Flow cytometric enumeration of DNA-stained oceanic planktonic protists

The aim of this study was to test the practicality of enumerating fixed, DNA-stained heterotrophic protists (H) and phototrophic protists (P) in contrasting regions of the Atlantic Ocean. Oceanic protists were enumerated using a standard flow cytometer (FACSort, BD) at an enhanced flow rate of up to 1.0 mL min⁻¹ to increase numbers of counted cells. The enumeration error of protists decreased hyperbolically from 30–40 to < 5% corresponding to the number (<100 to > 2000) of enumerated cells. H and P were discriminated using the extra red chlorophyll-derived plastidic fluorescence of the latter. The relationship between counts of stained and unstained fixed and unfixed P was statistically close to 1:1, confirming the accuracy of stained protist counting by flow cytometry and adequate discrimination of P from H cells. The estimated average abundance of H in the surface mixed layer of the southern and northern oligotrophic gyres was remarkably similar, with 400 ± 140 and 450 ± 60 cells mL⁻¹, respectively, adding further evidence to the suggestion that these regions are in steady state. In agreement with earlier studies in more productive aquatic environments, a significant correlation (correlation coefficient 0.84, P < 0.0001) was found between the H and the total bacterioplankton numbers, with an average ratio of ∼1300 prokaryotes to 1 H cell, suggesting a relatively constant trophic interaction between these two groups. This study demonstrates that flow cytometric enumeration of protists is ∼100 times faster compared with microscopy and, thus, represents a major improvement for quantifying protists in ocean waters, including oligotrophic gyres.