ASTM D4412-15

Standard Test Methods for Sulfate-Reducing Bacteria in Water and Water-Formed Deposits

SIGNIFICANCE AND USE
5.1 Sulfate-reducing bacteria are widely distributed in marine and fresh water muds which, in consequence, frequently are laden with the hydrogen sulfide produced by these organisms during dissimilatory sulfate reduction.  
5.2 It has been reported that Desulfovibrio spp. can form as much as 10 g of sulfide per litre during active multiplication. Sulfate-reducing bacteria can cause the external or internal corrosion of water or wastewater pipelines and pipelines for petroleum and natural gas. The formation of galvanic cells by massive growth of sulfate-reducing bacteria under suitable conditions makes the corrosion much worse than just the effect of the hydrogen sulfide on the metal or concrete.
SCOPE
1.1 These test methods cover the procedure for the detection and enumeration by the most probable number (MPN) technique of sulfate-reducing bacteria in water or water-formed deposits.  
1.2 Two media preparations are provided. Medium A which is prepared with reagent grade water, and Medium B which is prepared using the water to be sampled as the water source. Medium B is offered for those special conditions where sulfate-reducing bacterial strains have adapted to atypical non-fresh water environment.  
1.3 For the isolation and enumeration of thermophilic sulfate-reducing bacteria encountered in waters associated with oil and gas production, all broths, dilution blanks, and incubations must be maintained at temperatures of at least 45°C and preferably within 5°C at the sample temperature.  
1.4 The sensitivity of these test methods can be increased by purging the dilution blanks and tubes of media with nitrogen immediately prior to use.  
1.5 The analyst should be aware that adequate collaborative data for precision and bias statements as required by Practice D2777 are not provided. See Section 11 for details.  
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.7 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.

General Information

• Status: Historical
• Publication Date: 14-Jul-2015
• ICS: 07.100.20 - Microbiology of water
• Technical Committee: D19 - Water
• Drafting Committee: D19.24 - Water Microbiology

Relations

• Replaced By: ASTM D4412-19 - Standard Test Methods for Sulfate-Reducing Bacteria in Water and Water-Formed Deposits
• Effective Date: 01-Dec-2019

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: D4412 − 15
Standard Test Methods for
Sulfate-Reducing Bacteria in Water and Water-Formed
1
Deposits
This standard is issued under the fixed designation D4412; 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
1.1 Thesetestmethodscovertheprocedureforthedetection 2.1 ASTM Standards:
and enumeration by the most probable number (MPN) tech- D1129Terminology Relating to Water
nique of sulfate-reducing bacteria in water or water-formed D1193Specification for Reagent Water
deposits. D2777Practice for Determination of Precision and Bias of
Applicable Test Methods of Committee D19 on Water
1.2 Two media preparations are provided. MediumAwhich
D6503Test Method for Enterococci in Water Using Enter-
is prepared with reagent grade water, and Medium B which is
olert
prepared using the water to be sampled as the water source.
2.2 Other Standards:
Medium B is offered for those special conditions where
Standard Methods 9221Multiple-Tube Fermentation Tech-
sulfate-reducing bacterial strains have adapted to atypical
3
nique for Members of the Coliform Group
non-fresh water environment.
1.3 For the isolation and enumeration of thermophilic 3. Terminology
sulfate-reducingbacteriaencounteredinwatersassociatedwith
3.1 Definitions—For definitions of terms used in this
oil and gas production, all broths, dilution blanks, and incuba-
standard, refer to Terminology D1129.
tions must be maintained at temperatures of at least 45°C and
3.1.1 most probable number, n—statistical method for de-
preferably within 5°C at the sample temperature.
termining bacterial density based on the Poisson distribution.
D6503
1.4 Thesensitivityofthesetestmethodscanbeincreasedby
purging the dilution blanks and tubes of media with nitrogen
3.2 Acronyms:
immediately prior to use.
3.2.1 SRB, n—sulfate-reducing bacteria
1.5 The analyst should be aware that adequate collaborative
4. Summary of Test Methods
data for precision and bias statements as required by Practice
D2777 are not provided. See Section 11 for details. 4.1 Water and water deposit samples and dilutions of these
samplesaredispensedintotubesofStarkey’smedium(AorB)
1.6 The values stated in SI units are to be regarded as
followingfivetubeMPNprocedures.Thetubesaresealedwith
standard. No other units of measurement are included in this
4
liquid paraffin, and incubated at 20°C for 21 days. Positive
standard.
reactions are indicated by the deposit of a black precipitate.
1.7 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
5. Significance and Use
responsibility of the user of this standard to establish appro-
5.1 Sulfate-reducing bacteria are widely distributed in ma-
priate safety and health practices and determine the applica-
rine and fresh water muds which, in consequence, frequently
bility of regulatory limitations prior to use.
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
1
These test methods are under the jurisdiction of ASTM Committee D19 on contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Water and are the direct responsibility of Subcommittee D19.24 on Water Micro- Standards volume information, refer to the standard’s Document Summary page on
biology. the ASTM website.
3
Current edition approved July 15, 2015. Published August 2015. Originally Available from Standard Methods, http://standardmethods.org.
4
approved in 1984. Last previous edition approved in 2009 as D4412–84 (2009). Starkey, R. L., “Characteristics and Cultivation of Sulfate-Reducing Bacteria,”
DOI: 10.1520/D4412-15. Journal of the American Water Works Association, Vol 40, 1948, pp. 1291–1298.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

---------------------- Page: 1 ----------------------
D4412 − 15
are laden with the hydrogen sulfide produced by these organ- 7.3.4 pH of medium should be 7.2 after autoclave
isms during dissimilatory sulfate reduction. sterilization, at 121°C for 15 min.
5.2 It has been reported that Desulfovibrio spp. can form as
7.4 Starkey’s Medium B—The medium is similar to that
much as 10 g of sulfide per litre during active multiplication. described in 7.3, 7.3.1, and 7.3.2 with the following modifica-
Sulfate-reducing bacteria can cause the external or internal
tion:
corrosion of water or wastewater pipelines and pipelines for 7.4.1 Watercollectedfromthesamplecollectionsiteisused
petroleum and natural gas
...

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: D4412 − 84 (Reapproved 2009) D4412 − 15
Standard Test Methods for
Sulfate-Reducing Bacteria in Water and Water-Formed
1
Deposits
This standard is issued under the fixed designation D4412; 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 Scope*
1.1 These test methods cover the procedure for the detection and enumeration by the most probable number (MPN) technique
of sulfate-reducing bacteria in water or water-formed deposits.
1.2 Two media preparations are provided. Medium A which is prepared with reagent grade water, and Medium B which is
prepared using the water to be sampled as the water source. Medium B is offered for those special conditions where
sulfate-reducing bacterial strains have adapted to atypical non-fresh water environment.
1.3 For the isolation and enumeration of thermophilic sulfate-reducing bacteria encountered in waters associated with oil and
gas production, all broths, dilution blanks, and incubations must be maintained at temperatures of at least 45°C and preferably
within 5°C at the sample temperature.
1.4 The sensitivity of these test methods can be increased by purging the dilution blanks and tubes of media with nitrogen
immediately prior to use.
1.5 The analyst should be aware that adequate collaborative data for precision and bias statements as required by Practice D2777
are not provided. See Section 11 for details.
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.7 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.
2. Referenced Documents
2
2.1 ASTM Standards:
D1129 Terminology Relating to Water
D1193 Specification for Reagent Water
D2777 Practice for Determination of Precision and Bias of Applicable Test Methods of Committee D19 on Water
D6503 Test Method for Enterococci in Water Using Enterolert
2.2 APHA Standard:Other Standards:
Standard Methods for the Examination of Water and Wastewater, 9221 Fifteenth EditionMultiple-Tube Fermentation Technique
3
for Members of the Coliform Group
3. Terminology
3.1 Definitions—For definitions of terms used in these test methods, refer to Terminology D1129.
3.1 Definitions—For definitions of terms used in this standard, refer to Terminology D1129.
3.1.1 most probable number, n—statistical method for determining bacterial density based on the Poisson distribution. D6503
1
These test methods are under the jurisdiction of ASTM Committee D19 on Water and are the direct responsibility of Subcommittee D19.24 on Water Microbiology.
Current edition approved May 1, 2009July 15, 2015. Published June 2009August 2015. Originally approved in 1984. Last previous edition approved in 20022009 as
D4412 – 84 (2002).(2009). DOI: 10.1520/D4412-84R09.10.1520/D4412-15.
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
Available from American Public Health Association, 1015 18th St. N.W., Washington, DC 20036.Standard Methods, http://standardmethods.org.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

---------------------- Page: 1 ----------------------
D4412 − 15
4
3.2 Definitions—For a description of the term MPN used in these test methods, refer to literature.
3.2 Acronyms:
3.2.1 SRB, n—sulfate-reducing bacteria
4. Summary of Test Methods
4.1 Water and water deposit samples and dilutions of these samples are dispensed into tubes of Starkey’s medium (A or B)
4
following five tube MPN procedures. The tubes are sealed with liquid paraffin, and incubated at 20°C for 21 days. Positive
reactions are indicated by the deposit of a black precipitate.
5. Significance and Use
5.1 Sulfate-reducing bacteria are widely distributed in marine and fresh water muds which, in consequence, frequently are laden
with the hydrogen sulfide produced by t
...