ASTM D7464-08
(Practice)Standard Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological Testing
Standard Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological Testing
SIGNIFICANCE AND USE
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. As discussed in Guide D 6469, 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.
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.
The objective of sampling for microbiological testing is to obtain a representative sample that is likely to reflect the degree and nature of microbial contamination in the system from which the sample is collected. Manual 47 addresses the rational for and design of microbial contamination programs.
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.
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.
The guidance provided in Practice D 4057 generally applies to this practice as well. Consequently, this practice will address only those procedures that apply uniquely to microbiological sampling.
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 D 4057 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 and health practices and determine the applicability of regulatory limitations prior to use.
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Designation: D7464 − 08
StandardPractice 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 and health practices and determine the applica-
bility of regulatory limitations prior to use.
1.1 This practice covers aspects of sample device prepara-
tion and sample handling that prevent samples from becoming
2. Referenced Documents
contaminated with microorganisms not originally contained
2.1 ASTM Standards:
within the sample.
D396Specification for Fuel Oils
1.2 This practice also covers sample handling consider-
D910Specification for Aviation Gasolines
ations that reflect the perishability of samples collected for
D975Specification for Diesel Fuel Oils
microbiological testing.
D1129Terminology Relating to Water
1.3 This practice supplements Practice D4057 by providing
D1193Specification for Reagent Water
guidance specific to the manual sampling of fuels when
D1655Specification for Aviation Turbine Fuels
samples are to be tested for microbial contamination.
D2069Specification for Marine Fuels (Withdrawn 2003)
D2880Specification for Gas Turbine Fuel Oils
1.4 The values stated in SI units are to be regarded as
D3508Method for Evaluating Water Testing Membrane
standard. No other units of measurement are included in this
Filters for Fecal Coliform Recovery (Withdrawn 1995)
standard.
D3699Specification for Kerosine
1.5 This standard does not purport to address all of the
D4057Practice for Manual Sampling of Petroleum and
safety concerns, if any, associated with its use. It is the
Petroleum Products
responsibility of the user of this standard to establish appro-
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
This practice is under the jurisdiction ofASTM Committee D02 on Petroleum contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
ProductsandLubricantsandisthedirectresponsibilityofSubcommitteeD02.14on Standards volume information, refer to the standard’s Document Summary page on
Stability and Cleanliness of Liquid Fuels. the ASTM website.
Current edition approved July 1, 2008. Published August 2008. DOI: 10.1520/ The last approved version of this historical standard is referenced on
D7464-08. www.astm.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D7464 − 08
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
5. Significance and Use
D6974Practice for Enumeration of Viable Bacteria and
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:
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
the time of sampling and the time of testing.
3.2.1 aseptic, adj—sterile, free from viable microbiological
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 a representative sample that is likely to reflect the
asurfacebyforcefulstrokesofaninstrumentsuchasaspatula.
degree and nature of microbial contamination in the system
from which the sample is collected. Manual 47 addresses the
4. Summary of Practices
rational for and design of microbial contamination programs.
4.1 Liquid Sampling:
5.4 The physical, chemical and microbiological property
4.1.1 Fuel and fuel-associated bottom-water samples in-
tests to be performed on a sample will dictate the sampling
tended for microbiological testing are collected similarly to
procedures, the sample quantity required, and many of the
conventionalsamplesasdescribedinPracticeD4057,however
sample handling requirements.
specific measures are added to reduce the risk of sample
5.5 Fuel systems are not normally designed to facilitate
contamination.
optimal microbiological sampling. Consequently, the selection
4.1.2 Sampling devices are disinfected before collecting
of sampling device and sample source reflect compromises
microbiological samples.
between accessibility and suitability for meeting the sample
4.1.3 Sterile sample containers are used.
collection objective.
4.1.4 Unique chain of custody procedures are used to
5.6 The guidance provided in Practice D4057 generally
minimizethepotentialqualitative,quantitativeorbothtypesof
changes in the sample between sampling and testing. appliestothispracticeaswell.Consequently,thispracticewill
address only those procedures that apply uniquely to micro-
4.2 Surface Sampling:
biological sampling.
4.2.1 Sterile swabs are used to collect surface samples for
microbiological testing.
6. Apparatus
4.2.2 Swabbed areas are measured to facilitate test result
6.1 The general considerations provided in Practice D4057
normalization into parameter units per unit surface area (for
apply here. Sample containers come in a variety of shapes,
example CFU/cm ).
4.2.3 The post-sampling chain of custody procedures for
liquid samples apply.
Passman, F. J., McFarland, B. L., and Hillyer, M. J., “Oxygenated Gasoline
4.3 Filter Media:
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 − 08
sizes and materials. To paraphrase D4057, Paragraph 6.1, in 7.2 Water—Type I Reagent Grade or better (Specification
order to be able to select the right container for a given D1193; Terminology D1129).
application one must ensure that there will be no interaction
8. Manual Sampling Considerations
between the sampled material and the container which would
affect the integrity of the other. For general microbiological
8.1 TheconsiderationsdetailedinPracticeD4057Section7
testing, either glass or plastic containers are appropriate.
apply.
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.
6.2 Sampling Devices—Sampling devices are described in 8.2.1.3 Drain the alcohol thoroughly from the device into a
detail under each of the specific sampling procedures.
suitable disposal container.
6.2.1 Sampling Device Cleanliness: 8.2.1.4 Allow all residual alcohol to evaporate from device
6.2.1.1 Sampling devices shall be cleaned between use in
surfaces.
accordance with 8.2.1, except cleaning is not necessary be-
9. Special Precautions
tween repeated spot samples obtained either for the purpose of
filling a single sample container or filling multiple sample
9.1 The precautions enumerated in Practice D4057 Section
containers intended to be used as replicate spot samples. Such
8 apply to sampling for microbiological testing.
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 The normal microflora of healthy skin is >1 × 10
variability, or both.
bacteria/cm .Precautionsshallbetakentominimizetheriskof
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-
6.3 Funnel—20 to 25 cm diameter mouth; ≤1.9 cm diam-
placedorrinsedwith70%alcohol(7.1)betweensamples.(See
eter outlet (diameter small enough to fit into mouth of sample
Note 1.)
container).
9.2.2 Sampling Devices can become contaminated with
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
manufactured are incompatible with certain fuels, and can disintegrate on sample into sample container, wipe any debris from the
contact with such fuel, thereby creating a skin contact hazard. Where
sampler’s external surfaces and use alcohol to disinfect the
either spark, product incompatibility or both types of risk exist, use an
funnel surface over which the sample will flow.
alternative, clean, non-porous glove that has been disinfected in accor-
9.2.3 Drain and Tap Samples—Microbiological testing may
dance with 8.2 in order to address the explosion hazard risk and still
also be performed on drain samples. If sampling from a fluid
minimizetheriskofcontaminatingsampleswithmicrobesassociatedwith
human skin. drain line or dispenser nozzle, clean the area around the
discharge orifice and wipe the area with alcohol (7.1). Follow
6.6 Spatula—Stainless steel; 1.5 by 10 cm for collecting
the guidance provide in Practice D4057, Paragraph 13.6.
surface residue samples.
9.2.4 Sample Containers should remain closed until just
6.7 Swabs—Sterile, ATP-free.
before the sample is dispensed from the sampling device into
the container, and should be re-closed immediately after the
7.
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