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Two new fluorochromes, PicoGreenm and SYTOX Green@ stain (Molecular Probes, Inc.), are useful with flow cytometry for quantitative detection of cellular DNA in a variety of marina phytoplankton. The basic instrument con.iguration of modern lozupower Pow cytomters (15 mW, 488 nm excitation) is sensitive enough to detect the DNA signal in nearly all of the 121 strains @om 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 2 15 nm) showing no overlap with the autojuorescence of the plankton pigments in the red band, 3) high fluorescence yield of the DNA-dye complex with an increase in fluorescence > 1 00-fold compared to the unstained cell, and 4) dyes can be used to quanta2 double-stranded DNA. The high sensitivity allowed the quanta$cation 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. (Eustagmatophyceae) stained poorly, and a f e u members ofthe Chlorophyceae and Pelagophyceae showed poor staining occasionally. In general, maximal fluorescence was achieved within 15 min after addition of the dye. Although the PzcoGreen dye stained some living phytoplankton species, preservation is recommended f m quantitation. SYTOX Green did not stain live cells. The combination ofthe dyes, therefore, allows the discrimination between live and dead cells in some algal groups (Prochlorococcus, diatoms, prasinophytes, and pelagophytes). Parafaaldehyde was preferred over glutaraldehyde for fixation to avoid (induced) green autofluorescence.Total DNA values measured in 90 algal species (ca. 121 strains) varied a factor of 20,000. The lowest values were found in Prochlorococcus and the highest in a large dinojagellate (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 pelagtc phytoplankton.

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The respiratory activity of marine bacteria is an important indication of the ecological functioning of these organisms in marine ecosystems. The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) is reduced intracellularly in respiring cells to an insoluble, fluorescent precipitate. This product is detectable and quantifiable by flow cytometry in individual cells. We describe here an evaluation of flow cytometry for measuring CTC activity in natural assemblages of marine bacteria growing in dilution cultures. We found that more CTC-positive cells are detected by flow cytometry than by visual epifluorescence microscopy. Samples can be stored refrigerated or frozen in liquid nitrogen for at least 4 weeks without a significant loss of total cells, CTC-positive cells, or CTC fluorescence. Cytometry still may not detect all active cells, however, since the dimmest fluorescing cells are not clearly separated from background noise. Reduction of CTC is very fast in most active cells, and the number of active cells reaches 80% of the maximum number within 2 to 10 min. The proportion of active cells is correlated with the growth rate, while the amount of fluorescence per cell varies inversely with the growth rate. The CTC reduction kinetics in assemblages bubbled with nitrogen and in assemblages bubbled with air to vary the oxygen availability were the same, suggesting that CTC can effectively compete with oxygen for reducing power. A nonbubbled control, however, contained more CTC-positive cells, and the amount of fluorescence per cell was greater. Activity may have been reduced by bubble-induced turbulence. Addition of an artificial reducing agent, sodium dithionite, after CTC incubation and fixation resulted in a greater number of positive cells but did not “activate” a majority of the cells. This indicated that some of the negative cells actually transported CTC across their cell membranes but did not reduce it to a detectable level. Automated analysis by flow cytometry allows workers to study single-cell variability in marine bacterioplankton activity and changes in activity on a small temporal or spatial scale.

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