The overall goal of this procedure is to isolate magneto tactic bacteria from a freshwater environment. This is accomplished by first collecting water sediment samples from the edge of a river or lake. The second step is to isolate magneto tactic bacteria contained within the environmental sample using a bar magnet.
Next, the magneto tactic bacteria are enriched using a glass capillary racetrack and magnet. Finally, the magneto tactic bacteria are characterized using optical microscopy. Ultimately, transmission electron microscopy is used to show the size and shape of the bacteria and their magnetite containing magneto zones.
This method helps us understand key questions, environmental microbiology, such as how magneto evolved on earth, and how magneto tactic bacteria synthesized nanometer sized magnets. Demonstrating the procedure today will be Zachary a Stryker. Zachary is a PhD candidate in my laboratory.
He works with my brother and myself here at Ohio State University. When deciding on a freshwater site to collect magneto tactic bacteria or MTB, it is often best to start with a pond or slow moving stream that has a soft muddy sediment layer. In this demonstration, we collected a sample at the edge of the Oland Tangy River on the campus of the Ohio State University in Columbus, Ohio.
Find a location where the depth of the water is between 10 and 100 centimeters, and using a clear screw top container have fill the container with the uppermost layer of sediment and the remaining space with water, capping the container under water back in the lab, loosen the cap and leave it covering the container to reduce evaporation and store it in the dark for several hours to several days to allow the sediment and find particles to settle. To make racetracks for enriching MTB. Begin by using a diamond pen or file to cut off the top of a 5.75 inch glass pastier pipette, allowing enough room for one to two milliliters of water.
Next, use a and burner to seal the tip autoclave. Several raise tracks along with cotton and long metal needles. When ready to isolate MTB, place magnets on the sides of the plastic container, approximately one centimeter above the sediment water interface with the south and north poles on opposite sides of the container.
Take care not to disturb the sediment. Wait 30 minutes to several hours for the bacteria to swim to the magnet. Next, use a syringe to remove fluid from the sample container near the sediment water interface.
Attach a filter to the syringe and a metal needle to the filter, then ensuring that there are no bubbles in the capillary. Use the needle to add the filtered sample water to an autoclave racetrack with sterile cotton and the metal needle. Plug the racetrack by pushing the cotton inside the capillary to 0.5 to one centimeter away from the sealed end.
Using a sterile pipette, remove the magneto tactic bacteria from the container where they're clustered near the south pole of the magnet, and add the MTB containing water to the open end of the racetrack. Once the racetrack is filled with sample fluid, lay it on its side on a horizontal surface and place the south pole of a bar magnet next to the seal tip of the racetrack. Wait five to 30 minutes for the MTB to migrate through the cotton.
Use a diamond knife to gently make a little scratch near the cotton plug and snap off the end of the racetrack. Use a one milliliter syringe with a narrow needle to remove the fluid enriched with MTB from the tip of the racetrack. To observe the MTB by light microscopy, place a drop of the enriched sample onto a cover slip.
Place the cover slip onto an O-ring that is resting on a glass slide. This is called the hanging drop. The O-ring should have a slightly smaller diameter than the cover slip.
Next, place the slide onto a light microscope stage and focus on one edge of the drop. Then to attract the MTB to one side of the drop, place the south end of a bar magnet close to that side within a few minutes, many MTB should be at the edge of the drop. To observe the MTB by TEM, place a form var stabilized and carbon coated 200 mesh copper grid onto the drop of the enriched bacteria and allow the sample to settle for 10 minutes.
After washing the sample on a drop of water for 30 seconds, place it on a drop of 2%urinal acetate for 30 seconds to five minutes, and then use a piece of filter paper to dry completely analyze the samples using transmission electron microscopy at 200 kilovolts. Shown here is a transmission electron microscope image of a single magneto tactic bacterium enriched from an environmental sediment sample. The spiral and morphology of the cell and magneto zones are clearly visible along with a single flegel.
Once the magneto tactic bacteria have been collected from the environment, the magnetic separation technique will take approximately three hours if it is performed properly. Following this procedure, other methods like 16 s ribosomal RNA analysis can be performed, which will answer additional questions like, what is the phylogenetic relationship of the newly discovered magneto tactic bacteria to other, to other microorganisms.
