Abundance of Prokaryotes in deep-sea sediments analyzed by CARD-FISH

We fixed 0.5 g aliquots of sediment with a 4% formaldehyde solution for 2-4 h, washed the fixed sediments three times with 1x phosphate-buffered saline (PBS), before storing them in 50% ethanol/PBS at -20°C. For water samples, we fixed 10 ml (for samples from the deep chlorophyll maximum and 100 m water depth) and 30 ml (for meso- and bathypelagic samples) with formaldehyde to a final concentration of 2-4% for 2-4 h, then filtered over a 0.22 µm polycarbonate filter, and stored samples at -20°C. We performed total cell counts as described by Schauer et al. (2011, doi:10.1111/j.1462-2920.2011.02530.x) using the nucleic acid dye 4'-6-diamidino-2-phenylindole (DAPI). A minimum of 1,000 cells in 20 independent grids were counted using a Zeiss Axio Imager M1 epifluorescence microscope equipped with a 100x/1.25 oil plan-apochromat objective. We used Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization (CARD-FISH) according to Ishii et al. (2004) to count Gammaproteobacteria and JTB255 cells. We used the GAM42a oligonucleotide probe and the BET42a competitor probe to target members of the Gammaproteobacteria (Manz et al., 1992, doi:10.1016/S0723-2020(11)80121-9). We designed the JTB819a and JTB897 probes to target 16S rRNA gene sequences assigned to JTB255 in SILVA release 128 and the cJTB897 competitor probe to target all non-JTB255 16S sequences in SILVA release 128 that have a single mismatch to JTB819a and JTB897 probes. We obtained total gammaproteobacterial and JTB255 cell counts from duplicate filters derived from each sampling site.