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ASTM D8412-21

(Guide)

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

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

SIGNIFICANCE AND USE
5.1 This guide provides a protocol for detecting, characterizing, and quantifying nucleic acids (that is, DNA) of living and recently dead microorganisms in fuels and fuel-associated waters by means of a culture independent qPCR procedure. Microbial contamination is inferred when elevated DNA levels are detected in comparison to the expected background DNA level of a clean fuel and fuel system.  
5.2 A sequence of protocol steps is required for successful qPCR testing.  
5.2.1 Quantitative detection of microorganisms depends on the DNA-extraction protocol and selection of appropriate oligonucleotide primers.  
5.2.2 The preferred DNA extraction protocol depends on the type of microorganism present in the sample and potential impurities that could interfere with the subsequent qPCR reaction.  
5.2.3 Primers vary in their specificity. Some 16S and 18S RNA gene regions present in the DNA of prokaryotic and eukaryotic microorganisms appear to have been conserved throughout evolution and thus provide a reliable and repeatable target for gene amplification and detection. Amplicons targeting these conserved nucleotide sequences are useful for quantifying total population densities. Other target DNA regions are specific to a metabolic class (for example, sulfate reducing bacteria) or individual taxon (for example, the bacterial species Pseudomonas aeruginosa). Primers targeting these unique nucleotide sequences are useful for detecting and quantifying specific microbes or groups of microbes known to be associated with biodeterioration.  
5.3 Just as the quantification of microorganisms using microbial growth media employs standardized formulations of growth conditions enabling the meaningful comparison of data from different laboratories (Practice D6974), this guide seeks to provide standardization to detect, characterize, and quantify nucleic acids associated with living and recently dead microorganisms in fuel-associated samples using qPCR.
Note 3: Many primers, an...
SCOPE
1.1 This guide covers procedures for using quantitative polymerase chain reaction (qPCR), a genomic tool, to detect, characterize and quantify nucleic acids associated with microbial DNA present in liquid fuels 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 guide 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 and/or facilitate the recovery of hydrocarbons in oil and gas recovery, 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 To test a fuel sample, the live and recently dead microorganisms must be separated from the fuel phase which can include any DNA fragments by using one of various methods such as filtration or any other microbial capturing methods.  
1.1.4 Some of the protocol steps are universally required and are indicated by the use of the word must. Other protocol steps are testing-objective dependent. At those process steps, options are offered and the basis for choosing among them are explained.  
1.2 The guide describes 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 eucaryotes (that is, mold and yeast collectively termed fungi), respectively.  
1.3 This guide describes laboratory protocols. As described in Practice D7464, the...

General Information

Status
Published
Publication Date
30-Nov-2021
ICS
07.100.01 - Microbiology in general
Technical Committee
D02 - Petroleum Products, Liquid Fuels, and Lubricants
Drafting Committee
D02.14 - Stability, Cleanliness and Compatibility of Liquid Fuels
Current Stage
Ref Project

Relations

Refers
ASTM D6974-20 - Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels—Filtration and Culture Procedures
Effective Date
01-May-2020
Refers
ASTM D7464-20 - Standard Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological Testing
Effective Date
01-May-2020

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ASTM D8412-21 - Standard Guide for Quantification of Microbial Contamination in Liquid Fuels and Fuel-Associated Water by Quantitative Polymerase Chain Reaction (qPCR)ASTM D8412-21 - Standard Guide for Quantification of Microbial Contamination in Liquid Fuels and Fuel-Associated Water by Quantitative Polymerase Chain Reaction (qPCR)
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ASTM D8412-21 - Standard Guide for Quantification of Microbial Contamination in Liquid Fuels and Fuel-Associated Water by Quantitative Polymerase Chain Reaction (qPCR)
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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.
Designation:D8412 −21
Standard Guide for
Quantification of Microbial Contamination in Liquid Fuels
and Fuel-Associated Water by Quantitative Polymerase
1
Chain Reaction (qPCR)
This standard is issued under the fixed designation D8412; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope present in all prokaryotes (that is, bacteria and archaea) and
eucaryotes (that is, mold and yeast collectively termed fungi),
1.1 This guide covers procedures for using quantitative
respectively.
polymerase chain reaction (qPCR), a genomic tool, to detect,
1.3 This guide describes laboratory protocols.As described
characterize and quantify nucleic acids associated with micro-
in Practice D7464, the qualitative and quantitative relationship
bial DNA present in liquid fuels and fuel-associated water
between the laboratory results and actual microbial communi-
samples.
tiesinthesystemsfromwhichsamplesarecollectedisaffected
1.1.1 Watersamplesthatmaybeusedintestinginclude,but
by the time delay and handling conditions between the time of
are not limited to, water associated with crude oil or liquid
sampling and time that testing is initiated.
fuels in storage tanks, fuel tanks, or pipelines.
1.4 The values stated in SI units are to be regarded as
1.1.2 While the intent of this guide is to focus on the
standard. No other units of measurement are included in this
analysis of fuel-associated samples, the procedures described
standard with the exception of the concept unit of gene
here are also relevant to the analysis of water used in
copies/mL(that is, 16S or 18S gene copies/mL) to indicate the
hydrotesting of pipes and equipment, water injected into
starting concentration of microbial DNA for the intended
geologicalformationstomaintainpressureand/orfacilitatethe
microbial targets (that is, bacteria, archaea, fungi).
recovery of hydrocarbons in oil and gas recovery, water
1.5 This standard does not purport to address all of the
co-produced during the production of oil and gas, water in fire
protection sprinkler systems, potable water, industrial process safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
water, and wastewater.
priate safety, health, and environmental practices and deter-
1.1.3 To test a fuel sample, the live and recently dead
mine the applicability of regulatory limitations prior to use.
microorganisms must be separated from the fuel phase which
1.6 This international standard was developed in accor-
can include any DNA fragments by using one of various
dance with internationally recognized principles on standard-
methods such as filtration or any other microbial capturing
ization established in the Decision on Principles for the
methods.
Development of International Standards, Guides and Recom-
1.1.4 Some of the protocol steps are universally required
mendations issued by the World Trade Organization Technical
and are indicated by the use of the word must. Other protocol
Barriers to Trade (TBT) Committee.
steps are testing-objective dependent. At those process steps,
options are offered and the basis for choosing among them are
2. Referenced Documents
explained.
2
2.1 ASTM Standards:
1.2 The guide describes the application of quantitative
D1129Terminology Relating to Water
polymerase chain reaction (qPCR) technology to determine
D4175Terminology Relating to Petroleum Products, Liquid
total bioburden or total microbial population present in fuel-
Fuels, and Lubricants
associated samples using universal primers that allow for the
D6469GuideforMicrobialContaminationinFuelsandFuel
quantification of 16S and 18S ribosomal RNA genes that are
Systems
D6974Practice for Enumeration of Viable Bacteria and
1
This guide is under the jurisdiction of ASTM Committee D02 on Petroleum
2
Products, Liquid Fuels, and Lubricants and is the direct responsibility of Subcom- For referenced ASTM standards, visit the ASTM website, www.astm.org, or
mittee D02.14 on Stability, Cleanliness and Compatibility of Liquid Fuels. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Current edition approved Dec. 1, 2021. Published January 2022. DOI: 10.1520/ Standards volume information, refer to the standard’s Document Summary page on
D8412-21. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

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5.1 This practice is to help in the development of protocols to assess the survival, removal and/or inactivation of human pathogens or their surrogates in indoor air. It accommodates the testing of technologies based on physical (for example, UV light) and chemical agents (for example, vaporized hydrogen peroxide) or simple microbial removal by air filtration or a combination thereof.  
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1.1 This practice is to assess technologies for microbial decontamination of indoor air using a sealed, room-sized chamber (~24 m3) as recommended by the U.S. Environmental Protection Agency (3). The test microbe is aerosolized inside the chamber where a fan uniformly mixes the aerosols and keeps them airborne. Samples of the air are collected and assayed, firstly to determine the rates of physical and biological decay of the test microbe, and then to assess the air decontaminating activity of the technology under test as log10 or percentage reductions in viability per m3 (1). The air temperature and relative humidity (RH) in the chamber are measured and recorded during each test.  
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5.1 The exposure-chamber method is a quantitative procedure for determining the microbial-barrier properties of porous materials under the conditions specified by the test. Data obtained from this test is useful in assessing the relative potential of a particular porous material in contributing to the loss of sterility to the contents of the package versus another porous material. This test method is not intended to predict the performance of a given material in a specific sterile-packaging application. The maintenance of sterility in a particular packaging application will depend on a number of factors, including, but not limited to the following:  
5.1.1 The bacterial challenge (number and kinds of microorganisms) that the package will encounter in its distribution and use. This may be influenced by factors such as shipping methods, expected shelf life, geographic location, and storage conditions.  
5.1.2 The package design, including factors such as adhesion between materials, the presence or absence of secondary and tertiary packaging, and the nature of the device within the package.  
5.1.3 The rate and volume exchange of air that the porous package encounters during its distribution and shelf life. This can be influenced by factors including the free-air volume within the package and pressure changes occurring as a result of transportation, manipulation, weather, or mechanical influences (such as room door closures and HVAC systems).  
5.1.4 The microstructure of a porous material which influences the relative ability to adsorb or entrap microorganisms, or both, under different air-flow conditions.
SCOPE
1.1 This test method is used to determine the passage of airborne bacteria through porous materials intended for use in packaging sterile medical devices. This test method is designed to test materials under conditions that result in the detectable passage of bacterial spores through the test material.  
1.1.1 A round-robin study was conducted with eleven laboratories participating. Each laboratory tested duplicate samples of six commercially available porous materials to determine the Log Reduction Value (LRV) (see calculation in Section 12). Materials tested under the standard conditions described in this test method returned average values that range from LRV 1.7 to 4.3.  
1.1.2 Results of this round-robin study indicate that caution should be used when comparing test data and ranking materials, especially when a small number of sample replicates are used. In addition, further collaborative work (such as described in Practice E691) should be conducted before this test method would be considered adequate for purposes of setting performance standards.  
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5.2 The purpose of this test method is to direct a user in how to grow, sample, and analyze an E. coli biofilm in a urinary catheter under a constant flow of artificial urine. The test method incorporates operational parameters utilized in similar published methods (4). The E. coli biofilm that grows has a patchy appearance that varies across the catheter. Microscopically, the biofilm is heterogenous, with large clusters in some areas, and flat sheets of cells or even single cells in others. By 24 h, the biofilm is developed in the control catheters. If the goal is to monitor early stage biofilm development, then tubing and effluent samples need to be collected prior to the 24 h sample collection. Monitoring biofilm development requires sampling. The biofilm generated in the Intraluminal Catheter Model is suitable for comparison testing between antimicrobial and control catheters.
SCOPE
1.1 This test method specifies the operational parameters required to assess the ability of antimicrobial urinary catheters to prevent or control biofilm growth. Efficacy is reported as the log reduction in viable bacteria when compared to a repeatable (1)2  Escherichia coli biofilm grown in the intra-lumen of a urinary catheter under a constant flow of artificial urine.  
1.2 The test method is versatile and may also be used for growing and/or characterizing biofilms and suspended bacteria of different species, although this will require changing the operational parameters to optimize the method based upon the growth requirements of the new organism.  
1.3 This test method may be used to evaluate surface modified urinary catheters that contain no antimicrobial agent.  
1.4 This test method describes how to sample and analyze catheter segments and effluent for viable cells. Biofilm population density is recorded as log colony forming units per surface area. Suspended bacterial population density is reported as log colony forming units per volume.  
1.5 Basic microbiology training is required to perform this test method.  
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, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.8 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.

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ASTM
ASTM E3286-21(Practice)
Standard Practice for Preparation Of Cell Monolayers on Glass Surfaces for Evaluation of Microbicidal Properties of Non-Chemical Based Antimicrobial Treatment Technologies

SIGNIFICANCE AND USE
5.1 There are no reproducible standardized protocols for preparing specimens used to evaluate the microbicidal efficacy of non-chemical treatments such as ultraviolet (UV), highenergy electron beam, or other forms of non-chemical antimicrobial technologies.  
5.2 Conventional protocols for applying bioburdens to carriers (see Test Method E2197) cause cells to stack upon one another, thereby creating multiple cell layers in which cells in layers closer to the carrier are masked by cells in overlying layers, which makes relative comparison of different non-chemical antimicrobial treatments more difficult.  
5.3 Steel and other metal carriers have asperities that can shield a percentage of the applied cells from direct exposure to electromagnetic irradiation.  
5.4 The combined effects of 5.2 and 5.3 confound determination of the microbicidal effect of electromagnetic irradiation on test specimens.  
5.5 The practice addresses these two confounding factors by:  
5.5.1 Using glass microscope slides – the surfaces of which are asperity-free – as carriers.  
5.5.2 Reliably depositing bacterial cells onto the carrier as a monolayer.  
5.6 The resulting specimen ensures that all microbes deposited onto the carrier are exposed equally to the irradiation source thereby ensuring that the only variables are the controlled ones – starting inoculum concentration, wavelength (λ – in nm), exposure time(s), and resulting energy dose (J).
SCOPE
1.1 This practice provides a protocol for creating bacterial cell monolayers on a flat surface.  
1.2 The cultures used and culture preparation steps in this Practice are similar to AOAC Method 961.02 and US EPA MB-06. However, test bacteria are applied to the carrier using an automated deposition device (6.2) rather than as a suspension droplet.  
1.3 The carrier inspection protocol is similar to US EPA MB-03 except that carrier surfaces are inspected microscopically rather than visually, unaided.  
1.4 A monolayer of cells eliminates the confounding effect caused by the shadowing effect of outer layers of bacteria stacked upon other bacteria on test specimens – thereby attenuating directed energy beams (that is, ultraviolet light, high-energy electron beams) before they can reach underlying cells.  
1.5 An asperity-free surface eliminates the shadowing effect of specimen surface topology that can block direct exposure of target bacteria to non-chemical antimicrobial treatments.  
1.6 This practice provides a reproducible target microbe and surface specimen to minimize specimen variability within and between testing facilities. This facilitates direct data comparisons among various non-chemical antimicrobial technologies.  
1.6.1 Antimicrobial pesticides used in clinical and industrial applications are expected to overcome shadowing effects. However, this practice meets a need for a protocol that facilitates relative comparisons among non-chemical antimicrobial treatments.  
1.6.2 This practice is not intended to satisfy or replace existing test requirements for liquid chemical antimicrobial treatments (for example Test Methods E1153 and E2197) or established regulatory agency performance standards such as US EPA MB-06.  
1.7 This practice was validated using Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 15442) using a protocol based on AOAC Method 961.02. If other cultures are used, the suitability of this practice must be confirmed by inspecting prepared surfaces, by using scanning electron microscopy (SEM) or comparable high-resolution microscopy.  
1.8 The specimens prepared in accordance with this practice are not meant to simulate end-use conditions.  
1.8.1 Non-chemical technologies are only to be used on visibly clean, non-porous surfaces. Consequently, a soil load is not used.  
1.9 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.10 Th...

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