Cellular DNA content of marine phytoplankton using two new fluorochromes: Taxonomic and ecological implications

Two new fluorochromes, PicoGreen(R) and SYTOX Green(TM) stain (Molecular Probes, inc.), are useful with flow cytometry for quantitative detection of cellular DNA in a variety of marine phytoplankton. The basic instrument configuration of modern low-power flow cytometers (15 mW, 488 nm excitation) is sensitive enough to detect the DNA signal in nearly all of the 121 strains (from 12 taxonomic classes) examined. The major advantages of these dyes over others are 1) suitability for direct use in seawater, 2) green fluorescence emission of the DNA-dye complex (wavelength 525 +/- 15 nm) showing no overlap with the autofluorescence of the plankton pigments in the red band, 3) high fluorescence yield of the DNA-dye complex with an increase in fluorescence >100-fold compared to the unstained cell, and 4) dyes can be used to quantify double-stranded DNA. The high sensitivity allowed the quantification of the DNA of the smallest known phytoplankter (Prochlorococcus) as well as bacteria found in some of the algal cultures. Of the 12 taxonomic classes tested, only the 3 Nannochloropsis spp. (Eustigmatophyceae) stained poorly, and a few members of the Chlorophyceae and Pelagophyceae showed poor staining occasionally. In general, maximal fluorescence was achieved within 15 min after addition of the dye. Although the PicoGreen dye stained some living phytoplankton species, presentation is recommended for quantitation. SYTOX Green did not stain live cells. The combination of the dyes, therefore, allows the discrimination between live and dead cells in some algal groups (Prochlorococcus, diatoms, prasinophytes, and pelagophytes). Paraformaldehyde was preferred over glutaraldehyde for fixation to avoid (induced) green autofluorescence. Total DNA values measured in 90 algal species (ca. 121 strains) vaned by a factor of 20,000. The lowest values were found in Prochlorococcus and the highest in a large dinoflagellate (Prorocentrum micans). DNA content appears to be a scaleable cell component covarying with the carbon and nitrogen contents of the phytoplankton cells. This covariation allows the total DNA content to be used as an accurate, independent estimate of total cell carbon biomass in unicellular pelagic phytoplankton.