Fixation of Planktonic Samples
Add freshly prepared PFA fixative (see 'Fixing Cells with PFA Protocol') to water sample to a final concentration of 1-2%.Alternatively, use Formaldehyde, and fix for 12-24 hours at 4°C.
Place a moistened support filter (0.45 μm pore size, cellulose nitrate, 47 mm diameter) and a membrane filter (0.2 μm pore size, white polycarbonate (GTTP), 47 mm diameter) into a filtration tower.
Filter an appropriate volume of the fixed sample by applying gentle vacuum (~ 5 inch).
Place a moistened support filter (0.45 μm pore size, cellulose nitrate, 25 mm diameter; Sartorius, Germany) and a membrane filter (0.2 μm pore size, white polycarbonate (GTTP), 25 mm diameter; Millipore, Eschborn, Germany) into a filtration tower.
Filter 1 ml of the fixed sample by applying gentle vacuum (~5 inch).
Filter 2 ml of the fixed sample on a second filter.
After complete sample filtration, wash with 10-20 ml of sterile H2O.
Remove H2O by filtration.
Put membrane filter on blotting paper for drying and cover, e.g.
with the lid of a cryo box or a Petri dish.
Allow air-drying.
Store each filter in a separate labelled Petri dish.
Place membrane filter between separator sheets that the GTTP membrane filters were provided with.
Seal Petri dish with parafilm.
Store at -20°C until processing.
