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ABSTRACT Standard methods for calculating microbial growth rates (μ) through the use of proxies, such as in situ fluorescence, cell cycle, or cell counts, are critical for determining the magnitude of the role bacteria play in marine carbon (C) and nitrogen (N) cycles. Taxon-specific growth rates in mixed assemblages would be useful for attributing biogeochemical processes to individual species and understanding niche differentiation among related clades, such as found in Synechococcus and Prochlorococcus . We tested three novel DNA sequencing-based methods (iRep, bPTR, and GRiD) for evaluating the growth of light-synchronized Synechococcus cultures under different light intensities and temperatures. In vivo fluorescence and cell cycle analysis were used to obtain standard estimates of growth rate for comparison with those of the sequence-based methods (SBM). None of the SBM values were correlated with growth rates calculated by standard techniques despite the fact that all three SBM were correlated with the percentage of cells in S phase (DNA replication) over the diel cycle. Inaccuracy in determining the time of maximum DNA replication is unlikely to account entirely for the absence of a relationship between SBM and growth rate, but the fact that most microbes in the surface ocean exhibit some degree of diel cyclicity is a caution for application of these methods. SBM correlate with DNA replication but cannot be interpreted quantitatively in terms of growth rate. IMPORTANCE Small but abundant, cyanobacterial strains such as the photosynthetic Synechococcus spp. are important because they contribute significantly to primary productivity in the ocean. These bacteria generate oxygen and provide biologically available carbon, which is essential for organisms at higher trophic levels. The small size and diversity of natural microbial assemblages mean that taxon-specific activities (e.g., growth rate) are difficult to obtain in the field. It has been suggested that sequence-based methods (SBM) may be able to solve this problem. We find, however, that SBM can detect DNA replication and are correlated with phases of the cell cycle but cannot be interpreted in terms of absolute growth rate for Synechococcus cultures growing under a day-night cycle, like that experienced in the ocean. 

The recent development of biological sensors has extended marine plankton studies from conducting laboratory bench work to in vivo and real-time observations. Flow cytometry (FCM) has shed new light on marine microorganisms since the 1980s through its single-cell approach and robust detection of the smallest cells. FCM records valuable optical properties of light scattering and fluorescence from cells passing in a single file in front of a narrow-collimated light source, recording tens of thousands of cells within a few minutes. Depending on the instrument settings, the sampling strategy, and the automation level, it resolves the spatial and temporal distribution of microbial marine prokaryotes and eukaryotes. Cells are usually classified and grouped on cytograms by experts and are still lacking standards, reducing data sharing capacities. Therefore, the need to make FCM data sets FAIR (Findability, Accessibility, Interoperability, and Reusability of digital assets) is becoming critical. In this paper, we present a consensus vocabulary for the 13 most common marine microbial groups observed with FCM using blue and red-light excitation. The authors designed a common layout on two-dimensional log-transformed cytograms reinforced by a decision tree that facilitates the characterization of groups. The proposed vocabulary aims at standardising data analysis and definitions, to promote harmonisation and comparison of data between users and instruments. This represents a much-needed step towards FAIRification of flow cytometric data collected in various marine environments.