ASTM D5259-92(2000)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation:D5259–92(Reapproved2000)
Standard Test Method for
Isolation and Enumeration of Enterococci from Water by the
Membrane Filter Procedure
This standard is issued under the fixed designation D 5259; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope D 3870 Practice for Establishing Performance Characteris-
tics for Colony Counting Methods in Microbiology
1.1 This test method covers a membrane filter (MF) proce-
dure for the detection and enumeration of the enterococci
3. Terminology
bacteria in water. The enterococci, which include Entero-
3.1 Definitions—For definitions of terms used in this test
coccus faecalis (E. faecalis), E. faecium, and their varieties are
method, refer to Terminology D 1129.
commonly found in the feces of humans and other warm-
3.2 Definitions of Terms Specific to This Standard:
blooded animals.Although some strains are ubiquitous and not
3.2.1 Enterococcus—In this test method, Enterococcus spe-
relatedtofecalpollution,enterococciinwaterareanindication
ciesarethosebacteriathatproduceredtomarooncolonieswith
of fecal pollution and the possible presence of enteric patho-
black or reddish-brown precipitate on underside, after incuba-
gens. These bacteria are found in water and wastewater in a
tion on mE agar and subsequent transfer to EIA medium.
wide range of densities. The detection limit is one colony
Enterococci include E. faecalis, E. faecium, E. avium, and their
forming unit (CFU)/volume filtered.
variants.
1.2 This test method has been used successfully with
temperatefreshandmarineambientwaters,andwastewaters.It
4. Summary of Test Method
is the user’s responsibility to ensure the validity of this test
4.1 The procedure given in this test method provides a
method for waters of untested types.
direct count of bacteria in water based on the development of
1.3 The values stated in SI units are to be regarded as the
coloniesonthesurfaceofthemembranefilter. Awatersample
standard.
is filtered through the membrane that retains the bacteria.
1.4 This standard does not purport to address all of the
Following filtration, the membrane containing the bacterial
safety concerns, if any, associated with its use. It is the
cells is placed on a selective, medium, mE agar, and incubated
responsibility of the user of this standard to establish appro-
for 48 h at 41°C, then transferred to EIAagar and held at 41°C
priate safety and health practices and determine the applica-
for20min.Enterococcidevelopasredtomarooncolonieswith
bility of regulatory limitations prior to use. For specific hazard
black or reddish-brown precipitate on the underside of the
statements, see Section 9.
filter.
2. Referenced Documents
5. Significance and Use
2.1 ASTM Standards:
5.1 The enterococci are indicators of the bacteriological
D 1129 Terminology Relating to Water
2 quality for potable water, shellfish growing waters, ambient,
D 1193 Specification for Reagent Water
and recreational waters. A direct relationship between swim-
D 3370 Practices for Sampling Water from Closed Con-
2 ming, associated gastroenteritis, and enterococci has been
duits
established through epidemiological studies and marine and
1 3
This test method is under the jurisdiction of ASTM Committee D19 on Water Annual Book of ASTM Standards, Vol 11.02.
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology. Cabelli,V. J., Dufour,A. P., Levin, M.A., McCabe, L. J., and Haberman, P.W.,
Current edition approved June 15, 1992. Published October 1992. “RelationshipofMicrobialIndicatorstoHealthEffectsatMarineBathingBeaches,”
Annual Book of ASTM Standards, Vol 11.01. American Journal of Public Health, 69, 1979, pp. 690–696.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D5259–92 (2000)
fresh water bathing beaches. These studies have led to the 7.14 Bottles, milk dilution, borosilicate glass, screw-cap
development of criteria that can be used to establish bathing with neoprene liners, marked at 99 mL for 1 to 100 dilutions.
water standards based on established health-water quality Dilution bottles marked at 90 mLor tubes marked at 9 mLmay
relationships. be used for 1:10 dilutions.
7.15 Inoculation Loops, at least 3 mm diameter, and
5.2 Since small or large volumes of water or dilutions
needles, nichrome or platinum wire, 26 B and S gage, in
thereof, can be analyzed by the membrane filter technique, a
suitable holders.
wide range of levels of enterococci in water can be enumerated
7.16 Incubator maintained at 41 6 0.5°C.
and detected.
7.17 Waterbath maintained at 44 to 46°C for tempering
agar.
6. Interferences
7.18 Test Tubes, 150 by 20 mm, borosilicate glass or plastic.
6.1 Water with high levels of colloidal or suspended mate-
7.19 Caps, aluminum or autoclavable plastic, for 20 mm
rials can clog the membrane filter pores and prevent filtration.
diameter test tubes.
Also, suspended materials cause spreading colonies that could
7.20 Test Tubes, screw-cap, borosilicate glass, 125 by 16
interfere with target colonies and thereby prevent accurate
mm or other appropriate size.
counting.
6.2 Smaller sample size or sample dilution can be used to
8. Reagents and Materials
minimize the interference of turbidity or high-background
8.1 Purity of Reagents—Reagent grade chemicals shall be
(non-target) bacterial densities. Replicates of smaller sample
used in all tests. Unless otherwise indicated, it is intended that
volumes or dilutions of sample may be filtered and the results
all reagents conform to the specifications of the Committee on
combined. If the membrane filter technique is not applicable,
Analytical Reagents of the American Chemical Society where
the most probable number (MPN) method for fecal strepto- 5
such specifications are available. Other grades may be used,
cocci is recommended, with verification.
provided it is first ascertained that the reagent is of sufficiently
6.3 In some samples, chemicals may have toxic effects on
high purity to permit its use without lessening the accuracy of
the target organism.
the determination.
8.1.1 The agar used in preparation of culture media must be
7. Apparatus
of microbiological grade. Whenever possible, use commercial
culture media as a means of quality control.
7.1 Stereoscopic Microscope, wide-field type with magnifi-
8.1.2 Purity of Water— Unless otherwise indicated, refer-
cation of 10 to 15X.
ences to water shall be understood to mean reagent water as
7.2 Microscope Lamp, producing diffuse light from a cool,
defined by Type III of Specification D 1193.
white fluorescent lamp adjusted to give maximum visibility.
8.1.3 Ethanol, Methanol or Isopropanol, in a small, wide-
7.3 Counting Device, hand tally or electronic.
mouth container, for flame-sterilization of pipets.
7.4 Pipet Container, stainless steel, aluminum, or borosili-
8.2 Membrane Filters, sterile, white, grid marked, 47 mm
cate glass, for glass pipets.
diameter, with 0.45 6 0.02 µm pore size or other pore sizes for
7.5 Pipets, sterile tip delivery bacteriological or Mohr, glass
which the manufacturer provides data demonstrating equiva-
or plastic, of appropriate volume.
lency.
7.6 Graduated Cylinders, 100 to 1000 mL, covered with
8.3 Buffered Dilution Water/Buffered Rinse Water:
aluminum foil or kraft paper and sterile.
8.3.1 Composition/Litre:
7.7 Membrane Filtration Units, (filter base and funnel),
glass plastic or stainless steel, wrapped in aluminum foil or
Sodium dihydrogen phosphate (NaH PO)0.58g
2 4
Sodium monohydrogen phosphate (Na HPO)2.50g
kraft paper and sterilized. 2 4
Sodium chloride 8.50 g
7.8 Ultraviolet Unit, for disinfecting the filtration unit
8.3.2 Preparation—Dissolvetheingredientsin1Lofwater
(optional).
in a flask and dispense in appropriate amounts for dilutions in
7.9 Line Vacuum, Electric Vacuum Pump, or Aspirator, for
screw-cap bottles or culture tubes or into containers for use as
use as a vacuum source. In an emergency or in the field, a hand
rinse water, or both. Autoclave after preparation at 121°C (15
pump or a syringe equipped with a check valve to prevent the
lb pressure at sea level) for 15 min. The final pH should be 7.4
return flow or air, can be used.
6 0.2.
7.10 Flask, filter, vacuum, usually 1 L, with appropriate
8.4 mE Agar :
tubing. A filter manifold to hold a number of filter bases is
8.4.1 Composition of Basal Medium/Litre:
optional.
7.11 Forceps, straight or curved, with smooth tips to handle
filters without damage.
7.12 Thermometer, checked against a National Institute of
“Reagent Chemicals,American Chemical Society Specifications,”Am. Chemi-
Standards and Technology (NIST) certified thermometer, or cal Soc., Washington, DC. For suggestions on the testing of reagents not listed by
theAmerican Chemical Society, see “Analar Standards for Laboratory Chemicals,”
one traceable to an NIST thermometer.
BDH Ltd. Poole, Dorset, U.K. and the “United States Pharmacopeia.”
7.13 Petri Dishes, sterile, plastic, 50 by 12 mm, with
Difco 0333-15-1, available from Difco Laboratories, P.O. Box 1058, Detroit,
tight-fitting lids. MI 48232, or equivalent, has been found suitable for this purpose.
D5259–92 (2000)
8.7.1 Composition— BHI broth with 6.5 % NaCl is the
Peptone 10.0 g
Sodium chloride 15.0 g
same as BHI broth in 8.6 with additional NaCl.
Yeast extract 30.0 g
8.7.2 Preparation— Dissolve 60.0 g NaCl per litre of
Esculin 1.0
prepared BHI broth. Since most commercially available dehy-
Actidione 0.05 g
Sodium azide 0.15 g
dratedmediacontainsodiumchloride,thisamountistakeninto
Agar 15.0 g
consideration in determining the final NaCl percentage above.
Water 1000 mL
8.8 BHI Agar :
8.4.2 Preparation of Basal Medium—Add 71.2 g of the
8.8.1 Composition— BHI agar contains the same compo-
above mE basal medium to 1 L of water in a flask and heat to
nents as BHI (see 8.6) with the addition of 15.0 g of agar per
boilinguntilingredientsdissolve.Autoclaveat121°Cand15lb
litre of BHI Broth.
pressure for 15 min and cool in a 44 to 46°C water bath.
8.8.2 Preparation— Add 15.0 g of agar and 37.0 g of BHI
8.4.3 Reagents Added After Sterilization—Mix 0.25 g nali-
dehydrated broth to 1 L of water. Heat to boiling until
dixic acid in 5 mL water, add 0.2 mL of NaOH solution (400
ingredients are dissolved. Dispense 10 to 12 mL of medium in
g/L)todissolve,andaddtothelitreofbasalmedium.Add0.15
screw-cap test tubes and sterilize for 15 min at 121°C (15 lb
gtriphenyltetrazoliumchlorideseparatelytothebasalmedium
pressure at sea level). Slant after sterilization. The final pH
and mix.
should be 7.4 6 0.2.
8.4.4 Preparation of mE Agar Plates—Pour the mE agar
8.9 Bile Esculin Agar (BEA) :
into 50 mm petri plates to a 4 to 5 mm depth (approximately 4
8.9.1 Composition/Litre:
to 6 mL), and allow to solidify.The final pH of medium should
be 7.1 6 0.2. Store in a refrigerator.
Bacto beef extract 3.0 g
8.5 EIA Agar : Bacto peptone 5.0 g
Bacto oxgall 40.0 g
8.5.1 Composition of EIA Medium/Litre:
Bacto esculin 1.0 g
Ferric citrate 0.5 g
Bacto agar 15.0
Esculin 1.0 g
Water 1000 mL
Ferric citrate 0.5 g
Agar 15.0 g
8.9.2 Preparation— Add 64.5 g of dehydrated BEA to 1 L
Water 1000 mL
water and heat to boiling to dissolve. Dispense in 8 to 10 mL
8.5.2 Preparation—Add 16.5 g of dehydrated EIAmedium
volumes in tubes for slants or into flasks for subsequent
to 1 Lof water in flask and heat to boiling until ingredients are
plating.Autoclave at 121°C (15 lb pressure at sea level) for 15
dissolved.Autoclave the EIAmedium solution at 121°C (15 lb
min. Overheating may cause darkening of the medium. Cool to
pressure at sea level) for 15 min and cool in a 44 to 46°C water
44 to 46°C and dispense into sterile petri plates. The final pH
bath. After cooling, pour the medium into 50-mm petri dishes
should be 6.6 6 0.2. Store in a refrigerator.
to a depth of 4 to 5 mm (approximately 4 to 6 mLand allow to 11
8.10 Gram Stain—Prepare according toAPHAdocument.
solidify. The final pH should be 7.1 6 0.2 before autoclaving.
Store in a refrigerator.
9. Hazards
8.6 Brain Heart Infusion (BHI) Broth :
9.1 The analyst/technician must know and observe the
8.6.1 Composition:
normal good laboratory practices and safety procedures re-
quiredinamicrobiologylaboratorywhilepreparing,using,and
Calf brain infusion 200.0 g
disposing of cultures, reagents, and materials, and while
Beef heart infusion 250.0 g
operating sterilization and other equipment and instrumenta-
Peptone 10.0 g
Sodium chloride 5.0 g
tion.
Disodium phosphate 2.5 g
9.2 Mouth-pipetting is prohibited.
Dextrose 2.0 g
Water 1000 mL
10. Sample Collection, Preservation, and Holding Times
8.6.2 Preparation— Dissolve 37 g of dehydrated BHI broth
10.1 Sampling procedures are described in detail in Section
in 1 L of water. Dispense in 8 to 10 mL volumes in screw-cap
II, A of the USEPA manual and Practice D 3370 and
tubes and autoclave at 121°C (15 lb pressure at sea level) for
adherence to sample preservation procedure and holding time
15min.Ifthemediumisnotusedthesamedayaspreparedand
sterilized, heat in boiling water bath for several min to remove
absorbed oxygen, and cool quickly without agitation, remove
absorbed oxygen, and cool quickly without agitation, just prior 9
Difco 0418-02, BBL11064, available from Difco Laboratories, P.O. Box 1058,
to inoculation. The final pH should be 7.4 6 0.2.
Detroit, MI 48232, or equivalent, has been found suitable for this purpose.
Difco 0879, BBL 21838, available from Difco Laboratories, P.O. Box 1058,
8.7 BHI Broth with 6.5 % NaCl:
Detroit, MI 48232, (plates or equivalent), has been found suitable for this purpose.
Standard Methods for Examination of Water and Wastewater, 18th EJ.,
American Public Health Association, Washington, DC, 1992, pp.9-48.
Bordner, R., Winter, J. A., and Scarpino, P. V., (eds.),“ Microbiological
Difco 0488-15-4, available from Difco Laboratories, P.O. Box 1058, Detroit, Methods for Monitoring the Environment, Water and Wastes,” EPA-600/8-78-017,
MI 48232, or equivalent, has been found suitable for this purpose. U.S. Environmental Protection Agency, Office of Research and Development,
Difco 0037-02, BBL11058, available from Difco Laboratories, P.O. Box 1058, Environmental Monitoring and Support Laboratory—Cincinnati, Cincinnati, Ohio,
Detroit, MI 48232, or equivalent, has been found suitable for this purpose. 1978.
D5259–92 (2000)
limitsiscriticaltotheproductionofvaliddata.Samplesshould 13. Verification Procedure
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