ASTM D6974 - 09(2013)

    Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels—Filtration and Culture Procedures

    Active Standard ASTM D6974 | Developed by Subcommittee: D02.14

    Book of Standards Volume: 05.04


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    ASTM D6974

    Significance and Use

    5.1 Biodeteriogenic microbes infecting fuel systems typically are most abundant within slime accumulations on system surfaces or at the fuel-water interface (Guide D6469). However, it is often impractical to obtain samples from these locations within fuel systems. Although the numbers of viable bacteria and fungi recovered from fuel-phase samples are likely to be several orders of magnitude smaller than those found in water-phase samples, fuel-phase organisms are often the most readily available indicators of fuel and fuel system microbial contamination.

    5.2 Growth Medium Selectivity—Guide E1326 discusses the limitations of growth medium selection. Any medium selected will favor colony formation by some species and suppress colony formation by others. As noted in 6.3, physical, chemical and physiological variables can affect viable cell enumeration test results. Test Method D7463 provides a non-culture means of quantifying microbial biomass in fuels and fuel associated water.

    5.3 Since a wide range of sample sizes, or dilutions thereof, can be analyzed by the membrane filter technique (Test Methods D5259 and F1094), the test sensitivity can be adjusted for the population density expected in the sample.

    5.4 Enumeration data should be used as part of diagnostic efforts or routine condition monitoring programs. Enumeration data should not be used as fuel quality criteria.

    1. Scope

    1.1 This practice covers a membrane filter (MF) procedure for the detection and enumeration of Heterotrophic bacteria (HPC) and fungi in liquid fuels with kinematic viscosities 24 mm2 · s-1 at ambient temperature.

    1.2 This quantitative practice is drawn largely from IP Method 385 and Test Method D5259.

    1.3 This test may be performed either in the field or in the laboratory.

    1.4 The ability of individual microbes to form colonies on specific growth media depends on the taxonomy and physiological state of the microbes to be enumerated, the chemistry of the growth medium, and incubation conditions. Consequently, test results should not be interpreted as absolute values. Rather they should be used as part of a diagnostic or condition monitoring effort that includes other test parameters, in accordance with Guide D6469.

    1.5 This practice offers alternative options for delivering fuel sample microbes to the filter membrane, volumes or dilutions filtered, growth media used to cultivate fuel-borne microbes, and incubation temperatures. This flexibility is offered to facilitate diagnostic efforts. When this practice is used as part of a condition monitoring program, a single procedure should be used consistently.

    1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.

    1.7 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 standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.


    2. Referenced Documents (purchase separately) The documents listed below are referenced within the subject standard but are not provided as part of the standard.

    ASTM Standards

    D1129 Terminology Relating to Water

    D1193 Specification for Reagent Water

    D4175 Terminology Relating to Petroleum, Petroleum Products, and Lubricants

    D5259 Test Method for Isolation and Enumeration of Enterococci from Water by the Membrane Filter Procedure

    D6426 Test Method for Determining Filterability of Middle Distillate Fuel Oils

    D6469 Guide for Microbial Contamination in Fuels and Fuel Systems

    D7463 Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Fuel, Fuel/Water Mixtures and Fuel Associated Water

    D7464 Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological Testing

    E1326 Guide for Evaluating Nonconventional Microbiological Tests Used for Enumerating Bacteria

    F1094 Test Methods for Microbiological Monitoring of Water Used for Processing Electron and Microelectronic Devices by Direct Pressure Tap Sampling Valve and by the Presterilized Plastic Bag Method

    Energy Institute Standards

    IP385 Viable aerobic microbial content of fuels and fuel components boiling below 90C--Filtration and culture method


    ICS Code

    ICS Number Code 75.160.20 (Liquid fuels)

    UNSPSC Code

    UNSPSC Code 15101500(Petroleum and distillates)


    DOI: 10.1520/D6974

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