ASTM D4412-84(2002)
(Test Method)Standard Test Methods for Sulfate-Reducing Bacteria in Water and Water-Formed Deposits
Standard Test Methods for Sulfate-Reducing Bacteria in Water and Water-Formed Deposits
SIGNIFICANCE AND USE
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.
It has been reported that Desulfovibrio 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 D 2777 are not provided. See Section 11 for details.
1.6 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.
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Designation:D4412–84(Reapproved2002)
Standard Test Methods for
Sulfate-Reducing Bacteria in Water and Water-Formed
Deposits
This standard is issued under the fixed designation D 4412; 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 (e) indicates an editorial change since the last revision or reapproval.
1. Scope Standard Methods for the Examination of Water and Waste-
water, Fifteenth Edition
1.1 Thesetestmethodscovertheprocedureforthedetection
and enumeration by the most probable number (MPN) tech-
3. Terminology
nique of sulfate-reducing bacteria in water or water-formed
3.1 Definitions—For definitions of terms used in these test
deposits.
methods, refer to Terminology D 1129.
1.2 Two media preparations are provided. MediumAwhich
3.2 Definitions of Terms Specific to This Standard: —For a
is prepared with reagent grade water, and Medium B which is
description of the term MPN used in these test methods, refer
prepared using the water to be sampled as the water source.
to literature.
Medium B is offered for those special conditions where
sulfate-reducing bacterial strains have adapted to atypical
4. Summary of Test Methods
non-fresh water environment.
4.1 Water and water deposit samples and dilutions of these
1.3 For the isolation and enumeration of thermophilic
samples are dispensed into tubes of Starkey’s medium (Aor B)
sulfate-reducingbacteriaencounteredinwatersassociatedwith
following five tube MPN procedures.The tubes are sealed with
oil and gas production, all broths, dilution blanks, and incuba-
liquid paraffin, and incubated at 20°C for 21 days. Positive
tions must be maintained at temperatures of at least 45°C and
reactions are indicated by the deposit of a black precipitate.
preferably within 5°C at the sample temperature.
1.4 Thesensitivityofthesetestmethodscanbeincreasedby
5. Significance and Use
purging the dilution blanks and tubes of media with nitrogen
5.1 Sulfate-reducing bacteria are widely distributed in ma-
immediately prior to use.
rine and fresh water muds which, in consequence, frequently
1.5 The analyst should be aware that adequate collaborative
are laden with the hydrogen sulfide produced by these organ-
data for precision and bias statements as required by Practice
isms during dissimilatory sulfate reduction.
D 2777 are not provided. See Section 11 for details.
5.2 It has been reported that Desulfovibrio can form as
1.6 This standard does not purport to address all of the
much as 10 g of sulfide per litre during active multiplication.
safety concerns, if any, associated with its use. It is the
Sulfate-reducing bacteria can cause the external or internal
responsibility of the user of this standard to establish appro-
corrosion of water or wastewater pipelines and pipelines for
priate safety and health practices and determine the applica-
petroleum and natural gas. The formation of galvanic cells by
bility of regulatory limitations prior to use.
massive growth of sulfate-reducing bacteria under suitable
conditions makes the corrosion much worse than just the effect
2. Referenced Documents
of the hydrogen sulfide on the metal or concrete.
2.1 ASTM Standards:
D 1129 Terminology Relating to Water
6. Apparatus and Materials
D 1193 Specification for Reagent Water
6.1 Anaerobic Incubator,20°C,ifavailable,orconventional
D 2777 Practice for Determination of Precision and Bias of
2 20°C incubator.
Applicable Methods of Committee D19 on Water
6.2 Pipets, sterile, 1 mL and 10 mL, “calibrated” to deliver.
D 3370 Practices for Sampling Water from Closed Con-
2 6.3 Test Tubes, with close fitting or airtight caps; 16 by 150
duits
mm and 20 by 150 mm.
2.2 APHA Standard:
1 3
These test methods are under the jurisdiction of ASTM Committee D19 on Available from American Public Health Association, 1015 18th St. N.W.,
Water and are the direct responsibility of Subcommittee D19.24 on Water Micro- Washington, DC 20036.
biology. Bonde, G. J., “Bacterial Indicators of Water Pollution,” A Study of Quantitative
Current edition approved Oct. 26, 1984. Published February 1985. Estimation, Teknisk Forlag, Copenhagen, 1963.
2 5
Annual Book of ASTM Standards, Vol 11.01. For thermophilic organisms use a 45°C incubator.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D4412–84 (2002)
6.4 Test Tube Racks, of sufficient size to contain 16 and 7.5.2 p-Aminodimethylaniline Dihydrochloride Stock Solu-
20-mm tubes. tion
p-Aminodimethylaniline dihydrochloride 1.0 g
(C H N ·2HCl)
7. Reagents 8 12 2
HCl (6 N) 500 mL
7.1 Purity of Reagents—Reagent grade chemicals shall be
Dissolve1gof p-aminodimethylaniline dihydrochloride in
used in all tests. Unless otherwise indicated, it is intended that
500 mL of 6 N HCl. Store for up to 1 month in an amber
all reagents conform to the specifications of the Committee on
airtight container.
Analytical Reagents of theAmerican Chemical Society, when
such specifications are available. 7.6 Liquid Paraffın—Heavy, sterile, or sterile mineral oil.
7.2 Purity of Water—Unless otherwise indicated, references
7.7 Buffered Dilution Water—Stock Solution
to water shall be understood to mean Reagent Water Type II
7.7.1 Dissolve 34.0 g of KH PO in 500 mLof water, adjust
2 4
conforming to Specification D 1193. In addition, reagent water
pH to 7.2 with 1 N NaOH and dilute to 1 Lwith distilled water.
used for these test methods must be sterile.
This is called the stock phosphate solution.
7.3 Starkey’s Medium A— (modified):
7.7.2 Dissolve 38 g of MgCL in 1 L of distilled water.
Sodium lactate (C H NaO ) 3.5 g
3 5 3
7.8 Buffered Dilution Water, Working Solution—Add 1.25
Ammonium chloride (NH Cl) 1.0 g
Dipotassium, hydrogen orthophosphate 0.5 g
mL of stock buffered dilution water and 5 mL of MgCl
(K HPO )
2 4
solution to 500 mLof water. Bring to 1 Lwith water. Mix well
Magnesium sulfate (MgSO ·7H O) 2.0 g
4 2
and dispense as 90 mLdilution blanks in screw-capped bottles.
Sodium sulfate (Na SO ) 0.5 g
2 4
Calcium chloride (CaCl ·2H O) 0.1 g
2 2
Sterilize by autoclaving at 121°C for 15 min.
Thioglycollic acid 0.1 g
Ammonium ferrous sulfate or ferrous 0.001g
ammonium sulfate 8
...
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