ASTM D6974-20

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, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 30-Apr-2020
• ICS: 75.160.20 - Liquid fuels
• Technical Committee: D02 - Petroleum Products, Liquid Fuels, and Lubricants
• Drafting Committee: D02.14 - Stability, Cleanliness and Compatibility of Liquid Fuels

Relations

• Replaces: ASTM D6974-16 - Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels—Filtration and Culture Procedures
• Effective Date: 01-May-2020
• Refers: ASTM D5259-24 - Standard Test Method for Isolation and Enumeration of Enterococci from Water by the Membrane Filter Procedure
• Effective Date: 01-Apr-2024
• Refers: ASTM D4175-23a - Standard Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
• Effective Date: 15-Dec-2023
• Refers: ASTM D4175-23e1 - Standard Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
• Effective Date: 01-Jul-2023
• Refers: ASTM D1129-13(2020)e2 - Standard Terminology Relating to Water
• Effective Date: 01-May-2020
• Refers: ASTM F1094-87(2020) - Standard 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 (Withdrawn 2023)
• Effective Date: 15-Apr-2020
• Refers: ASTM D7464-19 - Standard Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological Testing
• Effective Date: 01-Nov-2019
• Refers: ASTM D5259-19 - Standard Test Method for Isolation and Enumeration of Enterococci from Water by the Membrane Filter Procedure
• Effective Date: 01-Apr-2019
• Refers: ASTM D7463-16 - Standard Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Fuel, Fuel/Water Mixtures, and Fuel Associated Water
• Effective Date: 01-Jun-2016
• Refers: ASTM D7463-16e1 - Standard Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Fuel, Fuel/Water Mixtures, and Fuel Associated Water
• Effective Date: 01-Jun-2016
• Refers: ASTM D7463-15 - Standard Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Fuel, Fuel/Water Mixtures, and Fuel Associated Water
• Effective Date: 01-Dec-2015
• Refers: ASTM E1326-15 - Standard Guide for Evaluating Non-culture Microbiological Tests Used for Enumerating Bacteria
• Effective Date: 01-May-2015
• Refers: ASTM D7464-14 - Standard Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological Testing
• Effective Date: 01-Oct-2014
• Refers: ASTM D5259-14 - Standard Test Method for Isolation and Enumeration of Enterococci from Water by the Membrane Filter Procedure
• Effective Date: 01-Aug-2014
• Refers: ASTM D7463-14a - Standard Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Fuel, Fuel/Water Mixtures, and Fuel Associated Water
• Effective Date: 01-Jun-2014

Overview

ASTM D6974-20: Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels-Filtration and Culture Procedures is a recognized guideline developed by ASTM for detecting and quantifying heterotrophic bacteria and fungi in liquid fuels. Using membrane filter techniques and culture procedures, this standard supports fuel quality professionals, environmental specialists, and laboratory personnel in assessing microbial contamination. It applies to fuels with kinematic viscosities ≤24 mm²·s⁻¹ at ambient temperature, providing flexible and adaptable methodologies for both field and laboratory settings.

Key Topics

• Microbial Contamination Detection: Focuses on the identification and enumeration of viable bacteria and fungi present in liquid fuels, which act as indicators of deteriorative microbial activity in fuel systems.
• Sampling and Filtration: Details procedures for sampling fuel-phase liquids, utilizing membrane filters to retain microorganisms, and offers options for filration apparatus and techniques to suit different operational environments.
• Growth Media Selectivity: Addresses the implications of nutrient media choices, as different media may favor or suppress the growth of certain microbial species-an essential consideration for accurate enumeration.
• Interpretation and Use of Data: Stresses that enumeration results should not be used as direct measures of fuel quality but as part of broader diagnostic or condition monitoring strategies.
• Test Method Flexibility: Provides alternative approaches for handling samples, filter types, growth media, and incubation temperatures, enabling adaptation based on available resources and monitoring requirements.
• Safety and Best Practices: Highlights the importance of health, safety, and environmental precautions when handling fuel samples and microorganisms.

Applications

ASTM D6974-20 is valuable in a range of operational scenarios, including:

• Routine Condition Monitoring: Used by fuel facility managers and maintenance teams to regularly assess microbial activity as part of preventive maintenance programs for storage tanks, pipelines, and distribution systems.
• Diagnostic Investigations: Supports in-depth analysis when fuel system issues such as filter plugging, corrosion, or unexpected degradation arise, helping to pinpoint microbial influences.
• Field and Laboratory Testing: Enables both rapid, on-site testing and more thorough laboratory-based analyses, making it suitable for remote operations as well as centralized testing facilities.
• Standardized Reporting: Provides transparent and reproducible methods for reporting viable bacteria and fungi counts. This ensures consistency in records and supports effective trend analysis over time.
• Fuel System Hygiene Optimization: Supports decision-making for biocide dosing, tank cleaning, and other corrective actions based on concrete microbial enumeration data.

Related Standards

The practice references and aligns with several other key ASTM and international standards related to fuel microbiology and quality monitoring:

• ASTM D6469 - Guide for Microbial Contamination in Fuels and Fuel Systems
• ASTM D5259 - Test Method for Isolation and Enumeration of Enterococci from Water by the Membrane Filter Procedure
• ASTM D6426 - Test Method for Determining Filterability of Middle Distillate Fuel Oils
• ASTM D7463 - Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Fuel, Fuel/Water Mixtures, and Fuel Associated Water
• ASTM D7464 - Practice for Manual Sampling of Liquid Fuels for Microbiological Testing
• ASTM E1326 - Guide for Evaluating Non-culture Microbiological Tests
• Energy Institute IP 385 - Determination of the Viable Aerobic Microbial Content of Fuels and Fuel Components

Conclusion

Implementing ASTM D6974-20 in fuel management practices enhances the early detection and management of microbial contamination in liquid fuels. By providing a standardized approach to viable cell enumeration through filtration and culture, this practice supports operational reliability, system longevity, and compliance with industry quality standards. For effective fuel system maintenance and diagnostic programs, adherence to ASTM D6974-20 ensures accurate, consistent microbial monitoring and informed decision making.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: D6974 − 20
Standard Practice for
Enumeration of Viable Bacteria and Fungi in Liquid Fuels—
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* ization established in the Decision on Principles for the
Development of International Standards, Guides and Recom-
1.1 This practice covers a membrane filter (MF) procedure
mendations issued by the World Trade Organization Technical
for the detection and enumeration of Heterotrophic bacteria
Barriers to Trade (TBT) Committee.
(HPC) and fungi in liquid fuels with kinematic viscosities
2 -1
≤24 mm ·s at ambient temperature.
2. Referenced Documents
1.2 This quantitative practice is drawn largely from IP
2.1 ASTM Standards:
Method 385 and Test Method D5259.
D1129 Terminology Relating to Water
1.3 This test may be performed either in the field or in the
D1193 Specification for Reagent Water
laboratory.
D4175 Terminology Relating to Petroleum Products, Liquid
Fuels, and Lubricants
1.4 The ability of individual microbes to form colonies on
D5259 Test Method for Isolation and Enumeration of En-
specific growth media depends on the taxonomy and physi-
terococci from Water by the Membrane Filter Procedure
ological state of the microbes to be enumerated, the chemistry
D6426 Test Method for Determining Filterability of Middle
of the growth medium, and incubation conditions.
Distillate Fuel Oils
Consequently, test results should not be interpreted as absolute
D6469 GuideforMicrobialContaminationinFuelsandFuel
values. Rather they should be used as part of a diagnostic or
Systems
condition monitoring effort that includes other test parameters,
D7463 Test Method forAdenosineTriphosphate (ATP) Con-
in accordance with Guide D6469.
tent of Microorganisms in Fuel, Fuel/Water Mixtures, and
1.5 This practice offers alternative options for delivering
Fuel Associated Water
fuel sample microbes to the filter membrane, volumes or
D7464 Practice for Manual Sampling of Liquid Fuels, As-
dilutions filtered, growth media used to cultivate fuel-borne
sociated Materials and Fuel System Components for
microbes, and incubation temperatures. This flexibility is
Microbiological Testing
offered to facilitate diagnostic efforts. When this practice is
E1326 Guide for Evaluating Non-culture Microbiological
used as part of a condition monitoring program, a single
Tests
procedure should be used consistently.
F1094 Test Methods for Microbiological Monitoring of
1.6 The values stated in SI units are to be regarded as
Water Used for Processing Electron and Microelectronic
standard. No other units of measurement are included in this Devices by Direct Pressure Tap Sampling Valve and by
standard.
the Presterilized Plastic Bag Method
2.2 Energy Institute Standards:
1.7 This standard does not purport to address all of the
IP 385 Determination of the Viable Aerobic Microbial Con-
safety concerns, if any, associated with its use. It is the
tent of Fuels and Fuel Components Boiling Below
responsibility of the user of this standard to establish appro-
390 °C—Filtration and Culture Method
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
3. Terminology
1.8 This international standard was developed in accor-
dance with internationally recognized principles on standard- 3.1 Definitions:
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 [e-mail]. ForAnnual Book ofASTM
mittee D02.14 on Stability, Cleanliness and Compatibility of Liquid Fuels. Standards volume information, refer to the standard’s Document Summary page on
Current edition approved May 1, 2020. Published May 2020. Originally theASTM website.
approved in 2003. Last previous edition approved in 2016 as D6974 – 16. DOI: Available from Energy Institute, 61 New Cavendish St., London, WIG 7AR,
10.1520/D6974-20. 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
D6974 − 20
3.1.1 For definition of terms used in this method refer to test results. Test Method D7463 provides a non-culture means
Terminologies D1129 and D4175, and Guide D6469. of quantifying microbial biomass in fuels and fuel associated
water.
3.1.2 aseptic, adj—sterile, free from viable microbiological
contamination.
5.3 Since a wide range of sample sizes, or dilutions thereof,
3.2 Acronyms: can be analyzed by the membrane filter technique (Test
3.2.1 CFU—colony forming unit MethodsD5259andF1094),thetestsensitivitycanbeadjusted
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
3.2.3 MF—membrane filter
efforts or routine condition monitoring programs. Enumeration
3.2.4 MEA—malt extract agar
data should not be used as fuel quality criteria.
3.2.5 TNTC—too numerous to count
6. Interferences
3.2.6 TSA—tryptone soy agar
6.1 High non-biological particulate loads (sediment) can
3.3 Symbols:
-1
clog the membrane and prevent filtration.
3.3.1 N—number of CFU · L
6.2 Each CFU is assumed to originate from a single micro-
3.3.2 CC—number of colonies on membrane filter
bial cell. In reality, microbes often form aggregates which
3.3.3 V—sample volume filtered, mL
appear as a single colony. Consequently, viable count data are
likely to underestimate the total number of viable organisms in
4. Summary of Practice
the original sample.
4.1 Any free water present in a fuel sample is removed by
6.3 The metabolic state of individual microbes may be
settling in a separatory funnel. After the water has been
affected by numerous physical-chemical variables in the fuel.
removed, a known volume of the remaining fuel is filtered
Injured cells or cells that have relatively long generation times
through a membrane filter aseptically by one of three methods.
may not form colonies within the time allotted for test
4.2 Thefiltermembraneretainsmicrobespresentinthefuel.
observations.Thisresultsinanunderestimationofthenumbers
Filter replicate fuel samples through fresh membranes to
of viable microbes in the original fuel sample.
permit replicate testing, growth on alternative nutrient media,
or both.
7. Apparatus
4.3 After filtration, place each membrane on one of two
7.1 Separatory Funnels, glass, nominal capacity 500 mL.
types of agar growth media, incubate at a designated tempera-
7.2 Measuring Cylinders, glass, nominal capacity 100 mL
ture for three days, and examine for the presence of CFU.
and 1 L.
4.4 Incubatethefiltermediaonagarfortwomoredays,then
7.3 Pipettes, glass or sterile disposable plastic, nominal
reexamine.
capacity 10 mL, or adjustable volume pipette and sterile
4.5 Count the colonies manually or by electronic counter.
disposable plastic tips.
4.5.1 If practical, identify colonies on each agar medium,
7.4 Membrane Filter, polyethersulfone (PES) or cellulose
based on colony color, morphology, and microscopic exami-
acetate (CA), presterilized, preferably gridded, 47 mm
nation.
diameter, nominal pore size 0.45 µm.
4.5.2 Convert bacterial and fungal colony counts to CFU
NOTE 1—The choice of filter material will depend on local availability
per litre of fuel.
or preference for use of a disposable filtration device, or both.
NOTE 2—CA filters will become translucent on wetting with fuel, but
5. Significance and Use this is not detrimental to the final recovery of microorganisms.
7.5 Filtration Unit, one of:
5.1 Biodeteriogenic microbes infecting fuel systems typi-
7.5.1 Unit, as described in Test Method D6426, with pre-
cally are most abundant within slime accumulations on system
sterilized in-line filter housing, or
surfaces or at the fuel-water interface (Guide D6469).
However, it is often impractical to obtain samples from these 7.5.2 Hypodermic Syringe, sterile, 100 mL, with pre-
sterilized in-line filter housing, or
locations within fuel systems. Although the numbers of viable
bacteria and fungi recovered from fuel-phase samples are 7.5.3 Filter Holder Assembly, single or manifold, glass,
likely to be several orders of magnitude smaller than those stainless steel, or polypropylene, pre-sterilized.
found in water-phase samples, fuel-phase organisms are often
NOTE 3—If the vacuum filtration option (7.5.3) is chosen, a vacuum
the most readily available indicators of fuel and fuel system
source, not more than –66 kPa will also be needed.
microbial contamination.
7.6 Forceps, blunt tipped.
5.2 Growth Medium Selectivity—Guide E1326 discusses the
7.7 Filter Flask, of sufficient capacity to receive the entire
limitations of growth medium selection.Any medium selected
sample being filtered plus washings.
will favor colony formation by some species and suppress
colonyformationbyothers.Asnotedin6.3,physical,chemical 7.8 Petri Dishes, disposable plastic or glass, nominal diam-
and physiological variables can affect viable cell enumeration eter ≥50 mm.
D6974 − 20
NOTE 4—Pre-poured Petri dishes, containing the growth media de-
Malt Extract 30 g
scribed below are available commercially and may be substituted for the
Mycological Peptone 5 g
dishes listed here. Agar 15 g
Water 1 L
7.9 Incubator, capable of maintaining a temperature of
8.8.2 Preparation—Suspend the malt extract, mycological
25 °C 6 2 °C or any other temperature (within the range–am-
peptone and agar in 1 L of water and boil to dissolve. Adjust
bient to 60 °C), as appropriate.
-1
the pH to 5.4 6 0.2 using either 1 mL · L hydrochloric acid
7.10 Water Bath, capable of maintaining a temperature of
(8.6) or sodium hydroxide 10 % w/v (8.10). Dispense 250 mL
47 °C 6 2 °C and receiving 500 mL bottles. Water bath
portions into 500 mL glass screw-cap bottles (7.11). Sterilize
capacityshouldbesufficienttoaccommodateatleastonebottle
byautoclavingat121 °C 62 °Cfor10 min.Coolandmaintain
of each type of agar growth medium used.
the sterilized agar in a water bath (7.10)at47°C 6 2 °C.
7.11 Glass Bottles, screw cap with gas-tight closures, Optionally, after the agar has cooled to 47 °C 6 2 °C, add
1 mL of a 0.1 % aqueous solution of chlorotetracycline (filter
500 mL nominal capacity.
sterilized by passing through a 0.2 µm filter, see 8.4) per
7.12 Culture Tubes, glass, 16 mm by 125 mm, screw cap.
100 mLMEAand mix by shaking. If the medium is required at
7.13 Autoclave, with capacity to hold 500 mL glass bottles
pH 3.5, add 10 % lactic acid (filter sterilized by passing
upright.
through a 0.2 µm filter, see 8.7) to adjust pH. Once acidified,
NOTE 5—Items 7.10 – 7.13 are not needed if using commercially
the MEA shall not be reheated. Make agar plates of the
prepared Petri dishes, as indicated in Note 4.
medium by pouring sufficient MEA into sterile petri dishes to
give a layer approximately 4 mm thick. Allow to cool and set.
8. Reagents and Materials
NOTE 6—MEA is available from various manufacturers in dehydrated
8.1 Purity of Reagents—Reagent grade chemicals shall be
form and in pre-poured plates with and without added antibiotic, either of
used in all tests. Unless otherwise indicated, it is intended that
which may be used. When sterilizing MEA prepared from commercial
all reagents conform to the specifications of the Committee on dehydrated media, follow the manufacturer’s instructions for sterilization.
Avoid overheating.
Analytical Reagents of the American Chemical Society where
NOTE 7—Alternative media to MEAmay be used, providing the ability
such specifications are available.
of any alternative medium to support comparable growth of yeast and
8.2 Theagarusedinpreparationofculturemediashallbeof
molds that are likely to be encountered in test samples can be demon-
microbiological grade. Whenever possible, use commercial strated.
NOTE 8—Alternative antibiotics may be used providing their ability to
culture media.
inhibit growth of bacteria but not yeast and molds has been validated.
8.3 Water Purity—Unless otherwise indicated, references to
8.9 Ringer’s Solution, One-Quarter Strength:
water shall be understood to mean reagent water as defined by
8.9.1 Composition/Litre:
Type III of Specification D1193.
Sodium chloride 2.25 g
8.4 Chlorotetracycline, 0.1 % (w/v) aqueous. Dissolve 0.1 g
Potassium chloride 0.105 g
chlorotetracycline in water and dilute to 100 mL. Sterilize by
Calcium chloride 0.12 g
Sodium bicarbonate 0.05 g
passing through a 0.2 µm filter.
Water 1 L
8.5 Detergent Solution 0.1 % by volume—Dissolve 1.0 mL
5 8.9.2 Preparation—Dissolve salts in 1 L of water and dis-
of polyoxyethylene (20) sorbitan monooleate in 999 mL
pense 10 mL portions into screw capped culture tubes (7.12).
water. Sterilize, either by passing through a 0.2 µm membrane
Sterilize by autoclaving at 121 °C f
...

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: D6974 − 16 D6974 − 20
Standard Practice for
Enumeration of Viable Bacteria and Fungi in Liquid Fuels—
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*
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 safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use.
1.8 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
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
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 Stability, Cleanliness and CleanlinessCompatibility of Liquid Fuels.
Current edition approved July 1, 2016May 1, 2020. Published July 2016May 2020. Originally approved in 2003. Last previous edition approved in 20132016 as
ɛ2
D6974 – 09 (2013)D6974 – 16. . DOI: 10.1520/D6974-16.10.1520/D6974-20.
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.
*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
D6974 − 20
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
2.2 Energy Institute Standards:
IP 385 Determination of the Viable Aerobic Microbial Content of Fuels and Fuel Components Boiling Below 390 °C—Filtration
and Culture Method
3. Terminology
3.1 Definitions:
3.1.1 For definition of terms used in this method refer to Terminologies D1129 and D4175, and Guide D6469.
3.1.2 aseptic, adj—sterile, free from viable microbiological contamination.
3.2 Acronyms:
3.2.1 CFU—colony forming unit
3.2.2 HPC—heterotrophic plate count
3.2.3 MF—membrane filter
3.2.4 MEA—malt extract agar
3.2.5 TNTC—too numerous to count
3.2.6 TSA—tryptone soy agar
3.3 Symbols:
-1
3.3.1 N—number of CFU · L
3.3.2 CC—number of colonies on membrane filter
3.3.3 V—sample volume filtered, mL
4. Summary of Practice
4.1 Any free water present in a fuel sample is removed by settling in a separatory funnel. After the water has been removed,
a known volume of the remaining fuel is filtered through a membrane filter aseptically by one of three methods.
4.2 The filter membrane retains microbes present in the fuel. Filter replicate fuel samples through fresh membranes to permit
replicate testing, growth on alternative nutrient media, or both.
4.3 After filtration, place each membrane on one of two types of agar growth media, incubate at a designated temperature for
three days, and examine for the presence of CFU.
4.4 Incubate the filter media on agar for two more days, then reexamine.
4.5 Count the colonies manually or by electronic counter.
4.5.1 If practical, identify colonies on each agar medium, based on colony color, morphology, and microscopic examination.
4.5.2 Convert bacterial and fungal colony counts to CFU per litre of fuel.
5. 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.
6. Interferences
6.1 High non-biological particulate loads (sediment) can clog the membrane and prevent filtration.
Available from Energy Institute, 61 New Cavendish St., London, WIG 7AR, U.K., http://www.energyinst.org.uk.
D6974 − 20
6.2 Each CFU is assumed to originate from a single microbial cell. In reality, microbes often form aggregates which appear as
a single colony. Consequently, viable count data are likely to underestimate the total number of viable organisms in the original
sample.
6.3 The metabolic state of individual microbes may be affected by numerous physical-chemical variables in the fuel. Injured
cells or cells that have relatively long generation times may not form colonies within the time allotted for test observations. This
results in an underestimation of the numbers of viable microbes in the original fuel sample.
7. Apparatus
7.1 Separatory Funnels, glass, nominal capacity 500 mL.
7.2 Measuring Cylinders, glass, nominal capacity 100 mL and 1 L.
7.3 Pipettes, glass or sterile disposable plastic, nominal capacity 10 mL, or adjustable volume pipette and sterile disposable
plastic tips.
7.4 Membrane Filter, mixed esters of cellulose, polyethersulfone (PES) or cellulose acetate (CA), presterilized, preferably
gridded, 47 mm diameter, nominal pore size 0.45 μm.
NOTE 1—While the recommended filter material is mixed esters of cellulose, the selection of membrane material will depend on individual preference
and fuel type.The choice of filter material will depend on local availability or preference for use of a disposable filtration device, or both.
NOTE 2—CA filters will become translucent on wetting with fuel, but this is not detrimental to the final recovery of microorganisms.
7.5 Filtration Unit, one of:
7.5.1 Unit, as described in Test Method D6426, with pre-sterilized in-line filter housing, or
7.5.2 Hypodermic Syringe, sterile, 100 mL, with pre-sterilized in-line filter housing, or
7.5.3 Filter Holder Assembly, single or manifold, glass, stainless steel, or polypropylene, pre-sterilized.
NOTE 3—If the vacuum filtration option (7.5.3) is chosen, a vacuum source, not more than –66 kPa will also be needed.
7.6 Forceps, blunt tipped.
7.7 Filter Flask, of sufficient capacity to receive the entire sample being filtered plus washings.
7.8 Petri Dishes, disposable plastic or glass, nominal diameter ≥50 mm.
NOTE 4—Pre-poured Petri dishes, containing the growth media described below are available commercially and may be substituted for the dishes listed
here.
7.9 Incubator, capable of maintaining a temperature of 25 °C 6 2 °C or any other temperature (within the range–ambient to
60 °C), as appropriate.
7.10 Water Bath, capable of maintaining a temperature of 47 °C 6 2 °C and receiving 500 mL bottles. Water bath capacity
should be sufficient to accommodate at least one bottle of each type of agar growth medium used.
7.11 Glass Bottles, screw cap with gas-tight closures, 500 mL nominal capacity.
7.12 Culture Tubes, glass, 16 mm by 125 mm, screw cap.
7.13 Autoclave, with capacity to hold 500 mL glass bottles upright.
NOTE 5—Items 7.10 – 7.13 are not needed if using commercially prepared Petri dishes, as indicated in Note 34.
8. Reagents and Materials
8.1 Purity of Reagents—Reagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that all
reagents conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society where such
specifications are available.
8.2 The agar used in preparation of culture media shall be of microbiological grade. Whenever possible, use commercial culture
media.
8.3 Water Purity—Unless otherwise indicated, references to water shall be understood to mean reagent water as defined by Type
III of Specification D1193.
8.4 Chlorotetracycline, 0.1 % (w/v) aqueous. Dissolve 0.1 g chlorotetracycline in water and dilute to 100 mL. Sterilize by
passing through a 0.2 μm filter.
Reagent Chemicals, American Chemical Society Specifications,ACS Reagent Chemicals, Specifications and Procedures for Reagents and Standard-Grade Reference
Materials, American Chemical Society, Washington, DC. For Suggestionssuggestions on the testing of reagents not listed by the American Chemical Society, see
AnnualAnalar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharmacopeial
Convention, Inc. (USPC), Rockville, MD.
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8.5 Detergent Solution 0.1 % by volume—Dissolve 1.0 mL of polyoxyethylene (20) sorbitan monooleate in 999 mL water.
Sterilize, either by passing through a 0.2 μm membrane filter into a sterile vessel, or autoclaving at 121 °C for 15 min.
-1
8.6 Hydrochloric Acid, 1 mol HCl · L .
8.7 Lactic Acid, 10 % (w/v) aqueous. Dissolve 10 g of lactic acid in water and dilute to 100 mL. Sterilize by passing through
a 0.2 μm filter.
8.8 Malt Extract Agar (MEA):
8.8.1 Composition/Litre:
Malt Extract 30 g
Mycological Peptone 5 g
Agar 15 g
Water 1 L
8.8.2 Preparation—Suspend the malt extract, mycological peptone and agar in 1 L of water and boil to dissolve. Adjust the pH
-1
to 5.4 6 0.2 using either 1 mL · L hydrochloric acid (8.6) or sodium hydroxide 10 % w/v (8.10). Dispense 250 mL portions into
500 mL glass screw-cap bottles (7.11). Sterilize by autoclaving at 121 °C 6 2 °C for 10 min. Cool and maintain the sterilized agar
in a water bath (7.10) at 47 °C 6 2 °C. Optionally, after the agar has cooled to 47 °C 6 2 °C, add 1 mL of a 0.1 % aqueous solution
of chlorotetracycline (filter sterilized by passing through a 0.2 μm filter, see 8.4) per 100 mL MEA and mix by shaking. If the
medium is required at pH 3.5, add 10 % lactic acid (filter sterilized by passing through a 0.2 μm filter, see 8.7) to adjust pH. Once
acidified, the MEA shall not be reheated. Make agar plates of the medium by pouring sufficient MEA into sterile petri dishes to
give a layer approximately 4 mm thick. Allow to cool and set.
NOTE 6—MEA is available from various
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