ASTM_D_6974_-_09_2013e2.pdf

Designation:D697409(Reapproved 2013)2Standard Practice forEnumeration of Viable Bacteria and Fungi in Liquid FuelsFiltration and Culture Procedures1This standard is issued under the fixed designation D6974;the number immediately following the designation indicates the year oforiginal adoption 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.1NOTESubsections 8.4 and 8.8.2 were editorially corrected in August 2014.2NOTEReferences in Section 9 and the title of IP 385 were editorially corrected in January 2015.1.Scope1.1 This practice covers a membrane filter(MF)procedurefor the detection and enumeration of Heterotrophic bacteria(HPC)and fungi in liquid fuels with kinematic viscosities24 mm2 s-1at ambient temperature.1.2 This quantitative practice is drawn largely from IPMethod 385 and Test Method D5259.1.3 This test may be performed either in the field or in thelaboratory.1.4 The ability of individual microbes to form colonies onspecific growth media depends on the taxonomy and physi-ological state of the microbes to be enumerated,the chemistryofthegrowthmedium,andincubationconditions.Consequently,test results should not be interpreted as absolutevalues.Rather they should be used as part of a diagnostic orcondition monitoring effort that includes other test parameters,in accordance with Guide D6469.1.5 This practice offers alternative options for deliveringfuel sample microbes to the filter membrane,volumes ordilutions filtered,growth media used to cultivate fuel-bornemicrobes,and incubation temperatures.This flexibility isoffered to facilitate diagnostic efforts.When this practice isused as part of a condition monitoring program,a singleprocedure should be used consistently.1.6 The values stated in SI units are to be regarded asstandard.No other units of measurement are included in thisstandard.1.7 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.Referenced Documents2.1 ASTM Standards:2D1129 Terminology Relating to WaterD1193 Specification for Reagent WaterD4175 Terminology Relating to Petroleum,PetroleumProducts,and LubricantsD5259 Test Method for Isolation and Enumeration of En-terococci from Water by the Membrane Filter ProcedureD6426 Test Method for Determining Filterability of MiddleDistillate Fuel OilsD6469 Guide for Microbial Contamination in Fuels and FuelSystemsD7463 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 TestingE1326 Guide for Evaluating Nonconventional Microbiologi-cal Tests Used for Enumerating BacteriaF1094 Test Methods for Microbiological Monitoring ofWater Used for Processing Electron and MicroelectronicDevices by Direct Pressure Tap Sampling Valve and bythe Presterilized Plastic Bag Method2.2 Energy Institute Standards:3IP 385 Determination of the Viable Aerobic Microbial Con-tent of Fuels and Fuel Components Boiling Below390CFiltration and Culture Method3.Terminology3.1 Definitions:3.1.1 For definition of terms used in this method refer toTerminologies D1129 and D4175,and Guide D6469.1This practice is under the jurisdiction of ASTM Committee D02 on PetroleumProducts,Liquid Fuels,and Lubricantsand is the direct responsibility of Subcom-mittee D02.14 on Stability and Cleanliness of Liquid Fuels.Current edition approved May 1,2013.Published August 2013.Originallyapproved in 2003.Last previous edition approved in 2009 as D6974 09.DOI:10.1520/D6974-09R13E02.2For 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 Summary page onthe ASTM website.3Available from Energy Institute,61 New Cavendish St.,London,WIG 7AR,U.K.,http:/www.energyinst.org.uk.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 3.1.2 aseptic,adjsterile,free from viable microbiologicalcontamination.3.2 Acronyms:3.2.1 CFUcolony forming unit3.2.2 HPCheterotrophic plate count3.2.3 MFmembrane filter3.2.4 MEAmalt extract agar3.2.5 TNTCtoo numerous to count3.2.6 TSAtryptone soy agar3.3 Symbols:3.3.1 Nnumber of CFU L-13.3.2 CCnumber of colonies on membrane filter3.3.3 Vsample volume filtered,mL4.Summary of Practice4.1 Any free water present in a fuel sample is removed bysettling in a separatory funnel.After the water has beenremoved,a known volume of the remaining fuel is filteredthrough a membrane filter aseptically by one of three methods.4.2 The filter membra