Colibri light technology

We performed FISH to identify the occurrence of prokaryotes on nine nematodes collected during the Momarsat, 2017 cruise. On board ship, some nematodes were fixed (see Nematode sorting and fixation). Later, at the laboratory, these specimens were hybridized with universal probes (Eurogentec, Liège, Belgium) (Table 1). Whole nematodes were rinsed in a 30 or 40% formamide-buffer. Then, they were incubated in a final volume of 30 μl of a hybridization buffer containing 30 or 40% formamide and 3 μl of each probe at 8 μM for 3.5 h at 46°C. The nematodes were then rinsed in a washing buffer for 45 min at 48°C. This step was ended by a final wash in milliQ water at room temperature for 10 min. After a quick drying period, the labeled organisms were mounted on a slide in an anti-fade mounting medium (SlowFade^® Gold anti-fade reagent, Invitrogen) containing DAPI (4′,6-diamidino-2-phenylindole, dilactate), a DNA intercalary agent. Observations were made on an Imager.Z2 microscope (Zeiss, Oberkochen, Germany) equipped with a slider module Apo-Tome (Zeiss) and Colibri light technology (Zeiss) and using an AxioCam MRm (Zeiss) camera. Micrographs were analyzed using Zen (Zeiss) software.

FISH was performed to confirm the occurrence of prokaryotes on 13 nematodes (Table 2). In the field or quickly after sampling, some nematodes were fixed (see Nematode sorting section). Later, in the laboratory, they were hybridized with universal probes (Eurogentec, Liège, Belgium) (Table 3). Nematodes were rinsed in a 30% formamide buffer^{53 (link)} and incubated in a final volume of 30 µl hybridization buffer containing 30% formamide and 3 µl of each probe (8 µM) for 3.5 h at 46 °C. After that, the nematodes were rinsed in a washing buffer for 45 min at 48 °C. This step was ended by a final wash in milliQ water at room temperature for 10 min. After a quick drying period, the labelled organisms were mounted on a slide in an anti-fade mounting medium (SlowFade® Gold anti-fade reagent, Invitrogen) containing DAPI (4′6-diamidino-2-phenylindole, dilactate), a DNA intercalary agent. Observations were performed using the Imager.Z2 microscope (Zeiss, Oberkochen, Germany) equipped with an Apo-Tome slider module (Zeiss) and Colibri light technology (Zeiss) and using an AxioCam MRm (Zeiss) camera. Micrographs were analysed using the Zen (Zeiss) software.