Hi, I'm David Walsh from the Laboratory of Steven Hallam in the Department of Microbiology and Immunology at the University of British Columbia. Today we'll show you a procedure for filtering small 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. Before filtering first, wipe away any condensation from the full bottles and weigh them. Place an autoclave bin on one side of the pump and the waste flask in an autoclave bin on the other.
Then place a full sample bottle in the autoclave bin. However, do not remove the cap of the bottle yet. Pass the tubing through the peristaltic pump so that the lure end of the tubing is in the beaker.
Using tweezers, remove the cotton from a sterile 25 milliliter pipette and attach the pipette to the other end of the tubing. To begin filtering sea water, open the bottle and insert the pipette. Turn on the pump and set the flow speed to one watch to ensure that water is being pumped in the proper direction.
Allow about 100 milliliters of water to pass through the entire tubing. Turn off the pump and attach a stev X filter to the male lure fitting at the free end of the tubing, insert filter into waste flask. Turn on the pump and set the flow speed to one.
Continue filtering until no water is left in the bottles. 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 stair of x filter with a small piece of perfil and label the filters with the date and sample identification. Store the filters in a pre-labeled 50 milliliter Falcon tube at minus 80 degrees Celsius.
Insert the 25 milliliter pipette into a flask containing 0.1%hydrochloric acid and switch on the pump. After the hydrochloric acid wash, rinse the tubing with autoclaved water. Finally, measure and record the mass of the now empty one liter bottles in order to calculate the volume of water passed through the filter.
Each one liter water sample yields one ster of X filter, which contains biomass greater than 0.2 microns. This is stored in lysis buffer for subsequent DNA. Extraction and analysis filters can be stored at minus 80 degrees Celsius prior to EDNA extraction.
We've just demonstrated a small volume filtration protocol for collecting seawater biomass. 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.
