ASTM D5259-24

Standard Test Method for Isolation and Enumeration of Enterococci from Water by the Membrane Filter Procedure

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.
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.

General Information

• Status: Published
• Publication Date: 31-Mar-2024
• ICS: 13.060.70 - Examination of biological properties of water
• Technical Committee: D19 - Water
• Drafting Committee: D19.24 - Water Microbiology

Relations

• Replaces: ASTM D5259-19 - Standard Test Method for Isolation and Enumeration of Enterococci from Water by the Membrane Filter Procedure
• Effective Date: 01-Apr-2024
• Referred By: ASTM D5465-16(2020) - Standard Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
• Effective Date: 01-Apr-2024
• Referred By: ASTM E3152-23 - Standard Guide for Standard Test Methods and Practices Available for Determining Antifungal Activity on Natural or Synthetic Substrates Treated with Antimicrobial Agents
• Effective Date: 01-Apr-2024
• Referred By: ASTM E1891-21 - Standard Guide for Determination of a Survival Curve for Antimicrobial Agents Against Selected Microorganisms and Calculation of a D-Value and Concentration Coefficient
• Effective Date: 01-Apr-2024
• Referred By: ASTM D6974-20 - Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels—Filtration and Culture Procedures
• Effective Date: 01-Apr-2024

Standards Content (Sample)

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.
Designation: D5259 − 24
Standard Test Method for
Isolation and Enumeration of Enterococci from Water by the
1
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 2. Referenced Documents
2
2.1 ASTM Standards:
1.1 This test method covers a membrane filter (MF) proce-
dure for the detection and enumeration of the enterococci D1129 Terminology Relating to Water
D1193 Specification for Reagent Water
bacteria in water. The enterococci, which include Entero-
coccus faecalis (E. faecalis), E. faecium, and their varieties are D3370 Practices for Sampling Water from Flowing Process
Streams
commonly found in the feces of humans and other warm-
blooded animals. Although some strains are ubiquitous and not D3870 Practice for Establishing Performance Characteristics
related to fecal pollution, enterococci in water are an indication for Colony Counting Methods in Microbiology (With-
3
of fecal pollution and the possible presence of enteric patho- drawn 2000)
gens. These bacteria are found in water and wastewater in a
3. Terminology
wide range of densities. The detection limit is one colony
forming unit (CFU)/volume filtered.
3.1 Definitions:
3.1.1 For definitions of terms used in this standard, refer to
1.2 This test method has been used successfully with
Terminology D1129.
temperate fresh and marine ambient waters, and wastewaters. It
is the user’s responsibility to ensure the validity of this test 3.2 Definitions of Terms Specific to This Standard:
method for waters of untested types.
3.2.1 Enterococcus, n—in this test method, Enterococcus
species are those bacteria that produce red to maroon colonies
1.3 The values stated in SI units are to be regarded as
with black or reddish-brown precipitate on underside, after
standard. The values given in parentheses after SI units are
incubation on mE agar and subsequent transfer to EIA medium.
included for information only and are not considered standard.
3.2.1.1 Discussion—Enterococci include E. faecalis, E.
1.4 This standard does not purport to address all of the
faecium, E. avium, and their variants.
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
4. Summary of Test Method
priate safety, health, and environmental practices and deter-
4.1 The procedure given in this test method provides a
mine the applicability of regulatory limitations prior to use.
direct count of bacteria in water based on the development of
For specific hazard statements, see Section 9.
4
colonies on the surface of the membrane filter. A water sample
1.5 This international standard was developed in accor-
is filtered through the membrane that retains the bacteria.
dance with internationally recognized principles on standard-
Following filtration, the membrane containing the bacterial
ization established in the Decision on Principles for the
cells is placed on a selective, medium, mE agar, and incubated
Development of International Standards, Guides and Recom-
mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee. 2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
1 3
This test method is under the jurisdiction of ASTM Committee D19 on Water The last approved version of this historical standard is referenced on
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology. www.astm.org.
4
Current edition approved April 1, 2024. Published April 2024. Originally Cabelli, V. J., Dufour, A. P., Levin, M. A., McCabe, L. J., and Haberman, P. W.,
approved in 1992. Last previous edition approved in 2019 as D5259 – 19. DOI: “Relationship of Microbial Indicators to Health Effects at Marine Bathing Beaches,”
10.1520/D5259-24. American Journal of Public Health, Vol 69, 1979, pp. 690–696.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

---------------------- Page: 1 ----------------------
D5259 − 24
for 48 h at 41 °C, then transferred to EIA agar and held at 41 °C pump or a syringe equipped with a check valve to prevent the
for 20 min. Enterococci develop as red to maroon colonies with r
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: D5259 − 19 D5259 − 24
Standard Test Method for
Isolation and Enumeration of Enterococci from Water by the
1
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
2.1 ASTM Standards:
D1129 Terminology Relating to Water
D1193 Specification for Reagent Water
D3370 Practices for Sampling Water from Flowing Process Streams
3
D3870 Practice for Establishing Performance Characteristics for Colony Counting Methods in Microbiology (Withdrawn 2000)
3. Terminology
3.1 Definitions:
1
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.
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
3
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
1

---------------------- Page: 1 ----------------------
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
4
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
...