Mounted specimens were then sputter coated with 10–15 nm C646 cell line of gold and palladium (60:40) using a Tousimis Samsputter 2A and visualized with a Hitachi S4800 scanning electron microscope. A minimum of 50 microscopic fields (0.5 × 1.0 mm) were observed to count ciliates in each of the samples, and ciliates were counted on at least three different filters. Fluorescence in situ hybridization In order to evaluate the relative abundance of ciliates as part of the protistan assemblages we used fluorescence in situ hybridization with a AZD4547 mw specific oligonucleotide probe. FISH followed the protocol of [103]. In short, 100–150 ml of paraformaldehyde-fixed (2% final concentration) seawater was filtered onto

0.65 μm filters and frozen at −20°C. Filters were thawed, and cut into small triangles before the hybridization step and ~20 μl of pre-heated 0.2% metaphor agarose were pipetted onto filters. After the metaphor agarose had dried, filter pieces were transferred to 0.5 ml sterile tubes containing the hybridization mix. All hybridizations were carried out using the universal eukaryotic FISH probe Euk1209 [104] with 40% formamide for 2 hours learn more at 46°C. After the hybridization step, filter pieces were incubated at 48°C in preheated washing buffer for 10 minutes in sterile

50 ml tubes. Filter pieces were then washed with distilled water and placed into sterile 0.5 ml tubes containing DAPI (2 μg/ml) and incubated for 5 minutes in the dark. Filter pieces were then washed with sterile water and incubated for 2 minutes in 70% ethanol, followed by a 2-minute wash with 100% ethanol. Filters were air dried and mounted on glass slides with a Citifluor/Vectashield mix (4:1) to prevent bleaching. Cells were enumerated under epifluorescence

using a Zeiss Axioplan 2 microscope and photographed with a Hamamatsu digital camera. Acknowledgements The authors would like to thank Dr. Maria Pachiadaki, Dr. Matthias Engel and Melanie Müller for assistance with sample collection during the R/V Urania cruise, and the captains and crew of the R/V Urania and R/V Oceanus for their tireless assistance with sample collection. We thank Dominik Forster for help with R. This work was funded by NSF grants OCE-0849578 and OCE-1061774 to VE and support selleck screening library from Carl Zeiss fellowship to AS and from the Deutsche Forschungsgemeinschaft (grants STO414/3-2 and STO414/7-1) to TS. Electronic supplementary material Additional file 1: Figure S1: Rarefaction curves of V4 SSU rRNA-amplicons that were assigned to ciliate genera for all eight samples. (PPTX 155 KB) Additional file 2: Figure S2: Proportion of rare versus abundant ciliate taxa. The number of detected taxa is opposed to the number of ciliate V4 SSU rRNA amplicons. (PDF 60 KB) Additional file 3: Table S1: Number of ciliate V4 SSU rRNA-amplicons in each sample assigned to described ciliate genera. The assigned genus represents the best BLAST hit of assigned amplicons to NCBIs GenBank nucleotide database 187.