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

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                 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

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                      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

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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

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                       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

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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

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      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

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                        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

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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

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                       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

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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

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                      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

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                       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

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         •  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

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                       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

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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

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                       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

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     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

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