Quantifying aquatic viruses using epifluorescence microscopy and SYBR staining has come to a standstill with the end of the production of Anodisc (0.02孩 filters. While flow cytometry is an alternative, not everyone has this expensive equipment and it requires considerable expertise, attention to detail and consistency across operators and environments. To quantify the number of bacteria and viruses in a range of natural waters from sub-tropical Australia we used a scanning fluorescence spectrophotometer, in conjunction with a DNA and RNA stain (SYBR Gold) to produce fluorescence excitation-emission matrices (EEM). We corrected for dissolved humic substances in water samples that sometimes changed the position and shape of the DNA/RNA SYBR spectral peak. Bacterial and viral abundance were determined using this EEM method across a range of environments and they correspond with abundance determined using the traditional method of SYBR staining of bacteria and viruses collected on (our last remaining) Anodisc filters and counted using epifluorescence microscopy.