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+ 0.5°C. Following
incubation, the filter is transferred to a differential medium, EIA agar, and incubated at 41+ 0.5°C
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 50°C 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 121°C (15 Ib pressure)
for 15 min. Final pH should be 7.4 ± 0.2.
7.5 Phosphate Buffered Dilution Water (Reference 18.2)
7.5.1 Composition of Stock Phosphate Buffer Solution:
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 121°C (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 121°C (15 Ib pressure) for 15 min, and cool in a 50°C
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 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.
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 121°C (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 121°C (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 121°C (15 Ib pressure) for 15
min. Overheating may cause darkening of the medium. Cool in a 50°C waterbath, and
dispense into sterile petri dishes. Final pH should be 6.6 ± 0.2. Store in a refrigerator.
8.0 Sample Collection, Preservation, and Storage
8.1 Sampling procedures are described in detail in the USEPA microbiology methods manual, Section
II, A (Reference 18.2). Adherence to sample preservation procedures and holding time limits is
critical to the production of valid data. Samples 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 1-4°C 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 ± 0.5°C 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 ± 0.5°C 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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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
for48hat35±0.5°C.
15.3 After a 24 h incubation, transfer a loopful of material from each BHIB tube to BEA, BHIB and
BHIB with 6.5%NaCl.
15.3.1 Incubate the BEA and BHIB with 6.5% Nad at 35 ± 0.5°C for 48 h.
15.3.2 Incubate the BHIB at 45 ± 0.5°C for 48 h.
15.4 Observe for growth 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 ± 0.5°C and BHIB with 6.5% NaCl at 35 ± 0.5°C are
verified as enterococci.
16.0 Pollution Prevention
16.1 The solutions and reagents used in this method pose little threat to the environment when recycled
and managed properly.
16.2 Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize
the volume of expired materials to be disposed.
17.0 Waste Management
17.1 It is the laboratory's responsibility to comply with all federal, state, and local regulations governing
waste management, particularly the biohazard and hazardous waste identification rules and land
disposal restrictions, and to protect the air, water, and land by minimizing and controlling all
releases from fume hoods and bench operations. Compliance with all sewage discharge permits
and regulations is also required.
17.2 Samples, reference materials, and equipment known or suspected to have viable enterococci
attached or contained must be sterilized prior to disposal.
17.3 Samples preserved with HC1 to pH <2 are hazardous and must be neutralized before being
disposed, or must be handled as hazardous waste.
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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