgments
This laboratory manual was prepared under the
direction of Latisha Parker, Health and Ecological Criteria
Division, U.S. Environmental Protection Agency (USEPA),
Office of Water, Washington, DC. This manual was prepared
under EPA Contract No. 68-C-98-141 by The COM Group,
Inc., Chevy Chase, Maryland.
For their technical contributions, special thanks are
extended to: Alfred Dufour and Kristen Brenner, Microbio-
logical and Chemical Exposure Assessment Research
Division, National Exposure Research Laboratory, Cincinnati,
Ohio; and Robin Oshiro, Health and Ecological Criteria
Division, Office of Water, Washington, DC.
In preparation of the two original methods, the major
contributions are acknowledged of Alfred Dufour and
Theodore Erickson of the Toxicology and Microbiology
Division (TMD), Health Effects Research Laboratory
(HERL), and their assistance and that of Robert Bordner,
Biological Methods Branch, and John Winter and Paul
Britton, Quality Assurance Branch, Environmental Monitor-
ing and Support Laboratory—Cincinnati (EMSL—Cincinnati),
U.S. Environmental Protection Agency (USEPA), in preparing
the final protocol and in completing the formal method
validation studies.
The revised enterococci method was developed under
the direction of James W Messer and Alfred P. Dufour of the
USEPA Microbiological and Chemical Exposure Assessment
Research Division, National Exposure Research Laboratory,
Cincinnati, Ohio. The method document was prepared under
EPA Contract No. 68-C3-0337 by the DynCorp Environmen-
tal Programs Division, Washington, DC. The revised E. coli
method was developed by Bennett G. Smith of the USEPA
Microbiological and Chemical Exposure Assessment Research
Division, National Exposure Research Laboratory, Cincinnati,
Ohio.
48. Acknowledgments
-------
Questions concerning these methods or their applica-
tion should be addressed to:
William A. Telliard, Director
Analytical Methods Staff
Engineering and Analysis Division (4303)
USEPA Office of Water
401 M Street, SW
Washington, DC 20460
Phone: 202-260-7120
Fax: 202-260-7185
Requests for additional copies of this manual (doc. no.
EPA/821/R-97/004) or videotape (doc. no. EPA/822/V-99/
001) should be directed to:
USEPA National Center for Environmental
Publications and Information (NCEPI)
11029 Kenwood Road
Cincinnati, OH 45242
Phone: 513-489-8190
Document No. EPA/821/R-97/004
This manual is also available on the Internet at:
www.EPA.gov/ OST/beaches
Acknowledgments 49
------- |