High-throughput viral enumeration of aquatic ecosystems via flow cytometry.

For the past 25 years, flow cytometry (FCM) has been a gold standard for the direct measurement of virus-like particles (VLPs) in aquatic ecosystems. Flow cytometry allows for higher throughput and costs less than alternative enumeration methods, leading to its broad usage in aquatic viral ecology. A major challenge associated with flow cytometry is the degradation of VLPs over time, making the use of high-throughput plates not possible, thus lowering the overall throughput. It has also been difficult to maintain a method with low contamination, high signal-to-noise ratios, and observations of real VLPs vs fake particles. For these reasons, the use of flow cytometry has rapidly declined over the years due to the advent of massively parallel sequencing. Here, we describe a high-throughput method (HTM) in a 96-well plate format that provides a hands-free approach to viral enumeration. In a standard run, 60 samples can be measured for VLPs within 2.25 h, which is ~1 h faster than the standard single-tube approach and ~1.5 h faster than epifluorescence microscopy (EFM). Direct measurement of VLPs still provides a window into the viral-host interactions within aquatic ecosystems, which can be rapidly measured and resolved in a high-throughput manner.IMPORTANCEViruses represent the most numerically abundant biological entity on planet Earth. Enumeration of viruses within aquatic ecosystems (e.g., lakes and oceans) is essential to viral ecology and our understanding of their role within ecosystems. However, reliable and time-efficient high-throughput methods (HTM) for their enumeration are still needed. Here, we present a time saving HTM to count virus-like particles (VLPs) in a consistent and robust manner.