PICOPLANKTON
- Flow Cytometry (glutaraldehyde preserved samples)
– 3 x 1 mL per depth (0.024 L total over 8 depths)
- Single Cell Genomics (glycerol preserved samples)
– 3 x 1 mL per depth (0.024 L total over 8 depths)
- qPCR and Metagenomics (sterivex filters)
– 6 x 500 mL* per depth (24 L total over 8 depths)
*amount of water can be reduced to minimum 100mL if supply is limited
- Filtration and sample preservation steps
– 30 minutes to drain the water and 2 h for post-sampling processing
Note: To make things simple (especially for inexperienced people), sampling may be limited to just sterivex filtration. This can be done relatively high-throughput with a peristaltic pump filtration rig.
NITROGEN FIXATION
- Collect 2 L of seawater per depth and filter. Details on filter type etc and storage to follow.
ARCHAEA
- Selection of depths:
– Choose depths based on the identification of water masses, using the same depths as collected for bioactive elements. For example – in the subtropical North Atlantic five depths were sampled : subsurface (100m), oxygen minimum zone (250-500m), Antarctic Intermediate Water (900m), North East Atlantic deep water (2750m), and lower deep water (3500-4000m). Samples can be collected either from stainless steel or titanium CTD.
- Abundance of bacteria and archaea determined by flow cytometry:
– 2ml samples are fixed with 1% paraformaldehyde, shock frozen in liquid nitrogen for 5 minutes and then stored at -80°C until analysis.
- Molecular characterisation of Bacteria, Crenarchaeota and Euryarchaeota:
– Collect 5 L of seawater from depths above 200m and 10 L of seawater from deeper depths from about 6 depths at each station for fingerprinting (T-RFLP, ARISA), Q-PCR for amoA and carboxylase (accA, accC) genes as well as on the 16S rRNA using specific primers for Bacteria, Crenarchaeota and Euryarchaeota. Some other genes or specific prokaryotic groups could also be quantified once we have DNA/RNA extracted from these samples.
– Filter through a 0.22 μm Sterivex filter GP unit (Millipore polysulfonate filters), add 1.8 ml of lysis buffer (40 mM EDTA, 50 mM Tris-HCl, 0.75 M sucrose) and store at -80°C until analysis.