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 .
