Hi, I'm David Walsh in the laboratory of Steven Hallam in the Department of Microbiology Immunology at the University of Bridge Columbia. Today we'll show you a procedure for filtering large volumes of seawater for microbial biomass. We use this procedure in our laboratory to study microbial diversity in the marine environment.
So let's get started. To set up the filtration apparatus, use tweezers to place a grade GFD filter into the pre-filter housing unit. Loosely tighten the housing by hand.
As a rule of thumb, do not overtighten or the unit will leak. Once the pumping starts, replace the carboy cap with the autoclave cap and attach tubing by carefully inserting the internal tubing into the car without touching it. If necessary, use tweezers to guide the tubing into the car.
Then tighten the cap, pass the external tubing through the peristaltic pump. Attach pre-filter housing units to the end of both external tubes. Tighten the connection with pliers to begin filtering the sea water.
Turn on the pump and set the flow speed to one watch to ensure that water is being pumped in the proper direction. Allow 500 milliliters of water to flow freely through the pre-filter unit and into a one liter beaker. Turn off the pump and attach a Stax filter to the male lure fitting at the free end of the pre-filter setup.
Tighten the pre-filter housing snugly by hand. Watch for leaks at the pre-filter housing and at the connection between the pre-filter housing and the tubing that leads to the car. Boy if leaks occur, turn the pump off and loosen and re-tighten the pre-filter housing accordingly, turn the pump back on.
Collect the filtrate in four liter flasks. Once they're full, empty the flasks into a clean carboy. Be sure to note the volume passed through each filter collected.
Filtrate can be further processed such as filtration for viruses. Continue filtering until no water is left in the carboys. Usual filtration times range between four to five hours per 20 liter car.
However, this depends dramatically on the amount of biomass within the water. Once filtration is complete, switch the pump off and disconnect the Stax filters. To store the filters.
First expel any remaining water with a 30 to 60 milliliter syringe. Using a pipette add 1.8 milliliters of lysis buffer to the filter, keeping about 200 microliters of space in the filter. For later edition of reagents, seal the bottom and top of the stex Filter with a small piece of para film and label the filters with a date and sample identification.
Store the filters in a pre-labeled 50 milliliter Falcon tube at minus 80 degrees Celsius. Remove the filters from the housing unit and place them in labeled 50 milliliter Falcon tubes containing two milliliters of lysis buffer. Store these at minus 80 degrees Celsius.
For final cleanup, place the tubing that was inside the car into a flask containing 0.1 molar hydrochloric acid. Direct the tubing that leads to the four liter flask into the original car. Switch on the pump and run it three, collecting the flow through in the original car.
Finally, rinse the tubing with autoclave water. Each 20 liter car yields two Stax filters, each containing microbial biomass in the size range between 0.22 and 2.7 microns from approximately 10 liters of seawater. In addition, biomass greater than 2.7 microns is collected and concentrated on the GFD pre-filters.
These filters and pre-filters are stored in lysis buffer and are ready for DNA extraction. If additional processing steps are taken, the 0.2 micron flow through can be concentrated down to collect the viral fraction as Well. We've just described a peristaltic pumping method for large volume filtration of planktonic biomass ranging in the size of 0.22 microns to 2.7 microns in diameter.
When doing this procedure, it's important to wipe down benches and pumps with a damp towel after use in order to remove any lingering salt water. They may otherwise corrode the equipment. Although the procedure is simple, the time requirement can be extensive depending on the concentration of biomass or debris in the sample being filtered.
So that's it. Thanks for watching and good luck with your experiments.
