United States
         Environmental Protection
         Agency
               Office of Water
               Washington DC
               20460
EPA821-R-02-021
September 2002
vvEPA
Method 1106.1:  Enterococci in
Water by Membrane Filtration
Using membrane-Enterococcus
Esculin Iron Agar (mE-EIA)

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U.S. Environmental Protection Agency
      Office of Water (4303T)
   1200 Pennsylvania Avenue, NW
      Washington, DC 20460
        EPA-821-R-02-021

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                                         Disclaimer

The Engineering and Analysis Division, of the Office of Science and Technology, has reviewed and
approved this report for publication. The Office of Science and Technology directed, managed, and
reviewed the work of DynCorp in preparing this report. Neither the United States Government nor any of
its employees, contractors, or their employees make any warranty, expressed or implied, or assumes any
legal liability or responsibility for any third party's use of or the results of such use of any information,
apparatus, product, or process discussed in this report, or represents that its use by such party would not
infringe on privately owned rights.  This document combines the information previously published in Test
Methods for Escherichia coli and Enterococci in Water by the Membrane Filter Procedure (EPA-600/4-
85-076) (Reference 18.7) and Improved Enumeration Methods for the Recreational Water Quality
Indicators: Enterococci and Escherichia coli (EPA/821/R-97/004) (Reference 18.5).  Mention of trade
names or commercial products does not constitute endorsement or recommendation for use.
Questions concerning this method or its application should be addressed to:

       Robin K. Oshiro
       Engineering and Analysis Division (4303T)
       U.S. EPA Office of Water, Office of Science and Technology
       1200 Pennsylvania Avenue, NW
       Washington, DC 20460
       oshiro.robin@epa.gov
       202-566-1075
       202-566-1053 (facsimile)

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                                   Table of Contents










1.0    Scope and Application	1




2.0    Summary of Method	1




3.0    Definitions 	1




4.0    Interferences and Contamination  	1




5.0    Safety	2




6.0    Equipment and Supplies  	2




7.0    Reagents and Standards	3




8.0    Sample Collection, Preservation,  and Storage	6




9.0    Quality Control	6




10.0   Calibration and Standardization	6




11.0   Procedure	6




12.0   Data Analysis and Calculations	8




13.0   Method Performance	8




14.0   Reporting Results	11




15.0   Verification Procedure	11




16.0   Pollution Prevention  	11




17.0   Waste Management	11




18.0   References	12

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   Method 1106.1: Enterococci in Water by Membrane  Filtration  Using
            membrane-Enterococcus-Esculin Iron Agar (mE-EIA)

                                   September 2002
1.0    Scope and Application

1.1    This method describes a membrane filter (MF) procedure for the detection and enumeration of the
       enterococci bacteria in water. The enterococci are commonly found in the feces of humans and
       other warm-blooded animals. Although some strains are ubiquitous and not related to fecal
       pollution, the presence of enterococci in water is an indication of fecal pollution and the possible
       presence of enteric pathogens.
1.2    The enterococci test measures the bacteriological quality of recreational waters. Epidemiological
       studies have led to the development of criteria which can be used to promulgate recreational water
       standards based on the established relationship between health effects and water quality. The
       significance of finding enterococci in recreational water samples is the direct relationship between
       the density of enterococci in the water and swimming-associated gastroenteritis studies  of marine
       and freshwater bathing beaches (Reference 18.3).

1.3    The test for enterococci can be applied to potable, fresh, estuarine, marine, and shellfish growing
       waters.
1.4    Since a wide range of sample volumes or dilutions can be analyzed by the MF  technique, a wide
       range of enterococci levels in water can be detected and enumerated.


2.0    Summary of Method

2.1    The MF method provides a direct count of bacteria in water based on the development of colonies
       on the surface of the membrane filter (Reference 18.4). A water sample is filtered through the
       membrane which retains the bacteria.  Following filtration, the membrane containing the bacterial
       cells is placed on a selective medium, mE agar, and incubated for 48 h at 41+ 0.5C. Following
       incubation, the filter is transferred to a differential medium, EIA agar, and incubated at 41+ 0.5C
       for and additional  20 min.  Pink to red enterococci colonies will develop a black or reddish-brown
       precipitate on the underside of the filter.  These colonies are counted with a fluorescent lamp and a
       magnifying lens.

3.0    Definitions

3.1    In this method, enterococci are those bacteria which produce pink to red colonies after incubation
       on mE agar and that form a with black or reddish-brown precipitate after subsequent transfer to
       EIA medium. Enterococci include Streptococcus faecalis, Streptococcus faecium, Streptococcus
       avium, and their variants.

4.0    Interferences and Contamination
                                                                            September 2002

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Method 1106.1
4.1    Water samples containing collodial or suspended participate materials can clog the membrane filter
       and prevent filtration, or cause spreading of bacterial colonies which could interfere with
       identification of target colonies.

5.0   Safety

5.1    The analyst/technician must know and observe the normal safety procedures required in a
       microbiology laboratory while preparing, using and disposing of cultures, reagents, and materials,
       and while operating sterilization equipment.

5.2    Mouth-pipetting is prohibited.
6.0    Equipment and Supplies

6.1     Glass lens with magnification of 2-5x or stereoscopic microscope.

6.2     Lamp, with a cool, white fluorescent tube.

6.3     Hand tally or electronic counting device.

6.4     Pipet container, stainless steel, aluminum or borosilicate glass, for glass pipets.

6.5     Pipets, sterile, T.D. bacteriological or Mohr,  glass or plastic, of appropriate volume.

6.6     Graduated cylinders, 100-1000 mL, covered with aluminum foil or kraft paper and sterile.

6.7     Membrane filtration units (filter base and funnel),  glass, plastic or stainless steel, wrapped with
        aluminum foil or kraft paper and sterilized.

6.8     Ultraviolet unit for sanitization of the filter funnel  between filtrations (optional).

6.9     Line vacuum, electric vacuum pump, or aspirator for use as a vacuum source. In an emergency or
        in the field,  a hand pump or a syringe equipped with a check valve to prevent the return flow of air,
        can be used.

6.10    Flask, filter, vacuum, usually 1 L, with appropriate tubing.  A filter manifold to  hold a number of
        filter bases is optional.

6.11    Flask for safety trap placed between the filter flask and the vacuum source.

6.12    Forceps, straight or curved, with smooth tips to handle filters without damage.

6.13    Ethanol, methanol or isopropanol in a small,  wide-mouth container, for flame-sterilizing forceps.

6.14    Burner, Bunsen or Fisher type, or electric incinerator unit for sterilizing loops and needles.

6.15    Thermometer, checked against a National Institute of Standards and Technology (NIST) certified
        thermometer, or one that meets the requirements of NIST Monograph SP 250-23.


September 2002                                 2

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                                                                                   Method 1106.1
6.16   Petri dishes, sterile, plastic, 9 x 50 mm, with tight-fitting lids.

6.17   Bottles, milk dilution, borosilicate glass, screw-cap with neoprene liners, marked at 99 mL for
       1:100 dilutions.  Dilution bottles marked at 90 mL or tubes marked at 9 mL may be used for 1:10
       dilutions.

6.18   Flasks, borosilicate glass, screw-cap, 250-2000 mL volume.

6.19   Membrane filters, sterile, white, grid marked, 47 mm diameter, with 0.45  0.02 um pore size.

6.20   Inoculation loops, at least 3 mm diameter, and needles, nichrome or platinum wire, 26 B & S
       gauge, in suitable holders. Sterile disposable applicator sticks or plastic loops are alternatives to
       inoculation loops.

6.21   Incub ator maintained at 41  0.5 C.

6.22   Waterbath maintained at 50C for tempering agar.

6.23   Test tubes, 20 x 150 mm, borosilicate glass  or plastic.

6.24   Caps, aluminum or autoclavable plastic, for 20 mm diameter test tubes.

6.25   Test tubes, screw-cap, borosilicate glass, 16 x 125 mm or other appropriate size.

6.26   Whirl-Pak bags.


7.0   Reagents and Standards

7.1    Purity of Reagents: Reagent grade chemicals shall be used in all tests. Unless otherwise indicated,
       reagents shall conform to the specifications of the Committee on Analytical Reagents of the
       American Chemical Society  (Reference 18.6). The agar used in preparation of culture media must
       be of microbiological grade.

7.2    Whenever possible, use commercial  culture media as a means of quality control.

7.3    Purity of Water: Reagent water conforming to Specification Dl 193, reagent water conforming
       Type II, Annual Book of ASTM Standards (Reference 18.1).

7.4    Phosphate Buffered Saline

       7.4.1   Composition:

                       Sodium Dihydrogen Phosphate          0.58 g
                       Sodium Monohydrogen Phosphate       2.5 g
                       Sodium Chloride                       8.5 g
                      Reagent-Grade Distilled Water          1.0 L

       7.4.2   Preparation: Dissolve the ingredients above in 1 L of reagent-grade distilled water in a
               flask, and dispense in appropriate amounts for dilutions in screw-cap bottles or culture
               tubes, and/or into containers for use as rinse water. Autoclave at  121C (15 Ib pressure)
               for 15 min. Final pH should be 7.4  0.2.

7.5    Phosphate Buffered Dilution Water (Reference 18.2)

       7.5.1   Composition of Stock Phosphate Buffer Solution:

                                                3                                 September 2002

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Method 1106.1
                       Phosphate dihydrogen phosphate         34.0 g
                       Reagent-Grade distilled water          500.0 mL

        7.5.2  Preparation: Adjust the pH of the solution to 7.2 with 1 N NaOH, and bring the volume to
               1 L with reagent-grade distilled water. Sterilize by filtration or autoclave at 121 C (15 Ib
               pressure) for 15 min.

        7.5.3  Preparation of Stock Magnesium Chloride Solution:  Add 38 g anhydrous MgCl2 or 81.1 g
               MgCl2-6H2O to 1 L reagent-grade distilled water.  Sterilize by filtration or autoclave at
               121 C (15 Ib pressure) for 15 min.

        7.5.4  Storage of Stock Solutions: After sterilization, store the stock solutions in the refrigerator
               until used. Handle aseptically. If evidence of mold or other contamination appears, the
               affected stock solution should be discarded and a fresh solution should be prepared.

        7.5.5  Working Phosphate Buffered Dilution Water: Mix 1.25 mL of the stock phosphate buffer
               and 5 mL of the MgCl2 stock per liter of reagent-grade distilled water. Dispense in
               appropriate amounts for dilutions in screw-cap bottles or culture tubes, and/or into
               containers for use as rinse water. Autoclave at 121C (15 Ib pressure) for 15 min. Final
               pH should be 7.0 0.2.

7.6     mE Agar (Difco 0333)

        7.6.1  Composition of Basal Medium Ingredients:

                       Peptone                               10.0  g
                       Sodium Chloride                       15.0  g
                       Yeast Extract                         30.0  g
                       Esculin                                1.0  g
                       Actidione (Cycloheximide)               0.05 g
                       Sodium Azide                          0.15 g
                       Agar                                 15.0  g
                       Reagent-Grade Distilled Water           1.0  L

        7.6.2  Preparation of Basal Medium: Add 71.2 g dehydrated mE basal medium to 1 L of reagent-
               grade distilled water in a flask, and heat to boiling until the ingredients dissolve using a
               magnetic stirrer.  Autoclave at 121C (15 Ib pressure) for 15 min, and cool in a 50C
               waterbath.

        7.6.3  Reagents Added After Sterilization: Mix 0.24 g nalidixic acid in 5 mL of reagent-grade
               distilled water, add 0.2 mL of 10 N NaOH.  Allow the  mixture to dissolve, and add the
               mixture to the basal medium. Add 0.15 g triphenyltetrazolium chloride to the basal
               medium and mix.

        7.6.4  Alternately, the following  solutions may be used:

               7.6.4.1 Nalidixic acid: Add 0.48 g of nalidixic acid and 0.4 mL  10 N NaOH to 10 mL of
                       reagent-grade distilled water  and mix.  Filter-sterilize the solution, and add 5.2 mL
                       per liter of medium.

               7.6.4.2 Triphenyltetrazolium chloride (TTC): Add 0.25 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.

        7.6.5  Preparation of mE Agar Plates: Pour the mE Agar into 9x50 mm petri dishes to a 4-5 mm
               depth (approximately 4-6  mL), and allow to solidify. Final pH of medium should be 7.1 
               0.2. Store in a refrigerator.

September 2002                                 4

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                                                                                   Method 1106.1
7.7    Esculin Iron Agar (EIA) (Difco 0488)

       7.7.1   Composition:

                      Esculin                               1.0 g
                      Ferric Citrate                          0.5 g
                      Agar                                15.0g
                      Reagent-Grade Distilled Water          1.0 L

       7.7.2   Preparation: Add 16.5  g dehydrated EIA to 1 L of reagent-grade distilled water in a flask,
               and heat to boiling until the ingredients are dissolved. Autoclave the medium at 121C (15
               Ib pressure) for 15 min, and cool in a 50C 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.

7.8    Brain Heart Infusion Broth (BHIB) (Difco 0037, BD 4311059)

       7.8.1   Composition:

                      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

       7.8.2   Preparation: Dissolve 37 g dehydrated BHIB in 1 L of reagent-grade distilled water.
               Dispense in 10-mL volumes in screwcap tubes, and autoclave at 121C (15 Ib pressure)
               for 15 min. If the medium is not used the same day as prepared and sterilized, heat in
               boiling water bath for several min to remove absorbed oxygen, and cool quickly without
               agitation, just prior to inoculation. Final pH should be 7.4  0.2.

7.9    Brain Heart Infusion Broth (BHIB) with 6.5% NaCl

       7.9.1   Composition:

                      BHIB with 6.5% NaCl is the same as BHIB broth above, but with additional
                      NaCl.

       7.9.2   Preparation: Add 60.0  g NaCl per liter of medium. Since most commercially available
               dehydrated media already contain sodium chloride, that amount is subtracted from the 65 g
               per liter required to make a final concentration of 6.5% NaCl.

7.10   Brain Heart Infusion Agar (BHIA) (Difco 0418, BD 4311065)

       7.10.1  Composition:

                      BHIA contains the same components as  BHIB (see above)  with the addition of
                      15.0 g agar per liter of BHIB.

       7.10.2 Preparation: Suspend 52 g dehydrated BHIA in 1 L of reagent-grade distilled water. Heat
               to boiling until the ingredients  are dissolved.  Dispense 10 mL of medium in screwcap test
               tubes, and sterilize for 15 min  at 121C (15 Ib pressure).  After sterilization, slant until
               solid. Final pH should be 7.4  0.2.

7.11   Bile Esculin Agar (BEA) (Difco 0879)
                                                                                 September 2002

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Method 1106.1
       7.11.1 Composition:

                     Bacto Beef Extract                    3.0 g
                     Bacto Peptone                        5.0 g
                     Bacto Oxgall                        40.0 g
                     Bacto Esculin                         l.Og
                     Ferric Citrate                         0.5 g
                     Bacto Agar                          15.0 g
                     Reagent-Grade Distilled Water          1.0 L

       7.11.2 Preparation: Add 64.0 g dehydrated BEA to 1 L reagent-grade distilled water, and heat to
              boiling to dissolve completely.  Dispense 10-mL volumes in tubes for slants or larger
              volumes into flasks for subsequent plating. Autoclave at 121C (15 Ib pressure) for 15
              min. Overheating may cause darkening of the medium. Cool in a 50C waterbath, and
              dispense into sterile petri dishes. Final pH should be 6.6  0.2. Store in a refrigerator.

8.0   Sample Collection,  Preservation,  and Storage

8.1    Sampling procedures are described in detail in the USEPA microbiology methods manual, Section
       II, A (Reference 18.2).  Adherence to sample preservation procedures and holding time limits is
       critical to the production of valid data.  Samples should not be analyzed if these conditions are not
       met.

       8.1.1  Storage Temperature and Handling Conditions

              Ice or refrigerate bacteriological samples at a temperature of 1-4C during transit to the
              laboratory. Use insulated containers to assure proper maintenance of storage temperature.
              Take care that sample bottles are not totally immersed in water during transit or storage.

       8.1.2  Holding Time Limitations

              Examine samples as  soon as possible after collection.  Do not hold samples longer than  6
              h between collection  and initiation of analyses.

9.0   Quality Control

9.1    See recommendations on quality control for microbiological analyses in the USEPA microbiology
       methods manual, Part IV, C (Reference 18.2).

10.0  Calibration and Standardization

10.1   Check temperatures in incubators daily to ensure operation within stated limits.

10.2   Check thermometers at least  annually against a NIST certified thermometer or one that meets the
       requirements of NIST monograph SP 250-23.  Check mercury columns for breaks.

11.0  Procedure

11.1   Prepare the mE Agar as directed in section 7.6.

11.2   Mark the petri dishes and report form with the sample identification and volume.
September 2002                               6

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                                                                                   Method 1106.1
11.3   Place a sterile membrane filter on the filter base, grid side up, and attach the funnel to the base so
       that the membrane filter is held between the funnel and the base.

11.4   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.

11.5   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).

11.6   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.

11.7   Filter the sample, and rinse the sides of the funnel at least twice with 20-30 mL of sterile buffered
       rinse water. Turn off the vacuum, and remove the funnel from the filter base.

11.8   Use sterile forceps to aseptically remove the membrane filter from the filter base, and roll it onto
       the mE 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  0.5C for 48 h
       (See Photo 1.).
Photo 1.      Enterococci on ME Agar. Colonies that are pink to dark red are considered to be
               presumptive enterococci.

11.9   After incubation, transfer the membranes to EIA plates that have been warmed up to room
       temperature for 20-30 min, and incubate at 41  0.5C for an additional 20-30 mia (See Photo 2.)

11.10 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.
                                                                                 September 2002

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Method 1106.1
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.

12.0  Data Analysis and Calculations

12.1   Use the following general rules to calculate the enterococci count per 100 ml of sample:

       12.1.1  Select the membrane filter with an acceptable number of pink-to-red colonies (20-60) that
               form a black or reddish-brown precipitate on the underside of the filter when placed on
               EIA. Calculate the number of enterococci per 100 mL according to the following general
               formula:
                                         Number of enterococci colonies
               Enterococci/100mL=    	X   100
                                         Volume of sample filtered (mL)

       12.1.2  See the USEPA microbiology methods manual, Part II, Section C, 3.5, for general
               counting rules (Reference 18.2).

13.0  Method Performance

13.1   Performance characteristics

       13.1.1  Precision - The degree of agreement of repeated measurements of the same parameter
               expressed quantitatively as the standard deviation or as the 95% confidence limits of the
               mean computed from the results of a series of controlled determinations. Precision of the
               mE method was established by Levin et al. (Reference 18.4) who indicated that the method
               did not exceed the expected limits for counts having the Poisson distribution.

       13.1.2  Bias - The persistent positive or negative deviation of the results from the assumed or
               accepted true value. The bias of the enterococci MF method with the mE Agar has been
               reported to be +2% of the true value (Reference 18.4).

       13.1.3  Specificity - The ability of a method to select and/or distinguish the target bacteria from
               other bacteria  in the same water sample. The specificity characteristic of a method is
               usually reported as the percent of false positive and false negative results. The specificity
               for this medium as reported for various environmental water samples was 10% false
               positive and 11.7% false negative (Reference 18.4).

13.2   Collaborative study data


September 2002                                8

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                                                                            Method 1106.1
13.2.1 A collaborative study was conducted among eleven volunteer laboratories, each with two
       analysts who independently tested local fresh and marine recreational waters and sewage
       treatment plant effluent samples, in duplicate. That data were reported to the
       Environmental Monitoring and Support Laboratory - Cincinnati, U.S. Environmental
       Protection Agency, for statistical analyses.

13.2.2 The results of the study are shown in Figure 1 where S0 equals the pooled standard
       deviation among replicate counts from a single analyst for three groupings (counts less
       than 30, counts from 30 to 50, and counts greater than 50) and SB equals the pooled
       standard deviation between means of duplicates from analysts in the same laboratory for
       the same groupings.  The precision estimates from this study did not differ with the water
       types tested.

13.2.3 By linear regression, the precision of the method can be  generalized as:

       S0 = 0.013 count/100 mL + 2.42 (dilution factor) and

       SB = 0.152 count/100 mL + 5.16 (dilution factor)

                                                  100
       Where dilution factor =
                                   Volume of original sample filtered
13.2.4 Because of the instability of microbial populations in water samples, each laboratory
       analyzed is own sample series and no full measure of recovery or bias was possible.
       However, all laboratories analyzed a single surrogate sample prepared from a freeze-dried
       culture of Streptococcus faecalis. The mean count (X) and the standard deviation of the
       counts (Sr) (including the variability among laboratories for this standardized enterococci
       sample) were 32.5 colonies/membrane and 9.42 colonies/membrane, respectively.
                                                                           September 2002

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Method 1106.1



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September 2002
10

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                                                                                Method 1106.1
14.0  Reporting Results

14.1   There should be at least three volumes tested per sample.  Report the results as enterococci per 100
       mL of sample.

15.0  Verification Procedure

15.1   Pink-to-red colonies on mE Agar that produce a black or reddish-brown precipitate after
       incubation on EIA agar can be verified as enterococci. Verification of colonies may be required in
       evidence gathering and it is also recommended as a means of quality control for the initial use of
       the test and for changes in sample sites, lots of commercial media, or major ingredients in media
       compounded in the laboratory. The verification procedure follows.

15.2   Using a sterile inoculating needle, transfer cells from the centers of at least 10 well-isolated typical
       colonies into a BHIB tube and onto a BHIA slant.  Incubate broth tubes for 24 h and agar slants
       for48hat350.5C.
15.3   After a 24 h incubation, transfer a loopful of material from each BHIB tube to BEA, BHIB and
       BHIB with 6.5%NaCl.

       15.3.1 Incubate the BEA and BHIB with 6.5% Nad at 35  0.5C for 48 h.

       15.3.2 Incubate the BHIB at 45  0.5C for 48 h.

15.4   Observe for growth on all media.

15.5   After 48 h incubation, apply a Gram stain to growth from each BHIA slant.

15.6   Gram-positive cocci that grow and hydrolyze esculin on BEA (i.e., produce a black or brown
       precipitate), and grow in BHIB at 45  0.5C and BHIB with 6.5% NaCl at 35  0.5C are
       verified as enterococci.

16.0  Pollution  Prevention

16.1   The solutions and reagents used in this method pose little threat to the environment when recycled
       and managed properly.

16.2   Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize
       the volume of expired materials to be disposed.

17.0  Waste Management

17.1   It is the laboratory's responsibility to comply with all federal, state, and local regulations governing
       waste management, particularly the biohazard and hazardous waste identification rules and land
       disposal restrictions, and to protect the air, water, and land by minimizing and controlling all
       releases from fume hoods and bench operations. Compliance with all sewage discharge permits
       and regulations is also required.

17.2   Samples, reference materials, and equipment known or suspected to have viable enterococci
       attached or contained must be sterilized prior to disposal.

17.3   Samples preserved with HC1 to pH <2 are hazardous and must be neutralized before being
       disposed, or must be handled as hazardous waste.

                                              11                                September 2002

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Method 1106.1
17.4   For further information on waste management, consult "The Waste Management Manual for
       Laboratory Personnel" and "Less Is Better: Laboratory Chemical Management for Waste
       Reduction," both available from the American Chemical Society's Department of Government
       Relations and Science Policy, 1155 16th Street NW, Washington, DC 20036.


18.0  References

18.1   Annual Book of ASTM Standards, Vol. 11.01, American Society for Testing and Materials,
       Philadelphia, PA 19103.

18.2   Bordner, R.,  J.A. Winter and P.V. Scarpino (eds.).  1978.  Microbiological Methods for
       Monitoring the Environment, Water and Wastes, EPA-600/8-78-017.  Office of Research and
       Development, USEPA.

18.3   Cabelli, V. J., A. P. Dufour,  M. A. Levin, L. J. McCabe, and P. W. Haberman, 1979. Relationship
       of Microbial Indicators to Health Effects at Marine Bathing Beaches. Amer. Jour. Public Health.
       69:690-696.
18.4   Levin, M.  A., J. R. Fischer and V. J. Cabelli. 1975. Membrane Filter Technique for Enumeration
       of Enterococci in Marine Waters. Appl. Microbiol.  30:66-71.

18.5   Improved Enumeration Methods for the Recreational Water Quality Indicators: Enterococci and
       Escherichia coli. 2000. EPA/821/R-97/004. Office of Science and Technology, Washington D.C.
18.6   Reagent Chemicals, American Chemical Society Specifications,  American Chemical Society,
       Washington, DC. For suggestions of the testing of reagents not listed by the American Chemical
       Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, UK and the
       United States Pharmacopeia.

18.7   Test Methods for Escherichia coli and Enterococci in Water by the Membrane Filter Procedure.
       1985. EPA-600/4-85/076. Environmental Monitoring and Support Laboratory, Cincinnati,
       USEPA.
September 2002                               12

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