1.1 该测试方法涵盖了一种捕获、提取和量化与实验室培养物和水中浮游生物和外周生物样品中常见微生物相关的细胞三磷酸腺苷（cATP）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ATP的浓度成比例。光以相对光单位（RLU）的形式产生和定量测量，通过与ATP标准进行比较并计算为pg-ATP/mL。 1.3 这种方法不能去除所有已知的化学干扰，已知的是530 nm±20 nm范围内发射的光或使在该范围内发射出的光熄灭。它不应用于测定溶解有机化合物、重金属或总溶解固体>1000 ppm的样品中的ATP浓度。已经开发了用于测定可能含有此类干扰的流体样品中ATP浓度的替代方法（试验方法 D7687 和 E2694 )。 1.4 ATP浓度的知识可能与微生物的活生物量或代谢活性有关( 附录X1 )。 1.5 该测试方法具有高度的灵敏度、快速性、准确性和再现性。 1.6 分析员应意识到，精度声明仅适用于试剂水中的测定，而不一定适用于被测基质中的测定。 1.7 这种测试方法同样适用于实验室或现场。 1.8 该方法通常检测0.1 pg cATP/mL范围内的cATP浓度（–1.0Log 10 [pg cATP/mL]）至 4000 000 pg cATP/mL（6.6对数 10 [pg-cATP/mL]）。 1.9 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定量ATP的方法，尽管该方法不能区分来自不同来源的ATP，例如来自不同类型微生物的ATP，如细菌、真菌、藻类和原生动物。 1.10 以国际单位制表示的数值应视为标准。本标准中不包括其他计量单位。 1.11 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。 1.12 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 测定培养物、水、废水以及从地表水中采集的浮游生物和周边生物样本中活微生物生物量的快速和常规程序通常至关重要。然而，诸如直接显微镜计数、浊度、有机化学分析、细胞标记和平板计数等经典技术是昂贵、耗时的，或者往往低估了总数。此外，其中一些方法不能区分活细胞和非活细胞。 5.2 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、藻类、原生动物和真菌。因此，细胞ATP的存在是水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。 5.3 ATP（荧光素-萤光素酶）法是一种快速、灵敏地测定活微生物生物量的方法。ATP是生命过程的主要能量供体，与非生命碎屑物质无关，单位生物量的ATP含量（以重量表示）相对恒定。 （每个细胞的ATP随物种和生物体的生理状态而变化。） 5.4 此测试方法可用于: 5.4.1 估计培养物和水中的可行微生物生物量。 5.4.2 估计浮游生物和周边生物样本中的总活生物量。 5.4.3 如果每个细胞的cATP含量（或cATP的平均量）已知，则估计单种培养物中活细胞的数量。 5.4.4 通过水样的大小分级来估计和区分浮游动物、浮游植物、细菌和真菌的cATP。 5.4.5 在夹带研究的毒性测试中，以及在微生物种群或组合处于压力下的其他情况下，测量微生物的死亡率。 5.5 该试验方法类似于试验方法 D7687 和 E2694 除了取样的体积，以及试验方法中使用的洗涤和干燥步骤的省略 D7687 和 E2694 删除干扰( 1.3 )。 5.6 尽管ATP数据通常与水样中的培养数据一致，但影响cATP浓度的因素与影响可培养性的因素不同。 6.5.6 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。 3. 5.6.2 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载（见 附录X1 )。 5.6.2.1 物种效应的一个例子是，活性真菌细胞的每个细胞的ATP量明显大于细菌( 附录X1 )。 5.6.2.2 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。 5.6.2.3 总生物负载越大，样品中的ATP浓度就越高。

1.1 This test method covers a protocol for capturing, extracting and quantifying the cellular adenosine triphosphate (cATP) content associated with microorganisms normally found in laboratory cultures and waters in plankton and periphyton samples from waters. 1.2 The ATP is measured using a bioluminescence enzyme assay, whereby light is generated in amounts proportional to the concentration of 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 and computation to pg ATP/mL. 1.3 This method does not remove all known chemical interferences, known to either luminesce in the 530 nm ± 20 nm range, or to quench light emitted in that range. It should not be used to determine ATP concentrations in samples with dissolved organic compounds, heavy metals or >10 000 ppm total dissolved solids. Alternative methods have been developed for determining ATP concentrations in fluids samples likely to contain such interferences (Test Methods D7687 and E2694 ). 1.4 Knowledge of the concentration of ATP can be related to viable biomass or metabolic activity of microorganisms ( Appendix X1 ). 1.5 This test method offers a high degree of sensitivity, rapidity, accuracy, and reproducibility. 1.6 The analyst should be aware that the precision statement pertains only to determinations in reagent water and not necessarily in the matrix being tested. 1.7 This test method is equally suitable for use in the laboratory or field. 1.8 The method normally detects cATP concentrations in the range of 0.1 pg cATP/mL (–1.0Log 10 [pg cATP/mL]) to 4 000 000 pg cATP/mL (6.6 Log 10 [pg cATP/mL]) in 50 mL water samples. 1.9 Providing interferences can be overcome, bioluminescence is a reliable and proven method for qualifying and quantifying ATP, although the method does not differentiate between ATP from different sources, for example, from different types of microorganisms, such as bacteria, fungi, algae and protozoa. 1.10 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. 1.11 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. 1.12 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 A rapid and routine procedure for determining biomass of the living microorganisms in cultures, waters, wastewaters, and in plankton and periphyton samples taken from surface waters is frequently of vital importance. However, classical techniques such as direct microscope counts, turbidity, organic chemical analyses, cell tagging, and plate counts are expensive, time-consuming, or tend to underestimate total numbers. In addition, some of these methods do not distinguish between living and nonliving cells. 5.2 This test method measures the concentration of cellular-ATP present in the sample. ATP is a constituent of all living cells, including bacteria, algae, protozoa, and fungi. Consequently, the presence of cellular-ATP is an indicator of total metabolically active microbial contamination in water. ATP is not associated with matter of non-biological origin. 5.3 The ATP (luciferin-luciferase) method is a rapid, sensitive determination of viable microbial biomass. ATP is the primary energy donor for life processes, does not exist in association with nonliving detrital material, and the amount of ATP per unit of biomass (expressed in weight) is relatively constant. (ATP per cell varies with species and physiological state of the organism.) 5.4 This test method can be used to: 5.4.1 Estimate viable microbial biomass in cultures and waters. 5.4.2 Estimate the amount of total viable biomass in plankton and periphyton samples. 5.4.3 Estimate the number of viable cells in a unispecies culture if the cATP content (or if the average amount of cATP) per cell is known. 5.4.4 Estimate and differentiate between zooplanktonic, phytoplanktonic, bacterial, and fungal cATP through size fractionation of water samples. 5.4.5 Measure the mortality rate of microorganisms in toxicity tests in entrainment studies, and in other situations where populations or assemblages of microorganisms are placed under stress. 5.5 This test method is similar to Test Methods D7687 and E2694 except for the volumes sampled, and omission of wash and drying steps used in Test Methods D7687 and E2694 to remove interferences ( 1.3 ). 5.6 Although ATP data generally covary with culture data in water samples, different factors affect cATP concentration than those that affect culturability. 5.6.1 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. 3 5.6.2 ATP concentration is affected by: the microbial species present, the physiological states of those species, and the total bioburden (see Appendix X1 ). 5.6.2.1 One example of the species effect is that the amount of ATP per cell is substantially greater for active fungal cells than bacteria ( Appendix X1 ). 5.6.2.2 Within a species, cells that are more metabolically active will have more ATP per cell than dormant cells, such as fungal spores. 5.6.2.3 The greater the total bioburden, the greater the ATP concentration in a sample.