1、Designation: D7978 14Standard Test Method forDetermination of the Viable Aerobic Microbial Content ofFuels and Associated WaterThixotropic Gel CultureMethod1This standard is issued under the fixed designation D7978; the number immediately following the designation indicates the year oforiginal adopt
2、ion or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method describes a procedure that can be usedin the field or in
3、 a laboratory to quantify culturable, viableaerobic microorganisms present as contaminants in liquidfuels, including those blended with synthesized hydrocarbonsor biofuels, with kinematic viscosities (at 40 C) of 24 mm2s-1and heavy and residual fuels with kinematic viscosities (at40 C) of 700 mm2s-1
4、and in fuel-associated water.1.1.1 This test method has been validated by an ILS for arange of middle distillate fuels meeting Specifications D975,D1655, ISO 8217 DMA, and NATO F-76.21.2 This test method quantitatively assesses culturable,viable aerobic microbial content present in the form ofbacter
5、ia, fungi, and fungal spores. Results are expressed as thetotal number of microbial colony forming units (CFU)/L offuel or total number of CFU/mL of associated water. Thenumber of CFU should not be interpreted as absolute valuesbut should be used as part of a diagnostic or conditionmonitoring effort
6、; for example, these values can be used toassess contamination as absent, light, moderate, or heavy.NOTE 1This test method is technically equivalent to IP 613, althoughthe two methods are not currently jointed.1.3 The values stated in SI units are to be regarded asstandard. No other units of measure
7、ment are included in thisstandard.1.4 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 limita
8、tions prior to use.2. Referenced Documents2.1 ASTM Standards:3D975 Specification for Diesel Fuel OilsD1129 Terminology Relating to WaterD1655 Specification for Aviation Turbine FuelsD4175 Terminology Relating to Petroleum, PetroleumProducts, and LubricantsD4176 Test Method for Free Water and Particu
9、late Contami-nation in Distillate Fuels (Visual Inspection Procedures)D6469 Guide for Microbial Contamination in Fuels and FuelSystemsD6974 Practice for Enumeration of Viable Bacteria andFungi in Liquid FuelsFiltration and Culture ProceduresD7464 Practice for Manual Sampling of Liquid Fuels, As-soci
10、ated Materials and Fuel System Components forMicrobiological TestingD7847 Guide for Interlaboratory Studies for MicrobiologicalTest MethodsE2756 Terminology Relating to Antimicrobial and AntiviralAgents2.2 Energy Institute Standards:4IP 385 Determination of the Viable Aerobic Microbial Con-tent of F
11、uels and Fuel Components Boiling Below 390CFiltration and Culture MethodIP 613 Determination of the Viable Aerobic Microbial Con-tent of Fuels and Associated WaterThixotropic GelCulture MethodGuidelines for the Investigation of the Microbial Content ofPetroleum Fuels and for the Implementation of Av
12、oidanceand Remedial Strategies, 2nd Edition, 2008, EnergyInstitute, London, ISBN 978 0 85293 524 82.3 Other Standards:ISO 8217 DMA Petroleum Products, Fuels (Class F), Speci-fications of Marine Fuels, 4th Edition 2010, ISO Switzer-land1This test method is under the jurisdiction of ASTM Committee D02
13、 onPetroleum Products, Liquid Fuels, and Lubricants and is the direct responsibility ofSubcommittee D02.14 on Stability and Cleanliness of Liquid Fuels.Current edition approved Dec. 15, 2014. Published February 2015. DOI:10.1520/D7978-14.2Defense Standard 91-4, Fuel, Naval, Distillate (NATO Code: F-
14、76, JointService designation DIESO F-76), Issue 9, 3 May 2013, UK Defense Standardiza-tion3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Sum
15、mary page onthe ASTM website.4Available from Energy Institute, 61 New Cavendish St., London, WIG 7AR,U.K., http:/www.energyinst.org.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1IATA Guidance Material on Microbiological Contaminatio
16、nin Aircraft Fuel Tanks, 4th Edition, December 201153. Terminology3.1 DefinitionsFor definitions of terms used in this testmethod, refer to Terminologies D1129, D4175, and E2756.3.2 Definitions:3.2.1 colony, na discreet visible aggregate of microorgan-isms that develops when a viable microorganism,
17、or particlecontaining viable microorganisms, is introduced into a gel-based nutritive culture medium and reproduces there.3.2.1.1 DiscussionA period of incubation is necessary toallow sufficient reproduction. This test method utilizes areactive compound that shortens the time for colonies tobecome v
18、isible and stains them so that they appear as red orpurple spots.3.2.1.2 DiscussionTypically, bacterial colonies becomevisible to the naked eye only after the colony contains 109individual cells. Consequently, the time required for a colonyto become visible is dependent on the organisms generation(d
19、oubling), which can range from 1 week.3.2.2 culture medium, nsolid, semi-solid, or liquid prepa-rations that contain nutrients that support microbial growth,and usually other chemical agents that can inhibit or stimulategrowth by specific microorganisms or that can indicate thepresence of all cultur
20、able or specific microorganisms.3.3 Definitions of Terms Specific to This Standard:3.3.1 thixotropic gel, na sheer thinning gel that is semi-solid while static and becomes a liquid when a sheer force isapplied.3.4 Acronyms and Abbreviations:3.4.1 CCnumber of colonies (colony count).3.4.2 CFUcolony f
21、orming unit.3.4.3 Nnumber of CFU/mL (in water) or CFU/L (in fuel).3.4.4 TNTCtoo numerous to count.3.4.5 Vvolume tested, mL4. Summary of Test Method4.1 A known volume of fuel or water is added to the testkit6, which consists of a rectangular, transparent glass bottlecontaining a patented sterile, thi
22、xotropic gel-based culturemedium capable of sustaining the growth of a wide range ofmicroorganisms encountered in liquid fuels and petroleumproducts (8.1).4.2 The gel liquefies when the bottle is shaken, dispersingthe fuel or water specimen containing any microorganisms.4.3 The gel is allowed to res
23、et into a flat layer on one of thelarger sides of the bottle.4.4 The bottle is incubated in the dark in this position forfour days. The gel contains components that sustain the growthof viable, culturable microorganisms and the fuel specimenitself contributes additional nutrients.4.5 Viable, cultura
24、ble microorganisms in the volume of fuelor water tested grow into visible colonies, and a reactivecompound changes the color of these colonies to red or purplesuch that they can be easily counted or their number estimated.4.6 The number of colonies formed is considered in relationto the volume of sp
25、ecimen added to the test, and expressed asCFU/L of fuel, or CFU/mL if the result relates to a test of waterin a fuel system sample.5. Significance and Use5.1 This test method is intended to provide a tool forassessing whether fuel storage and distribution facilities or enduser fuel tanks are subject
26、 to microbial growth and alert fuelsuppliers or users to the potential for fuel quality or operationalproblems and/or the requirement for preventative or remedialmeasures.5.2 This test method detects numbers of microbial colonyforming units (CFU), the same detection parameter used in thelaboratory s
27、tandard procedures Practice D6974 and IP 385.However, whereas Practice D6974 and IP 385 provide separateassessment of numbers of viable aerobic bacteria CFU andnumbers of viable fungal CFU, this test method provides acombined total count of viable aerobic bacteria and fungalCFU.5.3 This test method
28、is designed to detect a recognizedgroup of microorganisms of significance in relation to contami-nation of distillate fuels, but it is recognized that microbiologi-cal culture techniques do not detect all microorganisms that canbe present in a sample. Culturability is affected primarily bythe abilit
29、y of captured microbes to proliferate on the growthmedium provided, under specific growth conditions.Consequently, a proportion of the active or inactive microbialpopulation present in a sample can be viable but not detectedby any one culture test.7In this respect, the test is indicative ofthe exten
30、t of microbial contamination in a sample ,and it isassumed that when a fuel sample is significantly contaminated,some of the dominant microbial species present will bequantifiably detected, even if not all species present areculturable.5.4 Many samples from fuel systems can be expected tocontain a l
31、ow level of “background” microbial contamination,which is not necessarily of operational significance. Theminimum detection level of this test method is determined bythe volume of specimen tested and is set such that microbialcontamination will generally only be detected when it is atlevels indicati
32、ve of active proliferation.5.5 The test will detect culturable bacteria and fungi that aremetabolically active and dormant fungal spores. Presence of5Available from International Air Transport Assocation (IATA), 800 PlaceVictoria, PO Box 113, Montreal H4Z 1M1, Quebec, Canada or 33, Route delAeroport
33、, PO Box 416, 1215 Geneva 15Airport, Switzerland, http:/www.iata.org.6The sole source of supply of the test kit known to the committee at this time isECHA Microbiology Ltd., Cardiff, CF3 0EF, UK. If you are aware of alternativesuppliers, please provide this information to ASTM International Headquar
34、ters.Your comments will receive careful consideration at a meeting of the responsibletechnical committee,1which you may attend.7White, J. et al., “Culture-IndependentAnalysis of Bacterial Fuel ContaminationProvides Insight into the Level of Concordance with the Standard Industry Practiceof Aerobic C
35、ultivation,” Applied and Environmental Microbiology, Vol. 77, No. 13,July 2011, pp. 4527-4538.D7978 142fungal spores in a fuel sample can be indicative of activemicrobial proliferation within a fuel tank or system, but at apoint distant from the location sampled. Active microbialgrowth only occurs i
36、n free water, and this can be present onlyas isolated pockets at tank or system low points. Becausefungal spores are more hydrophobic than active cells andfungal material (mycelium), they disperse more readily in fuelphase and are thus more readily detected when low pointscannot be directly sampled
37、and only fuel phase is present insamples.5.6 This test method can determine whether microbialcontamination in samples drawn from fuel tanks and systems isabsent or present at light, moderate, and heavy levels.5.7 The categorization of light, moderate, and heavy levelsof contamination will depend on
38、the fuel type, the samplinglocation, the facility sampled, and its specific operating cir-cumstances.5.8 Further guidance or interpretation of test results can befound in Guide D6469, in the Energy Institute Guidelines forthe investigation of the microbial content of petroleum fuels,and for the impl
39、ementation of avoidance and remedial strate-gies and in the IATA Guidance Material on MicrobiologicalContamination in Aircraft Fuel Tanks.5.8.1 Further guidance on sampling can be found in PracticeD7464.5.9 Testing can be conducted on a routine basis or toinvestigate incidents.5.10 Microbiological t
40、ests are not intended to be used todetermine compliance with absolute fuel specifications orlimits. The implementation of specification limits for micro-biological contamination in fuels is generally not appropriate,and microbial contamination levels cannot be used alone ordirectly to make inference
41、s about fuel quality or fitness for use.5.11 When interpreting results, it must be appreciated thatthe test result applies only to the specific sample and specimentested and not necessarily to the bulk fuel. Microbiologicalcontamination usually shows a highly heterogeneous distribu-tion in fuel syst
42、ems, and therefore, analysis of a single samplewill rarely provide a complete assessment of the overall levelsof contamination present.5.12 Water phase will usually contain substantially highernumbers of microbial CFU than fuel phase and, consequently,a different interpretation of results is require
43、d.6. Interferences6.1 Some antioxidant additives that can be present in thefuel being tested can cause a uniform light peach or orangecolor in the gel culture medium (usually within 12 h). Thiscolor change will not interfere with the growth of anymicroorganisms, and in most cases, microbial colonies
44、 can becounted or estimated ignoring the background color. If anti-oxidants are present at very high concentration, the colorchange in the gel culture medium can be so strong that userscan find it difficult to distinguish from the appearance of a testin which more than 10 000 microbial colonies have
45、 grown. Theeffect can be compensated for by testing a smaller volume ofspecimen, as described in 12.5.2.3 in the test procedure.6.2 Some bacteria are motile and can, on prolongedincubation, spread through the gel culture medium, producinglarge irregularly-shaped colonies, streaks, or patches of red
46、orpurple color that are difficult to count. Procedures for compen-sating for this effect are described in 12.5.2.4 in the testprocedure.6.3 If microorganisms other than those in the specimen areintroduced into the culture medium, they can give rise tospurious colonies. To avoid this, the test should
47、 be conductedin as clean an environment as practicable, and care should betaken to avoid touching surfaces of dispensing apparatus,pipettes, and sample containers that come into direct contactwith the sample or culture medium.7. Apparatus7.1 Incubator, capable of operating at 25 C 6 3 C. Thedesign o
48、f the incubator shall ensure the culture medium is notexposed to light during incubation. The use of an incubator isnot essential (see 12.5.1.1).7.2 Temperature Measuring Device, accurate to 62 C, tomeasure incubation temperature.8. Reagents and Materials8.1 MicrobMonitor2,8commercially available te
49、st kit com-prised of a rectangular, sterile transparent glass bottle fittedwith a cap and seal and containing a sterile, thixotropicgel-based culture medium capable of sustaining the growth ofa wide range of microorganisms encountered in liquid fuelsand petroleum products and associated water.8.2 Disposable Syringe, sterile and fuel compatible, capac-ity 1 mL graduated in 0.1 mL units used for dispensing therecommended volume of middle distillate fuels described in12.3.4.8.3 Disposable Loop Dispenser, sterile and fuel compatible,capacity 0.01 mL, used for
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