ASTM D6974-04
(Practice)Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels—Filtration and Culture Procedures
Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels—Filtration and Culture Procedures
SIGNIFICANCE AND USE
Biodeteriogenic microbes infecting fuel systems typically are most abundant within slime accumulations on system surfaces or at the fuel-water interface (Guide D 6469). However, it is often impractical to obtain samples from these locations within fuel systems. Although the numbers of viable bacteria and fungi recovered from fuel-phase samples are likely to be several orders of magnitude smaller than those found in water-phase samples, fuel-phase organisms are often the most readily available indicators of fuel and fuel system microbial contamination.
Growth Medium Selectivity—Guide E 1326 discusses the limitations of growth medium selection. Any medium selected will favor colony formation by some species and suppress colony formation by others. As noted in 6.3, physical, chemical and physiological variables can affect viable cell enumeration test results.
Since a wide range of sample sizes, or dilutions thereof, can be analyzed by the membrane filter technique (Test Methods D 5259 and F 1094), the test sensitivity can be adjusted for the population density expected in the sample.
Enumeration data should be used as part of diagnostic efforts or routine condition monitoring programs. Enumeration data should not be used as fuel quality criteria.
SCOPE
1.1 This practice covers a membrane filter (MF) procedure for the detection and enumeration of Heterotrophic bacteria (HPC) and fungi in liquid fuels with kinematic viscosities ≤24 mm2 · s-1 at ambient temperature.
1.2 This quantitative practice is drawn largely from IP Method 385 and Test Method D 5259.
1.3 This test may be performed either in the field or in the laboratory.
1.4 The ability of individual microbes to form colonies on specific growth media depends on the taxonomy and physiological state of the microbes to be enumerated, the chemistry of the growth medium, and incubation conditions. Consequently, test results should not be interpreted as absolute values. Rather they should be used as part of a diagnostic or condition monitoring effort that includes other test parameters, in accordance with Guide D 6469.
1.5 This practice offers alternative options for delivering fuel sample microbes to the filter membrane, volumes or dilutions filtered, growth media used to cultivate fuel-borne microbes, and incubation temperatures. This flexibility is offered to facilitate diagnostic efforts. When this practice is used as part of a condition monitoring program, a single procedure should be used consistently.
1.6 The values stated in SI units are to be regarded as the 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.
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Standards Content (Sample)
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An American National Standard
Designation:D6974–04
Standard Practice for
Enumeration of Viable Bacteria and Fungi in Liquid Fuels—
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Filtration and Culture Procedures
This standard is issued under the fixed designation D 6974; 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 2. Referenced Documents
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1.1 This practice covers a membrane filter (MF) procedure 2.1 ASTM Standards:
for the detection and enumeration of Heterotrophic bacteria D 1129 Terminology Relating to Water
(HPC) and fungi in liquid fuels with kinematic viscosities#24 D 1193 Specification for Reagent Water
2 -1
mm ·s at ambient temperature. D 4057 Practice for Manual Sampling of Petroleum and
1.2 This quantitative practice is drawn largely from IP Petroleum Products
Method 385 and Test Method D 5259. D 4175 Terminology Relating to Petroleum, Petroleum
1.3 This test may be performed either in the field or in the Products, and Lubricants
laboratory. D 5259 Test Method for Isolation and Enumeration of
1.4 The ability of individual microbes to form colonies on EnterococcifromWaterbytheMembraneFilterProcedure
specific growth media depends on the taxonomy and physi- D 6426 Test Method for Determining Filterability of Distil-
ological state of the microbes to be enumerated, the chemistry late Fuel Oils
of the growth medium, and incubation conditions. Conse- D 6469 Guide for Microbial Contamination in Fuels and
quently, test results should not be interpreted as absolute Fuel Systems
values. Rather they should be used as part of a diagnostic or E 1326 Guide for Evaluating Nonconventional Microbio-
condition monitoring effort that includes other test parameters, logical Tests Used for Enumerating Bacteria
in accordance with Guide D 6469. F 1094 Test Methods for Microbiological Monitoring of
1.5 This practice offers alternative options for delivering Water Used for Processing Electronic and Microelectronic
fuel sample microbes to the filter membrane, volumes or Devices by Direct Pressure Tap Sampling Valve and by the
dilutions filtered, growth media used to cultivate fuel-borne Presterilized Plastic Bag Method
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microbes, and incubation temperatures. This flexibility is 2.2 Energy Institute Standards:
offered to facilitate diagnostic efforts. When this practice is IP 385 Viable aerobic microbial content of fuels and fuel
used as part of a condition monitoring program, a single components boiling below 90°C—Filtration and culture
procedure should be used consistently. method
1.6 The values stated in SI units are to be regarded as the
3. Terminology
standard.
1.7 This standard does not purport to address all of the 3.1 Definitions—For definition of terms used in this method
refer to Terminologies D 1129 and D 4175, and Guide D 6469.
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro- 3.1.1 aseptic, adj—sterile, free from viable microbiological
contamination.
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use. 3.2 Acronyms:
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For referenced ASTM standards, visit the ASTM website, www.astm.org, or
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This practice is under the jurisdiction ofASTM Committee D02 on Petroleum contactASTM Customer Service at service@astm.org. ForAnnual Book ofASTM
Products and Lubricants and is the direct responsibility of Subcommittee D02.14 on Standards volume information, refer to the standard’s Document Summary page on
Stability and Cleanliness of Liquid Fuels. the ASTM website.
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Current edition approved May 1, 2004. Published June 2004. Originally Available from Energy Institute, 61 New Cavendish St., London, WIG 7AR,
approved in 2003. Last previous edition approved in 2003 as D 6974–03. U.K.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
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D6974–04
3.2.1 CFU—colony forming unit 6. Interferences
3.2.2 HPC—heterotrophic plate count
6.1 High non-biological particulate loads (sediment) can
3.2.3 MF—membrane filter clog the membrane and prevent filtration.
6.2 Each CFU is assumed to originate from a single micro-
3.2.4 MEA—malt extract agar
bial cell. In reality, microbes often form aggregates which
3.2.5 TNTC—too numerous to count
appear as a single colony. Consequently, viable count data are
3.2.6 TSA—tryptone soy agar
likely to underestimate the total number of viable organisms in
3.3 Symbols:
the original sample.
-1
3.3.1 N—number of CFU · L
6.3 The metabolic state of individual microbes may be
3.3.2 CC—number of c
...
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