Outer Limits Of Flow Cytometry To Quantify Viruses In Water

The use of flow cytometry (FCM) with environmental or clinical samples to enumerate viruses (flow virometry) has become popular with the development of sensitive fluorescent dyes that bind to nucleic acids, yet there is no quantitative evidence of the sensitivity and accuracy for flow virometry as applied to aquatic environments. Rigorous background controls are missing. Here we address the gap in our knowledge of how the background interferes with interpreting flow virometry results. To remove background interference, we discovered it was essential to separate viruses from their water matrix and resuspended them in virus free Tris-EDTA buffer. Background substances and a SYBR Green dye colloid produce "virus-like" artifacts that generate false-positive viral counts. We show that neither human enteric viruses nor bacteriophage surrogates of a small genome size (<150 kbp) can be detected using standard FCM. We concluded that the current use of flow virometry is neither sensitive nor accurate enough to quantify most natural viral populations in aquatic environments. We recommend improved procedures for unambiguously proving the FCM signal is indeed viral. However, flow virometry is still limited by the inability of instruments to detect most natural viruses, yet other methods with the example of a sensitive prototype flow cytometer developed for nanomaterials (with a throughput of 10000 viruses per minute) could have the potential for online monitoring of viral abundance in both natural and engineered environments.