ASTM D6974-16
(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
5.1 Biodeteriogenic microbes infecting fuel systems typically are most abundant within slime accumulations on system surfaces or at the fuel-water interface (Guide D6469). 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.
5.2 Growth Medium Selectivity—Guide E1326 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. Test Method D7463 provides a non-culture means of quantifying microbial biomass in fuels and fuel associated water.
5.3 Since a wide range of sample sizes, or dilutions thereof, can be analyzed by the membrane filter technique (Test Methods D5259 and F1094), the test sensitivity can be adjusted for the population density expected in the sample.
5.4 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 D5259.
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 D6469.
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 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.
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Designation: D6974 − 16
Standard Practice for
Enumeration of Viable Bacteria and Fungi in Liquid Fuels—
1
Filtration and Culture Procedures
This standard is issued under the fixed designation D6974; 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* 2. Referenced Documents
2
2.1 ASTM Standards:
1.1 This practice covers a membrane filter (MF) procedure
D1129 Terminology Relating to Water
for the detection and enumeration of Heterotrophic bacteria
D1193 Specification for Reagent Water
(HPC) and fungi in liquid fuels with kinematic viscosities
2 -1
D4175 Terminology Relating to Petroleum Products, Liquid
≤24 mm ·s at ambient temperature.
Fuels, and Lubricants
1.2 This quantitative practice is drawn largely from IP
D5259 Test Method for Isolation and Enumeration of En-
Method 385 and Test Method D5259.
terococci from Water by the Membrane Filter Procedure
D6426 Test Method for Determining Filterability of Middle
1.3 This test may be performed either in the field or in the
Distillate Fuel Oils
laboratory.
D6469 GuideforMicrobialContaminationinFuelsandFuel
1.4 The ability of individual microbes to form colonies on
Systems
specific growth media depends on the taxonomy and physi-
D7463 Test Method forAdenosineTriphosphate (ATP) Con-
ological state of the microbes to be enumerated, the chemistry
tent of Microorganisms in Fuel, Fuel/Water Mixtures, and
of the growth medium, and incubation conditions.
Fuel Associated Water
Consequently, test results should not be interpreted as absolute
D7464 Practice for Manual Sampling of Liquid Fuels, As-
values. Rather they should be used as part of a diagnostic or
sociated Materials and Fuel System Components for
condition monitoring effort that includes other test parameters,
Microbiological Testing
in accordance with Guide D6469.
E1326 Guide for Evaluating Non-culture Microbiological
Tests
1.5 This practice offers alternative options for delivering
F1094 Test Methods for Microbiological Monitoring of
fuel sample microbes to the filter membrane, volumes or
Water Used for Processing Electron and Microelectronic
dilutions filtered, growth media used to cultivate fuel-borne
Devices by Direct Pressure Tap Sampling Valve and by
microbes, and incubation temperatures. This flexibility is
the Presterilized Plastic Bag Method
offered to facilitate diagnostic efforts. When this practice is
3
2.2 Energy Institute Standards:
used as part of a condition monitoring program, a single
IP 385 Determination of the Viable Aerobic Microbial Con-
procedure should be used consistently.
tent of Fuels and Fuel Components Boiling Below
1.6 The values stated in SI units are to be regarded as
390 °C—Filtration and Culture Method
standard. No other units of measurement are included in this
standard.
3. Terminology
1.7 This standard does not purport to address all of the
3.1 Definitions:
safety concerns, if any, associated with its use. It is the
3.1.1 For definition of terms used in this method refer to
responsibility of the user of this standard to establish appro-
Terminologies D1129 and D4175, and Guide D6469.
priate safety and health practices and determine the applica-
3.1.2 aseptic, adj—sterile, free from viable microbiological
bility of regulatory limitations prior to use.
contamination.
1 2
This practice is under the jurisdiction ofASTM Committee D02 on Petroleum For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Products, Liquid Fuels, and Lubricantsand is the direct responsibility of Subcom- contactASTM Customer Service at service@astm.org. ForAnnual Book ofASTM
mittee D02.14 on Stability and Cleanliness of Liquid Fuels. Standards volume information, refer to the standard’s Document Summary page on
Current edition approved July 1, 2016. Published July 2016. Originally approved the ASTM website.
ɛ2 3
in 2003. Last previous edition approved in 2013 as D6974 – 09 (2013) . DOI: Available from Energy Institute, 61 New Cavendish St., London, WIG 7AR,
10.1520/D6974-16. U.K., http://www.energyinst.org.uk.
*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 ----------------------
D6974 − 16
3.2 Acronyms: MethodsD5259andF1094),thetestsensitivitycanbeadjusted
3.2.1 CFU—colony forming unit for the population density expected in the sample.
3.2.2 HPC—heterotrophic plate count
5.4 Enumeration data should be used as part of diagnostic
efforts or routine condition monitoring programs. Enumeration
3.2.3 MF—membrane filter
...
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.
´2
Designation: D6974 − 09 (Reapproved 2013) D6974 − 16
Standard Practice for
Enumeration of Viable Bacteria and Fungi in Liquid Fuels—
1
Filtration and Culture Procedures
This standard is issued under the fixed designation D6974; 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
ε NOTE—Subsections 8.4 and 8.8.2 were editorially corrected in August 2014.
2
ε NOTE—References in Section 9 and the title of IP 385 were editorially corrected in January 2015.
1. Scope Scope*
1.1 This practice covers a membrane filter (MF) procedure for the detection and enumeration of Heterotrophic bacteria (HPC)
2 -1
and fungi in liquid fuels with kinematic viscosities ≤24 mm · s at ambient temperature.
1.2 This quantitative practice is drawn largely from IP Method 385 and Test Method D5259.
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 D6469.
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 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
D4175 Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
D5259 Test Method for Isolation and Enumeration of Enterococci from Water by the Membrane Filter Procedure
D6426 Test Method for Determining Filterability of Middle Distillate Fuel Oils
D6469 Guide for Microbial Contamination in Fuels and Fuel Systems
D7463 Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Fuel, Fuel/Water Mixtures, and Fuel
Associated Water
D7464 Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological
Testing
E1326 Guide for Evaluating Non-culture Microbiological Tests
F1094 Test Methods for Microbiological Monitoring of Water Used for Processing Electron and Microelectronic Devices by
Direct Pressure Tap Sampling Valve and by the Presterilized Plastic Bag Method
1
This practice is under the jurisdiction of ASTM Committee D02 on Petroleum Products, Liquid Fuels, and Lubricantsand is the direct responsibility of Subcommittee
D02.14 on Stability and Cleanliness of Liquid Fuels.
Current edition approved May 1, 2013July 1, 2016. Published August 2013July 2016. Originally approved in 2003. Last previous edition approved in 20092013 as
ɛ2
D6974 – 09.D6974 – 09 (2013) . DOI: 10.1520/D6974-09R13E02.10.1520/D6974-16.
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
*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 ----------------------
D6974 − 16
3
2.2 Energy Institute Standards:
IP 385 Determination of the Viable Aerobic Microbial Content of Fuels and Fuel Components Boiling Below 390°C—
Filtration390 °C—Filtration and Culture Method
3. Terminology
3.1 Definitions:
3.1.1 For definition of terms used in this method r
...
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