ASTM D5259-14

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 the 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 and health practices and determine the applicability of regulatory limitations prior to use. For specific hazard statements, see Section 9.

General Information

• Status: Historical
• Publication Date: 31-Jul-2014
• ICS: 13.060.70 - Examination of biological properties of water
• Technical Committee: D19 - Water
• Drafting Committee: D19.24 - Water Microbiology

Relations

• Replaces: ASTM D5259-92(2012) - Standard Test Method for Isolation and Enumeration of Enterococci from Water by the Membrane Filter Procedure
• Effective Date: 01-Aug-2014
• Replaced By: ASTM D5259-19 - Standard Test Method for Isolation and Enumeration of Enterococci from Water by the Membrane Filter Procedure
• Effective Date: 01-Apr-2019
• Referred By: ASTM D5465-16(2020) - Standard Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
• Effective Date: 01-Aug-2014
• Referred By: ASTM D6974-20 - Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels—Filtration and Culture Procedures
• Effective Date: 01-Aug-2014
• 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-Aug-2014
• 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-Aug-2014

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: D5259 − 14
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.Anumber 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-
D1129Terminology Relating to Water
dure for the detection and enumeration of the enterococci
D1193Specification for Reagent Water
bacteria in water. The enterococci, which include Entero-
D3370Practices for Sampling Water from Closed Conduits
coccus faecalis(E. faecalis), E. faecium,andtheirvarietiesare
D3870PracticeforEstablishingPerformanceCharacteristics
commonly found in the feces of humans and other warm-
for Colony Counting Methods in Microbiology (With-
bloodedanimals.Althoughsomestrainsareubiquitousandnot
3
drawn 2000)
relatedtofecalpollution,enterococciinwaterareanindication
of fecal pollution and the possible presence of enteric patho-
3. Terminology
gens. These bacteria are found in water and wastewater in a
3.1 Definitions:
wide range of densities. The detection limit is one colony
3.1.1 For definitions of terms used in this test method, refer
forming unit (CFU)/volume filtered.
to Terminology D1129.
1.2 This test method has been used successfully with
3.2 Definitions of Terms Specific to This Standard:
temperatefreshandmarineambientwaters,andwastewaters.It 3.2.1 Enterococcus—in this test method, Enterococcus spe-
is the user’s responsibility to ensure the validity of this test
ciesarethosebacteriathatproduceredtomarooncolonieswith
method for waters of untested types. black or reddish-brown precipitate on underside, after incuba-
tion on mE agar and subsequent transfer to EIA medium.
1.3 The values stated in SI units are to be regarded as the
3.2.1.1 Discussion—Enterococci include E. faecalis, E.
standard.
faecium, E. avium, and their variants.
1.4 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
4. Summary of Test Method
responsibility of the user of this standard to establish appro-
4.1 The procedure given in this test method provides a
priate safety, health, and environmental practices and deter-
direct count of bacteria in water based on the development of
mine the applicability of regulatory limitations prior to use.
4
coloniesonthesurfaceofthemembranefilter. Awatersample
For specific hazard statements, see Section 9.
is filtered through the membrane that retains the bacteria.
1.5 This international standard was developed in accor-
Following filtration, the membrane containing the bacterial
dance with internationally recognized principles on standard-
cells is placed on a selective, medium, mE agar, and incubated
ization established in the Decision on Principles for the
for48hat41°C,thentransferredtoEIAagarandheldat41°C
Development of International Standards, Guides and Recom-
for20min.Enterococcidevelopasredtomarooncolonieswith
mendations issued by the World Trade Organization Technical
black or reddish-brown precipitate on the underside of the
Barriers to Trade (TBT) Committee.
filter.
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 ofASTM 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 Aug. 1, 2014. Published October 2014. Originally Cabelli,V.J.,Dufour,A.P.,Levin,M.A.,McCabe,L.J.,andHaberman,P.W.,
approved in 1992. Last previous edition approved in 2012 as D5259–92 (2012). “RelationshipofMicrobialIndicatorstoHealthEffectsatMarineBathingBeaches,”
DOI: 10.1520/D5259-14. 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

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D5259 − 14
5. Significance and Use 7.10 Flask, filter, vacuum, usually 1 L, with appropriate
tubing. A filter manifold to hold a number of filter bases is
5.1 The enterococci are indicators of the bacteriological
optional.
quality for potable water, shellfish growing waters, ambient,
and recreational waters. A direct relationship between 7.11 Forceps, straight or curved, with smooth ti
...

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 − 92 (Reapproved 2012) D5259 − 14
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 the 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 and health practices and determine the applicability of regulatory
limitations prior to use. For specific hazard statements, see Section 9.
2. Referenced Documents
2
2.1 ASTM Standards:
D1129 Terminology Relating to Water
D1193 Specification for Reagent Water
D3370 Practices for Sampling Water from Closed Conduits
3
D3870 Practice for Establishing Performance Characteristics for Colony Counting Methods in Microbiology (Withdrawn 2000)
3. Terminology
3.1 DefinitionsDefinitions:—For
3.1.1 For definitions of terms used in this test method, refer to Terminology D1129. definitions of terms used in this test method,
refer to Terminology D1129.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 Enterococcus—Inin 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. Enterococci
include E. faecalis, E. faecium, E. avium, and their variants.
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 June 1, 2012Aug. 1, 2014. Published August 2012October 2014. Originally approved in 1992. Last previous edition approved in 20062012 as
D5259 – 92 (2006).(2012). DOI: 10.1520/D5259-92R12.10.1520/D5259-14.
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.
3.2.1.1 Discussion—
Enterococci include E. faecalis, E. faecium, E. avium, and their variants.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

---------------------- Page: 1 ----------------------
D5259 − 14
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
4
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,
then transferred to EIA agar and held at 41°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 fres
...