1、Designation: D7847 12Standard 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. A
2、 number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.INTRODUCTIONMicrobiological parameters present a number of unique challenges relative to chemical and physicaltest methods apropos of the developm
3、ent 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 balloted at the D02.14 subcommitteelevel in February and June 2002, were balloted
4、 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 us to change the titlefrom Method to Practice. The argument was that ASTM Me
5、thods 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 culturable populationdensity) and different growth media (based on the sub-po
6、pulation 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 microbiological Methods have been approved by ASTM:Method D7463 and Method D7687.
7、 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.Because these methods measure the concentration of a biomarker molecule, th
8、e 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 eitherimpractical for or not relevant to the methods that have been developed within D02:
9、 (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 guide will list key issues that must be addressed when designing ILS for Met
10、hods 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 proliferate, die off and continue to be metabolicallyactive in samples after th
11、ose 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 morecomplicated by the general absence of available water fromthese fluids.1.1.2
12、 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 variability.1.2 This guide addresses the unique considerations thanmust be accounted
13、for in the design and execution of inter-laboratory studies intended to determine the precision ofmicrobiological test methods designed to quantify microbialcontamination in fuels, lubricants and similar low water-content (water activity 0.8) fluids.1This test method is under the jurisdiction of AST
14、M Committee D02 onPetroleum Products and Lubricants and is the direct responsibility of SubcommitteeD02.14 on Stability and Cleanliness of Liquid Fuels.Current edition approved Dec. 1, 2012. Published January 2013. DOI: 10.1520/D7847-12.2Beckers, H. J., “Precision Testing of Standardized Microbiolog
15、ical 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 States11.3 This standard does not purport to address all of thesafety concerns, if any, associa
16、ted 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. Referenced Documents2.1 ASTM Standards:3D156 Test Method for Saybolt Color of Petroleum Products(Say
17、bolt Chromometer Method)D1129 Terminology Relating to WaterD4012 Test Method forAdenosine Triphosphate (ATP) Con-tent of Microorganisms in WaterD4175 Terminology Relating to Petroleum, PetroleumProducts, and LubricantsD6300 Practice for Determination of Precision and BiasData for Use in Test Methods
18、 for Petroleum Products andLubricantsD6469 Guide for Microbial Contamination in Fuels and FuelSystemsD6974 Practice for Enumeration of Viable Bacteria andFungi in Liquid FuelsFiltration and Culture ProceduresD7463 Test Method forAdenosine Triphosphate (ATP) Con-tent of Microorganisms in Fuel, Fuel/W
19、ater 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 Cellular AdenosineTriphosphate in Fuel, Fuel/Water Mixtures, and Fuel-Associated Water with Sample
20、 Concentration by FiltrationE1259 Practice for Evaluation of Antimicrobials in LiquidFuels Boiling Below 390CE1326 Guide for Evaluating Nonconventional Microbiologi-cal Tests Used for Enumerating BacteriaE1601 Practice for Conducting an Interlaboratory Study toEvaluate the Performance of an Analytic
21、al 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 culturemethod3. Terminology3.1 For definition of terms used in this guide refer toTerminologies D
22、1129, 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 phase or layer. D1563.2.2 specific concentration, nthe fraction of a cell con-stituent as determin
23、ed 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 relative to a reference standard.3.3 Acronyms:3.3.1 ATPadenosine triphosphate3.3.2 DNAdeoxyrib
24、onucleic 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 constituents or for culture enumeration byviability test methods.4.1.2 Consequently, bias cannot be determ
25、ined 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 are useful for benchmarkingnewly measure parameters against historically measure ones.4.1.3.2 Bec
26、ause 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 method cannot be used todetermine the bias of either method.4.2 Precision Testing:4.2.1 Repeatabili
27、ty 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 medium.(2) In contrast to inanimate particles, microbes typicallyform aggregates in which individu
28、al 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 follows Stokes law. Is this stillthe case with biodiesels with entrained water?(4) Heterogeneou
29、s 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 4.2.1.2).(1) In a given fuel system, microbial population densitiest
30、end to be greatest at interfaces; particularly the fuel-water andfuel-system-surface interfaces.(2) Population densities within these interface zones arealso heterogeneous.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For An
31、nual Book of ASTMStandards volume information, 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 TriphosphateConcentra
32、tion as a Measure of Fuel Treatment Microbicide 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 122(3) In order to minimize variability due to sampleheterogeneity, replica
33、te samples should be recovered from asclose to the same locus (location?) 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 systems, flow and shear
34、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 state of a populati
35、onalso 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 provides guidance on the
36、 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 population densities a
37、nd 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 include, but are not limi
38、ted 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 ILS either at a sing
39、le 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 Test Organisms:5.1.1 Mi
40、crobial 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-population of the to
41、tal 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 type culture collecti
42、onmost 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 obtained from one or morec
43、ontaminated 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 producing a reliable sign
44、al 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.5.1.2 Physiological S
45、tate (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 ensuring that challenge p
46、opulations 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 zones in which free-wa
47、ter 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 Culture Media:5.2.1
48、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.5.2.2.1 It is gener
49、ally 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 culturability.5.3.1.1 Nutr
