1.1 该测试方法涵盖了捕获、提取和量化与燃料和燃料相关水中微生物相关的细胞三磷酸腺苷（细胞ATP）含量的方案。 1.2 使用生物发光酶测定法测量ATP，由此产生的光的量与样品中细胞ATP的浓度成比例。光是以相对光单位（RLU）的形式产生和定量测量的，通过与ATP标准进行比较，将其转换为pg ATP/mL，并可选地进一步转换为Log 10 [pg ATP/mL]。 1.3 该测试方法同样适用于实验室或便携式方法。 1.4 此试验方法仅限于具有标称粘度的燃料 ≤ 75 试验温度下的cSt。 1.5 该测试方法检测的ATP浓度范围为5.0 pg ATP/mL( ≈ 0.699对数 10 [pg ATP/mL]）至100 000pg-ATP/mL( ≈ 5.000对数 10 [pg ATP/mL]）持续20 mL燃料样品和20份 pg ATP/毫升( ≈ 1.301对数 10 [pg ATP/mL]）至400 000pg-ATP/mL( ≈ 5.602日志 10 [pg ATP/mL]），用于5 mL燃料样品- 相关水。 注1: 使用中提供的以pg/mL为单位的样品ATP计算公式计算这些范围 12.1 基于1的最小推荐RLU 当使用第节中规定的试剂时，ng/mL ATP标准 7. 以及中规定的光度计 6.4 并用中确定的试剂法空白进行校正 附录X5 . 1.6 如果可以克服干扰，生物发光是一种可靠且经验证的鉴定和量化ATP的方法。 这种测试方法不会区分来自不同来源的ATP，例如:来自不同类型的微生物，如细菌和真菌。 1.7 以国际单位制表示的数值应视为标准。本标准不包括其他计量单位。 1.8 本标准并非旨在解决与其使用相关的所有安全问题（如有）。本标准的使用者有责任在使用前制定适当的安全、健康和环境实践，并确定监管限制的适用性。 1.9 本国际标准是根据世界贸易组织技术性贸易壁垒委员会发布的《关于制定国际标准、指南和建议的原则的决定》中确立的国际公认的标准化原则制定的。 ====意义和用途====== 5.1 该测试方法测量样品中存在的细胞ATP的浓度。ATP是所有活细胞的组成部分，包括细菌和真菌。 因此，细胞ATP的存在是燃料中总代谢活性微生物污染的指标。ATP与非生物来源的物质无关。 5.2 此测试方法类似于测试方法 第2694页 除了采样的体积之外。 5.3 此测试方法与测试方法不同 D4012型 因为它利用了过滤和洗涤步骤，旨在消除干扰，这些干扰在历史上使ATP测试无法与复杂的有机流体（如燃料和燃料）一起使用- 相关水。 5.4 此测试方法与测试方法不同 第7463页 在几个方面: 5.4.1 试验方法 第7463页 报告相对光单位（RLU）。与试验方法一致 D4012型 和 第2694页 ，该测试方法报告ATP浓度。 5.4.2 该测试方法仅检测细胞ATP，可用于检测少量水不易分离的燃料和燃料库存中的细胞ATP（例如，含有 ≥ 5. % v/v乙醇）。 试验方法 第7463页 不能用于从少量水不易分离的燃料中回收ATP（例如，含有 ≥ 5. % v/v乙醇）。 5.4.3 该测试方法在一次测量中测量细胞ATP（单位为pg-ATP/mL）。试验方法 第7463页 使用两种单独的分析检测总ATP（作为RLU）和细胞外ATP（作为RL U），并允许计算细胞ATP（作为RLU）作为总ATP和细胞外ATP之间的差异。 5.4.4 试验方法 第7463页 建议标称500 mL燃料样品体积。此测试方法建议标称20 mL燃料样品。 5.5 本试验方法可用于规范中规定的所有燃料 第396页 , D975型 , 第1655天 , 2006年2月 , 880英镑 , 第3699页 , D6751 和 第7467天 和其他具有标称粘度的燃料 ≤ 75 20°±2°时的cSt。 5.6 ATP测试提供了反映样本中总生物负载的快速测试结果。从而减少了测试启动和数据采集之间的延迟，从36 h至48 可培养菌落可见所需的h（或更长时间），约为5 最小值。 5.7 尽管ATP数据通常与燃料和燃料相关水中的培养数据一致，但影响ATP浓度的因素与影响可培养性的因素不同。 5.7.1 可培养性主要受捕获微生物在特定生长条件下在所提供的生长培养基上增殖的能力的影响。因此，样品中存在的一定比例的活性或非活性微生物种群可能是可行的，但不能通过任何一种培养测试检测到。 4. 5.7.2 ATP浓度受以下因素影响:存在的微生物物种、这些物种的生理状态和总生物负荷（见 附录X1 ). 5.7.2.1 物种效应的一个例子是，对于活性真菌细胞，每个细胞的ATP量显著大于细菌。 5.7.2.2 在一个物种中，新陈代谢更活跃的细胞每个细胞的ATP比休眠细胞（如真菌孢子）更多。由于真菌孢子比活性真菌物质（菌丝体）更疏水，当从一些燃料系统中提取燃料样本时，孢子可能是真菌增殖的唯一指标，但ATP测试不会检测到它们。 5.7.2.3 总生物负载越大，样品中的ATP浓度就越高。 5.7.3 冲洗步骤可能存在( 11.15 )可能无法消除所有可能干扰生物发光反应的化学物质( 11.37 ). 5.7.3.1 可通过执行标准添加试验系列或稀释系列来评估任何此类干扰的存在，如 附录X4 .第节中的精度声明 13 将不适用。 5.8 如测试方法中所述 第7978页 ，由于决定因素的固有可变性以及各种可确定和不可确定的不准确性来源，在评估燃料微生物程序的精度方面存在固有的困难（见指南 第7847页 ). 5.8.1 任何微生物分析方法的精度通常都远低于石油工业中广泛用于分析燃料物理和化学财产的方法。

1.1 This test method covers a protocol for capturing, extracting and quantifying the cellular adenosine triphosphate (cellular-ATP) content associated with microorganisms found in fuels and fuel-associated water. 1.2 The ATP is measured using a bioluminescence enzyme assay, whereby light is generated in amounts proportional to the concentration of cellular-ATP in the samples. The light is produced and measured quantitatively as relative light units (RLU) which are converted by comparison with an ATP standard, computation to pg ATP/mL and optional further transformation to Log 10 [pg ATP/mL]. 1.3 This test method is equally suitable for use as a laboratory or portable method. 1.4 This test method is limited to fuels with a nominal viscosity ≤ 75 cSt at test temperature. 1.5 This test method detects ATP concentrations in the range of 5.0 pg ATP/mL ( ≈ 0.699 log 10 [pg ATP/mL]) to 100 000 pg ATP/mL ( ≈ 5.000 log 10 [pg ATP/mL]) for 20 mL samples of fuel and 20 pg ATP/mL ( ≈ 1.301 log 10 [pg ATP/mL]) to 400 000 pg ATP/mL ( ≈ 5.602 log 10 [pg ATP/mL]) for 5 mL samples of fuel-associated water. Note 1: These ranges were calculated with the formula for calculating sample ATP in pg/mL provided in 12.1 based on the minimum recommended RLU for a 1 ng/mL ATP standard when using the reagents specified in Section 7 and the luminometer specified in 6.4 and corrected with a reagent-method blank as determined in Appendix X5 . 1.6 Providing interferences can be overcome, bioluminescence is a reliable and proven method for qualifying and quantifying ATP. This test method does not differentiate between ATP from different sources, for example: from different types of microorganisms, such as bacteria and fungi. 1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. 1.8 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. 1.9 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. ====== Significance And Use ====== 5.1 This test method measures the concentration of cellular-ATP present in the sample. ATP is a constituent of all living cells, including bacteria and fungi. Consequently, the presence of cellular-ATP is an indicator of total metabolically active microbial contamination in fuels. ATP is not associated with matter of non-biological origin. 5.2 This test method is similar to Test Method E2694 except for the volumes sampled. 5.3 This test method differs from Test Method D4012 in that it utilizes filtration and wash steps designed to eliminate interferences that have historically rendered ATP testing unusable with complex organic fluids such as fuel and fuel-associated water. 5.4 This test method differs from Test Method D7463 in several regards: 5.4.1 Test Method D7463 reports relative light units (RLU). Consistent with Test Methods D4012 and E2694 , this test method reports ATP concentration. 5.4.2 This test method detects only cellular-ATP and it can be used to detect cellular-ATP in fuels and fuel stocks from which small quantities of water do not separate readily (for example, ethanol blended gasoline containing ≥ 5 % v/v ethanol). Test Method D7463 cannot be used to recover ATP from fuels from which small quantities of water do not separate readily (for example, ethanol blended gasoline containing ≥ 5 % v/v ethanol). 5.4.3 This test method measures cellular-ATP in a single measurement (as pg ATP/mL). Test Method D7463 detects total ATP (as RLU) and extra-cellular ATP (as RLU) using two separate analyses and permits computation of cellular-ATP (as RLU) as the difference between total and extracellular ATP. 5.4.4 Test Method D7463 suggests a nominal 500 mL fuel sample volume. This test method suggests a nominal 20 mL fuel sample. 5.5 This test method can be used with all fuels specified in Specifications D396 , D975 , D1655 , D2069 , D2880 , D3699 , D6751 , and D7467 and other fuels with nominal viscosities ≤ 75 cSt at 20° ± 2°. 5.6 The ATP test provides rapid test results that reflect the total bioburden in the sample. It thereby reduces the delay between test initiation and data capture, from the 36 h to 48 h (or longer) required for culturable colonies to become visible, to approximately 5 min. 5.7 Although ATP data generally covary with culture data in fuel and fuel-associated water, different factors affect ATP concentration than those that affect culturability. 5.7.1 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 may be viable but not detected by any one culture test. 4 5.7.2 ATP concentration is affected by: the microbial species present, the physiological states of those species, and the total bioburden (see Appendix X1 ). 5.7.2.1 One example of the species effect is that the amount of ATP per cell is substantially greater for active fungal cells than bacteria. 5.7.2.2 Within a species, cells that are more metabolically active will have more ATP per cell than dormant cells, such as fungal spores. Because fungal spores are more hydrophobic than active fungal material (mycelium), spores may be the only indicator of fungal proliferation when fuel samples are taken from some fuel systems, but they will not be detected by a test for ATP. 5.7.2.3 The greater the total bioburden, the greater the ATP concentration in a sample. 5.7.3 The possibility exists that the rinse step ( 11.15 ) may not eliminate all chemical substances that can interfere with the bioluminescence reaction ( 11.37 ). 5.7.3.1 The presence of any such interferences can be evaluated by performing a standard addition test series or dilution series as described in Appendix X4 . The precision statement in Section 13 will not apply. 5.8 As explained in Test Method D7978 , there are inherent difficulties in assessing precision of microbiological procedures for fuels on account of the inherent variability of the determinant and various determinable and indeterminable sources of inaccuracy (see Guide D7847 ). 5.8.1 The precision of any microbiological analytical method will generally be considerably less than that of methods widely used in the petroleum industry for analysis of physical and chemical properties of fuels.