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 .
