ASTM D7847-22

Standard Guide for Interlaboratory Studies for Microbiological Test Methods

SCOPE
1.1 Microbiological test methods present challenges that are unique relative to chemical or physical parameters, because microbes proliferate, die off and continue to be metabolically active in samples after those samples have been drawn from their source.  
1.1.1 Microbial activity depends on the presence of available water. Consequently, the detection and quantification of microbial contamination in fuels and lubricants is made more complicated by the general absence of available water from these fluids.  
1.1.2 Detectability depends on the physiological state and taxonomic profile of microbes in samples. These two parameters are affected by various factors that are discussed in this guide, and contribute to microbial data variability.  
1.2 This guide addresses the unique considerations that must be accounted for in the design and execution of interlaboratory studies intended to determine the precision of microbiological test methods designed to quantify microbial contamination in fuels, lubricants and similar low water-content (water activity  
1.3 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.  
1.4 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-Jun-2022
• ICS: 07.100.01 - Microbiology in general
• ICS: 75.100 - Lubricants, industrial oils and related products
• Technical Committee: D02 - Petroleum Products, Liquid Fuels, and Lubricants
• Drafting Committee: D02.14 - Stability, Cleanliness and Compatibility of Liquid Fuels

Relations

• Refers: ASTM D6300-24 - Standard Practice for Determination of Precision and Bias Data for Use in Test Methods for Petroleum Products, Liquid Fuels, and Lubricants
• Effective Date: 01-Mar-2024
• Refers: ASTM D4175-23a - Standard Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
• Effective Date: 15-Dec-2023
• Refers: ASTM D6300-23a - Standard Practice for Determination of Precision and Bias Data for Use in Test Methods for Petroleum Products, Liquid Fuels, and Lubricants
• Effective Date: 01-Dec-2023
• Refers: ASTM D4012-23a - Standard Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Water
• Effective Date: 01-Nov-2023
• Refers: ASTM D156-23 - Standard Test Method for Saybolt Color of Petroleum Products (Saybolt Chromometer Method)
• Effective Date: 01-Oct-2023
• Refers: ASTM D4175-23e1 - Standard Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
• Effective Date: 01-Jul-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 D7464-20 - Standard Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological Testing
• Effective Date: 01-May-2020
• Refers: ASTM D8070-19a - Standard Test Method for Screening of Fuels and Fuel Associated Aqueous Specimens for Microbial Contamination by Lateral Flow Immunoassay
• Effective Date: 01-Dec-2019
• Refers: ASTM D6300-19a - Standard Practice for Determination of Precision and Bias Data for Use in Test Methods for Petroleum Products and Lubricants
• Effective Date: 01-Dec-2019
• Refers: ASTM E1601-19 - Standard Practice for Conducting an Interlaboratory Study to Evaluate the Performance of an Analytical Method
• Effective Date: 01-Nov-2019
• 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 E2756-19 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-Nov-2019
• Refers: ASTM D8070-19 - Standard Test Method for Screening of Fuels and Fuel Associated Aqueous Specimens for Microbial Contamination by Lateral Flow Immunoassay
• Effective Date: 01-Jul-2019

Overview

ASTM D7847-22 is the Standard Guide for Interlaboratory Studies for Microbiological Test Methods, developed by ASTM International. This guide focuses on best practices for designing and conducting interlaboratory studies (ILS) aimed at assessing the precision of microbiological test methods, specifically for analyzing microbial contamination in fuels, lubricants, and similar low water-content fluids. Microbiological testing in these matrices presents unique challenges compared to chemical or physical test methods, due to the complex and dynamic nature of microbes within such samples.

Key Topics

• Unique Challenges in Microbiological Testing

  • Microbial populations can grow, die, or remain metabolically active after sample collection, leading to variable data.
  • The detection and quantification of microbes in fuels and lubricants is complicated by the absence of available water, which is necessary for microbial activity.
  • Microbial detectability depends on both the physiological state and taxonomic variety of the organisms present in the sample.

• Design and Execution of Interlaboratory Studies

  • This guide details specific considerations for sample preparation, storage, and handling to minimize bias and variability among laboratories.
  • It differentiates precision and bias analysis in culture-based and non-culture-based microbiological methods, noting that reference standards for microbial biomass are lacking.

• Sample Heterogeneity and Variability

  • Microbes are often unevenly distributed in samples, particularly at fuel-water interfaces.
  • Proper sampling methods and replication are critical for reducing variability and improving repeatability in test results.

• Operator, Apparatus, and Laboratory Effects

  • The guide emphasizes the importance of controlling for differences in equipment and personnel, recommending that certain ILS may be best conducted within a single or closely located set of facilities to reduce variability in sample history and handling.

Applications

• Fuel and Lubricant Quality Control

  • The standard is valuable for companies and laboratories needing to verify the precision and reliability of microbiological methods for detecting contamination in diesel, biodiesel, gasoline, and other fuels or lubricants.
  • It supports the development and validation of robust test methods that contribute to improved fuel system maintenance and reduced risk of biodeterioration due to microbial contamination.

• Test Method Evaluation and Development

  • Laboratories use ASTM D7847-22 to design interlaboratory studies required when introducing new or modified microbiological test methods.
  • The guide helps laboratories understand sources of method variability and implement best practices for sample collection, storage, and analysis.

• Regulatory and Compliance Testing

  • Industries subject to quality control or regulatory requirements for microbial contamination in fuels or lubricants refer to this guide for ensuring data validity in precision studies.

Related Standards

ASTM D7847-22 complements and references several relevant standards in the field of microbiological and fuel testing, including:

• ASTM D6974 - Enumeration of Viable Bacteria and Fungi in Liquid Fuels
• ASTM D6469 - Guide for Microbial Contamination in Fuels and Fuel Systems
• ASTM D7463, D7687, D7978, D8070 - Various methods for quantifying microbial contamination or biomarker molecules in fuels and fuel-associated waters
• ASTM D6300, E1601 - General practices for determining precision and bias in test methods
• IP 385 (Energy Institute) - Method for viable aerobic microbial content of fuels

These standards, together with ASTM D7847-22, support robust microbiological test method validation, contribute to improved fuel management, and enhance microbial contamination control in industry.

Keywords: interlaboratory studies, microbiological testing, fuels, lubricants, microbial contamination, ASTM D7847-22, precision, repeatability, laboratory variability, sample heterogeneity.

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: D7847 − 22
Standard Guide for
Interlaboratory Studies for Microbiological Test Methods
This standard is issued under the fixed designation D7847; 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
Microbiologicalparameterspresentanumberofuniquechallengesrelativetochemicalandphysical
test methods apropos of the development of precision and bias terms. A number of these challenges
are discussed in Guide E1326.As a working group (WG) we first grappled directly with some of these
issues during the development of Practice D6974. The drafts balloted at the D02.14 subcommittee
level in February and June 2002, were balloted with the document identified as a Method. Moreover,
the proposed Method was drafted as a harmonized document with the Energy Institute’s (EI) Method
IP385.When the item was balloted at D02 level, members of D02.94 compelled us to change the title
from Method to Practice. The argument was that ASTM Methods list single series of steps that lead
to a measurable result (a bit of data; quantitative, semi-quantitative or qualitative). Because D6974
provides for the selection of different sample volumes (based on the estimated culturable population
density) and different growth media (based on the sub-population to be quantified), it would only be
accepted as an ASTM Practice; not a Method. This issue of performing interlaboratory studies for
culture methods will be discussed below.
Since Practice D6974 was approved, four microbiological test methods have been approved by
ASTM: D7463, D7687, D7978, and D8070.
Because these methods measure the concentration of a biomarker molecule or microorganisms, the
issues that are relevant to ILS are similar to, but somewhat different than those that affect ILS for
culture methods. Beckers investigated microbiological test method interlaboratory studies, but
advised several measures that are either impractical for or not relevant to the methods that have been
developed within D02: (1) Freeze inoculated samples after dispensing into portions for shipment to
participating labs; (2) Use a single organisms challenge; (3) Add the challenge microbe to a sample
matrix in which it is likely to proliferate.
This guide will list key issues that must be addressed when designing ILS for Methods intended to
measure the microbial properties of fuels and fuel-associated waters.
1. Scope* 1.1.1 Microbial activity depends on the presence of avail-
able water. Consequently, the detection and quantification of
1.1 Microbiological test methods present challenges that are
microbial contamination in fuels and lubricants is made more
unique relative to chemical or physical parameters, because
complicated by the general absence of available water from
microbes proliferate, die off and continue to be metabolically
these fluids.
active in samples after those samples have been drawn from
their source. 1.1.2 Detectability depends on the physiological state and
taxonomic profile of microbes in samples. These two param-
eters are affected by various factors that are discussed in this
guide, and contribute to microbial data variability.
This test method is under the jurisdiction of ASTM Committee D02 on
Petroleum Products, Liquid Fuels, and Lubricants and is the direct responsibility of
1.2 This guide addresses the unique considerations that
Subcommittee D02.14 on Stability, Cleanliness and Compatibility of Liquid Fuels.
must be accounted for in the design and execution of inter-
Current edition approved July 1, 2022. Published August 2022. Originally
approved in 2012. Last previous edition approved in 2017 as D7847 – 17. DOI:
laboratory studies intended to determine the precision of
10.1520/D7847-22.
microbiological test methods designed to quantify microbial
Beckers, H. J., “Precision Testing of Standardized Microbiological Methods,”
contamination in fuels, lubricants and similar low water-
Journal of Testing and Evaluation, JTEVA, Vol. 14, No. 6, November 1986, pp.
318–320. content (water activity <0.8) fluids.
*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
D7847 − 22
1.3 This standard does not purport to address all of the 2.2 Energy Institute Standard:
safety concerns, if any, associated with its use. It is the IP 385 Viable aerobic microbial content of fuels and fuel
components boiling below 390 °C—Filtration and culture
responsibility of the user of this standard to establish appro-
method
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
3. Terminology
1.4 This international standard was developed in accor-
3.1 For definition of terms used in this guide refer to
dance with internationally recognized principles on standard-
Terminologies D1129, D4175 and E2756, and Guide D6469.
ization established in the Decision on Principles for the
Development of International Standards, Guides and Recom-
3.2 Definitions:
mendations issued by the World Trade Organization Technical 3.2.1 free water, n—water in excess of that soluble in the
Barriers to Trade (TBT) Committee. sample and appearing in the sample as a haze or cloudiness, as
droplets, or as a separated phase or layer. D156
2. Referenced Documents
3.2.2 specific concentration, n—the fraction of a cell con-
stituent as determined on a per cell basis.
2.1 ASTM Standards:
3.2.2.1 Discussion—The specific concentration can be ex-
D156 Test Method for Saybolt Color of Petroleum Products
pressed as weight to weight, weight to volume or volume to
(Saybolt Chromometer Method)
volume basis. Enzymes are commonly reported in terms of
D1129 Terminology Relating to Water
their activity relative to a reference standard.
D4012 Test Method forAdenosineTriphosphate (ATP) Con-
tent of Microorganisms in Water
3.3 Acronyms:
D4175 Terminology Relating to Petroleum Products, Liquid 3.3.1 ATP—adenosine triphosphate
Fuels, and Lubricants
3.3.2 DNA—deoxyribonucleic acid
D6300 Practice for Determination of Precision and Bias
3.3.3 ILS—interlaboratory study
Data for Use in Test Methods for Petroleum Products,
3.3.4 RNA—ribonucleic acid
Liquid Fuels, and Lubricants
D6469 GuideforMicrobialContaminationinFuelsandFuel
4. Determining Precision and Bias
Systems
4.1 Bias Testing:
D6974 Practice for Enumeration of Viable Bacteria and
4.1.1 There are no generally accepted reference standards
Fungi in Liquid Fuels—Filtration and Culture Procedures
for microbial cell constituents or for culture enumeration by
D7463 Test Method forAdenosineTriphosphate (ATP) Con-
viability test methods.
tent of Microorganisms in Fuel, Fuel/Water Mixtures, and
4.1.2 Consequently, bias cannot be determined for non-
Fuel Associated Water
culture methods.
D7464 Practice for Manual Sampling of Liquid Fuels, As-
4.1.3 Data obtained from testing an accepted non-culture
sociated Materials and Fuel System Components for
parameter or culture method can be compared against data
Microbiological Testing
obtained using a proposed new method.
D7687 Test Method for Measurement of CellularAdenosine
4.1.3.1 Such comparisons are useful for benchmarking
Triphosphate in Fuel and Fuel-associated Water With
newly measure parameters against historically measure ones.
Sample Concentration by Filtration
4.1.3.2 Because bioburden is not a condition of state and
D7978 Test Method for Determination of theViableAerobic
because individual microbial parameters respond to sources of
Microbial Content of Fuels and Associated Water—
variation differently, comparison of a new method’s test results
Thixotropic Gel Culture Method
against those of a preexisting method cannot be used to
D8070 Test Method for Screening of Fuels and Fuel Asso-
determine the bias of either method.
ciated Aqueous Specimens for Microbial Contamination
4.2 Precision Testing:
by Lateral Flow Immunoassay
4.2.1 Repeatability Testing:
E1259 Practice for Evaluation of Antimicrobials in Liquid
4.2.1.1 Sample Heterogeneity:
Fuels Boiling Below 390 °C
(1) Unlike chemical and physical characteristics which are
E1326 Guide for Evaluating Non-culture Microbiological
generally uniform throughout a well-mixed sample, microbes
Tests
are discrete bodies that are dispersed in the medium.
E1601 Practice for Conducting an Interlaboratory Study to
(2) In contrast to inanimate particles, microbes typically
Evaluate the Performance of an Analytical Method
form aggregates in which individual cells are bound to one
E2756 Terminology Relating to Antimicrobial and Antiviral
another within a polymeric matrix that is difficult to remove
Agents
without also damaging cells.
(3) Microbes are similar to inanimate particles in that their
settling rate within a medium follows Stoke’s law.
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 Available from Energy Institute, 61 New Cavendish St., London, WIG 7AR,
the ASTM website. U.K., http://www.energyinst.org.uk.
D7847 − 22
(4) Heterogeneous distribution of microbes within a me- the statistical computations are the same as those prescribed in
dium is likely to be a significant source of variability relative Practice D6300, test plan for a single site study does not satisfy
to other factors affecting test method repeatability. all of the reproducibility conditions stipulated in D6300, test
(5) Microbes require free-water in order to be metaboli- result variability between operators and apparatus setups is not
cally active (see 1.2). the same as reproducibility. Consequently, the test method’s
(a) In a given fuel system, microbial population densities variability is reported as operator/apparatus repeatability.
tend to be greatest at interfaces; particularly the fuel-water and
fuel-system-surface interfaces. 5. Culture Methods
(b) Population densities within these interface zones are
5.1 Selecting Test Organisms:
also heterogeneous.
5.1.1 Microbial Diversity:
(c) In order to minimize variability due to sample
5.1.1.1 Thenumberofdifferenttypesofmicrobesrecovered
heterogeneity, replicate samples should be recovered from as
from microbially contaminated fuel and fuel-associated waters
close to the same locus as possible.
is known to range from single to dozens of different taxa.
4.2.1.2 Microbial Population’s Physiological State:
(1) The physiological state of a challenge population is 5.1.1.2 Any given nutrient medium and set of growth
largely dictated by physicochemical conditions, population
conditions will select for a sub-population of the total micro-
lifecycle stage in closed systems, flow and shear in open and bial population (5.2.1).
semi-opensystems,andthesimilaritiesbetweenthechallenged
5.1.1.3 Non-culture methods have identified the presence of
microcosm and source microcosm.
microbial contaminants that have yet to be cultivated on
(2) The specific concentration of many microbial cell
growth media.
constituents varies in response to the physiological state of a
5.1.1.4 Depending on the method’s scope, the appropriate
challenge population.
options for precision testing include:
(3) Factors affecting the physiological state of a population
(1) Single culture from type culture collection—most ap-
also tend to affect the population’s culturability.
propriate when the method is designed to detect a specific
(4) Guidance provided in Practices D6300 and E1601
microbial taxon.
minimize the impact of physiological state on repeatability
(2) Mixed population of type collection cultures—provides
statistics.
a basis for evaluating the recovery of microbes representing a
4.2.2 Intermediate Precision Testing:
more diverse population (Practice E1259).
4.2.2.1 Microbiological parameters are very perishable.
(3) Uncharacterized population obtained from one or more
(1) Practice D7464 provides guidance on the maximum
contaminated systems—most closely reflects field conditions.
acceptabledelaysbetweensamplecollectionandtestinitiation.
(4) Commercially available uncharacterized mixed popula-
However, individual methods can specify acceptable condi-
tion of microbes known to metabolize fuel components (for
tions and delays between sampling and the initiation of
example: fats, oils and greases).
analysis.
(5) A commercially available population of microbes that
(2) The history of a sample between time of collection and
are capable of producing a reliable signal detectable by the
test initiation can affect population densities and physiological
instrument detector and will survive at least for 24 h in fuel
state substantially.
(hydrocarbon) environment.
(3) Differences in sample histories (4.2.2.1(2)) can contrib-
(6) Field samples.
ute to variability that eclipses variability due to differences in
(7) Combinations of two or more of the above.
instrumentation, analytical technique or both.
(4) Factors affecting the state of microbial populations in NOTE 1—No collection of contaminated fuels or fuels and fuel-
associated waters is likely to be truly representative of microbial diversity
samples include, but are not limited to: temperature, oxygen
in fuel systems.
availability, chemical composition of sample medium, compo-
sition of sample container, degree of ullage space. 5.1.2 Physiological State (4.2.1.2):
4.2.2.2 In order to minimize the potential contribution of
5.1.2.1 When a challenge population is transferred from the
disparate sample histories to reproducibility variability, it is
source medium to the test sample, it is likely that the
advisabletoconductILSeitheratasinglelocationoratseveral
population will need to acclimate to its new physicochemical
closely located facilities.
environment.
4.2.2.3 The ILS design should include detailed instructions
5.1.2.2 This acclimation period can be reduced—but not
designed to minimize differences in sample histories between
totally eliminated—by ensuring that challenge populations are
the time that participant subsamples are prepared and testing is
pre-acclimated to conditions by preculturing them in micro-
initiated.
cosms that are as similar as possible to the conditions of the
4.2.2.4 When all testing is performed at a single facility,
sample that will be used for precision testing.
operator/apparatus repeatability can be determined. Although
NOTE 2—During the acclimatization period microbes are likely to
regain full metabolic activity in zones in which free-water is present
Passman, F. J., English, E., Lindhardt, C., “Using Adenosine Triphosphate
(4.2.1.1(5)). If there is no free-water in the sample, microbes are likely to
Concentration as a Measure of Fuel Treatment Microbicide Performance,” Morris,
become metabolically dormant.
R. E., Ed., Proceedings of the 10th International Conference on the Stability and
Handling of Liquid Fuels, Oct. 7-11, 2007, Tucson,AZ.Available at www.iash.net. 5.1.3 Generation Time:
D7847 − 22
5.1.3.1 Commonly, microbes with generation times≤1 h are 7.1.4 During non-culture testing, cells normally do not have
used for culture tests so that colonies are visible within 24 h to the opportunity to undergo the transformation described in
48 h. 7.1.3. Consequently the specific concentrations of many cell
constituents are typically less than they are in vegetative cells.
NOTE 3—The generation times of different microbes in uncharacterized
7.1.5 The phenomena listed in 7.1.1 through 7.1.4 explain
populations is neither known nor uniform among microbes. Generation
why fuel specifications do not include microbiological quality
times vary among types of microbes and environmental conditions.
control criteria.
5.2 Selecting Culture Media:
7.1.5.1 Theprobabilityofrecoveringmicrobesinfuel-phase
5.2.1 Given the physiological diversity of Eubacteria,
samples is small relative to the probability of recovering
Archeae, and Fungi, no single nutrient medium formulation or
microbes from fuel-associated waters.
set of incubation conditions will support the proliferation of all
7.1.5.2 Specifications are for fuels; not fuel-associated wa-
cells in a challenge population.
ter; although uncontrolled microbial contamination in fuel-
5.2.2 Consequently, a negative bias is assumed for all
associated water can contribute to fuel and fuel system
culture test methods.
deterioration (Guide D6469).
5.2.2.1 It is generally accepted that only a small fraction of
microbial taxa have been cultured.
7.2 ILS Options:
5.2.2.2 There are no reference standards against which to
7.2.1 Water-free Fuel Samples:
quantify a culture method’s bias (Guide E1326), consequently,
7.2.1.1 Suspending challenge microbes into water-free fuel
only precision statistics can be developed for culture methods.
is likely to cause substantial negative bias to both culture and
5.2.3 Culture media selection is typically defined within a
non-culture tests. Osmotic shock can cause cell lysis, and
microbiological test method to ensure that the test results are
induce non-lysed cells to transition into a dormant state (7.1).
consistent with the method’s objectives (IP 385 and Practice
7.2.1.2 Microbiology is primarily a science of population
D6974).
dynamics. The impact of suspending challenge microbes into
water-free fuel is to incr
...

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: D7847 − 17 D7847 − 22
Standard Guide for
Interlaboratory Studies for Microbiological Test Methods
This standard is issued under the fixed designation D7847; 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
Microbiological parameters present a number of unique challenges relative to chemical and physical
test methods apropos of the development of precision and bias terms. A number of these challenges
are discussed in Guide E1326. As a working group (WG) we first grappled directly with some of these
issues during the development of Practice D6974. The drafts balloted at the D02.14 subcommittee
level in February and June 2002, were balloted with the document identified as a Method. Moreover,
the proposed Method was drafted as a harmonized document with the Energy Institute’s (EI) Method
IP 385. When the item was balloted at D02 level, members of D02.94 compelled us to change the title
from Method to Practice. The argument was that ASTM Methods list single series of steps that lead
to a measurable result (a bit of data; quantitative, semi-quantitative or qualitative). Because D6974
provides for the selection of different sample volumes (based on the estimated culturable population
density) and different growth media (based on the sub-population to be quantified), it would only be
accepted as an ASTM Practice; not a Method. This issue of performing interlaboratory studies for
culture methods will be discussed below.
Since Practice D6974 was approved, four microbiological test methods have been approved by
ASTM: D7463, D7687, D7978, and D8070.
Because these methods measure the concentration of a biomarker molecule or microorganisms, the
issues that are relevant to ILS are similar to, but somewhat different than those that affect ILS for
culture methods. Beckers investigated microbiological test method interlaboratory studies, but
advised several measures that are either impractical for or not relevant to the methods that have been
developed within D02: (1) Freeze inoculated samples after dispensing into portions for shipment to
participating labs; (2) Use a single organisms challenge; (3) Add the challenge microbe to a sample
matrix in which it is likely to proliferate.
This guide will list key issues that must be addressed when designing ILS for Methods intended to
measure the microbial properties of fuels and fuel-associated waters.
1. Scope*
1.1 Microbiological test methods present challenges that are unique relative to chemical or physical parameters, because microbes
proliferate, die off and continue to be metabolically active in samples after those samples have been drawn from their source.
1.1.1 Microbial activity depends on the presence of available water. Consequently, the detection and quantification of microbial
contamination in fuels and lubricants is made more complicated by the general absence of available water from these fluids.
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 Stability, Cleanliness and CleanlinessCompatibility of Liquid Fuels.
Current edition approved June 1, 2017July 1, 2022. Published June 2017August 2022. Originally approved in 2012. Last previous edition approved in 20122017 as
ɛ1
D7847 – 12D7847 – 17. . DOI: 10.1520/D7847-17.10.1520/D7847-22.
Beckers, H. J., “Precision Testing of Standardized Microbiological Methods,” Journal of Testing and Evaluation, JTEVA, Vol. 14, No. 6, November 1986, pp. 318–320.
*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
D7847 − 22
1.1.2 Detectability depends on the physiological state and taxonomic profile of microbes in samples. These two parameters are
affected by various factors that are discussed in this guide, and contribute to microbial data variability.
1.2 This guide addresses the unique considerations that must be accounted for in the design and execution of interlaboratory
studies intended to determine the precision of microbiological test methods designed to quantify microbial contamination in fuels,
lubricants and similar low water-content (water activity <0.8) fluids.
1.3 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.
1.4 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:
D156 Test Method for Saybolt Color of Petroleum Products (Saybolt Chromometer Method)
D1129 Terminology Relating to Water
D4012 Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Water
D4175 Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
D6300 Practice for Determination of Precision and Bias Data for Use in Test Methods for Petroleum Products, Liquid Fuels, and
Lubricants
D6469 Guide for Microbial Contamination in Fuels and Fuel Systems
D6974 Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels—Filtration and Culture Procedures
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
D7687 Test Method for Measurement of Cellular Adenosine Triphosphate in Fuel and Fuel-associated Water With Sample
Concentration by Filtration
D7978 Test Method for Determination of the Viable Aerobic Microbial Content of Fuels and Associated Water—Thixotropic Gel
Culture Method
D8070 Test Method for Screening of Fuels and Fuel Associated Aqueous Specimens for Microbial Contamination by Lateral
Flow Immunoassay
E1259 Practice for Evaluation of Antimicrobials in Liquid Fuels Boiling Below 390 °C
E1326 Guide for Evaluating Non-culture Microbiological Tests
E1601 Practice for Conducting an Interlaboratory Study to Evaluate the Performance of an Analytical Method
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
2.2 Energy Institute Standard:
IP 385 Viable aerobic microbial content of fuels and fuel components boiling below 390 °C—Filtration and culture method
3. Terminology
3.1 For definition of terms used in this guide refer to Terminologies D1129, D4175 and E2756, and Guide D6469.
3.2 Definitions:
3.2.1 free water, n—water in excess of that soluble in the sample and appearing in the sample as a haze or cloudiness, as droplets,
or as a separated phase or layer. D156
3.2.2 specific concentration, n—the fraction of a cell constituent as determined on a per cell basis.
3.2.2.1 Discussion—
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.
Available from Energy Institute, 61 New Cavendish St., London, WIG 7AR, U.K., http://www.energyinst.org.uk.
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The specific concentration can be expressed as weight to weight, weight to volume or volume to volume basis. Enzymes are
commonly reported in terms of their activity relative to a reference standard.
3.3 Acronyms:
3.3.1 ATP—adenosine triphosphate
3.3.2 DNA—deoxyribonucleic acid
3.3.3 ILS—interlaboratory study
3.3.4 RNA—ribonucleic acid
4. Determining Precision and Bias
4.1 Bias Testing:
4.1.1 There are no generally accepted reference standards for microbial cell constituents or for culture enumeration by viability
test methods.
4.1.2 Consequently, bias cannot be determined for non-culture methods.
4.1.3 Data obtained from testing an accepted non-culture parameter or culture method can be compared against data obtained
using a proposed new method.
4.1.3.1 Such comparisons are useful for benchmarking newly measure parameters against historically measure ones.
4.1.3.2 Because bioburden is not a condition of state and because individual microbial parameters respond to sources of variation
differently, comparison of a new method’s test results against those of a preexisting method cannot be used to determine the bias
of either method.
4.2 Precision Testing:
4.2.1 Repeatability Testing:
4.2.1.1 Sample Heterogeneity:
(1) Unlike chemical and physical characteristics which are generally uniform throughout a well-mixed sample, microbes are
discrete bodies that are dispersed in the medium.
(2) In contrast to inanimate particles, microbes typically form aggregates in which individual cells are bound to one another
within a polymeric matrix that is difficult to remove without also damaging cells.
(3) Microbes are similar to inanimate particles in that their settling rate within a medium follows Stoke’s law.
(4) Heterogeneous distribution of microbes within a medium is likely to be a significant source of variability relative to other
factors affecting test method repeatability.
(5) Microbes require free-water in order to be metabolically active (see 1.2).
(a) In a given fuel system, microbial population densities tend to be greatest at interfaces; particularly the fuel-water and
fuel-system-surface interfaces.
(b) Population densities within these interface zones are also heterogeneous.
(c) In order to minimize variability due to sample heterogeneity, replicate samples should be recovered from as close to the
same locus as possible.
4.2.1.2 Microbial Population’s Physiological State:
(1) The physiological state of a challenge population is largely dictated by physicochemical conditions, population lifecycle
stage in closed systems, flow and shear in open and semi-open systems, and the similarities between the challenged microcosm
and source microcosm.
Passman, F. J., English, E., Lindhardt, C., “Using Adenosine Triphosphate Concentration as a Measure of Fuel Treatment Microbicide Performance,” Morris, R. E., Ed.,
Proceedings of the 10th International Conference on the Stability and Handling of Liquid Fuels, Oct. 7-11, 2007, Tucson, AZ. Available at www.iash.net.
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(2) The specific concentration of many microbial cell constituents varies in response to the physiological state of a challenge
population.
(3) Factors affecting the physiological state of a population also tend to affect the population’s culturability.
(4) Guidance provided in Practices D6300 and E1601 minimize the impact of physiological state on repeatability statistics.
4.2.2 Reproducibility Intermediate Precision Testing:
4.2.2.1 Microbiological parameters are very perishable.
(1) Practice D7464 provides guidance on the maximum acceptable delays between sample collection and test initiation.
However, individual methods can specify acceptable conditions and delays between sampling and the initiation of analysis.
(2) The history of a sample between time of collection and test initiation can affect population densities and physiological state
substantially.
(3) Differences in sample histories (4.2.2.1(2)) can contribute to variability that eclipses variability due to differences in
instrumentation, analytical technique or both.
(4) Factors affecting the state of microbial populations in samples include, but are not limited to: temperature, oxygen
availability, chemical composition of sample medium, composition of sample container, degree of ullage space.
4.2.2.2 In order to minimize the potential contribution of disparate sample histories to reproducibility variability, it is advisable
to conduct ILS either at a single location or at several closely located facilities.
4.2.2.3 The ILS design should include detailed instructions designed to minimize differences in sample histories between the time
that participant subsamples are prepared and testing is initiated.
4.2.2.4 When all testing is performed at a single facility, operator/apparatus repeatability can be determined. Although the
statistical computations are the same as those prescribed in Practice D6300, test plan for a single site study does not satisfy all of
the reproducibility conditions stipulated in D6300, test result variability between operators and apparatus setups is not the same
as reproducibility. Consequently, the test method’s variability is reported as operator/apparatus repeatability.
5. Culture Methods
5.1 Selecting Test Organisms:
5.1.1 Microbial Diversity:
5.1.1.1 The number of different types of microbes recovered from microbially contaminated fuel and fuel-associated waters is
known to range from single to dozens of different taxa.
5.1.1.2 Any given nutrient medium and set of growth conditions will select for a sub-population of the total microbial population
(5.2.1).
5.1.1.3 Non-culture methods have identified the presence of microbial contaminants that have yet to be cultivated on growth
media.
5.1.1.4 Depending on the method’s scope, the appropriate options for precision testing include:
(1) Single culture from type culture collection—most appropriate when the method is designed to detect a specific microbial
taxon.
(2) Mixed population of type collection cultures—provides a basis for evaluating the recovery of microbes representing a more
diverse population (Practice E1259).
(3) Uncharacterized population obtained from one or more contaminated systems—most closely reflects field conditions.
(4) Commercially available uncharacterized mixed population of microbes known to metabolize fuel components (for example:
fats, oils and greases).
(5) A commercially available population of microbes that are capable of producing a reliable signal detectable by the
instrument detector and will survive at least for 24 h in fuel (hydrocarbon) environment.
(6) Field samples.
(7) Combinations of two or more of the above.
NOTE 1—No collection of contaminated fuels or fuels and fuel-associated waters is likely to be truly representative of microbial diversity in fuel systems.
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5.1.2 Physiological State (4.2.1.2):
5.1.2.1 When a challenge population is transferred from the source medium to the test sample, it is likely that the population will
need to acclimate to its new physicochemical environment.
5.1.2.2 This acclimation period can be reduced—but not totally eliminated—by ensuring that challenge populations are
pre-acclimated to conditions by preculturing them in microcosms that are as similar as possible to the conditions of the sample
that will be used for precision testing.
NOTE 2—During the acclimatization period microbes are likely to regain full metabolic activity in zones in which free-water is present (4.2.1.1(5)). If
there is no free-water in the sample, microbes are likely to become metabolically dormant.
5.1.3 Generation Time:
5.1.3.1 Commonly, microbes with generation times ≤1 h are used for culture tests so that colonies are visible within 24 h to 48 h.
NOTE 3—The generation times of different microbes in uncharacterized populations is neither known nor uniform among microbes. Generation times vary
among types of microbes and environmental conditions.
5.2 Selecting Culture Media:
5.2.1 Given the physiological diversity of Eubacteria, Archeae, and Fungi, no single nutrient medium formulation or set of
incubation conditions will support the proliferation of all cells in a challenge population.
5.2.2 Consequently, a negative bias is assumed for all culture test methods.
5.2.2.1 It is generally accepted that only a small fraction of microbial taxa have been cultured.
5.2.2.2 There are no reference standards against which to quantify a culture method’s bias (Guide E1326), consequently, only
precision statistics can be developed for culture methods.
5.2.3 Culture media selection is typically defined within a microbiological test method to ensure that the test results are consistent
with the method’s objectives (IP 385 and Practice D6974).
5.3 Separating Microbes from Sample:
5.3.1 Sample carryover can interfere with culturability.
5.3.1.1 Nutrients carried over with the sample can enable microbes that might not otherwise elaborate into colonies to proliferate
on the chosen culture medium.
5.3.1.2 Inhibitory chemicals (including, but not limited to microbicides) can prevent viable microbes from elaborating into
colonies.
5.3.2 Method and practice protocols should include provisions to minimize interferences due to the presence of sample fluid.
6. Non-Culture Methods
6.1 Common Issues Shared with Culture Methods:
6.1.1 The factors discussed in 5.1.1, 5.1.2 and 5.3 also apply to non-culture methods.
6.2 Issues Unique to Non-culture Methods:
6.2.1 In particular, the specific concentration of individual constituents (for example ATP – Methods D4012, D7463, D7687, and
D8070) will vary with the organisms’ physiological state (4.2.1.2(2)).
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6.2.2 Additionally, genetic constituents (DNA and RNA) vary qualitatively as well as quantitatively based on the taxonomic make
up (diversity) of the microbial population in the sample.
7. Sample Types
7.1 Microbes in Fuels:
7.1.1 As discussed in 4.2.1.1(5), microbes concentrate where there is free-water.
7.
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