Dmrbe trinocular

Representative samples for bacteria and virus counting (covering all sample types and the abundance range of the whole sample set) were additionally enumerated using epifluorescence microscopy. Samples from filter fractions >0.22 μm were centrifuged at 2,000 × g for 5 min to reduce the background fluorescence of cell debris. Virus filters were prepared after Suttle and Fuhrman (2010) . In brief, samples were diluted using 0.02 μm-filtered PBS buffer, vacuum-filtered onto 0.02 μm Anodisc filters (Whatman), stained with SYBR Green I for 15 min in the dark and mounted onto microscopic slides with 0.1% p-phenylenediamine as antifade solution. Bacterial cells were filtered onto 0.22 μm polycarbonate filters (Nucleopore, Track-Etch) as described by Lunau et al. (2005) (link) and stained with a SYBR Green I staining solution for 30 min as described by Pohlner et al. (2017) (link). A minimum of 300 viruses and 100 bacterial cells, respectively, were counted per filter in at least 15 randomly chosen counting grids at a 1,000× magnification (Leica DMRBE Trinocular, Leica Microsystems).

Representative samples covering all ecosystem types and the abundance range of the whole sample set were additionally counted using epifluorescence microscopy to validate VLP numbers based on flow cytometry. Filters for virus quantification were prepared following the standard protocol by Suttle and Fuhrman⁹³ . In brief, samples were diluted using 0.02 μm-filtered phosphate-buffered saline (VWR, Darmstadt, Germany), filtered onto 0.02 μm Anodisc filters (Whatman, Maidstone, UK) by applying vacuum, stained with SYBR Green I (20 x concentration, Thermo Fisher Scientific, Waltham, MA, USA) for 15 min. in the dark and mounted onto microscopic slides with 0.1% p-phenylenediamine (Acros Organics, Geel, Belgium) as antifade solution. A minimum of 300 VLPs were counted per filter in at least 15 randomly chosen counting grids at a 1000× magnification on a Leica DMRBE Trinocular (Leica Microsystems, Wetzlar, Germany) using the software EOS Utility v.2.10.2.0. Analysis of microscopic images was performed in ImageJ v.1.5.2^{94 (link)}.