1、Designation: D7847 121Standard Guide forInterlaboratory Studies for Microbiological Test Methods1This standard is issued under the fixed designation D7847; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision.
2、A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1NOTESubsection 1.2 and Section 4 were corrected editorially in March 2015.INTRODUCTIONMicrobiological parameters present a number of unique chal
3、lenges relative to chemical and physicaltest methods apropos of the development of precision and bias terms. A number of these challengesare discussed in Guide E1326.As a working group (WG) we first grappled directly with some of theseissues during the development of Practice D6974. The drafts ballo
4、ted at the D02.14 subcommitteelevel 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 Institutes (EI) MethodIP 385. When the item was balloted at D02 level, members of D02.94 compelled
5、us to change the titlefrom Method to Practice. The argument was that ASTM Methods list single series of steps that leadto a measurable result (a bit of data; quantitative, semi-quantitative or qualitative). Because D6974provides for the selection of different sample volumes (based on the estimated c
6、ulturable populationdensity) and different growth media (based on the sub-population to be quantified), it would only beaccepted as an ASTM Practice; not a Method. This issue of performing interlaboratory studies forculture methods will be discussed below.Since Practice D6974 was approved, two micro
7、biological Methods have been approved by ASTM:Method D7463 and Method D7687. Although both methods measure adenosine triphosphate (ATP) infuel and fuel-associated water samples the method of obtaining the sample differs; ASTM D7463 usesa liquid to liquid extraction whereas ASTM D7687 uses filtration
8、.Because these methods measure the concentration of a biomarker molecule, the issues that arerelevant to ILS are similar to, but somewhat different than those that affect ILS for culture methods.Beckers2investigated microbiological test method ILS, but advised several measures that are eitherimpract
9、ical for or not relevant to the methods that have been developed within D02: (1) Freezeinoculated samples after dispensing into portions for shipment to participating labs; (2) Use a singleorganisms challenge; (3) Add the challenge microbe to a sample matrix in which it is likely toproliferate.This
10、guide will list key issues that must be addressed when designing ILS for Methods intended tomeasure the microbial properties of fuels and fuel-associated waters.1. Scope1.1 Microbiological test methods present challenges that areunique relative to chemical or physical parameters, becausemicrobes pro
11、liferate, die off and continue to be metabolicallyactive in samples after those samples have been drawn fromtheir source.1.1.1 Microbial activity depends on the presence of avail-able water. Consequently, the detection and quantification ofmicrobial contamination in fuels and lubricants is made more
12、complicated by the general absence of available water fromthese fluids.1.1.2 Detectability depends on the physiological state andtaxonomic profile of microbes in samples. These two param-eters are affected by various factors that are discussed in thisguide, and contribute to microbial data variabili
13、ty.1.2 This guide addresses the unique considerations thatmust be accounted for in the design and execution of inter-laboratory studies intended to determine the precision ofmicrobiological test methods designed to quantify microbial1This test method is under the jurisdiction of ASTM Committee D02 o
14、nPetroleum Products, Liquid Fuels, and Lubricants and is the direct responsibility ofSubcommittee D02.14 on Stability and Cleanliness of Liquid Fuels.Current edition approved Dec. 1, 2012. Published January 2013. DOI: 10.1520/D7847-12E01.2Beckers, H. J., “Precision Testing of Standardized Microbiolo
15、gical Methods,”Journal of Testing and Evaluation, JTEVA, Vol. 14, No. 6, November 1986, pp.318-320.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1contamination in fuels, lubricants and similar low water-content (water activity 0.8) f
16、luids.1.3 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Refere
17、nced Documents2.1 ASTM Standards:3D156 Test Method for Saybolt Color of Petroleum Products(Saybolt Chromometer Method)D1129 Terminology Relating to WaterD4012 Test Method forAdenosine Triphosphate (ATP) Con-tent of Microorganisms in WaterD4175 Terminology Relating to Petroleum, PetroleumProducts, an
18、d LubricantsD6300 Practice for Determination of Precision and BiasData for Use in Test Methods for Petroleum Products andLubricantsD6469 Guide for Microbial Contamination in Fuels and FuelSystemsD6974 Practice for Enumeration of Viable Bacteria andFungi in Liquid FuelsFiltration and Culture Procedur
19、esD7463 Test Method forAdenosine Triphosphate (ATP) Con-tent of Microorganisms in Fuel, Fuel/Water Mixtures, andFuel Associated WaterD7464 Practice for Manual Sampling of Liquid Fuels, As-sociated Materials and Fuel System Components forMicrobiological TestingD7687 Test Method for Measurement of Cel
20、lular AdenosineTriphosphate in Fuel, Fuel/Water Mixtures, and Fuel-Associated Water with Sample Concentration by FiltrationE1259 Practice for Evaluation of Antimicrobials in LiquidFuels Boiling Below 390CE1326 Guide for Evaluating Nonconventional Microbiologi-cal Tests Used for Enumerating BacteriaE
21、1601 Practice for Conducting an Interlaboratory Study toEvaluate the Performance of an Analytical MethodE2756 Terminology Relating to Antimicrobial and AntiviralAgents2.2 Energy Institute Standard:4IP 385 Viable aerobic microbial content of fuels and fuelcomponents boiling below 90CFiltration and cu
22、lturemethod3. Terminology3.1 For definition of terms used in this guide refer toTerminologies D1129, D4175 and E2756, and Guide D6469.3.2 Definitions:3.2.1 free water, nwater in excess of that soluble in thesample and appearing in the sample as a haze or cloudiness, asdroplets, or as a separated pha
23、se or layer. D1563.2.2 specific concentration, nthe fraction of a cell con-stituent as determined on a per cell basis.3.2.2.1 DiscussionThe specific concentration can be ex-pressed as weight to weight, weight to volume or volume tovolume basis. Enzymes are commonly reported in terms oftheir activity
24、 relative to a reference standard.3.3 Acronyms:3.3.1 ATPadenosine triphosphate3.3.2 DNAdeoxyribonucleic acid3.3.3 ILSinterlaboratory study3.3.4 RNAribonucleic acid4. Determining Precision and Bias4.1 Bias Testing:4.1.1 There are no generally accepted reference standardsfor microbial cell constituent
25、s or for culture enumeration byviability test methods.4.1.2 Consequently, bias cannot be determined for non-culture methods.4.1.3 Data obtained from testing an accepted non-cultureparameter or culture method can be compared against dataobtained using a proposed new method.4.1.3.1 Such comparisons ar
26、e useful for benchmarkingnewly measure parameters against historically measure ones.4.1.3.2 Because bioburden is not a condition of state andbecause individual microbial parameters respond to sources ofvariation differently, comparison of a new methods test resultsagainst those of a preexisting meth
27、od cannot be used todetermine 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 aregenerally uniform throughout a well-mixed sample, microbesare discrete bodies that are dispersed in the med
28、ium.(2) In contrast to inanimate particles, microbes typicallyform aggregates in which individual cells are bound to oneanother within a polymeric matrix that is difficult to removewithout also damaging cells.(3) Microbes are similar to inanimate particles in that theirsettling rate within a medium
29、follows Stokes law.(4) Heterogeneous distribution of microbes within a me-dium is likely to be a significant source of variability relativeto other factors affecting test method repeatability.5(5) Microbes require free-water in order to be metaboli-cally active (see 1.2).(a) In a given fuel system,
30、microbial population densitiestend to be greatest at interfaces; particularly the fuel-water andfuel-system-surface interfaces.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information
31、, refer to the standards Document Summary page onthe ASTM website.4Available from Energy Institute, 61 New Cavendish St., London, WIG 7AR,U.K., http:/www.energyinst.org.uk.5Passman, F. J., English, E., Lindhardt, C., “Using Adenosine TriphosphateConcentration as a Measure of Fuel Treatment Microbici
32、de Performance,” Morris,R. E., Ed., Proceedings of the 10th International Conference on the Stability andHandling of Liquid Fuels, Oct. 7-11, 2007, Tucson, AZ. Available at .D7847 1212(b) Population densities within these interface zones arealso heterogeneous.(c) In order to minimize variability due
33、 to sampleheterogeneity, replicate samples should be recovered from asclose to the same locus as possible.4.2.1.2 Microbial Populations Physiological State:(1) The physiological state of a challenge population islargely dictated by physicochemical conditions, populationlifecycle stage in closed syst
34、ems, flow and shear in open andsemi-open systems, and the similarities between the challengedmicrocosm and source microcosm.(2) The specific concentration of many microbial cellconstituents varies in response to the physiological state of achallenge population.(3) Factors affecting the physiological
35、 state of a populationalso tend to affect the populations culturability.(4) Guidance provided in Practices D6300 and E1601minimize the impact of physiological state on repeatabilitystatistics.4.2.2 Reproducibility Testing:4.2.2.1 Microbiological parameters are very perishable.(1) Practice D7464 prov
36、ides guidance on the maximumacceptable delays between sample collection and test initiation.However, individual methods can specify acceptable condi-tions and delays between sampling and the initiation ofanalysis.(2) The history of a sample between time of collection andtest initiation can affect po
37、pulation densities and physiologicalstate substantially.(3) Differences in sample histories (4.2.2.1(2) can contrib-ute to variability that eclipses variability due to differences ininstrumentation, analytical technique or both.(4) Factors affecting the state of microbial populations insamples inclu
38、de, but are not limited to: temperature, oxygenavailability, chemical composition of sample medium, compo-sition of sample container, degree of ullage space.4.2.2.2 In order to minimize the potential contribution ofdisparate sample histories to reproducibility variability, it isadvisable to conduct
39、ILS either at a single location or at severalclosely located facilities.4.2.2.3 The ILS design should include detailed instructionsdesigned to minimize differences in sample histories betweenthe time that participant subsamples are prepared and testing isinitiated.5. Culture Methods5.1 Selecting Tes
40、t Organisms:5.1.1 Microbial Diversity:5.1.1.1 The number of different types of microbes recoveredfrom microbially contaminated fuel and fuel-associated watersis known to range from single to dozens of different taxa.5.1.1.2 Any given nutrient medium and set of growthconditions will select for a sub-
41、population of the total micro-bial population (5.2.1).5.1.1.3 Non-culture methods have identified the presence ofmicrobial contaminants that have yet to be cultivated ongrowth media.5.1.1.4 Depending on the methods scope, the appropriateoptions for precision testing include:(1) Single culture from t
42、ype culture collectionmost ap-propriate when the method is designed to detect a specificmicrobial taxon.(2) Mixed population of type collection culturesprovidesa basis for evaluating the recovery of microbes representing amore diverse population (Practice E1259).(3) Uncharacterized population obtain
43、ed from one or morecontaminated systemsmost closely reflects field conditions.(4) Commercially available uncharacterized mixed popula-tion of microbes known to metabolize fuel components (forexample: fats, oils and greases).(5) A commercially available population of microbes thatare capable of produ
44、cing a reliable signal detectable by theinstrument detector and will survive at least for 24 h in fuel(hydrocarbon) environment.(6) Field samples.NOTE 1No collection of contaminated fuels or fuels and fuel-associated waters is likely to be truly representative of microbial diversityin fuel systems.5
45、.1.2 Physiological State (4.2.1.2):5.1.2.1 When a challenge population is transferred from thesource medium to the test sample, it is likely that thepopulation will need to acclimate to its new physicochemicalenvironment.5.1.2.2 This acclimation period can be reducedbut nottotally eliminatedby ensur
46、ing that challenge populations arepre-acclimated to conditions by preculturing them in micro-cosms that are as similar as possible to the conditions of thesample that will be used for precision testing.NOTE 2During the acclimatization period microbes are likely toregain full metabolic activity in zo
47、nes in which free-water is present(4.2.1.1(5). If there is no free-water in the sample, microbes are likely tobecome metabolically dormant.5.1.3 Generation Time:5.1.3.1 Commonly, microbes with generation times 1 h areused for culture tests so that colonies are visible within 24 to 48h.5.2 Selecting
48、Culture Media:5.2.1 Given the physiological diversity of Eubacteria,Archeae, and Fungi, no single nutrient medium formulation orset of incubation conditions will support the proliferation of allcells in a challenge population.5.2.2 Consequently, a negative bias is assumed for allculture test methods
49、.5.2.2.1 It is generally accepted that only a small fraction ofmicrobial taxa have been cultured.5.2.2.2 There are no reference standards against which toquantify a culture methods bias (Guide E1326), consequently,only precision statistics can be developed for culture methods.5.2.3 Culture media selection is typically defined within amicrobiological test method to ensure that the test results areconsistent with the methods objectives (IP 385 and PracticeD6974).5.3 Separating Microbes from Sample:5.3.1 Sample carryover can interfere with cu
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