1.1 本试验方法提供了捕获、提取和量化与水溶性金属加工液（MWF）中发现的微生物相关的三磷酸腺苷（ATP）含量的方案。 1.2 使用生物发光酶测定法测量ATP，由此产生的光量与样品中ATP的浓度成比例。光作为相对光单位（RLU）进行定量产生和测量，相对光单位通过与ATP标准进行比较并计算为pg-ATP/mL进行转换。 1.3 本试验方法同样适用于实验室或现场。 1.4 该测试方法检测的ATP浓度范围为4.0 pg ATP/mL至40万pg ATP/mL。 1.5 如果可以克服干扰，生物发光是一种可靠且经验证的ATP鉴定和定量方法。该方法不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。 1.6 以国际单位制表示的数值应视为标准值。本标准不包括其他计量单位。 1.7 本标准并非旨在解决与其使用相关的所有安全问题（如有）。 本标准的用户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。 1.8 本国际标准是根据世界贸易组织技术性贸易壁垒（TBT）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》中确立的国际公认标准化原则制定的。 ====意义和用途====== 5.1 该方法测量样品中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，ATP的存在是金属加工液中总微生物污染的指标。ATP与非生物来源的物质无关。 5.2 试验方法 D4012年 验证ATP作为可培养细菌数据的替代物（指南 E1326 ). 5.3 该方法与试验方法不同 D4012年 因为它消除了以往导致ATP测试无法使用复杂有机液体（如MWF）的干扰。 5.4 ATP测试提供快速测试结果，反映样本中的总生物负荷。 因此，它将测试启动和数据采集之间的延迟从可培养菌落可见所需的36小时到48小时（或更长）减少到约5分钟。 5.5 虽然ATP数据通常与MWF中的培养数据共变， 4. 影响ATP浓度的因素不同于影响可培养性的因素。 5.5.1 可培养性主要受捕获的微生物在特定生长条件下在提供的生长培养基上增殖的能力影响。据估计，不到1 % 在任何给定的生长条件下，存在于环境样本中的物种将形成菌落。 5. 5.5.2 ATP浓度受以下因素影响:存在的微生物种类、这些种类的生理状态和总生物负荷（见 附录X1 ). 5.5.2.1 物种效应的一个例子是，真菌的每个细胞的ATP量远远大于细菌。 5.5.2.2 在一个物种内，代谢更活跃的细胞比休眠细胞具有更多的ATP。 5.5.2.3 总生物负荷越大，样品中的ATP浓度越高。 5.5.3 存在冲洗步骤的可能性( 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( 11.39 ). 5.5.3.1 任何此类干扰的存在都可以通过执行中所述的标准添加测试系列来评估 附录X3 . 5.5.3.2 干扰化学品的任何影响将反映为相对于从不包含干扰化学品的流体中获得的数据的偏差。

1.1 This test method provides a protocol for capturing, extracting, and quantifying the adenosine triphosphate (ATP) content associated with microorganisms found in water-miscible metalworking fluids (MWFs). 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 (RLUs) which are converted by comparison with an ATP standard and computation to pg ATP/mL. 1.3 This test method is equally suitable for use in the laboratory or field. 1.4 The test method detects ATP concentrations in the range of 4.0 pg ATP/mL to 400 000 pg ATP/mL. 1.5 Providing interferences can be overcome, bioluminescence is a reliable and proven method for qualifying and quantifying ATP. The method does not differentiate between ATP from different sources, for example, from different types of microorganisms, such as bacteria and fungi. 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. ====== Significance And Use ====== 5.1 This method measures the concentration of ATP present in the sample. ATP is a constituent of all living cells, including bacteria and fungi. Consequently, the presence of ATP is an indicator of total microbial contamination in metalworking fluids. ATP is not associated with matter of non-biological origin. 5.2 Test Method D4012 validated ATP as a surrogate for culturable bacterial data (Guide E1326 ). 5.3 This method differs from Test Method D4012 in that it eliminates interferences that have historically rendered ATP testing unusable with complex organic fluids such as MWFs. 5.4 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. 5.5 Although ATP data generally covary with culture data in MWF, 4 different factors affect ATP concentration than those that affect culturability. 5.5.1 Culturability is affected primarily by the ability of captured microbes to proliferate on the growth medium provided, under specific growth conditions. It has been estimated that less than 1 % of the species present in an environmental sample will form colonies under any given set of growth conditions. 5 5.5.2 ATP concentration is affected by: the microbial species present, the physiological states of those species, and the total bioburden (see Appendix X1 ). 5.5.2.1 One example of the species effect is that the amount of ATP per cell is substantially greater for fungi than bacteria. 5.5.2.2 Within a species, cells that are more metabolically active will have more ATP per cell than dormant cells. 5.5.2.3 The greater the total bioburden, the greater the ATP concentration in a sample. 5.5.3 The possibility exists that the rinse step ( 11.15 ) may not eliminate all chemical substances that can interfere with the bioluminescence reaction ( 11.39 ). 5.5.3.1 The presence of any such interferences can be evaluated by performing a standard addition test series as described in Appendix X3 . 5.5.3.2 Any impact of interfering chemicals will be reflected as bias relative to data obtained from fluid that does not contain interfering chemicals.