ASTM D5259-24

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Designation: D5259 − 19 D5259 − 24
Standard Test 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
1.1 This test method covers a membrane filter (MF) procedure for the detection and enumeration of the enterococci bacteria in
water. The enterococci, which include Entero-coccus faecalis (E. faecalis), E. faecium, and their varieties are commonly found in
the feces of humans and other warm-blooded animals. Although some strains are ubiquitous and not related to fecal pollution,
enterococci in water are an indication of fecal pollution and the possible presence of enteric pathogens. These bacteria are found
in water and wastewater in a wide range of densities. The detection limit is one colony forming unit (CFU)/volume filtered.
1.2 This test method has been used successfully with temperate fresh and marine ambient waters, and wastewaters. It is the user’s
responsibility to ensure the validity of this test method for waters of untested types.
1.3 The values stated in SI units are to be regarded as standard. The values given in parentheses after SI units are included for
information only and are not considered standard.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of
regulatory limitations prior to use. For specific hazard statements, see Section 9.
1.5 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
D1129 Terminology Relating to Water
D1193 Specification for Reagent Water
D3370 Practices for Sampling Water from Flowing Process Streams
D3870 Practice for Establishing Performance Characteristics for Colony Counting Methods in Microbiology (Withdrawn 2000)
3. Terminology
3.1 Definitions:
This test method is under the jurisdiction of ASTM Committee D19 on Water and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
Current edition approved April 1, 2019April 1, 2024. Published April 2019April 2024. Originally approved in 1992. Last previous edition approved in 20142019 as
D5259 – 14.D5259 – 19. DOI: 10.1520/D5259-19.10.1520/D5259-24.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
The last approved version of this historical standard is referenced on www.astm.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D5259 − 24
3.1.1 For definitions of terms used in this standard, refer to Terminology D1129.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 Enterococcus, n—in this test method,Enterococcus species are those bacteria that produce red to maroon colonies with black
or reddish-brown precipitate on underside, after incubation on mE agar and subsequent transfer to EIA medium.
3.2.1.1 Discussion—
Enterococci include E. faecalis, E. faecium, E. avium, and their variants.
4. Summary of Test Method
4.1 The procedure given in this test method provides a direct count of bacteria in water based on the development of colonies on
the surface of the membrane filter. A water sample is filtered through the membrane that 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°C,41 °C, then
transferred to EIA agar and held at 41°C41 °C for 20 min. Enterococci develop as red to maroon colonies with black or
reddish-brown precipitate on the underside of the filter.
5. Significance and Use
5.1 The enterococci are indicators of the bacteriological quality for potable water, shellfish growing waters, ambient, and
recreational waters. A direct relationship between swimming, associated gastroenteritis, and enterococci has been established
through epidemiological studies and marine and 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 relationships.
5.2 Since small or large volumes of water or dilutions thereof, can be analyzed by the membrane filter technique, a wide range
of levels of enterococci in water can be enumerated and detected.
6. Interferences
6.1 Water with high levels of colloidal or suspended materials can clog the membrane filter pores and prevent filtration. Also,
suspended materials cause spreading colonies that could interfere with target colonies and thereby prevent accurate counting.
6.2 Smaller sample size or sample dilution can be used to minimize the interference of turbidity or high-background (non-target)
bacterial densities. Replicates of smaller sample volumes or dilutions of sample may be filtered and the results combined. If the
membrane filter technique is not applicable, the most probable number (MPN) method for fecal streptococci is recommended, with
verification.
6.3 In some samples, chemicals may have toxic effects on the target organism.
7. Apparatus
7.1 Stereoscopic Microscope, wide-field type with magnification of 1010× to 15×.
7.2 Microscope Lamp, producing diffuse light from a cool, white fluorescent lamp adjusted to give maximum visibility.
7.3 Counting Device, hand tally or electronic.
7.4 Pipet Container, stainless steel, aluminum, or borosilicate glass, for glass pipets.
7.5 Pipets, sterile tip delivery bacteriological or Mohr, glass or plastic, of appropriate volume.
Cabelli, V. J., Dufour, A. P., Levin, M. A., McCabe, L. J., and Haberman, P. W., “Relationship of Microbial Indicators to Health Effects at Marine Bathing Beaches,”
American Journal of Public Health, Vol 69, 1979, pp. 690–696.
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7.6 Graduated Cylinders, 100 to 1000 mL, 100 mL to 1000 mL, covered with aluminum foil or kraft paper and sterile.
7.7 Membrane Filtration Units, (filter base and funnel), glass plastic or stainless steel, wrapped in aluminum foil or kraft paper
and sterilized.sterilized or disposable sterile plastic units.
7.8 Ultraviolet Unit, Unit (254 nm), for disinfecting the filtration unit (optional).
7.9 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 or air, can be used.
7.10 Flask, filter, vacuum, usually 1 L, with appropriate tubing. A filter manifold to hold a number of filter bases is optional.
7.11 Forceps, straight or curved, with smooth tips to handle filters without damage.
7.12 Thermometer, checked against a National Institute of Standards and Technology (NIST) certified thermometer, or one
traceable to an NIST thermometer.
7.13 Petri Dishes, sterile, plastic, 5050 mm by 12 mm, with tight-fitting lids.
7.14 Bottles, milk dilution, borosilicate glass, screw-cap with neoprene liners, marked at 99 mL for 1 to 100 dilutions. Dilution
bottles marked at 90 mL or tubes marked at 9 mL may be used for 1:10 dilutions.
7.15 Inoculation Loops, at least 3-mm3 mm diameter, and needles, nichrome or platinum wire, 26 B and S gage,gauge, in suitable
holders.
7.16 Incubator maintained at 41 6 0.5°C.41 °C 6 0.5 °C.
7.17 Waterbath maintained at 44 to 46°C44 °C to 46 °C for tempering agar.
7.18 Test Tubes, 150150 mm by 20 mm, borosilicate glass or plastic.
7.19 Caps, aluminum or autoclavable plastic, for 20-mm20 mm diameter test tubes.
7.20 Test Tubes, screw-cap, borosilicate glass, 125125 mm by 16 mm or other appropriate size.
8. Reagents and Materials
8.1 Purity of Reagents—Reagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that all
reagents conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society where such
specifications are available. Other grades may be used, provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without lessening the accuracy of the determination.
8.1.1 The agar used in preparation of culture media must be of microbiological grade. Whenever possible, use commercial culture
media as a means of quality control.
8.1.2 Purity of Water—Unless otherwise indicated, references to water shall be understood to mean reagent water as defined by
Type III of Specification D1193.
Reagent Chemicals, American Chemical Society Specifications,ACS Reagent Chemicals, Specifications and Procedures for Reagents and Standard-Grade Reference
Materials, American Chemical Society, Washington, DC. For suggestions on the testing of reagents not listed by the American Chemical Society, see Analar Standards for
Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC),
Rockville, MD.
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8.1.3 Ethanol, Methanol or Isopropanol, in a small, wide-mouth container, for flame-sterilization of pipets.
8.2 Membrane Filters, sterile, white, grid marked, 47-mm47 mm diameter, with 0.45 6 0.02 μm 0.45 μm 6 0.02 μm pore size or
other pore sizes for which the manufacturer provides data demonstrating equivalency.
8.3 Buffered Dilution Water/Buffered Rinse Water:
8.3.1 Composition/Litre:
Sodium dihydrogen phosphate (NaH PO ) 0.58 g
2 4
Sodium monohydrogen phosphate (Na HPO ) 2.50 g
2 4
Sodium chloride 8.50 g
8.3.2 Preparation—Dissolve the ingredients in 1 L of water in a flask and dispense in appropriate amounts for dilutions in
screw-cap bottles or culture tubes or into containers for use as rinse water, or both. Autoclave after preparation at 121°C (15 lb
121 °C (15 lb pressure at sea level) for 15 min. The final pH should be 7.4 6 0.2.
8.4 mE Agar:
8.4.1 Composition of Basal Medium/Litre:
Peptone 10.0 g
Sodium chloride 15.0 g
Yeast extract 30.0 g
Esculin 1.0
Actidione 0.05 g
Sodium azide 0.15 g
Agar 15.0 g
Water 1000 mL
8.4.2 Preparation of Basal Medium—Add 71.2 g of the above mE basal medium to 1 L of water in a flask and heat to boiling until
ingredients dissolve. Autoclave at 121°C and 15 lb 121 °C and 15 lb pressure for 15 min and cool in a 44 to 46°C44 °C to 46 °C
water bath.
8.4.3 Reagents Added After Sterilization—Mix 0.25 g nalidixic acid in 5 mL water, add 0.2 mL of NaOH solution (400(400 g g/L)
⁄L) to dissolve, and add to the litre of basal medium. Add 0.15 g 0.15 g triphenyl tetrazolium chloride separately to the basal
medium and mix.
8.4.4 Preparation of mE Agar Plates—Pour the mE agar into 50 mm petri plates to a 44 mm to 5 mm depth (approximately 44 mL
to 6 mL), and allow to solidify. The final pH of medium should be 7.1 6 0.2. Store in a refrigerator.
8.5 EIA Agar:
8.5.1 Composition of EIA Medium/Litre:
Esculin 1.0 g
Ferric citrate 0.5 g
Agar 15.0 g
Water 1000 mL
8.5.2 Preparation—Add 16.5 g of dehydrated EIA medium to 1 L of water in flask and heat to boiling until ingredients are
dissolved. Autoclave the EIA medium solution at 121°C121 °C (15 lb pressure at sea level) for 15 min and cool in a 44 to
46°C44 °C to 46 °C water bath. After cooling, pour the medium into 50-mm50 mm petri dishes to a depth of 44 mm to 5 mm
(approximately 44 mL to 6 mL and allow to solidify. The final pH should be 7.1 6 0.2 before autoclaving. Store in a refrigerator.
This is available commercially and is recommended to purchase and not prepare from individual components.
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8.6 Brain Heart Infusion (BHI) Broth:
8.6.1 Composition:
Calf brain infusion 200.0 g
Beef heart infusion 250.0 g
Peptone 10.0 g
Sodium chloride 5.0 g
Disodium phosphate 2.5 g
Dextrose 2.0 g
Water 1000 mL
8.6.2 Preparation—Dissolve 37 g of dehydrated BHI broth in 1 L of water. Dispense in 88 mL to 10 mL volumes in screw-cap
tubes and autoclave at 121°C121 °C (15 lb pressure at sea level) 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, remove
absorbed oxygen, and cool quickly without agitation, just prior to inoculation. The final pH sh
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