ASTM WK65790

    New Practice for Quantification of Microbial Contamination in Liquid Fuels and Fuel-Associated Water by Quantitative Polymerase Chain Reaction (qPCR)

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    Developed by Subcommittee: D02.14 | Committee D02 | Contact Staff Manager


    1. Scope

    1.1 This practice provides a procedure for using genomic tools to quantify microbial genomic deoxyribonucleic acid (DNA) in fuel and fuel-associated water samples. 1.1.1 Water samples that may be used in testing include, but are not limited to, water associated with crude oil or liquid fuels in storage tanks, fuel tanks, or pipelines. 1.1.2 While the intent of this recommended practice is to focus on the analysis of fuel-associated samples the procedures described here are also relevant to the analysis of water used in hydrotesting of pipes and equipment, water injected into geological formations to maintain pressure or facilitate the recovery of hydrocarbons in oil and gas recovery or both, water co-produced during the production of oil and gas, water in fire protection sprinkler systems, potable water, industrial process water, and wastewater. 1.1.3 Fuel samples can also be tested by separation of the microorganism from the fuel phase using various methods such as microbial filtration, microbial liquid-liquid based extractions, and other microbial-capturing methods. 1.2 This practice entails the application of quantitative polymerase chain reaction (qPCR) technology to determine total bioburden or total microbial population present in fuel-associated samples using universal primers that allow for the quantification of 16S and 18S ribosomal RNA genes that are present in all prokaryotes (that is, bacteria and archaea) and fungi, respectively. 1.3 This practice is a laboratory protocol. As described in Practice D7464, the qualitative and quantitative relationship between the laboratory results and actual microbial communities in the systems from which samples are collected are affected by the time delay and handling conditions between the time of sampling and time that testing is initiated. 1.4 Units--The values stated in SI units are to be regarded as the standard. No other units of measurement are included in this standard with the exception of the concept unit of gene copies/mL (that is,S or 18S gene copies/mL to indicate the starting concentration of microbial DNA for the intended microbial targets that is, bacteria, archaea, and fungi). 1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this practice to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. 1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.


    fuel; microorganisms; microbiology, bacteria; fungi; archaea; quantitative polymerase chain reaction; qPCR; DNA; nucleic acids; genomics; metagenomics


    This standard will provide an accurate method to detect and quantify microorganisms in fuel through the analysis of microbial DNA signatures using the quantitative PCR technique. The method will provide a simplified qPCR and sample preparation procedure to facilitate its use and application by users with minimal training.

    The title and scope are in draft form and are under development within this ASTM Committee.

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    Work Item Status

    Date Initiated:

    Technical Contact:
    Oscar Ruiz


    D02.14 (21-03)

    Withdrawn From Balloting


    D02 (21-09)

    In Balloting