Can Crusher - Culley Protocol
Using sterile technique , remove the plunger from the 60mL syringe .
Attach the 0.02 µm filter ( Anotop ) to the end of the syringe .
Pour 50 mL of 0.2 µm filtrate into the 60 - mL syringe .
Replace plunger and mount the syringe and filter into the can crusher .
Push the 0.2 µm filtrate very slowly through the Anotop , using the can crusher and applying gentle , steady pressure .
Watch carefully for the last drop of water to go into the Anotop , and then stop pushing .
Using sterile technique , remove the Anotop from the syringe .
Do not touch the filter inlet or outlet .
Suck up the remaining 50mL of 0.2 µm filtrate for this depth into the 60 - mL syringe .
Re - attach the Anotop to the end of the syringe .
Repeat Step 5 : Push the 0.2 µm filtrate slowly through the Anotop , using the can crusher .
Remove the Anotop from the syringe .
Write on the Anotop itself using a waterproof Sharpie .
Wrap the Anotop in clean parafilm , covering both the inlet and outlet .
Label a piece of aluminum foil with date , depth , and location ( using waterproof Sharpie ) .
Wrap the parafilmed Anotop in the labeled aluminum foil .
Label a Ziploc bag with date , depths , and location .
Collect several Anotops to put into one labeled Ziploc : one location / sample site per bag .
Put the foil - wrapped Anotops into the labeled Ziploc bag .
Freeze the Ziploc in - 80°C freezer ( if no - 80°C freezer is available , use a - 20°C freezer ) .
Discard the syringe and filtrate ( unless you need some sterile water ) .
