CELLULAR DNA CONTENT OF MARINE-PHYTOPLANKTON USING 2 NEW FLUOROCHROMES - TAXONOMIC AND ECOLOGICAL IMPLICATIONS

Two new fluorochromes, PicoGreen(R) and SYTOX Green(TM) stain (Molecul ar Probes, inc.), are useful with flow cytometry for quantitative dete ction of cellular DNA in a variety of marine phytoplankton. The basic instrument configuration of modern low-power flow cytometers (15 mW, 4 88 nm excitation) is sensitive enough to detect the DNA signal in near ly all of the 121 strains (from 12 taxonomic classes) examined. The ma jor advantages of these dyes over others are 1) suitability for direct use in seawater, 2) green fluorescence emission of the DNA-dye comple x (wavelength 525 +/- 15 nm) showing no overlap with the autofluoresce nce of the plankton pigments in the red band, 3) high fluorescence yie ld of the DNA-dye complex with an increase in fluorescence >100-fold c ompared to the unstained cell, and 4) dyes can be used to quantify dou ble-stranded DNA. The high sensitivity allowed the quantification of t he DNA of the smallest known phytoplankter (Prochlorococcus) as well a s bacteria found in some of the algal cultures. Of the 12 taxonomic cl asses tested, only the 3 Nannochloropsis spp. (Eustigmatophyceae) stai ned poorly, and a few members of the Chlorophyceae and Pelagophyceae s howed poor staining occasionally. In general, maximal fluorescence was achieved within 15 min after addition of the dye. Although the PicoGr een dye stained some living phytoplankton species, presentation is rec ommended for quantitation. SYTOX Green did not stain live cells. The c ombination of the dyes, therefore, allows the discrimination between l ive and dead cells in some algal groups (Prochlorococcus, diatoms, pra sinophytes, and pelagophytes). Paraformaldehyde was preferred over glu taraldehyde for fixation to avoid (induced) green autofluorescence. To tal DNA values measured in 90 algal species (ca. 121 strains) vaned by a factor of 20,000. The lowest values were found in Prochlorococcus a nd the highest in a large dinoflagellate (Prorocentrum micans). DNA co ntent appears to be a scaleable cell component covarying with the carb on and nitrogen contents of the phytoplankton cells. This covariation allows the total DNA content to be used as an accurate, independent es timate of total cell carbon biomass in unicellular pelagic phytoplankt on.