ASTM D7978-14(2019)
(Test Method)Standard Test Method for Determination of the Viable Aerobic Microbial Content of Fuels and Associated Water—Thixotropic Gel Culture Method
Standard Test Method for Determination of the Viable Aerobic Microbial Content of Fuels and Associated Water—Thixotropic Gel Culture Method
SIGNIFICANCE AND USE
5.1 This test method is intended to provide a tool for assessing whether fuel storage and distribution facilities or end user fuel tanks are subject to microbial growth and alert fuel suppliers or users to the potential for fuel quality or operational problems and/or the requirement for preventative or remedial measures.
5.2 This test method detects numbers of microbial colony forming units (CFU), the same detection parameter used in the laboratory standard procedures Practice D6974 and IP 385. However, whereas Practice D6974 and IP 385 provide separate assessment of numbers of viable aerobic bacteria CFU and numbers of viable fungal CFU, this test method provides a combined total count of viable aerobic bacteria and fungal CFU.
5.3 This test method is designed to detect a recognized group of microorganisms of significance in relation to contamination of distillate fuels, but it is recognized that microbiological culture techniques do not detect all microorganisms that can be present in a sample. Culturability is affected primarily by the ability of captured microbes to proliferate on the growth medium provided, under specific growth conditions. Consequently, a proportion of the active or inactive microbial population present in a sample can be viable but not detected by any one culture test.7 In this respect, the test is indicative of the extent of microbial contamination in a sample ,and it is assumed that when a fuel sample is significantly contaminated, some of the dominant microbial species present will be quantifiably detected, even if not all species present are culturable.
5.4 Many samples from fuel systems can be expected to contain a low level of “background” microbial contamination, which is not necessarily of operational significance. The minimum detection level of this test method is determined by the volume of specimen tested and is set such that microbial contamination will generally only be detected when it is at levels indicative of active ...
SCOPE
1.1 This test method describes a procedure that can be used in the field or in a laboratory to quantify culturable, viable aerobic microorganisms present as contaminants in liquid fuels, including those blended with synthesized hydrocarbons or biofuels, with kinematic viscosities (at 40 °C) of ≤24 mm2 s-1 and heavy and residual fuels with kinematic viscosities (at 40 °C) of ≤700 mm2 s-1 and in fuel-associated water.
1.1.1 This test method has been validated by an ILS for a range of middle distillate fuels meeting Specifications D975, D1655, ISO 8217 DMA, and NATO F-76.2
1.2 This test method quantitatively assesses culturable, viable aerobic microbial content present in the form of bacteria, fungi, and fungal spores. Results are expressed as the total number of microbial colony forming units (CFU)/L of fuel or total number of CFU/mL of associated water. The number of CFU should not be interpreted as absolute values but should be used as part of a diagnostic or condition monitoring effort; for example, these values can be used to assess contamination as absent, light, moderate, or heavy.
Note 1: This test method is technically equivalent to IP 613, although the two methods are not currently jointed.
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 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.
1.5 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 Bar...
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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: D7978 − 14 (Reapproved 2019)
Standard Test Method for
Determination of the Viable Aerobic Microbial Content of
Fuels and Associated Water—Thixotropic Gel Culture
Method
This standard is issued under the fixed designation D7978; 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 test method describes a procedure that can be used
mendations issued by the World Trade Organization Technical
in the field or in a laboratory to quantify culturable, viable
Barriers to Trade (TBT) Committee.
aerobic microorganisms present as contaminants in liquid
fuels, including those blended with synthesized hydrocarbons
2 2. Referenced Documents
or biofuels, with kinematic viscosities (at 40 °C) of ≤24 mm
-1
2.1 ASTM Standards:
s and heavy and residual fuels with kinematic viscosities (at
2 -1
40 °C) of ≤700 mm s and in fuel-associated water. D975 Specification for Diesel Fuel
D1129 Terminology Relating to Water
1.1.1 This test method has been validated by an ILS for a
range of middle distillate fuels meeting Specifications D975, D1655 Specification for Aviation Turbine Fuels
D4175 Terminology Relating to Petroleum Products, Liquid
D1655, ISO 8217 DMA, and NATO F-76.
Fuels, and Lubricants
1.2 This test method quantitatively assesses culturable,
D4176 Test Method for FreeWater and Particulate Contami-
viable aerobic microbial content present in the form of
nation in Distillate Fuels (Visual Inspection Procedures)
bacteria, fungi, and fungal spores. Results are expressed as the
D6469 GuideforMicrobialContaminationinFuelsandFuel
total number of microbial colony forming units (CFU)/L of
Systems
fuel or total number of CFU/mL of associated water. The
D6974 Practice for Enumeration of Viable Bacteria and
number of CFU should not be interpreted as absolute values
Fungi in Liquid Fuels—Filtration and Culture Procedures
but should be used as part of a diagnostic or condition
D7464 Practice for Manual Sampling of Liquid Fuels, As-
monitoring effort; for example, these values can be used to
sociated Materials and Fuel System Components for
assess contamination as absent, light, moderate, or heavy.
Microbiological Testing
NOTE 1—This test method is technically equivalent to IP613, although
D7847 GuideforInterlaboratoryStudiesforMicrobiological
the two methods are not currently jointed.
Test Methods
1.3 The values stated in SI units are to be regarded as
E2756 Terminology Relating to Antimicrobial and Antiviral
standard. No other units of measurement are included in this
Agents
standard.
2.2 Energy Institute Standards:
1.4 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 390
responsibility of the user of this standard to establish appro-
°C—Filtration and Culture Method
priate safety, health, and environmental practices and deter-
IP 613 Determination of the Viable Aerobic Microbial Con-
mine the applicability of regulatory limitations prior to use.
tent of Fuels and Associated Water—Thixotropic Gel
1.5 This international standard was developed in accor-
Culture Method
dance with internationally recognized principles on standard-
Guidelines for the Investigation of the Microbial Content of
Petroleum Fuels and for the Implementation ofAvoidance
This test method 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 July 1, 2019. Published August 2019. Originally For referenced ASTM standards, visit the ASTM website, www.astm.org, or
approved in 2014. Last previous edition approved in 2014 as D7978 – 14. DOI: contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
10.1520/D7978-14R19. Standards volume information, refer to the standard’s Document Summary page on
Defense Standard 91-4, Fuel, Naval, Distillate (NATO Code: F-76, Joint the ASTM website.
Service designation DIESO F-76), Issue 9, 3 May 2013, UK Defense Standardiza- Available from Energy Institute, 61 New Cavendish St., London, WIG 7AR,
tion U.K., http://www.energyinst.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D7978 − 14 (2019)
and Remedial Strategies, 2nd Edition, 2008, Energy medium capable of sustaining the growth of a wide range of
Institute, London, ISBN 978 0 85293 524 8 microorganisms encountered in liquid fuels and petroleum
products (8.1).
2.3 Other Standards:
ISO 8217 DMA Petroleum Products, Fuels (Class F), Speci-
4.2 The gel liquefies when the bottle is shaken, dispersing
fications of Marine Fuels, 4th Edition 2010, ISO Switzer-
the fuel or water specimen containing any microorganisms.
land
4.3 The gel is allowed to reset into a flat layer on one of the
IATA Guidance Material on Microbiological Contamination
larger sides of the bottle.
in Aircraft Fuel Tanks, 4th Edition, December 2011
4.4 The bottle is incubated in the dark in this position for
four days.The gel contains components that sustain the growth
3. Terminology
of viable, culturable microorganisms and the fuel specimen
3.1 Definitions—For definitions of terms used in this test
itself contributes additional nutrients.
method, refer to Terminologies D1129, D4175, and E2756.
4.5 Viable, culturable microorganisms in the volume of fuel
3.2 Definitions:
or water tested grow into visible colonies, and a reactive
3.2.1 colony, n—a discreet visible aggregate of microorgan-
compound changes the color of these colonies to red or purple
isms that develops when a viable microorganism, or particle
such that they can be easily counted or their number estimated.
containing viable microorganisms, is introduced into a gel-
based nutritive culture medium and reproduces there. 4.6 The number of colonies formed is considered in relation
3.2.1.1 Discussion—A period of incubation is necessary to to the volume of specimen added to the test, and expressed as
allow sufficient reproduction. This test method utilizes a CFU/Loffuel,orCFU/mLiftheresultrelatestoatestofwater
reactive compound that shortens the time for colonies to in a fuel system sample.
become visible and stains them so that they appear as red or
purple spots. 5. Significance and Use
3.2.1.2 Discussion—Typically, bacterial colonies become
5.1 This test method is intended to provide a tool for
visible to the naked eye only after the colony contains ≥10
assessing whether fuel storage and distribution facilities or end
individual cells. Consequently, the time required for a colony
user fuel tanks are subject to microbial growth and alert fuel
to become visible is dependent on the organism’s generation
suppliersoruserstothepotentialforfuelqualityoroperational
(doubling), which can range from <30 min to >1 week.
problems and/or the requirement for preventative or remedial
3.2.2 culture medium, n—solid, semi-solid, or liquid prepa-
measures.
rations that contain nutrients that support microbial growth,
5.2 This test method detects numbers of microbial colony
and usually other chemical agents that can inhibit or stimulate
forming units (CFU), the same detection parameter used in the
growth by specific microorganisms or that can indicate the
laboratory standard procedures Practice D6974 and IP 385.
presence of all culturable or specific microorganisms.
However, whereas Practice D6974 and IP385 provide separate
3.3 Definitions of Terms Specific to This Standard:
assessment of numbers of viable aerobic bacteria CFU and
3.3.1 thixotropic gel, n—a sheer thinning gel that is semi-
numbers of viable fungal CFU, this test method provides a
solid while static and becomes a liquid when a sheer force is
combined total count of viable aerobic bacteria and fungal
applied.
CFU.
3.4 Acronyms and Abbreviations:
5.3 This test method is designed to detect a recognized
3.4.1 CC—number of colonies (colony count).
groupofmicroorganismsofsignificanceinrelationtocontami-
3.4.2 CFU—colony forming unit. nation of distillate fuels, but it is recognized that microbiologi-
calculturetechniquesdonotdetectallmicroorganismsthatcan
3.4.3 N—number of CFU/mL(in water) or CFU/L(in fuel).
be present in a sample. Culturability is affected primarily by
3.4.4 TNTC—too numerous to count.
the ability of captured microbes to proliferate on the growth
3.4.5 V—volume tested, mL
medium provided, under specific growth conditions.
Consequently, a proportion of the active or inactive microbial
4. Summary of Test Method
population present in a sample can be viable but not detected
by any one culture test. In this respect, the test is indicative of
4.1 A known volume of fuel or water is added to the test
the extent of microbial contamination in a sample ,and it is
kit , which consists of a rectangular, transparent glass bottle
assumed that when a fuel sample is significantly contaminated,
containing a patented sterile, thixotropic gel-based culture
some of the dominant microbial species present will be
quantifiably detected, even if not all species present are
culturable.
Available from International Air Transport Assocation (IATA), 800 Place
Victoria, PO Box 113, Montreal H4Z 1M1, Quebec, Canada or 33, Route de
l’Aeroport, PO Box 416, 1215 Geneva 15Airport, Switzerland, http://www.iata.org.
The sole source of supply of the test kit known to the committee at this time is
ECHA Microbiology Ltd., Cardiff, CF3 0EF, UK. If you are aware of alternative White,J.etal.,“Culture-IndependentAnalysisofBacterialFuelContamination
suppliers, please provide this information to ASTM International Headquarters. Provides Insight into the Level of Concordance with the Standard Industry Practice
Your comments will receive careful consideration at a meeting of the responsible ofAerobic Cultivation,” Applied and Environmental Microbiology, Vol. 77, No. 13,
technical committee, which you may attend. July 2011, pp. 4527-4538.
D7978 − 14 (2019)
5.4 Many samples from fuel systems can be expected to color in the gel culture medium (usually within 12 h). This
contain a low level of “background” microbial contamination, color change will not interfere with the growth of any
which is not necessarily of operational significance. The microorganisms, and in most cases, microbial colonies can be
minimum detection level of this test method is determined by counted or estimated ignoring the background color. If anti-
the volume of specimen tested and is set such that microbial oxidants are present at very high concentration, the color
contamination will generally only be detected when it is at change in the gel culture medium can be so strong that users
levels indicative of active proliferation. can find it difficult to distinguish from the appearance of a test
inwhichmorethan10 000microbialcolonieshavegrown.The
5.5 The test will detect culturable bacteria and fungi that are
effect can be compensated for by testing a smaller volume of
metabolically active and dormant fungal spores. Presence of
specimen, as described in 12.5.2.3 in the test procedure.
fungal spores in a fuel sample can be indicative of active
microbial proliferation within a fuel tank or system, but at a 6.2 Some bacteria are motile and can, on prolonged
point distant from the location sampled. Active microbial incubation, spread through the gel culture medium, producing
growth only occurs in free water, and this can be present only large irregularly-shaped colonies, streaks, or patches of red or
as isolated pockets at tank or system low points. Because purple color that are difficult to count. Procedures for compen-
fungal spores are more hydrophobic than active cells and sating for this effect are described in 12.5.2.4 in the test
fungal material (mycelium), they disperse more readily in fuel procedure.
phase and are thus more readily detected when low points
6.3 If microorganisms other than those in the specimen are
cannot be directly sampled and only fuel phase is present in
introduced into the culture medium, they can give rise to
samples.
spurious colonies. To avoid this, the test should be conducted
5.6 This test method can determine whether microbial in as clean an environment as practicable, and care should be
contaminationinsamplesdrawnfromfueltanksandsystemsis taken to avoid touching surfaces of dispensing apparatus,
absent or present at light, moderate, and heavy levels. pipettes, and sample containers that come into direct contact
with the sample or culture medium.
5.7 The categorization of light, moderate, and heavy levels
of contamination will depend on the fuel type, the sampling
7. Apparatus
location, the facility sampled, and its specific operating cir-
7.1 Incubator, capable of operating at 25 °C 6 3 °C. The
cumstances.
design of the incubator shall ensure the culture medium is not
5.8 Further guidance or interpretation of test results can be
exposed to light during incubation. The use of an incubator is
found in Guide D6469, in the Energy Institute Guidelines for
not essential (see 12.5.1.1).
the investigation of the microbial content of petroleum fuels,
7.2 Temperature Measuring Device, accurate to 62 °C, to
and for the implementation of avoidance and remedial strate-
measure incubation temperature.
gies and in the IATA Guidance Material on Microbiological
Contamination in Aircraft Fuel Tanks.
8. Reagents and Materials
5.8.1 FurtherguidanceonsamplingcanbefoundinPractice
8.1 MicrobMonitor2, commercially available test kit com-
D7464.
prised of a rectangular, sterile transparent glass bottle fitted
5.9 Testing can be conducted on a routine basis or to
with a cap and seal and containing a sterile, thixotropic
investigate incidents.
gel-based culture medium capable of sustaining the growth of
a wide range of microorganisms encountered in liquid fuels
5.10 Microbiological tests are not intended to be used to
and petroleum products and associated water.
determine compliance with absolute fuel specifications or
limits. The implementation of specification limits for micro-
8.2 Disposable Syringe, sterile and fuel c
...
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