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, and primer and probe combinations that are not covered in this guide may be used to perform qPCR. This guide does not attempt to cover all of the possible qPCR assays and does not suggest nor imply that the qPCR assays (that is, combinations of primers and probes, and reaction conditions) discussed here are better suited for qPCR than other qPCR assays not presented here. Additional, primers, primers and probes combination, and qPCR assay conditions may be added in the future to this guide as they become available to the ASTM scientific community. Guide D6469 reviews the types of damage that uncontrolled microbial growth in fuels and fuel systems can cause.5.4 Culture-based microbiological tests depend on the ability of microbes to proliferate in liquid, solid or semisolid nutrient media, in order for microbes in a sample to be detected.5.5 There is general consensus among microbiologists that only a fraction of the microbes believed to be present in the environment have been cultured successfully.5.6 Since the mid-1990s, genetic test methods that do not rely on cultivation have been increasingly favored for the detection and quantification of microorganisms in environmental samples.5.7 qPCR is a quantitative, culture-independent method that is currently used in the medical, food, and cosmetic industries for the detection and quantification of microorganisms.5.8 Since the early 2000s, qPCR methodology has evolved and is now frequently used to quantify microorganisms in fuel-associated samples, but there is currently no standardized methodology for employing qPCR for this application (1-6).3 The purpose of this guide is to provide guidance and standardization for genetic testing of samples using qPCR to quantify total microbial populations present in fuel-associated samples.5.9 Although this guide focuses on describing recommended protocols for the quantification of total microorganisms present in fuel-associated samples using qPCR, the procedures described here can also be applied to the standardization of qPCR assays for other genetic targets and environmental matrices.5.10 Genetic techniques have great flexibility so that it is possible to design a nearly infinite number of methods to detect and quantify each and every gene. Because of this flexibility of genetic techniques, it is important to provide a standard protocol for qPCR so that data generated by different laboratories can be compared.5.11 This guide provides recommendations for primers sequences and experimental methodology for qPCR assays for the quantification of total microorganisms present in fuel-associated samples.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 qualitative and quantitative relationship between the laboratory results and actual microbial communities in the systems from which samples are collected is affected by the time delay and handling conditions between the time of sampling and time that testing is initiated.1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard with the exception of the concept unit of gene copies/mL (that is, 16S or 18S gene copies/mL) to indicate the starting concentration of microbial DNA for the intended microbial targets (that is, bacteria, archaea, 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 standard to establish appropriate safety, health, and environmental 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.

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4.1 Numerous ASTM test methods and practices (for example: Test Methods D5259 and D5392, and Practices D6974 and E2563) report colony counts as their measured parameter.4.2 These practices provide a uniform set of counting, calculating, and reporting procedures for ASTM test methods in microbiology.  SectionA—Counting Colonies on Membrane Filters 6B—Counting Colonies on Pour Plates 7C—Counting Colonies on Spread Plates 84.3 The counting rules provide a best attainable estimate of microorganisms in the sample, since the samples cannot be held and reanalyzed at a later date.1.1 These practices cover recommended procedures for counting colonies and reporting colony-forming units (CFU) on membrane filters (MF) and standard pour and spread plates.1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.3 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.1.4 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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5.1 This guide provides information addressing the conditions that lead to turbine oil microbial contamination and biodeterioration, the general characteristics of and strategies for controlling microbial contamination. It compliments and amplifies information provided in Practices D4378 and D6224 on condition monitoring of lubricating oils.5.2 This guide focuses on microbial contamination in turbine oils and power generation turbine oil systems. Uncontrolled microbial contamination in turbine oils and lubrication systems remains a largely unrecognized but potentially costly problem in power generation systems.5.2.1 Examples of turbine oil and system biodeterioration include, but are not limited to:5.2.1.1 Filter plugging,5.2.1.2 Oil line and orifice fouling,5.2.1.3 Increased oil acidity,5.2.1.4 Increased oil corrosivity,5.2.1.5 Oil additive depletion,5.2.1.6 Water emulsification,5.2.1.7 Lubricity loss, and5.2.1.8 Decreased oxidative stability and increased sludge generation.5.3 This guide introduces the fundamental concepts of turbine oil microbiology and biodeterioration control.5.4 This guide provides personnel who are responsible for turbine oil system stewardship with the background necessary to make informed decisions regarding the possible economic or safety, or both, impact of microbial contamination in their products or systems.1.1 This guide provides personnel who have a limited microbiological background with an understanding of the symptoms, occurrence, and consequences of chronic microbial contamination. The guide also suggests means for detection and control of microbial contamination in turbine oils and turbine oil systems. This guide applies primarily to turbine lubricants (see Specifications D4293 and D4304) and turbine oil systems. However, the principles discussed herein also apply generally to lubricating oils with viscosities <100 mm2/s (for example, see Specification D6158).1.2 This guide focuses on turbine system and turbine oil microbiology. Despite considerable differences in turbine systems (for example, gas and steam driven turbines; power generation and propulsion; etc.) as ecosystems for microbial communities – with the exception of temperature – these differences are largely irrelevant. Ambient temperatures are typically similar. Recirculating turbine oil temperatures are commonly >40 °C. However, generally speaking, all systems in which accumulations of free water can develop, share properties that are considered in this guide.1.2.1 Steam turbines, and to a greater extent hydro turbines, are continuously exposed to water ingression. Diligence is needed to ensure seals and bearings are in good condition to prevent water ingression or conditions that are conducive to biodeterioration. However, due to the risk of the accumulation of condensation, all equipment can become susceptible when shut down for extended periods.1.3 This guide complements Energy Institute’s Guidelines on detecting, controlling, and mitigating microbial growth in oils and fuels used at power generation facilities (2.2). The Energy Institute’s guidance document provides greater detail than the overview provided in this guide.1.4 Microbial contamination in turbine oil systems shares common features with microbial contamination in fuel systems (See Guide D6469). However, there are also relevant differences. Although the chemistry of the fluids is different, this Guide draws heavily on D6469 but highlights unique aspects of turbine oil and turbine oil system biodeterioration and microbial contamination.1.5 This guide is not a compilation of all of the concepts and terminology used by microbiologists. It provides basic explanations of microbial contamination and biodeterioration in turbine oils and turbine oil systems.1.6 The values in SI units are to be regarded as the 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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6.1 This test method has been developed as a result of research performed by Air Dispersion Limited (Manchester, UK) and funded by the Barrier Test Consortium Limited. The results of this research have been published in a peer-reviewed journal.4 This research demonstrated that testing the barrier performance of porous packaging materials using microorganisms correlates with measuring the filtration efficiency of the materials.6.2 This test method does not require the use of microbiological method; in addition, the test method can be conducted in a rapid and timely manner.6.3 When measuring the filtration efficiency of porous packaging materials a typical filtration efficiency curve is determined (see Fig. 1). Since the arc of these curves is dependent upon the characteristics of each individual material, the appropriate way to make comparison among materials is using the parameter that measures maximum penetration through the material.FIG. 1 A Typical Curve Showing Penetration as a Function of Flow RateNOTE 1: The point of maximum penetration is indicated by the upward pointing triangle.6.4 The particle filtration method is a quantitative procedure for determining the microbial barrier properties of materials using a challenge of 1.0 µm particles over range of pressure differentials from near zero to approximately 30 cm water column (WC) (2942 Pa). This test method is based upon the research of Tallentire and Sinclair4 and uses physical test methodology to allow for a rapid determination of microbial barrier performance.1.1 This test method measures the aerosol filtration performance of porous packaging materials by creating a defined aerosol of 1.0 μm particles and assessing the filtration efficiency of the material using either single or dual particle counters.1.2 This test method is applicable to porous materials used to package terminally sterilized medical devices.1.3 The intent of this test apparatus is to determine the flow rate through a material at which maximum penetration occurs.1.4 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only.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 standard to establish appropriate safety, health, and environmental 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.

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4.1 The static chambers have several different applications:4.1.1 The static chambers can be used to compare the susceptibility of different materials to the colonization and amplification of various microorganisms under defined conditions.4.1.2 Chambers operated at high relative humidities may be used to perform worst case scenario screening tests on materials by providing an atmosphere where environmental conditions may be favorable for microbial growth.4.1.3 Use of multiple chambers with different environmental parameters, such as a range of relative humidities, permits the evaluation of multiple microenvironments and allows investigation of materials under differing environmental conditions.4.1.4 Drying requirements for wetted materials may also be investigated. This information may be relevant for determining material resistance to microbial growth after becoming wet. These conditions may simulate those where materials are subjected to water incursion through leaks as well as during remediation of a building after a fire.4.1.5 Growth rates of microorganisms on the material may also be investigated. Once it has been established that organisms are able to grow on a particular material under defined conditions, investigations into the rate of organism growth may be performed. These evaluations provide base line information and can be used to evaluate methods to limit or contain amplification of microorganisms.4.2 These techniques should be performed by personnel with training in microbiology. The individual must be competent in the use of sterile technique, which is critical to exclude external contamination of materials.1.1 Many different types of microorganisms (for example, bacteria, fungi, viruses, algae) can occupy indoor spaces. Materials that support microbial growth are potential indoor sources of biocontaminants (for example, spores and toxins) that can become airborne indoor biopollutants. This guide describes a simple, relatively cost effective approach to evaluating the ability of a variety of materials to support microbial growth using a small chamber method.1.2 This guide is intended to assist groups in the development of specific test methods for a definite material or groups of materials.1.3 Static chambers have certain limitations. Usually, only small samples of indoor materials can be evaluated. Care must be taken that these samples are representative of the materials being tested so that a true evaluation of the material is performed.1.4 Static chambers provide controlled laboratory microenvironment conditions. These chambers are not intended to duplicate room conditions, and care must be taken when interpreting the results. Static chambers are not a substitute for dynamic chambers or field studies.1.5 A variety of microorganisms, specifically bacteria and fungi, can be evaluated using these chambers. This guide is not intended to provide human health effect data. However, organisms of clinical interest, such as those described as potentially allergenic, may be studied using this approach.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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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.5.2 While this practice is designed primarily for work with aerobic, mesophilic vegetative bacteria, it can be readily adapted to handle other classes of microbial pathogens or their surrogates.5.3 The pieces of equipment given here are as examples only. Other similar devices may be used as appropriate.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.1.2 The chamber can be used to assess microbial survival in indoor air as well as to test the ability of physical (for example, ultraviolet light) and chemical agents (for example, vaporized hydrogen peroxide) to inactivate representative pathogens or their surrogates in indoor air.1.3 This practice does not cover testing of microbial contamination introduced into the chamber as a dry powder.1.4 This practice does not cover work with human pathogenic viruses, which require additional safety and technical considerations.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 standard to establish appropriate safety, health, and environmental 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.

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5.1 This test method is intended to provide a tool for assessing whether fuel storage and distribution facilities or end user fuel tanks are subject to microbial growth and alert fuel suppliers or users to the potential for fuel quality or operational problems and/or the requirement for preventative or remedial measures.5.2 This test method detects numbers of microbial colony forming units (CFU), the same detection parameter used in the laboratory standard procedures Practice D6974 and IP 385. However, whereas Practice D6974 and IP 385 provide separate assessment of numbers of viable aerobic bacteria CFU and numbers of viable fungal CFU, this test method provides a combined total count of viable aerobic bacteria and fungal CFU.5.3 This test method is designed to detect a recognized group of microorganisms of significance in relation to contamination of distillate fuels, but it is recognized that microbiological culture techniques do not detect all microorganisms that can be present in a sample. Culturability is affected primarily by the ability of captured microbes to proliferate on the growth medium provided, under specific growth conditions. Consequently, a proportion of the active or inactive microbial population present in a sample can be viable but not detected by any one culture test.7 In this respect, the test is indicative of the extent of microbial contamination in a sample ,and it is assumed that when a fuel sample is significantly contaminated, some of the dominant microbial species present will be quantifiably detected, even if not all species present are culturable.5.4 Many samples from fuel systems can be expected to contain a low level of “background” microbial contamination, which is not necessarily of operational significance. The minimum detection level of this test method is determined by the volume of specimen tested and is set such that microbial contamination will generally only be detected when it is at levels indicative of active proliferation.5.5 The test will detect culturable bacteria and fungi that are metabolically active and dormant fungal spores. Presence of fungal spores in a fuel sample can be indicative of active microbial proliferation within a fuel tank or system, but at a point distant from the location sampled. Active microbial growth only occurs in free water, and this can be present only as isolated pockets at tank or system low points. Because fungal spores are more hydrophobic than active cells and fungal material (mycelium), they disperse more readily in fuel phase and are thus more readily detected when low points cannot be directly sampled and only fuel phase is present in samples.5.6 This test method can determine whether microbial contamination in samples drawn from fuel tanks and systems is absent or present at light, moderate, and heavy levels.5.7 The categorization of light, moderate, and heavy levels of contamination will depend on the fuel type, the sampling location, the facility sampled, and its specific operating circumstances.5.8 Further guidance or interpretation of test results can be found in Guide D6469, in the Energy Institute Guidelines for the investigation of the microbial content of petroleum fuels, and for the implementation of avoidance and remedial strategies and in the IATA Guidance Material on Microbiological Contamination in Aircraft Fuel Tanks.5.8.1 Further guidance on sampling can be found in Practice D7464.5.9 Testing can be conducted on a routine basis or to investigate incidents.5.10 Microbiological tests are not intended to be used to determine compliance with absolute fuel specifications or limits. The implementation of specification limits for microbiological contamination in fuels is generally not appropriate, and microbial contamination levels cannot be used alone or directly to make inferences about fuel quality or fitness for use.5.11 When interpreting results, it must be appreciated that the test result applies only to the specific sample and specimen tested and not necessarily to the bulk fuel. Microbiological contamination usually shows a highly heterogeneous distribution in fuel systems, and therefore, analysis of a single sample will rarely provide a complete assessment of the overall levels of contamination present.5.12 Water phase will usually contain substantially higher numbers of microbial CFU than fuel phase and, consequently, a different interpretation of results is required.1.1 This test method describes a procedure that can be used in the field or in a laboratory to quantify culturable, viable aerobic microorganisms present as contaminants in liquid fuels, including those blended with synthesized hydrocarbons or biofuels, with kinematic viscosities (at 40 °C) of ≤24 mm2 s-1 and heavy and residual fuels with kinematic viscosities (at 40 °C) of ≤700 mm2 s-1 and in fuel-associated water.1.1.1 This test method has been validated by an ILS for a range of middle distillate fuels meeting Specifications D975, D1655, ISO 8217 DMA, and NATO F-76.21.2 This test method quantitatively assesses culturable, viable aerobic microbial content present in the form of bacteria, fungi, and fungal spores. Results are expressed as the total number of microbial colony forming units (CFU)/L of fuel or total number of CFU/mL of associated water. The number of CFU should not be interpreted as absolute values but should be used as part of a diagnostic or condition monitoring effort; for example, these values can be used to assess contamination as absent, light, moderate, or heavy.NOTE 1: This test method is technically equivalent to IP 613, although the two methods are not currently jointed.1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.4 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.1.5 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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3.1 These results may be used to compare the susceptibility of materials when exposed to this test procedure.3.2 Microbiological susceptibility may be reflected by a number of changes including staining, weight loss, or reduction in tensile or flexural strength.3.3 This practice may be considered an inoculation with a mixed culture of fungi and bacteria.1.1 This practice is limited to the method of conducting an evaluation of a nonmetallic material's microbiological susceptibility when in contact with the natural environment of the soil under use conditions. This practice is intended for use on solid material test specimens that are no larger than approximately 2 cm (0.79 in.) thick and 100 cm 2 (15.5 in.2) or on film forming materials such as coatings which may be tested in the form of films at least 50 by 50 mm (2 by 2 in.) in size. This practice may be applied to articles that do not spend the majority of their service life in soil.1.2 A wide variety of properties may be affected by microbial attack depending on material or item characteristics. Standard methods (where available) should be used for each different property to be evaluated. This practice does not attempt to enumerate all of the possible properties of interest nor specify the most appropriate test for those properties. Test methods must, however, be appropriate to the material being tested.1.3 Materials intended for use in soil burial applications are often subjected to periods of exposure to solar radiation and other elements of weather for some time before they are buried. Because these exposures may alter the ability of a material to resist the effects of soil-borne microorganisms, it is recommended that this practice be combined with appropriate environmental exposures (for example, solar simulating weathering devices, the hydrolytic effects of extended aqueous contact, or extraneous nutrients) or fabrication into articles (for example, adhesive bonding of seams) which may promote microbiological susceptibility during the service life of the material.1.4 The values stated in SI units are to be regarded as standard. The values given in parentheses are provided for information purposes only.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 standard to establish appropriate safety, health, and environmental 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.

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3.1 Spoilage of paint in the container is often related to the use of contaminated raw materials, water (particularly recycled washwater), vessels, piping, and equipment in the manufacturing plant. There is a need for a simple method to determine the presence or absence of microorganisms in plants that manufacture paints and coatings. Such a determination enables the manufacturer to establish the point of contamination (that is, raw materials or problem housekeeping areas in the plant) to help in solving the spoilage problem.NOTE 1: Some contamination in plant areas is to be expected, since microorganisms are ubiquitous and cannot generally be eliminated practically (it is what an in-can preservative is supposed to control). Excessive levels of contamination or contaminated raw materials can exceed the capability of the preservative. If you have excessive contamination in the plant, there are methods for decontamination including steam, preservatives, bleach, etc. These should be discussed with your biocide supplier and used with care. Recovery of spoiled or contaminated products is often not feasible, so an adequate level of the appropriate biocide in conjunction with good plant housekeeping practices are essential. Your biocide supplier can also help here.3.2 This test method may be used by persons without basic microbiological training, but some training on aseptic techniques would be recommended.NOTE 2: The reliability of the results obtained from this test method is extremely dependent on the techniques employed. Improper techniques can result in a sterile sample appearing to be contaminated, and even worse, a contaminated sample appearing to be sterile (see also 5.1). It is recommended that you consult with your biocide supplier, raw material supplier, or an independent testing laboratory to confirm questionable results.1.1 This test method covers a procedure for the determination of the microbial condition (contamination or sterility) of raw materials used in the manufacture of paint, and the microbial condition of paint and paint manufacturing areas.1.2 The values in SI units are to be regarded as the standard. The values given in parentheses are for information only.1.3 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.1.4 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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4.1 This test method utilizes filter paper as a medium for evaluating touch-transfer of bacteria to material surfaces. Sample surfaces are exposed to filter papers saturated with a Staphylococcus aureus suspension, followed by recovery of the bacteria from the surface with Replicate Organism Detection and Counting (RODAC) plates. This test method reports the log10 reduction of bacterial transfer on an intended test surface compared to a control surface. The test and control surfaces can differ by texture, coating, treatment, or any other desired variables, as long as they are the same material.1.1 This test method is designed to evaluate the contact-mediated microbial transference on two-dimensional material surfaces. Material surfaces intended to reduce microorganism adherence or contamination may be evaluated using this test method. Additionally, this test method can be utilized on a wide variety of material surfaces for other downstream processes.1.2 This test method is designed to quantify the fomite transfer of bacteria on material surfaces through touch-transfer, be inexpensive, require limited training in practice, and be adaptable to many surfaces or microorganisms. Non-porous surfaces are contaminated with inoculated filter paper as a carrier and surface contamination is quantified by sampling with Replicate Organism Detection and Counting (RODAC) plates.1.3 Basic microbiology training is required to perform this test method.1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.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 standard to establish appropriate safety, health, and environmental 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.

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The objectives of the respiration inhibition tests may be defined by the interests of the user, but the test method is designed primarily for examination of the inhibition response with operating microbial systems such as an activated sludge process treating domestic or industrial wastes. Different apparatus exist that facilitate continuous or continual measurement of respiration in microbial systems and each may be used as the tool to observe respiration in this test method. Respirometry may utilize any apparatus and technique that will achieve the determination of respiration rate. A number of devices are presented in Appendix X1. Equivalency in the experimental capability of each device is not implied. The analyst should select the respirometric approach that best suits his needs. The inhibitory effect of a test candidate is identified more completely by examining inhibition over a range of concentrations, such as determining the EC50. The use of aerated containers permits concurrent management of a series of cell suspensions. A respirometer for each cell suspension might also be used.1.1 This test method covers a batch procedure that evaluates the impact of selected wastewaters, materials, or specific compounds on the respiration rate of an aqueous microbial culture, such as activated sludge. 1.2 Alternative procedures for measurement of microbial activity, such as adenosine 5′ triphosphate (ATP), specific substrate utilization, etc. are not within the scope of this test method. 1.3 The results obtained are based on comparisons in a specific test series that examines a range of concentrations of the potentially inhibitory test candidate using batch methods in a laboratory. Results are completed in a short time frame (a few hours). 1.4 The test results are specific to the microbial culture used. Microbial culture from different wastewater treatment plants will differ in kinds and numbers of organisms, and performance capability. Thus, there is no basis for comparing results for microbial cultures from different treatment facilities. 1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. 1.6 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.

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5.1 The use of plastics aboard ships is on the rise and the use of the sea as a trash dumping site is no longer a possibility; consequently, the disposal of plastic materials while at sea remains a major issue. It is possible that biodegradable plastics will help to allay public concern by allowing for the safe disposal of plastic materials at sea. This test method has been developed to assess the rate and degree of aerobic biodegradation of plastics exposed to marine microorganisms. Aerobic biodegradation is determined by measuring the amount of biogas (carbon dioxide) produced during such an exposure.5.2 It is acceptable to use the degree and rate of aerobic biodegradability of a plastic under the conditions of this test method to estimate the persistence of that plastic in biologically active marine environments, for example, seashore and open-ocean. However, it shall be recognized that predicting long-term environmental fate and effects from the results of short-term exposure to a simulated marine environment is difficult. Thus, caution shall be exercised when extrapolating the results obtained from this or any other controlled-environment test to disposal in the natural environment.1.1 This test method is used to determine the degree and rate of aerobic biodegradation of plastic materials (including formulation additives) exposed to pre-grown population of at least ten aerobic marine microorganisms of known genera or the indigenous population existing in natural seawater. The test method is conducted under controlled laboratory conditions.1.2 This test method is designed to index polymer materials that are possibly biodegradable, relative to a positive reference material, in an aerobic environment.1.3 This test method is applicable to all polymer materials containing at least 20 % carbon that are not inhibitory to the microorganisms present in a marine environment.1.4 The values stated in SI units are to be regarded as the standard.1.5 There is no known ISO equivalent to this standard.1.6 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.1.7 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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3.1 Microbial growth is a major cause of discoloration and deterioration of paint films. This guide describes techniques used to distinguish fungi and algae from other surface contaminants.3.2 Repainting a surface contaminated with fungi or algae generally causes more rapid infestation of the new paint than repainting a surface from which fungal or algal growth has been removed. In addition, poor adhesion, staining/bleeding, and many other problems may result. This guide describes methods for removing fungal or algal growth prior to repainting.NOTE 1: It is extremely important that all steps and notes be read and followed. In particular the washing steps outlined in 5.2.2 and 5.2.4 must be fully carried out, since residual sodium hypochlorite (bleach) can cause many of the same problems, or even loss of coloration after repainting, and residual phosphate detergents can encourage further fungal and algal growth.NOTE 2: This procedure is intended for use when recoating the surface. Reports of using this procedure to remove fungi and algae without recoating necessitate inclusion of a warning about the adverse effects of bleach on a coating film. The oxidative effects of bleach can degrade film properties, including particularly the “bleaching” (loss of color) in films containing organic coloring pigments (such as quinacridones, etc.), loss of gloss, chalking, etc. A small patch must be tested prior to use of this procedure for that purpose.1.1 This guide describes techniques used for determining the presence of fungal or algal growth on paint and related coatings and methods for removal of such growth prior to recoating.1.2 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only.1.3 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.1.4 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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5.1 This test method is intended to provide a tool for assessing whether fuel storage and distribution facilities, or end user fuel tanks, are subject to microbial growth, and to alert fuel suppliers or users to the potential for fuel quality or operational problems or the requirement for preventative or remedial measures, or both.5.2 This test method allows assessment of whether antigens generated by microbial activity in the specimens are present within specific defined ranges.5.3 This test method measures the presence of microbial and metabolite antigens in a specimen. The antigens are generated from the living cells and metabolites created by fungi and bacteria during growth on fuel. Consequently, the presence of antigens is an indicator of microbial contamination in fuel systems. Antigens are not associated with matter of nonbiological origin.5.3.1 Some of the antigens detected by this test method can persist after treatment with a biocide. See 11.4.5.4 This test method is semi-quantitative and can be used to determine whether contamination in samples drawn from fuel tanks and systems is negligible or present at moderate or heavy levels.5.4.1 Further information on using the test to assess biodeterioration risk is provided in Appendix X1.5.5 The significance of these levels to the operator will depend on the fuel type, the sampling location, the equipment or facility sampled, and the specific operating circumstances.5.6 Further guidance on interpretation of test results can be found in Guide D6469, in Energy Institute guidelines for the investigation of the microbial content of petroleum fuels, and in the IATA Guidance Material on Microbial Contamination in Aircraft Fuel Tanks.5.7 Further guidance on sampling can be found in Practice D7464.5.8 Testing can be conducted on a routine basis or to investigate incidents.5.9 Microbiological tests are not intended to be used to determine compliance with fuel specifications or limits. The implementation of specification limits for microbiological contamination in fuels is generally not appropriate, and microbial contamination levels cannot be used alone or directly to make inferences about fuel quality or fitness for use.5.10 When interpreting results, it must be appreciated that the test result applies only to the specific sample and specimen tested and not necessarily to bulk fuel. Microbiological contamination usually shows a highly heterogeneous distribution in fuel systems, and therefore, analysis of a single sample will rarely provide a complete assessment of the overall levels of contamination present.5.11 Water phase will usually contain substantially higher amounts of microbial contamination than fuel phase and, consequently, a different interpretation of results is required. This is why this test method reports antigen concentration per mL for water and per L for fuel.5.12 This test method differs from some other methods (for example Test Methods D7687 and D7978) and practices (for example Practice D6974) in that it detects microbial activity in fuels or associated aqueous specimens in the field and does not need to be performed in a laboratory or in an aseptic environment. It may be used in a laboratory.5.13 This test method does not require specialist microbiological experience or knowledge.5.14 This test method provides rapid results that reflect the total active microbial contamination in the specimen, and enables result to be obtained within 15 min.5.15 This test method differentiates among three ranges of contamination for H. resinae, other fungi, and aerobic bacteria (see 1.3).1.1 This test method describes a procedure that can be used in the field or in a laboratory to detect antigens indicative of microbial contamination in liquid fuels, including those blended with synthesized hydrocarbons or biofuels, with kinematic viscosities (at 40 °C) of ≤24 mm2s–1 (for example, Specifications D396, D975, and D1655) and in fuel-associated water.1.1.1 This test method has been validated by an ILS for a range of middle distillate fuels meeting Specification D1655, EN590, Specification D975, and ISO 8217:2012.1.2 This test method semi-quantitatively assesses the concentration of specific antigens generated by commonly recovered, fuel-associated, aerobic microorganisms during active growth in fuel systems.1.2.1 A proprietary formulation of antibodies and antibody mixtures is used to detect three types of microbial antigen contamination: antigens generally found in aerobic bacteria, antigens generally present in common fungi (yeast and molds), and an antigen that is characteristic of Hormoconis resinae (the fungus most commonly associated with fuel biodeterioration).1.2.2 Although the antibodies and antibody mixtures are characteristic of diverse types of bacteria and fungi, it is unlikely that they are universal. Recognizing that for every microbe that has been isolated and characterized, it is likely that there are a billion that have not. Consequently, as is the case with all microbiological test methods, this test method does not purport to detect 100 % of the microbes present in a fuel or fuel-associated water sample.1.3 For each of the three sets of antigen detected (H. resinae, common fungi, and aerobic bacteria), the test detects whether the antigen concentration present is within set ranges representing negligible, moderate, or heavy microbial contamination.1.3.1 For fuel specimens, the antigen concentration ranges detected are <150 µg/L (negligible), 150 µg/L to 750 µg/L (moderate), and >750 µg/L (heavy).1.3.2 For specimens of water associated with fuel, the antigen concentration ranges detected are <33 µg/mL (negligible), 33 µg/mL to 166 µg/mL (moderate), and >166 µg/mL (heavy).1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.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 standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. For a specific hazard statement, see Section 8.1.6 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.

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This test method provides a rapid means of determining the acute toxicity of an aqueous waste, or waste extract, prior to and following biological treatment, and contributes to assessing the potential biodegradability of the waste (see 1.1, 1.2, and Note 1). The change in toxicity to the marine bacterium P. phosphoreum with respect to time may serve as an indication of the biodegradation potential. Sample analyses are usually obtained in 45 to 60 min, with as little as 5 mL of sample required (5). Samples with high suspended solids concentrations may test nontoxic to the bacteria, while still exhibiting significant toxicity to freshwater organisms, due to those suspended solids. The absorbance correction procedure included in this test method allows for the analysis of highly colored lightabsorbing samples, by providing a means for mathematically adjusting the light output readings to account for light lost due to absorption. 1.1 This test method (1) covers a procedure for the rapid evaluation of the toxicity of wastewaters and aqueous extracts from contaminated soils and sediments, to the luminescent marine bacterium Photobacterium phosphoreum, prior to and following biological treatment. This test method is meant for use as a means to assess samples resulting from biotreatability studies. Sensitivity data for P. phosphoreum to over 1300 chemicals have been reported in the literature (2). Some of the publications are very relevant to this test method (3). The data obtained from this test method, when combined with respirometry, total organic carbon (TOC), biochemical oxygen demand (BOD), chemical oxygen demand (COD), or spectrophotometric data, can assist in the determination of the degree of biodegradability of a contaminant in water, soil, or sediment (3). The percentage difference between the IC20 of treated and untreated sample is used to assess the progress of detoxification. 1.2 This test method is applicable to the evaluation of the toxicity (to a specific microbe) and its implication on the biodegradation of aqueous samples from laboratory research bio-reactors (liquid or soil), pilot-plant biological treatment systems, full-scale biological treatment systems, and land application processes (see Notes 1 and 2). Note 1—If the biologically treated material is to be discharged in such a manner as to potentially impact surface waters and ground water, or both, then the user must consult appropriate regulatory guidance documents to determine the proper test species for evaluating potential environmental impact (4). Correlations between data concerning reduction in toxicity produced by this test method and by procedures for acute or short-term chronic toxicity tests, or both, utilizing invertebrates and fish (see Guides E729 and E1192E729E1192), should be established, wherever possible. Note 2—Color (especially red and brown), turbidity, and suspended solids interfere with this test method by absorbing or reflecting light. In these situations data are corrected for these effects by use of an absorbance correction procedure included in this test method (see 5.3, 6.1, and 6.2). 1.3 The results of this test method are reported in terms of an inhibitory concentration (IC), which is the calculated concentration of sample required to produce a specific quantitative and qualitative inhibition. The inhibition measured is the quantitative reduction in light output of luminescent marine bacteria (that is, IC20 represents the calculated concentration of sample that would produce a 20 % reduction in the light output of exposed bacteria over a specified time). 1.4 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only. 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 standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. Specific hazard statements are given in Section 9.

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