Designation: D 4412 – 84 (Reapproved 2002)

Standard Test Methods for

Sulfate-Reducing Bacteria in Water and Water-Formed
Deposits1
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 water, Fifteenth Edition3

1.1 These test methods cover the procedure for the detection 3. Terminology
and enumeration by the most probable number (MPN) tech-
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. Medium A which 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.4
Medium B is offered for those special conditions where 4. Summary of Test Methods
sulfate-reducing bacterial strains have adapted to atypical
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 (A or B)
sulfate-reducing bacteria encountered in waters associated with 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.4 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. 5. Significance and Use
1.4 The sensitivity of these test methods can be increased by
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.

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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: 6. Apparatus and Materials
D 1129 Terminology Relating to Water2
D 1193 Specification for Reagent Water2 6.1 Anaerobic Incubator, 20°C, if available, or conventional
D 2777 Practice for Determination of Precision and Bias of 20°C incubator.5
Applicable Methods of Committee D19 on Water2 6.2 Pipets, sterile, 1 mL and 10 mL,“ calibrated” to deliver.
D 3370 Practices for Sampling Water from Closed Con- 6.3 Test Tubes, with close fitting or airtight caps; 16 by 150
duits2 mm and 20 by 150 mm.
2.2 APHA Standard: 6.4 Test Tube Racks, of sufficient size to contain 16 and
Standard Methods for the Examination of Water and Waste- 20-mm tubes.

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.
4
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.

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 D 4412 – 84 (2002)
7. Reagents p-Aminodimethylaniline dihydrochloride 1.0 g
(C8H12N2·2HCl)
7.1 Purity of Reagents—Reagent grade chemicals shall be HCl (6 N) 500 mL
used in all tests. Unless otherwise indicated, it is intended that
all reagents conform to the specifications of the Committee on Dissolve 1 g of p-aminodimethylaniline dihydrochloride in
Analytical Reagents of the American Chemical Society,6 when 500 mL of 6 N HCl. Store for up to 1 month in an amber
such specifications are available. airtight container.
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 KH2PO4 in 500 mL of water, adjust
conforming to Specification D 1193. In addition, reagent water
pH to 7.2 with 1 N NaOH and dilute to 1 L with distilled water.
used for these test methods must be sterile.
This is called the stock phosphate solution.
7.3 Starkey’s Medium A—7 (modified): 7.7.2 Dissolve 38 g of MgCL2 in 1 L of distilled water.
Sodium lactate (C3H5NaO3) 3.5 g 7.8 Buffered Dilution Water, Working Solution—Add 1.25
Ammonium chloride (NH4Cl) 1.0 g
Dipotassium, hydrogen orthophosphate 0.5 g mL of stock buffered dilution water and 5 mL of MgCl2
(K2HPO4) solution to 500 mL of water. Bring to 1 L with water. Mix well
Magnesium sulfate (MgSO4·7H2O) 2.0 g and dispense as 90 mL dilution blanks in screw-capped bottles.
Sodium sulfate (Na2SO4) 0.5 g
Calcium chloride (CaCl2·2H2O) 0.1 g Sterilize by autoclaving at 121°C for 15 min.
Thioglycollic acid 0.1 g
Ammonium ferrous sulfate or ferrous 0.001g 8. Procedure
ammonium sulfate
((NH4)2SO4·FeSO4·6H2O) 8.1 Clean and disinfect the area with a cleaning solution that
Water (H2O) 1 L leaves no residue.
7.3.1 Double strength medium (23) is prepared as above 8.2 Set out and label five replicate tubes of 10-mL double-
except 500 mL of water are used instead of 1 L. strength Starkey’s medium, A or B, in the test tube rack.
8.3 Set out and label five replicate tubes of 10-mL single-
7.3.2 Heat to dissolve and dispense 9 mL of medium per
strength Starkey’s medium, A or B, for each mL of sample or
single strength tube, and 10 mL per double strength tube.
mL of sample dilution to be tested. Use two sets of five
7.3.3 Tubes should be of sufficient capacity to contain 1 mL replicate 10-mL tubes, each to contain 1 mL of sample or 1 mL
of inoculum plus 9 mL of single strength medium or 10 mL of of 1/10 dilution of sample.
inoculum plus 10 mL of 23 medium. 8.4 Prior to sample inoculation, heat tubes of media and
7.3.4 pH of medium should be 7.2 after autoclave steriliza- dilution blanks in a water bath to 60°C then cool rapidly to
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tion, at 121°C for 15 min. 20°C to ensure minimal oxygen levels.

7.4 Starkey’s Medium B—The medium is similar to that 8.5 Shake sample thoroughly,8 at least 25 times; make
described in 7.3, 7.3.1, and 7.3.2 with the following modifica- dilutions starting with 10 mL of sample into one 90-mL
tion: dilution blank.
7.4.1 Water collected from the sample collection site is used 8.6 Pipet 10 mL of sample into each double-strength broth
to prepare the medium outlined in 7.3. The water sample is and 1 mL of sample or diluted sample into each set of five
filtered to remove particulates (1.2 µm membrane filter) and the single-strength broths.
pH is recorded. 8.7 Maintain anaerobic conditions by layering 2 to 3 ml of
7.4.1.1 After preparing the Medium B following 7.3.1, sterile liquid paraffin in each tube.
7.3.2, and 7.3.3, and prior to dispensing, check and adjust pH, 8.8 Recap tubes and incubate at 20°C for 21 days.
if necessary to that of the original water used, then filter 8.9 Include sterile water samples with each test as negative
sterilize the medium by passage through 0.2-µm filter and controls.
asceptically dispense into presterilized tubes. 8.10 Positive reaction is indicated by the deposit of a black
7.5 Hydrogen Sulfide Test Reagent: precipitate (sulfide).
7.5.1 Ferric Chloride Stock Solution (FeCl3·6H2O)— 8.11 Confirm dubious results by the addition of 0.5 mL of
Dissolve 13.5 g of ferric chloride in a mixture of 250 mL of ferric chloride reagent followed by 0.5 mL of
water and 250 mL of HCl (sp gr 1.19). Store in an airtight p-aminodimethylaniline reagent to the MPN tube. Add reagent
amber container. Prepare fresh monthly. to the bottom of the tube using syringe or long Pasteur pipet. A
positive reaction, blue color, occurs within 10 min if H2S is
7.5.2 p-Aminodimethylaniline Dihydrochloride Stock Solu-
present.
tion
9. Calculation
9.1 Compute the number of positive findings resulting from
6
“Reagent Chemicals, American Chemical Society Specifications,” American multiple-portion decimal dilution planting as the combination
Chemical Society, Washington, DC. For suggestions on the testing of reagents not of positives and recorded in terms of the Most Probable
listed by the American Chemical Society, see “Reagent Chemicals and Standards,” Number3 (MPN).
by Joseph Rosin, D. Van Nostrand Co., Inc., New York, NY, and the “United States
Pharmacopeia.”
7
Starkey, R. L., “Characteristics and Cultivation of Sulfate-Reducing Bacteria,”
8
Journal of the American Water Works Association, Vol 40, 1948, pp. 1291–1298. Organisms do not appear to be hypersensitive to small amounts of oxygen.

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 D 4412 – 84 (2002)
9.2 When more than three series of tubes are employed in a testing can not be carried out. Statements can only be made on
decimal series of dilutions, use the results from only three of the precision of the MPN procedure.
these used in computing the MPN, for example: 11.2 Unless a large number of portions of sample are
10 1 0.1 0.01 examined, the precision of the MPN is rather low. For example,
mL mL mL mL
5/5 5/5 2/5 0/5 = 5-2-0 3 10 = 490/100 mL even when the sample contains one organism per millilitre,
5/5 4/5 2/5 0/5 = 5-4-2 = 220/100 mL about 37 % of tubes inoculated with 1 mL of sample may be
5/5 3/5 1/5 1/5 = 5-3-2 = 140/100 mL
5/5 0/5 0/5 0/5 = 5-0-0 = 23/100 mL
expected to yield negative results because of irregular distri-
bution of the bacteria in the sample and the multiple attachment
10. Report of bacteria to particles. When five tubes, each with 1 mL of
10.1 Report the results as number of sulfate-reducing bac- sample, are employed under these conditions, a completely
teria per 100 mL of sample. negative result may be expected less than 1 % of the time.
Thus, even when five tubes are employed for each dilution, the
11. Precision and Bias 9
precision of the results obtained is not of a high order.
11.1 Due to the instability of the organisms, round robin
11.3 See the Research Report for results of a single labora-
tory, two operator study.
9
Supporting data for these test methods have been filed at ASTM Headquarters.
Request RR: D-19-1116.

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