Flow cytometric enumeration of marine viral populations at low abundanceS

Flow cytometric enumeration has advanced our ability to analyze aquatic virus samples and thereby our understanding of the ecological role viruses play in the oceans. However, low virus abundances are underestimated using the current flow cytometry (FCM) protocol. Our results revealed that low dilutions (<30-fold) not only decreased the total virus count but also limited the ability to differentiate between virus clusters. Here we report a simple and efficient method optimization for improving virus counts and optical resolution at low abundances. Raising the pH of the Tris-EDTA (TE) buffer to 8.2 successfully countered the effect of insufficient buffering capacity at low dilutions, which is caused by the higher proportion of acidic glutaraldehyde fixative in the final sample. The higher buffer pH did not interfere with virus counts at higher dilutions. We therefore recommend amendment of the standard FCM aquatic virus enumeration protocol using a TE buffer with pH 8.2 as a simple and efficient improvement.