ASTM D5259-92(2006)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: D5259 − 92 (Reapproved 2006)
StandardTest Method for
Isolation and Enumeration of Enterococci from Water by the
Membrane Filter Procedure
This standard is issued under the fixed designation D5259; 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 (´) indicates an editorial change since the last revision or reapproval.
1. Scope for Colony Counting Methods in Microbiology (With-
drawn 2000)
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 D1129.
commonly found in the feces of humans and other warm-
blooded animals.Although some strains are ubiquitous and not
3.2 Definitions of Terms Specific to This Standard:
relatedtofecalpollution,enterococciinwaterareanindication
3.2.1 Enterococcus—In this test method, Enterococcus spe-
of fecal pollution and the possible presence of enteric patho-
ciesarethosebacteriathatproduceredtomarooncolonieswith
gens. These bacteria are found in water and wastewater in a
black or reddish-brown precipitate on underside, after incuba-
wide range of densities. The detection limit is one colony
tion on mE agar and subsequent transfer to EIA medium.
forming unit (CFU)/volume filtered.
Enterococciinclude E. faecalis, E. faecium, E. avium,andtheir
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 4
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.
5. Significance and Use
2. Referenced Documents
5.1 The enterococci are indicators of the bacteriological
2.1 ASTM Standards:
quality for potable water, shellfish growing waters, ambient,
D1129 Terminology Relating to Water
and recreational waters. A direct relationship between
D1193 Specification for Reagent Water
swimming,associatedgastroenteritis,andenterococcihasbeen
D3370 Practices for Sampling Water from Closed Conduits
established through epidemiological studies and marine and
D3870 PracticeforEstablishingPerformanceCharacteristics
fresh water bathing beaches. These studies have led to the
development of criteria that can be used to establish bathing
water standards based on established health-water quality
This test method is under the jurisdiction of ASTM Committee D19 on Water
relationships.
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
Current edition approved July 1, 2006. Published July 2006. Originally approved
in 1992. Last previous edition approved in 2000 as D5259 – 92 (2000). DOI:
10.1520/D5259-92R06. The last approved version of this historical standard is referenced on
For referenced ASTM standards, visit the ASTM website, www.astm.org, or www.astm.org.
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Cabelli,V. J., Dufour,A. P., Levin, M.A., McCabe, L. J., and Haberman, P.W.,
Standards volume information, refer to the standard’s Document Summary page on “RelationshipofMicrobialIndicatorstoHealthEffectsatMarineBathingBeaches,”
the ASTM website. 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 (Reapproved 2006)
5.2 Since small or large volumes of water or dilutions 7.14 Bottles, milk dilution, borosilicate glass, screw-cap
thereof, can be analyzed by the membrane filter technique, a with neoprene liners, marked at 99 mL for 1 to 100 dilutions.
wide range of levels of enterococci in water can be enumerated Dilution bottles marked at 90 mLor tubes marked at 9 mLmay
and detected. be used for 1:10 dilutions.
7.15 Inoculation Loops, at least 3 mm diameter, and
6. Interferences
needles, nichrome or platinum wire, 26 B and S gage, in
suitable holders.
6.1 Water with high levels of colloidal or suspended mate-
rials can clog the membrane filter pores and prevent filtration.
7.16 Incubator maintained at 41 6 0.5°C.
Also, suspended materials cause spreading colonies that could
7.17 Waterbath maintained at 44 to 46°C for tempering
interfere with target colonies and thereby prevent accurate
agar.
counting.
7.18 Test Tubes, 150 by 20 mm, borosilicate glass or plastic.
6.2 Smaller sample size or sample dilution can be used to
7.19 Caps, aluminum or autoclavable plastic, for 20 mm
minimize the interference of turbidity or high-background
diameter test tubes.
(non-target) bacterial densities. Replicates of smaller sample
volumes or dilutions of sample may be filtered and the results
7.20 Test Tubes, screw-cap, borosilicate glass, 125 by 16
combined. If the membrane filter technique is not applicable,
mm or other appropriate size.
the most probable number (MPN) method for fecal strepto-
cocci is recommended, with verification.
8. Reagents and Materials
6.3 In some samples, chemicals may have toxic effects on
8.1 Purity of Reagents—Reagent grade chemicals shall be
the target organism.
used in all tests. Unless otherwise indicated, it is intended that
all reagents conform to the specifications of the Committee on
7. Apparatus Analytical Reagents of the American Chemical Society where
such specifications are available. Other grades may be used,
7.1 Stereoscopic Microscope, wide-field type with magnifi-
provided it is first ascertained that the reagent is of sufficiently
cation of 10 to 15X.
high purity to permit its use without lessening the accuracy of
7.2 Microscope Lamp, producing diffuse light from a cool,
the determination.
white fluorescent lamp adjusted to give maximum visibility.
8.1.1 The agar used in preparation of culture media must be
of microbiological grade. Whenever possible, use commercial
7.3 Counting Device, hand tally or electronic.
culture media as a means of quality control.
7.4 Pipet Container, stainless steel, aluminum, or borosili-
8.1.2 Purity of Water—Unless otherwise indicated, refer-
cate glass, for glass pipets.
ences to water shall be understood to mean reagent water as
defined by Type III of Specification D1193.
7.5 Pipets, sterile tip delivery bacteriological or Mohr, glass
or plastic, of appropriate volume. 8.1.3 Ethanol, Methanol or Isopropanol, in a small, wide-
mouth container, for flame-sterilization of pipets.
7.6 Graduated Cylinders, 100 to 1000 mL, covered with
8.2 Membrane Filters, sterile, white, grid marked, 47 mm
aluminum foil or kraft paper and sterile.
diameter, with 0.45 6 0.02 µm pore size or other pore sizes for
7.7 Membrane Filtration Units, (filter base and funnel),
which the manufacturer provides data demonstrating equiva-
glass plastic or stainless steel, wrapped in aluminum foil or
lency.
kraft paper and sterilized.
8.3 Buffered Dilution Water/Buffered Rinse Water:
7.8 Ultraviolet Unit, for disinfecting the filtration unit
8.3.1 Composition/Litre:
(optional).
Sodium dihydrogen phosphate (NaH PO)0.58g
2 4
Sodium monohydrogen phosphate (Na HPO)2.50g
7.9 Line Vacuum, Electric Vacuum Pump, or Aspirator, for
2 4
Sodium chloride 8.50 g
use as a vacuum source. In an emergency or in the field, a hand
8.3.2 Preparation—Dissolve the ingredients in 1 Lof water
pump or a syringe equipped with a check valve to prevent the
in a flask and dispense in appropriate amounts for dilutions in
return flow or air, can be used.
screw-cap bottles or culture tubes or into containers for use as
7.10 Flask, filter, vacuum, usually 1 L, with appropriate
rinse water, or both. Autoclave after preparation at 121°C (15
tubing. A filter manifold to hold a number of filter bases is
lb pressure at sea level) for 15 min. The final pH should be 7.4
optional.
6 0.2.
7.11 Forceps, straight or curved, with smooth tips to handle
8.4 mE Agar:
filters without damage.
8.4.1 Composition of Basal Medium/Litre:
7.12 Thermometer, checked against a National Institute of
Standards and Technology (NIST) certified thermometer, or
one traceable to an NIST thermometer.
“Reagent Chemicals,American Chemical Society Specifications,”Am. Chemi-
cal Soc., Washington, DC. For suggestions on the testing of reagents not listed by
7.13 Petri Dishes, sterile, plastic, 50 by 12 mm, with
theAmerican Chemical Society, see “Analar Standards for Laboratory Chemicals,”
tight-fitting lids. BDH Ltd. Poole, Dorset, U.K. and the “United States Pharmacopeia.”
D5259 − 92 (Reapproved 2006)
8.8.1 Composition—BHI agar contains the same compo-
Peptone 10.0 g
Sodium chloride 15.0 g
nents as BHI (see 8.6) with the addition of 15.0 g of agar per
Yeast extract 30.0 g
litre of BHI Broth.
Esculin 1.0
8.8.2 Preparation—Add 15.0 g of agar and 37.0 g of BHI
Actidione 0.05 g
Sodium azide 0.15 g
dehydrated broth to 1 L of water. Heat to boiling until
Agar 15.0 g
ingredients are dissolved. Dispense 10 to 12 mL of medium in
Water 1000 mL
screw-cap test tubes and sterilize for 15 min at 121°C (15 lb
8.4.2 Preparation of Basal Medium—Add 71.2 g of the
pressure at sea level). Slant after sterilization. The final pH
above mE basal medium to 1 L of water in a flask and heat to
should be 7.4 6 0.2.
boilinguntilingredientsdissolve.Autoclaveat121°Cand15lb
8.9 Bile Esculin Agar (BEA):
pressure for 15 min and cool in a 44 to 46°C water bath.
8.9.1 Composition/Litre:
8.4.3 Reagents Added After Sterilization—Mix 0.25 g nali-
dixic acid in 5 mL water, add 0.2 mL of NaOH solution (400 Bacto beef extract 3.0 g
Bacto peptone 5.0 g
g/L)todissolve,andaddtothelitreofbasalmedium.Add0.15
Bacto oxgall 40.0 g
gtriphenyltetrazoliumchlorideseparatelytothebasalmedium
Bacto esculin 1.0 g
Ferric citrate 0.5 g
and mix.
Bacto agar 15.0
8.4.4 Preparation of mE Agar Plates—Pour the mE agar
Water 1000 mL
into 50 mm petri plates to a 4 to 5 mm depth (approximately 4
8.9.2 Preparation—Add 64.5 g of dehydrated BEA to 1 L
to 6 mL), and allow to solidify.The final pH of medium should
water and heat to boiling to dissolve. Dispense in 8 to 10 mL
be 7.1 6 0.2. Store in a refrigerator.
volumes in tubes for slants or into flasks for subsequent
8.5 EIA Agar:
plating.Autoclave at 121°C (15 lb pressure at sea level) for 15
8.5.1 Composition of EIA Medium/Litre:
min. Overheating may cause darkening of the medium. Cool to
Esculin 1.0 g 44 to 46°C and dispense into sterile petri plates. The final pH
Ferric citrate 0.5 g
should be 6.6 6 0.2. Store in a refrigerator.
Agar 15.0 g
Water 1000 mL
8.10 Gram Stain—Prepare according to APHA document.
8.5.2 Preparation—Add 16.5 g of dehydrated EIA medium
9. Hazards
to 1 Lof water in flask and heat to boiling until ingredients are
dissolved.Autoclave the EIAmedium solution at 121°C (15 lb
9.1 The analyst/technician must know and observe the
pressure at sea level) for 15 min and cool in a 44 to 46°C water
normal good laboratory practices and safety procedures re-
bath. After cooling, pour the medium into 50-mm petri dishes
quiredinamicrobiologylaboratorywhilepreparing,using,and
to a depth of 4 to 5 mm (approximately 4 to 6 mLand allow to
disposing of cultures, reagents, and materials, and while
solidify. The final pH should be 7.1 6 0.2 before autoclaving.
operating sterilization and other equipment and instrumenta-
Store in a refrigerator.
tion.
8.6 Brain Heart Infusion (BHI) Broth:
9.2 Mouth-pipetting is prohibited.
8.6.1 Composition:
Calf brain infusion 200.0 g
10. Sample Collection, Preservation, and Holding Times
Beef heart infusion 250.0 g
10.1 Sampling procedures are described in detail in Section
Peptone 10.0 g
Sodium chloride 5.0 g
II,AoftheUSEPAmanual andPracticeD3370andadherence
Disodium phosphate 2.5 g
to sample preservation procedure and holding time limits is
Dextrose 2.0 g
critical to the production of valid data. Samples should not be
Water 1000 mL
analyzed if these conditions are not met.
8.6.2 Preparation—Dissolve 37 g of dehydrated BHI broth
10.1.1 Storage Temperature and Handling Conditions—Ice
in 1 L of water. Dispense in 8 to 10 mL volumes in screw-cap
or refrigerate bacteriological samples at a temperature of 1 to
tubes and autoclave at 121°C (15 lb pressure at sea level) for
4°C during transit to the laboratory. Use insulated containers to
15min.Ifthemediumisnotusedthesamedayaspreparedand
ensure proper maintenance of storage temperature. Take care
sterilized, heat in boiling water bath for several min to remove
that sample bottles are not totally immersed in water during
absorbed oxygen, and cool quickly without agitation, remove
transit or storage.
absorbed oxygen, and cool quickly without agitation, just prior
10.1.2 Holding Time Limitations—Examine samples as
to inoculation. The final pH should be 7.4 6 0.2.
soon as possible after collection. Do not hold samples longer
8.7 BHI Broth with 6.5 % NaCl:
than 6 h between collection and initiation of analyses.
8.7.1 Composition—BHI broth with 6.5 % NaCl is the same
as BHI broth in 8.6 with additional NaCl.
8.7.2 Preparation—Dissolve 60.0 g NaCl per litre of pre-
Standard Methods for Examination of Water and Wastewater, 18th EJ.,
pared BHI broth. Since most commercially available dehy-
American Public Health Association, Washington, DC, 1992, pp.9-48.
dratedmediacontainsodiumchloride,thisamountistakeninto Bordner,R.,Winter,J.A.,andScarpino,P.V.,(eds.),“MicrobiologicalMethods
for Monitoring the Environment, Water and Wastes,” EPA-600/8-78-017, U.S.
consideration in determining the final NaCl percentage above.
Environmental Protection Agency, Office of Research and Development, Environ-
8.8 BHI Agar: mental Monitoring and Support Laboratory—C
...