A comparison of TO-PRO-1 iodide and 5-CFDA-AM staining methods for assessing viability of planktonic algae with epifluorescence microscopy

Two fluorescent dyes, TO-PRO-1 iodide and 5-CFDA-AM, were evaluated for LIVE/DEAD assessment of unicellular marine algae Brachiomonas submarina and Tetraselmis suecica. Epifluorescence microscopy was used to estimate cell viability in predetermined mixtures of viable and non-viable algal cells and validated using microplate growth assay as reference measurements. On average, 5-CFDA-AM underestimated live cell abundance by ~25% compared with viability estimated by the growth assay, whereas TO-PRO-1 iodide provided accurate viability estimates. Furthermore, viability estimates based on staining with TO-PRO-1 iodide were not affected by a storage period of up to one month in −80°C, making the assay a good candidate for routine assessment of phytoplankton populations in field and laboratory studies. ► We compared TO-PRO-1 iodide and 5-CFDA-AM for LIVE/DEAD assessment of algae. ► Using radiation-killed cells, test mixtures with 0–100% dead cells were prepared. ► Viability was estimated with epifluorescence microscopy and microplate growth assay. ► Whereas TO-PRO-1 provided accurate estimate, CFDA underestimated viability by ~25%. ► Staining with TO-PRO-1 was not affected by a storage for ≤1month in −80°C.