Treatments and Preservation of Seawater Samples for FISH
Add formaldehyde at a final concentration of 1-2% to filtrate.
Fix for 12-24 hr at 4°C.
Setup frittered glass support (25mm diameter) in filtration manifold.
Place a drop of Milli Q water (~250-500µl) on support, float 0.45µm cellulose nitrate support filter (25mm diameter) on top, turn on vacuum (~ 5 in of Hg) to lay filter flat with no air bubbles.
Turn off vacuum and release pressure from chamber.
Add another drop of Milli Q to support filter, float 0.2µm polycarbonate membrane filter (25mm diameter) on support filter (shiny side facing up!).
Save filter separator sheets for storage later.
Turn on vacuum (~ 5 in of Hg) to lay filter flat with no air bubbles.
Leave vacuum on.
Place filter tower on membrane and clamp.
Filter 1ml of fixed sample by applying gentle vacuum (~5 inch Hg); filter 2 ml of the fixed sample onto a second filter.
The support filter may be utilized for both samples.
Remove filter from filter holder and put it on Kimwipes to dry.
Cover, e.g. with the lid of a cryo box or a Petri dish.
Allow to air-dry.
Label membrane filter with pencil; place membrane filter between separator sheets (will prevent the membrane filters from sticking to each other or to the Petri dish).
Seal Petri dish with parafilm, prevent dish from opening with tape, and put Petri dishes into a Ziploc bag. 
Store at -20°C until processing.
Filters can be stored frozen for several months without apparent loss of hybridization signal.
Setup frittered glass (47mm diameter) support in filtration manifold.
Place a drop of Milli Q water (~1ml) on support, float 0.45µm cellulose nitrate support filter (47mm diameter) on top, turn on vacuum (~ 5 in of Hg) to lay filter flat with no air bubbles.
Turn off vacuum and release pressure from chamber.
Add another drop of Milli Q to support filter, float 0.2µm polycarbonate membrane filter (47mm diameter) on support filter (shiny side facing up!).
Save filter separator sheets for storage later.
Turn on vacuum (~ 5 in of Hg) to lay filter flat with no air bubbles.
Leave vacuum on.
Place filter tower on membrane and clamp.
Filter appropriate volume (see Table I) of fixed sample by applying gentle vacuum (~5 inch Hg).
The support filter may be utilized for several samples.
After complete sample filtration, wash filter and tower with 20-30 ml of sterile H2O; remove H2O by vacuum.
Remove filter from filter holder and put it on Kimwipes to dry.
Cover, e.g.
with the lid of a cryo box or a Petri dish.
Allow to air-dry.
Label membrane filter with pencil; place membrane filter between separator sheets (will prevent the membrane filters from sticking to each other or to the Petri dish).
Seal Petri dish with parafilm, prevent dish from opening with tape, and put Petri dishes into a Ziploc bag. 
Store at -20°C until processing.
Filters can be stored frozen for several months without apparent loss of hybridization signal.
