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 ≤ 1 month in − 80 °C.