Goooooodmooooorning....

Hello, how was your week? Great, mine was interesting as well. On Monday Dr. Rainey was in Mexico so we went to the Microscopy lab in the basement of Life Science. It was very interesting at first when Mrs. Cindy was telling all of the things that we were going to do. We were did DAPI stains, looked at our organisms under the light microscope, and also under the TEM. To prepare our cells for viewing under the light microscope we had to do a little preparation; that involved suspending the cells in 0.5 mL of water and then mix them. We also noted that if any of our organisms were adapted to high salt content that they may explode when added to the water because the osmotic pressure may be too high outside the cell. Lucky for us, this wasn’t the case. We then transferred only 2 uL of the organismal mix onto a slide for viewing. We only used 2uL because if more than that was used the cells may “swim” around the slide even if they aren’t motile. The water would cause them to move around. Before viewing the cells we stained them with DAPI, 4',6-diamidino-2-phenylindole, a florescent stain that intercalates between DNA. To load the DAPI into the cells we added at one end of the slide and that way it worked its way through the entire slide. Below are the pictures from the light micrscope, numbers 37 and 40, respectively. In looking at 37 you can see lots of cells uniformly shaped. They also look lumpy. Also, in looking at 40 we saw the cells moving around. This organism is probably motile because there were cells moving in a different direction than the cells that were all drifting in one direction because of all the water that was used. You can see that some cells look like spirochetes and some occurred in pairs. It was hard to get a good picture because they were moving so fast.When looking at our cells under the TEM we didn’t see very many. So, she made a slide from some gooey fish tank water that she had, and boy, did it have lots of things to see! To prepare the slide for viewing under the TEM we put a collidian on the slide along with the cells. The collidian has 2 sides, a light color and also a dark copper color. We then touched the collidian with the liquid (uranium acetate) which creates a halo around the bacteria. It is useful to use the TEM when you need a higher resolution. You can actually see the difference between two spots on the TV like screen located on the microscope. The resolution is improved so much in the TEM because electron beams improve resolution because the wavelength is shorter. We also added liquid nitrogen to the TEM to keep the vacuum cool. The liquid nitrogen does this by evaporating all of the vapors. Take a look below to see what we saw. In 37 we saw oval, lumpy shaped organisms again. In 40 we saw oval shaped organisms with flagella, and other weird shapes. We also could see some of the flagella that were separated from the organism.On Wednesday were supposed to go and get Jambalaya from outside of Williams and Choppin at 230. I thought Dr. Rainey remembered and that he would tell us what time we could go. Unfortunately, he didn’t remember so we missed the Jambalaya. So for that, he’s bringing us Krispy Kreme on Monday. On a heavier note, since DAPI didn’t work very well with our cells we learned that it could be because they are hydrophobic. To prepare these cells for viewing with DAPI you would have to get rid of the polysaccharide wall. Things that be used are toluene, alcohol, and formaldehyde.We also looked at our temperature, cellulose, xylan, and avircell plates. We stained the xylan plates with iodine and the AC and CG plates with Congo Red. Then once they the stains were there for 15 minutes we poured it off of the plates and added NaCl for 15 min. None of our plates tested positive. While DP and I were doing the hard work, Larry was over on the other side of the room running our PCR out on a gel. Once he was done we could see that our organisms ran out at about 560. This is right on target for Geodermatophilaceae. Look at the red arrows below. They are pointing to some of the organisms that ran out at 560.