DOI: 10.1594/pangaea.963430
title: Flow cytometry dataset from CTD casts showing the abundance of microorganisms (smaller than 20 µm) during the Arctic MOSAiC expedition

description: This dataset gives an overview of the abundance of microorganisms (smaller than 20 µm) enumerated using flow cytometry (FCM) during the Multidisciplinary drifting observatory for the study of Arctic Climate (MOSAiC) sampled from ship-based and on-ice CTD rosettes during leg 1, 2, 3, 4 and 5 (November 2019 – September 2020). Additional expedition and sampling details can be found in the ECO-overview paper (Fong et al., to be submitted to Elementa). We thank all persons involved in the expedition of the Research Vessel Polarstern during MOSAiC in 2019-2020 (AWI_PS122_00) as listed in Nixdorf et al. (2021).Flow cytometry (FCM) is a fast, high-throughput method to enumerate the abundance of microorganism (smaller than 20 µm). FCM uses the hydrodynamic focusing of a laminar flow to separate and line up microscopic particles. When particles pass a laser beam, the generated light scattering can be used to estimate their cell size, obtain information about cell granularity and surface characteristics and determine fluorescence from inherent pigments or applied stains, such as DNA binding ones. Photosynthetic microorganisms have auto-fluorescent pigments, such as chlorophylls which in combination with the light scattering properties (cell size) or surface properties, can be used to group them into clusters of similar or identical organism types. Heterotrophic microorganisms, including archaea, bacteria and heterotrophic nanoflagellates, and virus do not have fluorescent pigments and require staining, for example using SYBR Green to stain Nucleic Acids (DNA/RNA) in order to distinguish these cells from other organic and inorganic particles in the sample. Samples for flow cytometric analysis were taken in triplicates or quadruplicates of 1.8 mL of sample water and fixed with 36 μL 25 % glutaraldehyde (0.5 % final concentration) at 4 °C in the dark for approximately 2 hours, then flash frozen in liquid nitrogen and stored at -80 °C until analysis. The abundance of pico- and nano-sized phytoplankton and heterotrophic nanoflagellates (HNF) were determined using an Attune® NxT, Acoustic Focusing Cytometer (Invitrogen by Thermo Fisher Scientific) with a 20 mW 488 nm (blue) laser. Autotrophic pico-and nano-sized plankton were counted directly after thawing and the various groups discriminated based on their red fluorescence (BL3) vs. orange fluorescence (BL2), red fluorescence (BL3) vs. side scatter (SSC) and orange fluorescence (BL2) vs. side scatter (SSC). For HNF analysis, the samples were stained with SYBR Green I for 2 h in the dark and 1-2 mL were subsequently measured at a flow rate of 500 µl min-1 following the protocol of Zubkov et al. 2007. The abundance of virus and bacteria was determined using a FACS Calibur (Becton Dickinson) flow cytometer with a 15 mW 480 nm (blue) laser. Prior analysis of virus and bacteria, samples were first thawed, diluted x10 and x100 with 0.2 μm filtered TE buffer (Tris 10 mM, EDTA 1 mM, pH 8), stained with a green fluorescent nucleic acid dye (SYBR Green I ; Molecular Probes, Eugene, Oregon, USA) and then incubated for 10 min at 80°C in a water bath (Marie et al. 1999). Stained samples were counted at a flow rate of around 60 µL min-1 and different groups discriminated on a biparametric plot of green florescence (BL1) vs. side scatter (SSC). This allowed to distinguish virus particles of different sizes, and different bacterial groups including low nuclear acid (LNA) and high nuclear acid (HNA) bacteria. Names of size groups of photosynthetic and heterotrophic organisms are in accordance to "Standards and Best Practices For Reporting Flow Cytometry Observations: a technical manual (Version 1.1)" (Neeley et al., 2023). A short summary is listed here: RedPico = picophytoplankton (1-2 µm); RedNano = Nanophytoplankton (2-20µm), which includes subgroups RedNano_small (2-5 µm), RedNano_large (5-20 µm); OraPico = Nanophytoplankton with more orange fluorescence; OraNano = Cryptophytes; OraPicoProk = Synechococcus; HetNano = heterotrophic nanoflagellates; HetProk = bacteria (and when present archaea); HetLNA = low nucleic acid (LNA) containing bacteria; HetHNA = high nucleic acid (HNA) containing bacteria with the subgroups HetProk_medium = HNA-bacteria subgroup with less fluorescence signal, HetProk_large = HNA-bacteria subgroup with more fluorescence signal and HetProk_verylarge = HNA-bacteria subgroup with very strong fluorescence signal; Virus = virus-like particles, including size refined subgroups: LFV (low fluorescence virus or small virus); MFV (medium fluorescence virus or medium virus); HFV (high fluorescence virus or large virus) according to Larsen et al., 2008. Exemplary plots showing the gating strategies that were followed can be found in "Interoperable vocabulary for marine microbial flow cytometry" (Thyssen et al., 2022).