ASTM_D_8412_-_21.pdf

Designation:D841221Standard Guide forQuantification of Microbial Contamination in Liquid Fuelsand Fuel-Associated Water by Quantitative PolymeraseChain Reaction(qPCR)1This standard is issued under the fixed designation D8412;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.1.Scope1.1 This guide covers procedures for using quantitativepolymerase chain reaction(qPCR),a genomic tool,to detect,characterize and quantify nucleic acids associated with micro-bial DNA present in liquid fuels and fuel-associated watersamples.1.1.1 Water samples that may be used in testing include,butare not limited to,water associated with crude oil or liquidfuels in storage tanks,fuel tanks,or pipelines.1.1.2 While the intent of this guide is to focus on theanalysis of fuel-associated samples,the procedures describedhere are also relevant to the analysis of water used inhydrotesting of pipes and equipment,water injected intogeological formations to maintain pressure and/or facilitate therecovery of hydrocarbons in oil and gas recovery,waterco-produced during the production of oil and gas,water in fireprotection sprinkler systems,potable water,industrial processwater,and wastewater.1.1.3 To test a fuel sample,the live and recently deadmicroorganisms must be separated from the fuel phase whichcan include any DNA fragments by using one of variousmethods such as filtration or any other microbial capturingmethods.1.1.4 Some of the protocol steps are universally requiredand are indicated by the use of the word must.Other protocolsteps are testing-objective dependent.At those process steps,options are offered and the basis for choosing among them areexplained.1.2The guide describes the application of quantitativepolymerase chain reaction(qPCR)technology to determinetotal bioburden or total microbial population present in fuel-associated samples using universal primers that allow for thequantification of 16S and 18S ribosomal RNA genes that arepresent in all prokaryotes(that is,bacteria and archaea)andeucaryotes(that is,mold and yeast collectively termed fungi),respectively.1.3 This guide describes laboratory protocols.As describedin Practice D7464,the qualitative and quantitative relationshipbetween the laboratory results and actual microbial communi-ties in the systems from which samples are collected is affectedby the time delay and handling conditions between the time ofsampling and time that testing is initiated.1.4 The values stated in SI units are to be regarded asstandard.No other units of measurement are included in thisstandard with the exception of the concept unit of genecopies/mL(that is,16S or 18S gene copies/mL)to indicate thestarting concentration of microbial DNA for the intendedmicrobial targets(that is,bacteria,archaea,fungi).1.5 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,health,and environmental practices and deter-mine the applicability of regulatory limitations prior to use.1.6 This international standard was developed in accor-dance with internationally recognized principles on standard-ization established in the Decision on Principles for theDevelopment of International Standards,Guides and Recom-mendations issued by the World Trade Organization TechnicalBarriers to Trade(TBT)Committee.2.Referenced Documents2.1 ASTM Standards:2D1129 Terminology Relating to WaterD4175 Terminology Relating to Petroleum Products,LiquidFuels,and LubricantsD6469 Guide for Microbial Contamination in Fuels and FuelSystemsD6974 Practice for Enumeration of Viable Bacteria and1This guide is under the jurisdiction of ASTM Committee D02 on PetroleumProducts,Liquid Fuels,and Lubricants and is the direct responsibility of Subcom-mittee D02.14 on Stability,Cleanliness and Compatibility of Liquid Fuels.Current edition approved Dec.1,2021.Published January 2022.DOI:10.1520/D8412-21.2For 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.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards,Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade(TBT)Committee.1?Fungi in Liquid FuelsFiltration and Culture ProceduresD7464 Practice for Manual Sampling of Liquid Fuels,As-sociated Materials and Fuel System Comp