1.1 本试验方法旨在评估试验物质灭活营养细菌、病毒、真菌、分枝杆菌和细菌孢子的能力 ( 1. 7. ) 拉丝不锈钢磁盘载体，代表坚硬、无孔的环境表面和医疗器械。它还设计了幸存者，可以与不少于三个控制载体的平均值进行比较，以确定是否达到了性能标准。为了对结果进行适当的统计评估，试验接种物中活生物体的数量应足够高，以考虑性能标准和结果中的实验变化。 1.2 测试方案不包括任何擦拭或摩擦动作。 因此，它不是为测试湿巾而设计的。 1.3 本试验方法应由受过微生物学培训的人员在设计和配备用于在适当的生物安全水平下处理传染源的设施中进行 ( 8. ) . 1.4 研究者有责任确定是否需要《药物非临床研究质量管理规范》（GLP），并在适当的情况下遵守这些规范（EPA提交的40 CFR第160部分和FDA提交的21 CFR第58部分）。 1.5 在本试验方法中，所有应用均使用国际单位制，但距离除外，在这种情况下，使用英寸，公制单位紧随其后。 1.6 本标准并不旨在解决与其使用相关的所有安全问题（如果有的话）。 本标准的使用者有责任在使用前建立适当的安全、健康和环境实践，并确定监管限制的适用性。 1.7 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 该测试的设计消除了通过冲洗造成的活生物体的任何损失，从而可以使用比基于简单MPN估计的方法所需的更少的测试载体来产生统计上有效的数据。 5.2 测试的严格性是通过使用土壤负载、拉丝不锈钢载体表面的微观形貌以及许多消毒剂应用中典型的测试物质与表面积的较小比率来提供的。因此，被评估的受试物质面临着合理的挑战，同时允许从接种的载体中有效回收受试生物。基本测试中的金属盘也与各种活性物质兼容。 5.3 载体的设计使得可以在每个载体上放置精确测量体积的测试生物体（10μL）以及对照液或测试物质（50μL）。 5.4 接种物放置在每个圆盘的中心，而受试物质的体积几乎覆盖了整个圆盘表面，从而几乎消除了任何未暴露的生物体的风险。 5.5 在所有测试中，除病毒测试外，在接触时间结束时，添加10毫升洗脱液/稀释剂可立即将测试物质稀释1:200。虽然这一步骤本身可能足以阻止大多数活性物质的杀菌活性，但测试方案允许在需要时向洗脱液/稀释剂中添加特定的中和剂。除病毒外，膜过滤步骤还允许处理来自测试载体的全部洗脱液，从而捕获并随后检测可能存在的少量活生物体。随后用盐水冲洗膜过滤器也降低了将任何抑制性残留物带到回收介质上的风险。 通过用少量试验生物进行挑战，需要验证试验物质的中和过程。 5.6 在针对病毒的测试中，在接触时间结束时添加1 mL缓冲液可实现测试物质的1:20稀释，同时保持洗脱液的体积合理较小，以便滴定细胞培养物中的大部分或全部洗脱液。通过用低数量的试验病毒感染单位挑战残留消毒负荷，需要确认试验物质的中和。由于病毒检测系统是间接的，因此需要额外的步骤来证明适当的细胞系预先暴露于任何残留的消毒剂或消毒剂/中和剂混合物不会干扰低水平病毒攻击的检测（见 附录X1 ). 注1: In 5.5 和 5.6 ，建议分别使用10 mL和1 mL的体积，而不是9.95 mL和950μL，以便于分配洗脱液。 5.7 本试验中的土壤负荷是三种蛋白质（高分子量蛋白质、低分子量肽和粘液物质）的混合物，旨在代表杀微生物化学物质在田间条件下可能遇到的身体分泌物、排泄物或其他外来物质。它适用于与这里包括的所有类型的测试生物一起工作。土壤负荷的成分很容易获得，并且比动物血清的变异性小得多。 5.8 如果产品标签上没有指定蒸馏水或其他稀释剂，则假设试验物质的稀释剂是自来水。 由于自来水的质量在地理和时间上差异很大，因此该测试方法使用具有指定和记录的硬度水平的水来制备测试物质的稀释液，这些稀释液在使用前需要在水中稀释。而硬度至少为300ppm（以CaCO计）的水 3. 建议在测试前咨询当地有关硬水使用的法规。 5.9 附件列出了在评估用于环境表面或医疗器械的消毒剂的杀菌活性时经常使用的微生物。每种生物体的培养条件也包括在附件中。根据所需的标签声明和目标监管机构的要求，可以选择一种或多种列出的生物体进行测试。 如果要使用所列以外的生物体（例如，在乳制品或酿造行业），必须提供明确的理由，必须验证培养基和生长条件的细节，并在测试报告中明确说明。

1.1 This test method is designed to evaluate the ability of test substances to inactivate vegetative bacteria, viruses, fungi, mycobacteria, and bacterial spores ( 1- 7 ) on disk carriers of brushed stainless steel that represent hard, nonporous environmental surfaces and medical devices. It is also designed to have survivors that can be compared to the mean of no less than three control carriers to determine if the performance standard has been met. For proper statistical evaluation of the results, the number of viable organisms in the test inoculum should be sufficiently high to take into account both the performance standard and the experimental variations in the results. 1.2 The test protocol does not include any wiping or rubbing action. It is, therefore, not designed for testing wipes. 1.3 This test method should be performed by persons with training in microbiology in facilities designed and equipped for work with infectious agents at the appropriate biosafety level ( 8 ) . 1.4 It is the responsibility of the investigator to determine whether Good Laboratory Practice Regulations (GLPs) are required and to follow them where appropriate (40 CFR, Part 160 for EPA submissions and 21 CFR, Part 58 for FDA submissions). 1.5 In this test method, SI units are used for all applications, except for distance in which case inches are used and metric units follow. 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. ====== Significance And Use ====== 5.1 The design of this test eliminates any loss of viable organisms through wash off, thus making it possible to produce statistically valid data using many fewer test carriers than needed for methods based on simple MPN estimates. 5.2 The stringency in the test is provided by the use of a soil load, the microtopography of the brushed stainless steel carrier surface, and the smaller ratio of test substance to surface area typical for many disinfectant applications. Thus, the test substance being assessed is presented with a reasonable challenge while allowing for efficient recovery of the test organisms from the inoculated carriers. The metal disks in the basic test are also compatible with a wide variety of actives. 5.3 The design of the carriers makes it possible to place onto each a precisely measured volume of the test organism (10 μL) as well as the control fluid or test substance (50 μL). 5.4 The inoculum is placed at the center of each disk whereas the volumes of the test substance covers nearly the entire disk surface, thus virtually eliminating the risk of any organisms remaining unexposed. 5.5 In all tests, other than those against viruses, the addition of 10 mL of an eluent/diluent gives a 1:200 dilution of the test substance immediately at the end of the contact time. While this step in itself may be sufficient to arrest the microbicidal activity of most actives, the test protocol permits the addition of a specific neutralizer to the eluent/diluent, if required. Except for viruses, the membrane filtration step also allows processing of the entire eluate from the test carriers and, therefore, the capture and subsequent detection of even low numbers of viable organisms that may be present. Subsequent rinsing of the membrane filters with saline also reduces the risk of carrying any inhibitory residues over to the recovery medium. Validation of the process of neutralization of the test substance is required by challenge with low numbers of the test organism. 5.6 In tests against viruses, addition of 1 mL of buffer at the end of the contact time achieves a 1:20 dilution of the test substance while keeping the volume of the eluate reasonably small to allow for the titration of most or all of the eluate in cell cultures. Confirmation of neutralization of the test substance is required by challenge of a residual disinfection load with low numbers of infective units of the test virus. Since the virus assay system is indirect, an additional step is required to demonstrate that prior exposure of the appropriate cell line to any residual disinfectant or disinfectant/neutralizer mixture does not interfere with the detection of a low level of virus challenge (See Appendix X1 ). Note 1: In 5.5 and 5.6 , volumes of 10 mL and 1 mL are recommended instead of 9.95 mL and 950 μL, respectively, for ease of dispensing the eluent. 5.7 The soil load in this test is a mixture of three types of proteins (high molecular weight proteins, low molecular weight peptides, and mucous material) designed to represent body secretions, excretions, or other extraneous substances that microbicidal chemicals may encounter under field conditions. It is suitable for working with all types of test organisms included here. The components of the soil load are readily available and subject to much less variability than animal sera. 5.8 If distilled water or other diluent is not to be specified on the product label, the diluent for the test substance is assumed to be tap water. Since the quality of tap water varies considerably both geographically and temporally, this test method incorporates the use of water with a specified and documented level of hardness to prepare use-dilutions of test substance that require dilution in water before use. While water with a hardness of at least 300 ppm as CaCO 3 is recommended consult local regulations regarding use of hard water prior to testing. 5.9 The Annex contains a list of those organisms that are often used in assessing the microbicidal activities of disinfectants for use on environmental surfaces or medical devices. Culture conditions for each organism are also included in the Annex. Depending on the label claim(s) desired and the requirements of the target regulatory agency, one or more of the organisms listed may be selected for the testing. If organisms other than those listed are to be used (for example, in the dairy or brewing industries), a clear justification must be provided and details of the culture media and growth conditions must be validated and clearly specified in test reports.