ASTM D7847-12
(Guide)Standard Guide for Interlaboratory Studies for Microbiological Test Methods
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 than 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 and health practices and determine the applicability of regulatory limitations prior to use.
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Designation: D7847 − 12
StandardGuide 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, two microbiological Methods have been approved byASTM:
Method D7463 and Method D7687.Although both methods measure adenosine triphosphate (ATP) in
fuel and fuel-associated water samples the method of obtaining the sample differs;ASTM D7463 uses
a liquid to liquid extraction whereas ASTM D7687 uses filtration.
Because these methods measure the concentration of a biomarker molecule, 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 ILS, 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
1.1.2 Detectability depends on the physiological state and
their source.
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
1.2 This guide addresses the unique considerations than
Petroleum Products and Lubricants and is the direct responsibility of Subcommittee
must be accounted for in the design and execution of inter-
D02.14 on Stability and Cleanliness of Liquid Fuels.
Current edition approved Dec. 1, 2012. Published January 2013. DOI: 10.1520/
laboratory studies intended to determine the precision of
D7847-12.
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.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D7847 − 12
1.3 This standard does not purport to address all of the 3.2.2 specific concentration, n—the fraction of a cell con-
safety concerns, if any, associated with its use. It is the stituent as determined on a per cell basis.
responsibility of the user of this standard to establish appro- 3.2.2.1 Discussion—The specific concentration can be ex-
priate safety and health practices and determine the applica- pressed as weight to weight, weight to volume or volume to
bility of regulatory limitations prior to use. volume basis. Enzymes are commonly reported in terms of
their activity relative to a reference standard.
2. Referenced Documents
3.3 Acronyms:
2.1 ASTM Standards:
3.3.1 ATP—adenosine triphosphate
D156 Test Method for Saybolt Color of Petroleum Products
3.3.2 DNA—deoxyribonucleic acid
(Saybolt Chromometer Method)
3.3.3 ILS—interlaboratory study
D1129 Terminology Relating to Water
3.3.4 RNA—ribonucleic acid
D4012 Test Method forAdenosineTriphosphate (ATP) Con-
tent of Microorganisms in Water
4. Determining Precision and Bias
D4175 Terminology Relating to Petroleum, Petroleum
Products, and Lubricants
4.1 Bias Testing:
D6300 Practice for Determination of Precision and Bias
4.1.1 There are no generally accepted reference standards
Data for Use in Test Methods for Petroleum Products and
for microbial cell constituents or for culture enumeration by
Lubricants
viability test methods.
D6469 GuideforMicrobialContaminationinFuelsandFuel
4.1.2 Consequently, bias cannot be determined for non-
Systems
culture methods.
D6974 Practice for Enumeration of Viable Bacteria and
4.1.3 Data obtained from testing an accepted non-culture
Fungi in Liquid Fuels—Filtration and Culture Procedures
parameter or culture method can be compared against data
D7463 Test Method forAdenosineTriphosphate (ATP) Con-
obtained using a proposed new method.
tent of Microorganisms in Fuel, Fuel/Water Mixtures and
4.1.3.1 Such comparisons are useful for benchmarking
Fuel Associated Water
newly measure parameters against historically measure ones.
D7464 Practice for Manual Sampling of Liquid Fuels, As-
4.1.3.2 Because bioburden is not a condition of state and
sociated Materials and Fuel System Components for
because individual microbial parameters respond to sources of
Microbiological Testing
variation differently, comparison of a new method’s test results
D7687 Test Method for Measurement of CellularAdenosine
against those of a preexisting method cannot be used to
Triphosphate in Fuel, Fuel/Water Mixtures, and Fuel-
determine the bias of either method.
AssociatedWaterwithSampleConcentrationbyFiltration
4.2 Precision Testing:
E1259 Practice for Evaluation of Antimicrobials in Liquid
4.2.1 Repeatability Testing:
Fuels Boiling Below 390°C
4.2.1.1 Sample Heterogeneity:
E1326 GuideforEvaluatingNonconventionalMicrobiologi-
(1) Unlike chemical and physical characteristics which are
cal Tests Used for Enumerating Bacteria
generally uniform throughout a well-mixed sample, microbes
E1601 Practice for Conducting an Interlaboratory Study to
are discrete bodies that are dispersed in the medium.
Evaluate the Performance of an Analytical Method
(2) In contrast to inanimate particles, microbes typically
E2756 Terminology Relating to Antimicrobial and Antiviral
form aggregates in which individual cells are bound to one
Agents
another within a polymeric matrix that is difficult to remove
2.2 Energy Institute Standard:
without also damaging cells.
IP 385 Viable aerobic microbial content of fuels and fuel
(3) Microbes are similar to inanimate particles in that their
components boiling below 90°C—Filtration and culture
settling rate within a medium follows Stoke’s law. Is this still
method
the case with biodiesels with entrained water?
(4) Heterogeneous distribution of microbes within a me-
3. Terminology
dium is likely to be a significant source of variability relative
3.1 For definition of terms used in this guide refer to to other factors affecting test method repeatability.
Terminologies D1129, D4175 and E2756, and Guide D6469. (5) Microbes require free-water in order to be metaboli-
cally active (see 4.2.1.2).
3.2 Definitions:
(1) In a given fuel system, microbial population densities
3.2.1 free water, n—water in excess of that soluble in the
tend to be greatest at interfaces; particularly the fuel-water and
sample and appearing in the sample as a haze or cloudiness, as
fuel-system-surface interfaces.
droplets, or as a separated phase or layer. D156
(2) Population densities within these interface zones are
also heterogeneous.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on Passman, F. J., English, E., Lindhardt, C., “Using Adenosine Triphosphate
the ASTM website. Concentration as a Measure of Fuel Treatment Microbicide Performance,” Morris,
Available from Energy Institute, 61 New Cavendish St., London, WIG 7AR, R. E., Ed., Proceedings of the 10th International Conference on the Stability and
U.K., http://www.energyinst.org.uk. Handling of Liquid Fuels, Oct. 7-11, 2007, Tucson,AZ.Available at www.iash.net.
D7847 − 12
(3) In order to minimize variability due to sample (1) Single culture from type culture collection—most ap-
heterogeneity, replicate samples should be recovered from as propriate when the method is designed to detect a specific
close to the same locus (location?) as possible. microbial taxon.
(2) Mixed population of type collection cultures—provides
4.2.1.2 Microbial Population’s Physiological State:
a basis for evaluating the recovery of microbes representing a
(1) The physiological state of a challenge population is
more diverse population (Practice E1259).
largely dictated by physicochemical conditions, population
(3) Uncharacterized population obtained from one or more
lifecycle stage in closed systems, flow and shear in open and
contaminated systems—most closely reflects field conditions.
semi-opensystems,andthesimilaritiesbetweenthechallenged
(4) Commercially available uncharacterized mixed popula-
microcosm and source microcosm.
tion of microbes known to metabolize fuel components (for
(2) The specific concentration of many microbial cell
example: fats, oils and greases).
constituents varies in response to the physiological state of a
(5) A commercially available population of microbes that
challenge population.
are capable of producing a reliable signal detectable by the
(3) Factors affecting the physiological state of a population
instrument detector and will survive at least for 24 h in fuel
also tend to affect the population’s culturability.
(hydrocarbon) environment.
(4) Guidance provided in Practices D6300 and E1601
(6) Field samples.
minimize the impact of physiological state on repeatability
statistics.
NOTE 1—No collection of contaminated fuels or fuels and fuel-
4.2.2 Reproducibility Testing: associated waters is likely to be truly representative of microbial diversity
in fuel systems.
4.2.2.1 Microbiological parameters are very perishable.
5.1.2 Physiological State (4.2.1.2):
(1) Practice D7464 provides guidance on the maximum
5.1.2.1 When a challenge population is transferred from the
acceptabledelaysbetweensamplecollectionandtestinitiation.
source medium to the test sample, it is likely that the
However, individual Methods can specify acceptable condi-
population will need to acclimate to its new physicochemical
tions and delays between sampling and the initiation of
environment.
analysis.
5.1.2.2 This acclimation period can be reduced—but not
(2) The history of a sample between time of collection and
totally eliminated—by ensuring that challenge populations are
test initiation can affect population densities and physiological
pre-acclimated to conditions by preculturing them in micro-
state substantially.
cosms that are as similar as possible to the conditions of the
(3) Differences in sample histories (4.2.2.1(2)) can contrib-
sample that will be used for precision testing.
ute to variability that eclipses variability due to differences in
instrumentation, analytical technique or both.
NOTE 2—During the acclimatization period microbes are likely to
(4) Factors affecting the state of microbial populations in 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
samples include, but are not limited to: temperature, oxygen
become metabolically dormant.
availability, chemical composition of sample medium, compo-
5.1.3 Generation Time:
sition of sample container, degree of ullage space.
5.1.3.1 Commonly, microbes with generation times≤1 h are
4.2.2.2 In order to minimize the potential contribution of
usedforculturetestssothatcoloniesarevisiblewithin24to48
disparate sample histories to reproducibility variability, it is
h.
advisabletoconductILSeitheratasinglelocationoratseveral
closely located facilities. 5.2 Selecting Culture Media:
5.2.1 Given the physiological diversity of Eubacteria,
4.2.2.3 The ILS design should include detailed instructions
Archeae, and Fungi, no single nutrient medium formulation or
designed to minimize differences in sample histories between
set of incubation conditions will support the proliferation of all
the time that participant subsamples are prepared and testing is
cells in a challenge population.
initiated.
5.2.2 Consequently, a negative bias is assumed for all
culture test methods.
5. Culture Methods
5.2.2.1 It is generally accepted that only a small fraction of
5.1 Selecting Test Organisms:
microbial taxa have been cultured.
5.1.1 Microbial Diversity:
5.2.2.2 There are no reference standards against which to
5.1.1.1 Thenumberofdifferenttypesofmicrobesrecovered
quantify a culture method’s bias (Guide E1326), consequently,
from microbially contaminated fuel and fuel-associated waters
only precision statistics can be developed for culture methods.
is known to range from single to dozens of different taxa.
5.2.3 Culture media selection is typically defined within a
microbiological test method to ensure that the test results are
5.1.1.2 Any given nutrient medium and set of growth
conditi
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