ASTM D7464-20

Standard Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological Testing

SIGNIFICANCE AND USE
5.1 Representative samples of fuel products and associated substances are required for the determination of microbial contamination in fuels and fuel systems in order to accurately assess the biodeterioration risk posed to the fuel, fuel-system components or both. Uncontrolled microbial contamination can affect fuel specification properties adversely.6 As discussed in Guide D6469, microbes can cause a variety of operational problems, including filter plugging and microbially influenced corrosion (MIC), the latter of which causes valve failure, tank and pipeline failure.  
5.2 These practices for microbiological sampling decrease the risk of contaminating samples with extraneous microbes, thereby increasing the probability that the original microbial population in the sample does not change significantly between the time of sampling and the time of testing.  
5.3 The objective of sampling for microbiological testing is to obtain representative samples that are likely to reflect the degree and nature of microbial contamination in the system from which the samples are collected. Manual 477 addresses the rationale for and design of microbial contamination programs. Recognizing that microbiological contamination is not distributed uniformly throughout fuel systems, both the number and types of samples collected will normally be different from the samples collected per Practice D4057 in order to determine whether product meets specifications.  
5.4 The physical, chemical and microbiological property tests to be performed on a sample will dictate the sampling procedures, the sample quantity required, and many of the sample handling requirements.  
5.5 Fuel systems are not normally designed to facilitate optimal microbiological sampling. Consequently, the selection of sampling device and sample source reflect compromises between accessibility and suitability for meeting the sample collection objective.  
5.6 The guidance provided in Practice D4057 generally applies to ...
SCOPE
1.1 This practice covers aspects of sample device preparation and sample handling that prevent samples from becoming contaminated with microorganisms not originally contained within the sample.  
1.2 This practice also covers sample handling considerations that reflect the perishability of samples collected for microbiological testing.  
1.3 This practice supplements Practice D4057 by providing guidance specific to the manual sampling of fuels when samples are to be tested for microbial contamination.  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 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.  
1.6 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: 07.100.99 - Other standards related to microbiology
• 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 D7464-19 - Standard Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological Testing
• Effective Date: 01-May-2020
• Refers: ASTM D5245-19(2024) - Standard Practice for Cleaning Laboratory Glassware, Plasticware, and Equipment Used in Microbiological Analyses
• Effective Date: 01-Apr-2024
• Refers: ASTM D1655-24 - Standard Specification for Aviation Turbine Fuels
• Effective Date: 15-Mar-2024
• Refers: ASTM D910-24 - Standard Specification for Leaded Aviation Gasolines
• Effective Date: 01-Mar-2024
• Refers: ASTM D6227-24 - Standard Specification for Unleaded Aviation Gasoline Containing a Non-hydrocarbon Component
• Effective Date: 01-Mar-2024
• Refers: ASTM D4814-24 - Standard Specification for Automotive Spark-Ignition Engine Fuel
• Effective Date: 01-Jan-2024
• Refers: ASTM D975-23a - Standard Specification for Diesel Fuel
• Effective Date: 15-Dec-2023
• Refers: ASTM D4814-23a - Standard Specification for Automotive Spark-Ignition Engine Fuel
• Effective Date: 01-Dec-2023
• Refers: ASTM D1655-23a - Standard Specification for Aviation Turbine Fuels
• Effective Date: 01-Oct-2023
• Refers: ASTM D6974-20 - Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels—Filtration and Culture Procedures
• Effective Date: 01-May-2020
• Refers: ASTM D1129-13(2020)e2 - Standard Terminology Relating to Water
• Effective Date: 01-May-2020
• Refers: ASTM D4814-20 - Standard Specification for Automotive Spark-Ignition Engine Fuel
• Effective Date: 01-Feb-2020
• Refers: ASTM D396-19a - Standard Specification for Fuel Oils
• Effective Date: 15-Dec-2019
• Refers: ASTM D4814-19a - Standard Specification for Automotive Spark-Ignition Engine Fuel
• Effective Date: 01-Dec-2019
• Refers: ASTM D910-19 - Standard Specification for Leaded Aviation Gasolines
• Effective Date: 01-Dec-2019

Overview

ASTM D7464-20 is a standard practice developed by ASTM International that provides detailed guidance for the manual sampling of liquid fuels, associated materials, and fuel system components for microbiological testing. Effective microbiological sampling is crucial for identifying microbial contamination in fuels and fuel systems, which helps in accurately assessing biodeterioration risks. Microbial contamination can lead to various operational challenges, such as filter plugging and microbially influenced corrosion (MIC), potentially resulting in equipment failure and compromised product quality.

The standard addresses all critical aspects of sample collection, including device preparation, sample handling, and prevention of sample contamination by extraneous microbes. It also builds on and supplements the widely used ASTM D4057 practice for manual sampling by providing unique considerations required specifically for microbiological analysis.

Key Topics

• Sample Device Preparation: Guidance on selecting, cleaning, and disinfecting sampling devices and containers to prevent non-indigenous microbial contamination.
• Sample Handling: Specific instructions reflecting the perishability of microbiological samples, ensuring samples reflect the true microbial population at the time of collection.
• Sampling Objectives: Steps to obtain representative samples to:
  • Detect microbial contamination in fuel products and associated substances
  • Evaluate biodeterioration potential in tank bottoms and at fuel-water interfaces
  • Assess system component integrity via surface and filter media sampling
• Sampling Procedures:
  • Manual procedures for collecting liquid, surface, filter media, and component samples
  • Aseptic techniques to limit exposure to external contaminants
• Sample Labeling and Documentation: Requirements for accurate sample identification, chain of custody, and documentation to ensure traceability and reliability of results.

Applications

Implementing ASTM D7464-20 is essential across industries reliant on liquid fuels such as petroleum, diesel, aviation fuels, and biodiesel. Practical applications include:

• Fuel Storage and Distribution: Minimizing operational disruptions by detecting early signs of microbial activity in storage tanks, pipelines, and at fuel dispensers.
• Infrastructure Maintenance: Identifying and mitigating risks of microbially influenced corrosion in tanks, valves, and pipelines, thus preventing costly failures.
• Quality Assurance Programs: Ensuring delivered fuel meets microbial cleanliness specifications, supporting compliance and customer satisfaction.
• Contamination Incident Response: Providing a standardized approach for sampling during contamination investigations, supporting remediation and follow-up actions.
• Research and Development: Supporting fuel system research into biodeterioration mechanisms and the effect of microbial growth in varied operating environments.

Related Standards

ASTM D7464-20 is closely linked with several international and industry standards to ensure consistency in sampling and analysis:

• ASTM D4057 – Practice for Manual Sampling of Petroleum and Petroleum Products
• ASTM D6469 – Guide for Microbial Contamination in Fuels and Fuel Systems
• ASTM D5245 – Practice for Cleaning Laboratory Glassware, Plasticware, and Equipment Used in Microbiological Analyses
• ASTM D6974 – Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels
• American Petroleum Institute (API) Manual of Petroleum Measurement Standards, Chapter 3
• Petroleum Equipment Institute (PEI) 900-08 – Recommended Practices for the Inspection and Maintenance of UST Systems

By following ASTM D7464-20, organizations can improve the accuracy and reliability of microbiological testing in fuel systems, helping to prevent operational problems, ensure compliance with fuel quality standards, and extend asset life.

Keywords: microbiological sampling, liquid fuels, ASTM D7464, biodeterioration, fuel system contamination, microbiology, sampling procedures, fuel quality, chain of custody, sample handling, corrosion prevention.

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: D7464 − 20
Standard Practice for
Manual Sampling of Liquid Fuels, Associated Materials and
Fuel System Components for Microbiological Testing
This standard is issued under the fixed designation D7464; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
There are several important characteristics that distinguish microbiological parameters from other
parameters for which manually collected fuel samples are tested.
Microbes, when present in fuels or fuel systems are invariably present as contaminants. Similarly
to particulates, microbes are discrete entities rather than dissolved solutes in fuel, however, unlike
inanimate particles; microbes can proliferate or die during the interval between sampling and testing.
An important consequence of this is that microbes introduced into the sample from sources other
than the sample itself, can proliferate and potentially eclipse the population indigenous to the sample.
Although microbes can be transported in fuel, they require free-water in order to grow and
proliferate. Consequently, microbes tend to form colonies that are embedded in hydrophilic matrices.
These matrices are most likely to form at system interfaces, including: fuel-water, fuel-structure,
bottom-water-structure and air and fuel-vapor to structure. Microbes growing within these colonies
producechemicals(metabolitesandbiomoleculardetritus)thataredeteriogenic(candegradefueland
fuel system components) and diffuse into fuel.
These factors combine to require unique practices specific to the collection of samples that are
intended for microbiological testing.
1. Scope* priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
1.1 This practice covers aspects of sample device prepara-
1.6 This international standard was developed in accor-
tion and sample handling that prevent samples from becoming
dance with internationally recognized principles on standard-
contaminated with microorganisms not originally contained
ization established in the Decision on Principles for the
within the sample.
Development of International Standards, Guides and Recom-
1.2 This practice also covers sample handling consider-
mendations issued by the World Trade Organization Technical
ations that reflect the perishability of samples collected for
Barriers to Trade (TBT) Committee.
microbiological testing.
2. Referenced Documents
1.3 This practice supplements Practice D4057 by providing
guidance specific to the manual sampling of fuels when
2.1 ASTM Standards:
samples are to be tested for microbial contamination.
D396Specification for Fuel Oils
D910Specification for Leaded Aviation Gasolines
1.4 The values stated in SI units are to be regarded as
D975Specification for Diesel Fuel
standard. No other units of measurement are included in this
D1129Terminology Relating to Water
standard.
D1193Specification for Reagent Water
1.5 This standard does not purport to address all of the
D1655Specification for Aviation Turbine Fuels
safety concerns, if any, associated with its use. It is the
D2069Specification for Marine Fuels (Withdrawn 2003)
responsibility of the user of this standard to establish appro-
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 Lubricants and is the direct responsibility of Subcom- contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
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 June 2020. Originally the ASTM website.
approved in 2008. Last previous edition approved in 2019 as D7464–19. DOI: The last approved version of this historical standard is referenced on
10.1520/D7464-20. www.astm.org.
*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
D7464 − 20
D2880Specification for Gas Turbine Fuel Oils 4.2.2 Swabbed areas are measured to facilitate test result
D3508Method for Evaluating Water Testing Membrane normalization into parameter units per unit surface area (for
3 2
Filters for Fecal Coliform Recovery (Withdrawn 1995) example CFU/cm ).
D3699Specification for Kerosine 4.2.3 The post-sampling chain of custody procedures for
D4057Practice for Manual Sampling of Petroleum and liquid samples apply.
Petroleum Products
4.3 Filter Media:
D4814Specification for Automotive Spark-Ignition Engine
4.3.1 Canister Elements:
Fuel
4.3.1.1 Filter elements are transferred aseptically to sterile
D5245 Practice for Cleaning Laboratory Glassware,
plastic bags.
Plasticware, and Equipment Used in Microbiological
4.3.1.2 The post-sampling chain of custody procedures for
Analyses
liquid samples apply.
D6227Specification for Unleaded Aviation Gasoline Con-
4.3.2 Depth Media:
taining a Non-hydrocarbon Component
4.3.2.1 Media core-samples are collected aseptically and
D6469GuideforMicrobialContaminationinFuelsandFuel
transferred to tared, sterile containers.
Systems
4.3.2.2 The post-sampling chain of custody procedures for
D6751Specification for Biodiesel Fuel Blend Stock (B100)
liquid samples apply.
for Middle Distillate Fuels
D6974Practice for Enumeration of Viable Bacteria and
5. Significance and Use
Fungi in Liquid Fuels—Filtration and Culture Procedures
5.1 Representative samples of fuel products and associated
2.2 American Petroleum Institute (API) Standard:
substances are required for the determination of microbial
Manual of Petroleum Measurement Standards Chapter
contamination in fuels and fuel systems in order to accurately
3—Tank Gauging, section 1A—Standard Practice for the
assess the biodeterioration risk posed to the fuel, fuel-system
Manual Gauging of Petroleum and Petroleum Products
components or both. Uncontrolled microbial contamination
2.3 Petroleum Equipment Institute (PEI) Standard: 6
canaffectfuelspecificationpropertiesadversely. Asdiscussed
900-08Recommended Practices for the Inspection and
in Guide D6469, microbes can cause a variety of operational
Maintenance of UST Systems
problems, including filter plugging and microbially influenced
corrosion (MIC), the latter of which causes valve failure, tank
3. Terminology
and pipeline failure.
3.1 For definition of terms used in this method refer to
5.2 These practices for microbiological sampling decrease
Terminologies D1129 and D4175, Practice D4057 and Guide
the risk of contaminating samples with extraneous microbes,
D6469.
thereby increasing the probability that the original microbial
3.2 Definitions:
populationinthesampledoesnotchangesignificantlybetween
3.2.1 aseptic, adj—sterile, free from viable microbiological
the time of sampling and the time of testing.
contamination.
5.3 The objective of sampling for microbiological testing is
3.2.2 scrape sample, n—a portion of residue removed from
to obtain representative samples that are likely to reflect the
asurfacebyforcefulstrokesofaninstrumentsuchasaspatula.
degree and nature of microbial contamination in the system
from which the samples are collected. Manual 47 addresses
4. Summary of Practices
the rationale for and design of microbial contamination pro-
4.1 Liquid Sampling:
grams. Recognizing that microbiological contamination is not
4.1.1 Fuel and fuel-associated bottom-water samples in-
distributed uniformly throughout fuel systems, both the num-
tended for microbiological testing are collected similarly to
ber and types of samples collected will normally be different
conventionalsamplesasdescribedinPracticeD4057,however
from the samples collected per Practice D4057 in order to
specific measures are added to reduce the risk of sample
determine whether product meets specifications.
contamination.
5.4 The physical, chemical and microbiological property
4.1.2 Sampling devices are disinfected before collecting
tests to be performed on a sample will dictate the sampling
microbiological samples.
procedures, the sample quantity required, and many of the
4.1.3 Sterile sample containers are used.
sample handling requirements.
4.1.4 Unique chain of custody procedures are used to
5.5 Fuel systems are not normally designed to facilitate
minimizethepotentialqualitative,quantitativeorbothtypesof
optimal microbiological sampling. Consequently, the selection
changes in the sample between sampling and testing.
4.2 Surface Sampling:
4.2.1 Sterile swabs are used to collect surface samples for
Passman, F. J., McFarland, B. L., and Hillyer, M. J., “Oxygenated Gasoline
microbiological testing.
Biodeterioration and its Control in Laboratory Microcosms,” International Biode-
terioration and Biodegradation, Vol 47, No. 2, 2001, pp. 95-106.
Hill, G., “Sampling Methods for Detecting Microbial Contamination in Fuels
Available from American Petroleum Institute (API), 1220 L. St., NW, and Fuel Systems,” in Passman, F. J., Ed., ASTM Manual 47–Fuel and Fuel System
Washington, DC 20005-4070, http://www.api.org. Microbiology: Fundamentals, Diagnosis and Contamination Control, ASTM
Available from Petroleum Equipment Institute website, www.pei.org. International, West Conshohocken, PA, 2003.
D7464 − 20
alternative, clean, non-porous glove that has been disinfected in accor-
of sampling device and sample source reflect compromises
dance with 8.2 in order to address the explosion hazard risk and still
between accessibility and suitability for meeting the sample
minimizetheriskofcontaminatingsampleswithmicrobesassociatedwith
collection objective.
human skin.
5.6 The guidance provided in Practice D4057 generally
6.6 Spatula—Stainless steel; 1.5cm by 10cm for collecting
appliestothispracticeaswell.Consequently,thispracticewill
surface residue samples.
address only those procedures that apply uniquely to micro-
6.7 Swabs—Sterile, ATP-free.
biological sampling.
7. Reagents
6. Apparatus
7.1 Alcohol, ≥70% methanol, ethanol or isopropanol, tech-
6.1 The general considerations provided in Practice D4057
nical grade.
apply here. Sample containers come in a variety of shapes,
7.2 Water—Type I Reagent Grade or better (Specification
sizes and materials. To paraphrase D4057, Paragraph 6.1, in
D1193; Terminology D1129).
order to be able to select the right container for a given
application one must ensure that there will be no interaction
8. Manual Sampling Considerations
between the sampled material and the container which would
8.1 TheconsiderationsdetailedinPracticeD4057Section7
affect the integrity of the other. For general microbiological
apply.
testing, either glass or plastic containers are appropriate.
However, containers should be appropriate for the specific
8.2 Sampling Device Disinfection:
method of analysis intended.
8.2.1 Before collecting a sample, the sampling device shall
6.1.1 Sample Container Cleanliness:
becleanedanddisinfected.Duetotheriskoffireandexplosion
6.1.1.1 Sample containers must be clean and should be
when handling liquid fuels with boiling points below 90°C,
sterile.
procedures generally used to disinfect apparatus used for
6.1.1.2 For the purposes of most microbiological testing,
microbiological sampling cannot be used in the liquid fuel
previously unused containers that are received in original
environment. The following procedure shall be used instead:
manufacturer’s packaging are sufficiently clean to substitute
8.2.1.1 Clean the device, taking particular care to remove
for sterile containers.
any liquid and particulate residue remaining from previous
6.1.1.3 Practice D5245 provides details on cleaning previ-
samples.
ously used glassware, plasticware and equipment.
8.2.1.2 Rinse device with alcohol (7.1) by filling the device
6.1.1.4 Method D3508 specifies the protocol for sterilizing 1 1
approximately ⁄4 to ⁄3 with alcohol and shaking the closed
containers and labware.
device for 30 s.
8.2.1.3 Drain the alcohol thoroughly from the device into a
6.2 Sampling Devices—Sampling devices are described in
suitable disposal container.
detail under each of the specific sampling procedures.
8.2.1.4 Allow all residual alcohol to evaporate from device
6.2.1 Sampling Device Cleanliness:
surfaces.
6.2.1.1 Sampling devices shall be cleaned between use in
accordance with 8.2.1, except cleaning is not necessary be-
9. Special Precautions
tween repeated spot samples obtained either for the purpose of
9.1 The precautions enumerated in Practice D4057 Section
filling a single sample container or filling multiple sample
8 apply to sampling for microbiological testing.
containers intended to be used as replicate spot samples. Such
replicates may be used to test the sample for different
9.2 Contamination Control—Additional caution is required
parameters, when the contents of a single sample container are
to prevent the contamination of samples with non-indigenous
used for a single analysis (for example Practice D6974), for
microbes.
obtaining replicate data in order to determine parameter
9.2.1 Thenormalmicrofloraofhealthyskinis>1×10 bac-
variability, or both.
teria⁄cm . Precautions shall be taken to minimize the risk of
6.2.1.2 It can be impractical to sterilize some types of
contaminating samples with skin microflora. Wearing surgical
sampling devices used to obtain liquid petroleum, petroleum
glovesprovidesanadequatebarrierbetweentheskin,sampling
product or fuel-associated, free-water samples (see 8.2).
devices and sample containers. Gloves should either be re-
placedorrinsedwith70%alcohol(7.1)betweensamples.(See
6.3 Funnel—20cm to 25cm diameter mouth; ≤1.9cm di-
Note 1.)
ameter outlet (diameter small enough to fit into mouth of
9.2.2 Sampling Devices can become contaminated with
sample container).
residuefromcollectedsamples.Theproceduredescribedin8.2
6.4 Absorbent Spill Pads.
minimizes the risk of cross-contamination. Device disinfection
6.5 Gloves; Surgical—Usedtopreventthecontaminationof should be completed just before sample collection in order to
samples with microorganisms indigenous to human skin.
reduce the risk of contamination from airborne microbes. All
NOTE 1—The use of surgical gloves may create a static electricity
surfaces with which the sample will come into contact shall be
discharge risk that presents an explosion hazard when handling certain
disinfected. After collecting sample and before dispensing
fuels. Additionally, polymers from which some surgical gloves are
sample into sample container, wipe any debris from the
manufactured are incompatible with certain fuels, and can disintegrate on
sampler’s external surfaces and use alcohol to disinfect the
contact with such fuel, thereby creating a skin contact hazard. Where
either spark, product incompatibility or both types of risk exist, use an funnel surface over which the sample will flow.
D7464 − 20
9.2.3 Drain and Tap Samples—Microbiological testing may preventspillage.Beforetransferringasamplefromthesampler
also be performed on drain samples. If sampling from a fluid to the container, wipe any debris from the sampler’s external
drain line or dispenser nozzle, clean the area around the surfaces and use alcohol to disinfect the funnel surface over
discharge orifice and wipe the area with alcohol (7.1). which the sample will flow. In accordance with 8.2.1.4, allow
9.2.4 Sample Containers should remain closed until just all residual alcohol to evaporate from funnel after disinfecting
before the sample is dispensed from the sampling device into itandbeforeusingittodecantsamplefromdevicetocontainer.
the container, and should be re-closed immediately after the Label sample container in accordance with 15.1. Store sample
sample has been dispensed. This reduces the risk of contami- in dark for transport to testing facility.
nation from airborne particles or during sample container
10.3.2 System Components—Aseptically remove compo-
handling. nent from system and transfer to a plastic bag for transport to
testing facility. Smaller components may be stored in self-
9.3 Sample Perishability—Microbes are living organisms.
sealingstoragebags.Largercomponentsmaybestoredinlarge
Consequently, samples for microbiological testing are highly
garbagebags.Labelsamplecontainerinaccordancewith15.5.
perishable.Optimally,microbiologicaltestsareinitiatedwithin
Store sample in dark for transport to testing facility.
4 h after sample collection. If testing is to be performed within
4h,samplesmaybestoredatambienttemperature.Ifthedelay
NOTE3—Ifpossible,donotpermitcomponenttodrycompletelybefore
will be longer, store samples on ice, in insulated containers
transferring it to storage bag. Desiccation can kill microbes that were
previously viable.
containing ice packs or under refrigeration at 5°C to 10°C.
NOTE 4—Before using plastic bags for component sample storage, test
NOTE 2—Be careful to avoid freezing the sample as the freeze-thaw
for compatibility with the fuel. Petroleum products differ in their aggres-
process is likely to kill microbes.
siveness against the polymers from which storage bags are manufactured.
10. Sampling Procedures (General)
11. Tank Sampling
10.1 Sample Selection—Microbes are not distributed uni-
formly throughout fuel systems. Consequently, the probability 11.1 Core Thief Spot Sampling Procedure:
of detecting microbial contamination depends substantially on
11.1.1 Application—This procedure may be used for sam-
thetypeofsampleandthelocationwithinthefuelsystemfrom
pling liquids, including fuel grades defined under Specifica-
which the sample is collected. Hill provides details of the
tions D396, D910, D975, D1655, D2069, D2880, D3699,
essential sample selection considerations. The most critical
D4814, D6227 and D6751; for free-water and sludge/sediment
issues are listed here:
at the bottom of tanks containing these liquid fuel products.
11.1.2 Apparatus:
10.2 Objective—The three primary objectives of microbio-
11.1.2.1 Innage gauging tape and plumb bob for measuring
logical sampling are:
innage and bottom-water depth.
10.2.1 Product Condition—The bioburden being trans-
11.1.2.2 Water-detection paste appropriate for petroleum
ported in the liquid fuel product. Spot samples of product are
most appropriate to meet this objective. Tap or thief samples productcontainedintankfromwhichsampleistobecollected.
are appropriate. Thief samples should be taken from the 11.1.2.3 A typical closed-core type thief is shown in Fig.
bottom-third of tanks. Tap samples may be obtained from
1a-d and a typical bailer sampler is shown in Fig. 1e-g. The
dispenser nozzles, tank drains, or sampling taps. thief shall be designed so that a sample can be obtained from
10.2.2 Product Biodeterioration Potential—the presence of
the tank bottom. The size of the thief should be selected
a bioburden with potential to change product properties. As depending upon the internal diameter of the fitting or pipe
noted in the Introduction, microbial communities tend to throughwhichitwillbeloweredandthevolumeofthesample
develop at interfaces. For example, microbial population den- required. The thief should be capable of penetrating the liquid
3 6
sities at the fuel-water interface are typically 10 to 10 times fuel product in the tank. It should also be capable of penetrat-
population densities in the overlying fuel. Consequently, in
ing any membranous interface-layer that may have developed
tankswithmeasurablebottom-water,fuelwaterinterfacelayer, between the fuel and bottom-water. The thief shall inc
...

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: D7464 − 19 D7464 − 20
Standard Practice for
Manual Sampling of Liquid Fuels, Associated Materials and
Fuel System Components for Microbiological Testing
This standard is issued under the fixed designation D7464; 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.
INTRODUCTION
There are several important characteristics that distinguish microbiological parameters from other
parameters for which manually collected fuel samples are tested.
Microbes, when present in fuels or fuel systems are invariably present as contaminants. Similarly
to particulates, microbes are discrete entities rather than dissolved solutes in fuel, however, unlike
inanimate particles; microbes can proliferate or die during the interval between sampling and testing.
An important consequence of this is that microbes introduced into the sample from sources other
than the sample itself, can proliferate and potentially eclipse the population indigenous to the sample.
Although microbes can be transported in fuel, they require free-water in order to grow and
proliferate. Consequently, microbes tend to form colonies that are embedded in hydrophilic matrices.
These matrices are most likely to form at system interfaces, including: fuel-water, fuel-structure,
bottom-water-structure and air and fuel-vapor to structure. Microbes growing within these colonies
produce chemicals (metabolites and biomolecular detritus) that are deteriogenic (can degrade fuel and
fuel system components) and diffuse into fuel.
These factors combine to require unique practices specific to the collection of samples that are
intended for microbiological testing.
1. Scope*
1.1 This practice covers aspects of sample device preparation and sample handling that prevent samples from becoming
contaminated with microorganisms not originally contained within the sample.
1.2 This practice also covers sample handling considerations that reflect the perishability of samples collected for
microbiological testing.
1.3 This practice supplements Practice D4057 by providing guidance specific to the manual sampling of fuels when samples
are to be tested for microbial contamination.
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.5 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.
1.6 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:
D396 Specification for Fuel Oils
This practice is under the jurisdiction of ASTM Committee D02 on Petroleum Products, Liquid Fuels, and Lubricants and is the direct responsibility of Subcommittee
D02.14 on Stability, Cleanliness and Compatibility of Liquid Fuels.
Current edition approved Nov. 1, 2019May 1, 2020. Published November 2019June 2020. Originally approved in 2008. Last previous edition approved in 20142019 as
D7464 – 14.D7464 – 19. DOI: 10.1520/D7464-19.10.1520/D7464-20.
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
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D910 Specification for Leaded Aviation Gasolines
D975 Specification for Diesel Fuel
D1129 Terminology Relating to Water
D1193 Specification for Reagent Water
D1655 Specification for Aviation Turbine Fuels
D2069 Specification for Marine Fuels (Withdrawn 2003)
D2880 Specification for Gas Turbine Fuel Oils
D3508 Method for Evaluating Water Testing Membrane Filters for Fecal Coliform Recovery (Withdrawn 1995)
D3699 Specification for Kerosine
D4057 Practice for Manual Sampling of Petroleum and Petroleum Products
D4814 Specification for Automotive Spark-Ignition Engine Fuel
D5245 Practice for Cleaning Laboratory Glassware, Plasticware, and Equipment Used in Microbiological Analyses
D6227 Specification for Unleaded Aviation Gasoline Containing a Non-hydrocarbon Component
D6469 Guide for Microbial Contamination in Fuels and Fuel Systems
D6751 Specification for Biodiesel Fuel Blend Stock (B100) for Middle Distillate Fuels
D6974 Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels—Filtration and Culture Procedures
2.2 American Petroleum Institute (API) Standard:
Manual of Petroleum Measurement Standards Chapter 3—Tank Gauging, section 1A—Standard Practice for the Manual
Gauging of Petroleum and Petroleum Products
2.3 Petroleum Equipment Institute (PEI) Standard:
900-08 Recommended Practices for the Inspection and Maintenance of UST Systems
3. Terminology
3.1 For definition of terms used in this method refer to Terminologies D1129 and D4175, Practice D4057 and Guide D6469.
3.2 Definitions:
3.2.1 aseptic, adj—sterile, free from viable microbiological contamination.
3.2.2 scrape sample, n—a portion of residue removed from a surface by forceful strokes of an instrument such as a spatula.
4. Summary of Practices
4.1 Liquid Sampling:
4.1.1 Fuel and fuel-associated bottom-water samples intended for microbiological testing are collected similarly to conventional
samples as described in Practice D4057, however specific measures are added to reduce the risk of sample contamination.
4.1.2 Sampling devices are disinfected before collecting microbiological samples.
4.1.3 Sterile sample containers are used.
4.1.4 Unique chain of custody procedures are used to minimize the potential qualitative, quantitative or both types of changes
in the sample between sampling and testing.
4.2 Surface Sampling:
4.2.1 Sterile swabs are used to collect surface samples for microbiological testing.
4.2.2 Swabbed areas are measured to facilitate test result normalization into parameter units per unit surface area (for example
CFU/cm ).
4.2.3 The post-sampling chain of custody procedures for liquid samples apply.
4.3 Filter Media:
4.3.1 Canister Elements:
4.3.1.1 Filter elements are transferred aseptically to sterile plastic bags.
4.3.1.2 The post-sampling chain of custody procedures for liquid samples apply.
4.3.2 Depth Media:
4.3.2.1 Media core-samples are collected aseptically and transferred to tared, sterile containers.
4.3.2.2 The post-sampling chain of custody procedures for liquid samples apply.
5. Significance and Use
5.1 Representative samples of fuel products and associated substances are required for the determination of microbial
contamination in fuels and fuel systems in order to accurately assess the biodeterioration risk posed to the fuel, fuel-system
The last approved version of this historical standard is referenced on www.astm.org.
Available from American Petroleum Institute (API), 1220 L. St., NW, Washington, DC 20005-4070, http://www.api.org.
Available from Petroleum Equipment Institute website, www.pei.org.
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components or both. Uncontrolled microbial contamination can affect fuel specification properties adversely. As discussed in
Guide D6469, microbes can cause a variety of operational problems, including filter plugging and microbially influenced corrosion
(MIC), the latter of which causes valve failure, tank and pipeline failure.
5.2 These practices for microbiological sampling decrease the risk of contaminating samples with extraneous microbes, thereby
increasing the probability that the original microbial population in the sample does not change significantly between the time of
sampling and the time of testing.
5.3 The objective of sampling for microbiological testing is to obtain representative samples that are likely to reflect the degree
and nature of microbial contamination in the system from which the samples are collected. Manual 47 addresses the rationale for
and design of microbial contamination programs. Recognizing that microbiological contamination is not distributed uniformly
throughout fuel systems, both the number and types of samples collected will normally be different from the samples collected per
Practice D4057 in order to determine whether product meets specifications.
5.4 The physical, chemical and microbiological property tests to be performed on a sample will dictate the sampling procedures,
the sample quantity required, and many of the sample handling requirements.
5.5 Fuel systems are not normally designed to facilitate optimal microbiological sampling. Consequently, the selection of
sampling device and sample source reflect compromises between accessibility and suitability for meeting the sample collection
objective.
5.6 The guidance provided in Practice D4057 generally applies to this practice as well. Consequently, this practice will address
only those procedures that apply uniquely to microbiological sampling.
6. Apparatus
6.1 The general considerations provided in Practice D4057 apply here. Sample containers come in a variety of shapes, sizes and
materials. To paraphrase D4057, Paragraph 6.1, in order to be able to select the right container for a given application one must
ensure that there will be no interaction between the sampled material and the container which would affect the integrity of the other.
For general microbiological testing, either glass or plastic containers are appropriate. However, containers should be appropriate
for the specific method of analysis intended.
6.1.1 Sample Container Cleanliness:
6.1.1.1 Sample containers must be clean and should be sterile.
6.1.1.2 For the purposes of most microbiological testing, previously unused containers that are received in original
manufacturer’s packaging are sufficiently clean to substitute for sterile containers.
6.1.1.3 Practice D5245 provides details on cleaning previously used glassware, plasticware and equipment.
6.1.1.4 Method D3508 specifies the protocol for sterilizing containers and labware.
6.2 Sampling Devices—Sampling devices are described in detail under each of the specific sampling procedures.
6.2.1 Sampling Device Cleanliness:
6.2.1.1 Sampling devices shall be cleaned between use in accordance with 8.2.1, except cleaning is not necessary between
repeated spot samples obtained either for the purpose of filling a single sample container or filling multiple sample containers
intended to be used as replicate spot samples. Such replicates may be used to test the sample for different parameters, when the
contents of a single sample container are used for a single analysis (for example Practice D6974), for obtaining replicate data in
order to determine parameter variability, or both.
6.2.1.2 It can be impractical to sterilize some types of sampling devices used to obtain liquid petroleum, petroleum product or
fuel-associated, free-water samples (see 8.2).
6.3 Funnel—20 cm to 25 cm diameter mouth; ≤1.9 cm diameter outlet (diameter small enough to fit into mouth of sample
container).
6.4 Absorbent Spill Pads.
6.5 Gloves; Surgical—Used to prevent the contamination of samples with microorganisms indigenous to human skin.
NOTE 1—The use of surgical gloves may create a static electricity discharge risk that presents an explosion hazard when handling certain fuels.
Additionally, polymers from which some surgical gloves are manufactured are incompatible with certain fuels, and can disintegrate on contact with such
fuel, thereby creating a skin contact hazard. Where either spark, product incompatibility or both types of risk exist, use an alternative, clean, non-porous
glove that has been disinfected in accordance with 8.2 in order to address the explosion hazard risk and still minimize the risk of contaminating samples
with microbes associated with human skin.
6.6 Spatula—Stainless steel; 1.5 cm by 10 cm for collecting surface residue samples.
6.7 Swabs—Sterile, ATP-free.
Passman, F. J., McFarland, B. L., and Hillyer, M. J., “Oxygenated Gasoline Biodeterioration and its Control in Laboratory Microcosms,” International Biodeterioration
and Biodegradation, Vol 47, No. 2, 2001, pp. 95-106.
Hill, G., “Sampling Methods for Detecting Microbial Contamination in Fuels and Fuel Systems,” in Passman, F. J., Ed., ASTM Manual 47–Fuel and Fuel System
Microbiology: Fundamentals, Diagnosis and Contamination Control, ASTM International, West Conshohocken, PA, 2003.
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7. Reagents
7.1 Alcohol, ≥70 % methanol, ethanol or isopropanol, technical grade.
7.2 Water—Type I Reagent Grade or better (Specification D1193; Terminology D1129).
8. Manual Sampling Considerations
8.1 The considerations detailed in Practice D4057 Section 7 apply.
8.2 Sampling Device Disinfection:
8.2.1 Before collecting a sample, the sampling device shall be cleaned and disinfected. Due to the risk of fire and explosion
when handling liquid fuels with boiling points below 90 °C, procedures generally used to disinfect apparatus used for
microbiological sampling cannot be used in the liquid fuel environment. The following procedure shall be used instead:
8.2.1.1 Clean the device, taking particular care to remove any liquid and particulate residue remaining from previous samples.
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8.2.1.2 Rinse device with alcohol (7.1) by filling the device approximately ⁄4 to ⁄3 with alcohol and shaking the closed device
for 30 s.
8.2.1.3 Drain the alcohol thoroughly from the device into a suitable disposal container.
8.2.1.4 Allow all residual alcohol to evaporate from device surfaces.
9. Special Precautions
9.1 The precautions enumerated in Practice D4057 Section 8 apply to sampling for microbiological testing.
9.2 Contamination Control—Additional caution is required to prevent the contamination of samples with non-indigenous
microbes.
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9.2.1 The normal microflora of healthy skin is >1 × 10 bacteria ⁄cm . Precautions shall be taken to minimize the risk of
contaminating samples with skin microflora. Wearing surgical gloves provides an adequate barrier between the skin, sampling
devices and sample containers. Gloves should either be replaced or rinsed with 70 % alcohol (7.1) between samples. (See Note
1.)
9.2.2 Sampling Devices can become contaminated with residue from collected samples. The procedure described in 8.2
minimizes the risk of cross-contamination. Device disinfection should be completed just before sample collection in order to
reduce the risk of contamination from airborne microbes. All surfaces with which the sample will come into contact shall be
disinfected. After collecting sample and before dispensing sample into sample container, wipe any debris from the sampler’s
external surfaces and use alcohol to disinfect the funnel surface over which the sample will flow.
9.2.3 Drain and Tap Samples—Microbiological testing may also be performed on drain samples. If sampling from a fluid drain
line or dispenser nozzle, clean the area around the discharge orifice and wipe the area with alcohol (7.1).
9.2.3.1 Do not flush drain lines before sampling. The objective is to detect microbial contamination if it is present. Flushing lines
can remove the analyte—microbial contaminants—before they are detected.
NOTE 2—Water and biomass tend to accumulate in drain lines. Flushing lines is likely to remove the analyte targeted for detection, and thereby degrade
the utility of the sample. 9.2.3 and 9.2.3.1 reflect a balance between collecting samples that include microbial contaminants that are from outside the tank
and inadvertently removing relevant contaminants before collecting the sample.
9.2.4 Sample Containers should remain closed until just before the sample is dispensed from the sampling device into the
container, and should be re-closed immediately after the sample has been dispensed. This reduces the risk of contamination from
airborne particles or during sample container handling.
9.3 Sample Perishability—Microbes are living organisms. Consequently, samples for microbiological testing are highly
perishable. Optimally, microbiological tests are initiated within 4 h after sample collection. If testing is to be performed within 4
h, samples may be stored at ambient temperature. If the delay will be longer, store samples on ice, in insulated containers
containing ice packs or under refrigeration at 5 °C to 10 °C.
NOTE 2—Be careful to avoid freezing the sample as the freeze-thaw process is likely to kill microbes.
10. Sampling Procedures (General)
10.1 Sample Selection—Microbes are not distributed uniformly throughout fuel systems. Consequently, the probability of
detecting microbial contamination depends substantially on the type of sample and the location within the fuel system from which
the sample is collected. Hill provides details of the essential sample selection considerations. The most critical issues are listed
here:
10.2 Objective—The three primary objectives of microbiological sampling are:
10.2.1 Product Condition—The bioburden being transported in the liquid fuel product. Spot samples of product are most
appropriate to meet this objective. Tap or thief samples are appropriate. Thief samples should be taken from the bottom-third of
tanks. Tap samples may be obtained from dispenser nozzles, tank drains, or sampling taps.
10.2.2 Product Biodeterioration Potential—the presence of a bioburden with potential to change product properties. As noted
in the Introduction, microbial communities tend to develop at interfaces. For example, microbial population densities at the
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fuel-water interface are typically 10 to 10 times population densities in the overlying fuel. Consequently, in tanks with
measurable bottom-water, fuel water interface layer, or alternatively, bottom-samples are best suited for detecting microbial
contamination. Samples from the surfaces of system components, including filter media, are also useful for diagnosing product
biodeterioration risk.
10.2.3 System Component Biodeterioration—MIC can result in tank, valve or pipeline failure. Flocs of biomass can plug fuel
filters. The preferred samples for assessing component biodeterioration risk are surface samples. Removable pipeline spools,
corrosion coupons, valve components and filter media provide accessible surfaces for sampling. Bottom-samples (10.2.2) are also
useful for assessing system biodeterioration risk.
10.3 Sample Handling:
10.3.1 Liquid Samples—Samples should be dispensed into sample containers via a funnel or other suitable device to prevent
spillage. Before transferring a sample from the sampler to the container, wipe any debris from the sampler’s external surfaces and
use alcohol to disinfect the funnel surface over which the sample will flow. In accordance with 8.2.1.4, allow all residual alcohol
to evaporate from funnel after disinfecting it and before using it to decant sample from device to container. Label sample container
in accordance with 15.1. Store sample in dark for transport to testing facility.
10.3.2 System Components—Aseptically remove component from system and transfer to a plastic bag for transport to testing
facility. Smaller components may be stored in self-sealing storage bags. Larger components may be stored in large garbage bags.
Label sample container in accordance with 15.5. Store sample in dark for transport to testing facility.
NOTE 3—If possible, do not permit component to dry completely before transferring it to storage bag. Desiccation can kill microbes that were
previously viable.
NOTE 4—Before using plastic bags for component sample storage, test for compatibility with the fuel. Petroleum products differ in their aggressiveness
against the polymers from which storage bags are manufactured.
11. Tank Sampling
11.1 Core Thief Spot Sampling Procedure:
11.1.1 Application—This proced
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