Activity of Individual Aquatic Bacteria by Flow Cytometry; Instrumentation and Protocols

该奖项将支持流式细胞仪和相关协议的开发，这些仪器和相关协议可以分析水生细菌，重点是它们的生长和生存状态。流式细胞术已经成为研究血细胞和各种可以在培养中生长的细胞和生物体的成熟研究工具。然而，它用于分析自然细菌种群，例如在水生系统中发现的那些细菌，由于这种细菌典型的小细胞尺寸，它的使用变得复杂。很少有商业仪器专门用于探测这些小目标。该项目的主要工作是对商用流式细胞仪进行额外的改装，以增强其确定水生细菌生长和存活状态的能力。输出信号将被组织起来进行处理，以反映根据DNA含量判断的混合种群的增长速度。另外两个活性探针将在系统中进行测试，一个是RNA含量，另一个是膜电位，这两个都是细菌生长和生存的重要指标。该设备在研究高纬度地区温室气体的微生物生成方面将特别重要，因为那里的冰盖和细菌活动对温度高度敏感。

This award will support the development of flow cytometry instrumentation and associated protocols that can analyze aquatic bacteria with emphasis on their growth and viability status. Flow cytometry has become an established research tool for study of blood cells and of a variety of cells and organisms that can be grown in culture. However, its use to analyze natural populations of bacteria such as those found in aquatic systems is complicated by the small cell size typical of such bacteria. Few commercial instruments are dedicated to the detection of these small targets. The main effort of this project is to design additional modifications to a commercial flow cytometer to enhance its ability to determine the growth and viability status of aquatic bacteria. Output signals will be organized for manipulation to reflect the rate of growth in mixed populations as judged by DNA content. Two additional probes for activity will be tested in the system, one for RNA content and the other for membrane potential, both important indicators of bacterial growth and viability. The equipment will be particularly important in study of the microbial generation of greenhouse gasses at high latitudes where both ice cover and bacterial activity is highly temperature sensitive.