United States Office of Science and EPA/821/R-97/004

Environmental Protection Technology March 2000

Agency Washington DC 20460

EPA

Improved Enumeration Methods

for the Recreational Water

Quality Indicators: Enterococci

and Escherichia coli
i

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

9.2.2.3 mE Agar ................................. 9

9.2.2.4 Esculin Iron Agar................... 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

9.2.2.8 Bile Esculin Agar ................... 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
ii

9.3.2.2 Phosphate Buffered Dilution

Water................................... 18

9.3.2.3 mEI Agar .............................. 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

9.3.6Verification Procedure ....................... 22

9.3.7 Method Performance......................... 23

10 TEST METHODS FOR E. COLI ...................... 23

10.1 Summary .................................................... 23

10.2 Original E. coli Method (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 mTEC Agar ...................... 28

10.2.2.4 Urea Substrate Medium ..... 28

10.2.2.5 Nutrient Agar ................... 29

10.2.2.6Tryptic Soy Broth; Trypticase Soy Broth ....... 30

10.2.2.7 Simmons Citrate Agar ....... 30

10.2.2.8Tryptone 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. coli Test Procedure............ 31

10.2.4 Calculation of Results........................ 33

10.2.5 Reporting Results .............................. 34

10.2.6Verification Procedure ....................... 34

10.2.7 Method Performance......................... 35

10.3 Modified E. coli Method ............................... 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 Nutrient Agar ................... 40
iii

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 E. coli 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 coli colonies 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
Introduction 1

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. coli, for fresh waters only).

These organisms were chosen based on epidemiological studies conducted

at various beaches in the United States that showed a strong positive

correlation between the organisms and the occurrence of swimmingassociated 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 Agar for

enterococci and mTEC Agar for E. coli) have been improved, resulting

in faster and easier enumeration. The modified media are mEI Agar

for enterococci and modified mTEC Agar for 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
2 Introduction

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 et al., 1981; Levin et al., 1975; USEPA, 1985) for

ambient water, namely, mE Agar for enterococci and mTEC

Agar for E. coli. 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 et al., 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-ß-D-glucoside.

The mTEC method (Dufour et al., 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
Introduction 3

containing urea is used to distinguish urease-negative E. coli

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-ß-D-glucuronide, which is catabolized to glucuronic

acid and a red- or magenta-colored compound by E. coli

that produce the enzyme ß-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 al., 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 et al., 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 temperature. Take care that sample bottles are not totally immersed in

water from melting ice during transit or storage.
4 Calibration and Standardization

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 D1193-91, Type II water, as specified by the

American Society for Testing and Materials (1993).

7 QUALITY CONTROL

See recommendations on quality control for microbiological analyses in the USEPA microbiology methods manual,

Part IV, A–C (Bordner et al., 1978).
Method Performance Characteristics 5

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 enterococci in water are presented here: the original method and a

revised method, both using a membrane filter (MF) procedure. 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 et al., 1975; USEPA,

1985), a water sample is filtered through the membrane,

which retains the bacteria. Following filtration, the membrane 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
6 Original Enterococci Method

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)

9.2.1 Equipment and Supplies

» 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.
Original Enterococci Method 7

» 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, widemouth 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 tightfitting 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 μm 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
8 Original Enterococci Method

9.2.2 Reagents and Media

Preparation of the following reagents and media used

in the original enterococci test are described below:

» Phosphate buffered saline or phosphate buffered

dilution water

» mE 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 reagentgrade 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 lb pressure) for 15 min. Final pH should be

7.4±0.2.

9.2.2.2 Phosphate Buffered Dilution Water

(APHA, 1998; Bordner et al., 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 lb

pressure) for 15 min.
Original Enterococci Method 9

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 lb

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.

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

appropriate amounts for dilutions in screwcap bottles or

culture tubes and/or into containers for use as rinse water.

Autoclave at 121°C (15 lb pressure) for 15 min. Final pH

should be 7.0±0.2.

9.2.2.3 mE Agar (Difco 0333-17)

Basal medium ingredients:

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

Prepare basal medium: 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 lb pressure) for 15

min, and cool in a 50°C waterbath.

Reagents added after sterilization: Mix 0.24 g nalidixic 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
10 Original Enterococci Method

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 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.

(b) Triphenyltetrazolium chloride (TTC): 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.

Prepare mE Agar: Pour the mE 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.

9.2.2.4 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

Preparation: 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 lb 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.
Original Enterococci Method 11

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-mL volumes

in screwcap tubes, and autoclave at 121°C (15 lb 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 lb
12 Original Enterococci Method

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 lb 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 Original Enterococci 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).
Original Enterococci Method 13

» 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

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 EIA

plates that have been warmed up at room temperature 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. Colonies that are pink to

dark red are considered to be presumptive enterococci.
14 Original Enterococci Method

9.2.4 Calculation of Results

Use the following general rules to calculate the enterococci per 100 mL 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 enterococci colonies)

Enterococci/100 mL = ______________________________

 

 (volume of sample filtered, in mL)

See the USEPA microbiology methods manual, Part II,

Section C, 3.5, for general counting rules (Bordner et al.,

1978).

9.2.5 Reporting Results

There should be at least three volumes tested per

sample. Report the results as enterococci per 100 mL of

sample.

9.2.6 Verification 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.
Modified Enterococci Method 15

» 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

48 h at 35±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.

9.2.7 Method Performance (USEPA, 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)

9.3.1 Equipment and Supplies

» 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,
16 Modified Enterococci Method

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 tightfitting 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 μm pore size.

» Inoculation loops, at least 3-μm diameter, and
Modified Enterococci Method 17

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.

9.3.2 Reagents and Media

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 1.0L

Preparation: Dissolve ingredients in 1 L of reagentgrade 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 lb pressure) for 15 min. Final pH should be

7.4±0.2.
18 Modified Enterococci Method

9.3.2.2 Phosphate Buffered Dilution Water

(APHA, 1998; Bordner et al., 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 lb

pressure) for 15 min.

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 lb

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.

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

appropriate amounts for dilutions in screwcap bottles or

culture tubes and/or into containers for use as rinse water.

Autoclave at 121°C (15 lb pressure) for 15 min. Final pH

should be 7.0±0.2.

9.3.2.3 mEI Agar

Ingredients of basal medium (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
Modified Enterococci Method 19

Preparation of mEI medium: Add 71.2 g dehydrated mE

basal medium plus 0.75 g indoxyl-ß-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 lb

pressure) for 15 min, and cool in a 50°C waterbath.

Reagents added after sterilization: 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 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.

(b) 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.

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.

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
20 Modified Enterococci Method

Preparation: Dissolve 37 g dehydrated BHIB in 1 L of

reagent-grade distilled water. Dispense in 10-mL volumes in

screwcap tubes, and autoclave at 121°C (15 lb pressure) for 15

min. Final pH should be 7.4±0.2.

9.3.2.5 Brain Heart Infusion Broth (BHIB)

with 6.5% NaCl

Ingredients: BHIB with 6.5% NaCl is the same as

BHIB described 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 deducted

from the 65 g per liter required to make a final concentration 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.

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 lb

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
Modified Enterococci Method 21

larger volumes into flasks for subsequent plating. Autoclave

at 121°C (15 lb 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.

9.3.3 Modified Enterococci 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.)
22 Modified Enterococci Method

» 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 Calculation of Results

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:

 100 (number of enterococci colonies)

Enterococci/100 mL = _____________________________

 

 (volume of sample filtered, in mL)

See the USEPA microbiology methods manual, Part II,

Section C, 3.5, for general counting rules (Bordneret al., 1978).

9.3.5 Reporting Results

There should be at least three volumes tested per

sample. Report the results as enterococci per 100 mL of

sample.

9.3.6 Verification Procedure

Colonies with a blue halo, regardless of color, can be

verified as enterococci. Verification of colonies may be

Photo 3. Enterococci on mEI Agar.

Colonies with a

blue halo are

considered to be

enterococci.
Test Methods for E. coli 23

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

48 h at 35±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.

9.3.7 Method Performance (Messer and Dufour,

1998; USEPA, 1997)

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 E. COLI

10.1 Summary

Two test methods for the detection and enumeration

of Escherichia coli in water are presented here. The original

mTEC Agar enumeration method (Dufour et al., 1981) for
24 Original E. coli Method

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. coli 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 et al., 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 membrane filter to another substrate. The target colonies on

modified mTEC Agar are red in color after the incubation

period.

10.2 Original E. coli Method (Method 1103.1)

10.2.1 Equipment and Supplies

» 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, 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).
Original E. coli Method 25

» 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 tightfitting 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-μm 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.
26 Original E. coli Method

» 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.

10.2.2 Reagents and Media

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
Original E. coli Method 27

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 lb pressure) for 15 min. Final pH

should be 7.4±0.2.

10.2.2.2 Phosphate Buffered Dilution Water

(APHA, 1998; Bordner et al., 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 lb

pressure) for 15 min.

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 lb

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.

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

appropriate amounts for dilutions in screwcap bottles or

culture tubes and/or into containers for use as rinse water.

Autoclave at 121°C (15 lb pressure) for 15 min. Final pH

should be 7.0±0.2.
28 Original E. coli Method

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

NaCl 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 15.0 g

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

lb 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 100.0mL

Preparation: Add dry ingredients to 100 mL reagentgrade distilled water in a flask. Stir to dissolve, and adjust to

pH 3–4 with 1 N HCl. The substrate solution should be a

straw-yellow color at this pH. (See photo 4.)
Original E. coli Method 29

10.2.2.5 Nutrient Agar (Difco 0001-17-0,

BD 4311472)

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 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 lb 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.
30 Original E. coli 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

Preparation: 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 lb

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)

Ingredients:

magnesium sulfate 0.2 g

monoammonium phosphate 1.0 g

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 into screwcap tubes, and autoclave at

121°C (15 lb 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
Original E. coli Method 31

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 into tubes, and

autoclave at 121°C (15 lb pressure) for 15 min. Final pH

should be 7.2±0.2.

10.2.2.9 EC Broth (Difco 0314-01-0,

BD 4311187)

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 completely. Dispense into fermentation tubes (20x150 mm tubes

containing inverted 10x75 mm vials). Autoclave at 121°C (15

lb pressure) for 15 min. Final pH should be 6.9±0.2.

10.2.2.10 Oxidase Reagent

Ingredients:

N, N, N’, N’-tetramethyl-ρ-phenylenediamine

dihydrochloride, 1% aqueous solution

(1 g per 100 mL sterile reagent-grade distilled water).

10.2.2.11 Kovacs Indole Reagent

Ingredients:

ρ-dimethylaminobenzaldehyde 10.0 g

amyl or isoamyl alcohol 150.0 mL

concentrated (12 M) hydrochloric acid 50.0 mL

Preparation: Dissolve ρ-dimethylaminobenzaldehyde

in alcohol, slowly add hydrochloric acid, and mix.

10.2.3 Original E. coli Test Procedure

» Prepare mTEC Agar and Urea Substrate Medium

as directed above in the “Reagents and Media”

section.
32 Original E. coli Method

» 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. coli colonies on the membranes. Sample

volumes of 1–100 mL are normally tested at halflog 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 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 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
Original E. coli Method 33

Photo 5. Escherichia

coli colonies on

mTEC Agar. Colonies that are yellow,

yellow-green, or

yellow-brown are

E. coli.

Photo 6. Escherichia

coli colonies on an

absorbent pad

saturated with Urea

Substrate Medium.

E. coli 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.

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.)

10.2.4 Calculation of Results

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. coli per 100

mL according to the following general formula:
34 Original E. coli Method

 

 100 (number of E. coli colonies counted) E. coli/100 mL = __________________________________

 

 (volume of sample filtered, in mL)

See the USEPA microbiology methods manual, Part II,

Section C, 3.5, for general counting rules (Bordner et al.,

1978).

10.2.5 Reporting Results

There should be at least three volumes filtered per

sample. Report the results as E. coli per 100 mL of sample.

10.2.6 Verification Procedure

Yellow, yellow-green, or yellow-brown colonies from

the urease test 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 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 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
Modified E. coli Method 35

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. coli is 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 Enterobacteriaceae that

includes lactose fermentation, σ-nitrophenyl-ß-Dgalactopyranoside (ONPG), and cytochrome oxidase

test reactions.

10.2.7 Method Performance (Dufour et al., 1981;

USEPA, 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. coli. The

mean count (X) and the overall standard deviation of the

counts (ST) (including the variability among laboratories for

this standardized E. coli 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. coli 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. coli

on mTEC Agar has been reported as 80 colonies per filter.

10.3 Modified E. coli Method

The revised Escherichia coli method is a single-step

method that uses one medium, modified mTEC Agar, and
36 Modified E. coli Method

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-ß-Dglucuronide), which is catabolized to glucuronic acid and a

red- or magenta-colored compound by E. coli that produce

the enzyme ß-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.

10.3.1 Equipment and Supplies

» 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, 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.

» 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.
Modified E. coli Method 37

» 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 tightfitting 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 μm 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® bags.
38 Modified E. coli Method

10.3.2 Reagents and Media

Preparation of the following reagents and media used

in the revised E. coli 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 lb pressure) for 15 min. Final pH

should be 7.4±0.2.

10.3.2.2 Phosphate Buffered Dilution Water

(APHA, 1998; Bordner et al., 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 lb

pressure) for 15 min.
Modified E. coli Method 39

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 lb

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.

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

appropriate amounts for dilutions in screwcap bottles or

culture tubes and/or into containers for use as rinse water.

Autoclave at 121°C (15 lb 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

NaCl 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-ß-D-glucuronide) 0.5 g

agar 15.0 g

reagent-grade distilled water 1.0 L

Preparation: 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 lb pressure) for 15 min, and cool in a 50°C

waterbath. Pour the medium into each 9x50 mm culture dish
40 Modified E. coli Method

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,

BD 4311472)

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 boiling to dissolve the agar completely. Dispense in screwcap

tubes, and autoclave at 121°C (15 lb 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

Preparation: 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 lb

pressure) for 15 min. Final pH should be 7.3±0.2.
Modified E. coli Method 41

10.3.2.6 Simmons Citrate Agar (BD 4311620,

Difco 0091-17-1)

Ingredients:

magnesium sulfate 0.2 g

monoammonium phosphate 1.0 g

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 lb 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 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

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 lb pressure) for 15 min. Final pH

should be 7.2±0.2.

10.3.2.8 EC Broth (Difco 0314-01-0,

BD 4311187)

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
42 Modified E. coli Method

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 lb pressure) for 15 min. Final pH should be

6.9±0.2.

10.3.2.9 Oxidase Reagent

Ingredients:

N, N, N’, N’-tetramethyl-ρ-phenylenediamine

dihydrochloride, 1% aqueous solution (1 g per 100 mL sterile

reagent-grade distilled water).

10.3.2.10 Kovacs Indole Reagent

Ingredients:

ρ-dimethylaminobenzaldehyde 10 g

amyl or isoamyl alcohol 150 mL

concentrated (12 M) hydrochloric acid 50 mL

Preparation: Dissolve ρ-dimethylaminobenzaldehyde

in alcohol, slowly add hydrochloric acid, and mix.

10.3.3 Modified E. coli 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 halflog intervals (e.g., 100, 30, 10, 3 mL).
Modified E. coli Method 43

» 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. Escherichia

coli colonies on

modified mTEC Agar.

E. coli colonies are

red to magenta.
44 Modified E. coli Method

10.3.4 Calculation of Results

Select the membrane filter with an acceptable number

of magenta or red colonies (20–80), and calculate the number

of E. coli per 100 mL according to the following general

formula:

 

 100 (number of E. coli colonies counted) E. coli/100 mL = __________________________________

 

 (volume of sample filtered, in mL)

See the USEPA microbiology methods manual, Part II,

Section C, 3.5 for general counting rules (Bordner et al., 1978).

10.3.5 Reporting Results

There should be at least three volumes filtered per

sample. Report the results as E. coli per 100 mL of sample.

10.3.6 Verification 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.
Modified E. coli Method 45

• 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. coli is 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 Enterobacteriaceae that

includes lactose fermentation, σ-nitrophenyl-β-Dgalactopyranoside (ONPG), and cytochrome

oxidase test reactions.

10.3.7 Method Performance

The false-positive and false-negative rates, reported

for various environmental water samples, were <1% and

4%, respectively.
46 References

REFERENCES

American Public Health Association (APHA), American

Water Works Association, Water Environment Federation. (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: American Chemical Society.

Dufour AP, Strickland ER, Cabelli VJ. (1981). Membrane filter

method for enumerating Escherichia coli. Appl Environ

Microbiol 41:1152-1158.

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.
Disclaimer 47

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 coli 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 bacteria1986. 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 endorsement or recommendation for use.
48 Acknowledgments

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 CDM Group,

Inc., Chevy Chase, Maryland.

For their technical contributions, special thanks are

extended to: Alfred Dufour and Kristen Brenner, Microbiological 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 Monitoring 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 Environmental 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.
Acknowledgments 49

Questions concerning these methods or their application 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