Week 3

I cannot believe that it is already my third week here at CMOP. Everything is going really well; I could not have asked for a better lab to work in and people to work with. Before I tell you the riveting details of the last week, I would like to issue a correction for last weeks blog. Apparently I was incorrect in my description of how tetrazolium works in regards to cells. I had previously described it as working in the glycolysis pathway, but it seems to actually act as a substrate analogue for reductases and hydrogenases in mitochondria. It seems that by binding preferentially to these enzymes, the cell slowly exhausts itself because it uses up NADH and NADPH, thereby decreasing its reducing power.
Also, the product of this reaction is formazan, which is nasty, and may work to kill off the cells.

So now that this is all cleared up, here is a recap of my week...
At the beginning of the week, I counted and recorded the rest of the CFU data and put the information into a spreadsheet. The data was graphed via Excel (which was a feat in and of itself! I still have not gotten the hang of MAC's and only having one button on the mouse continues to throw me off). Thank goodness Craig was here to save me from breaking the Mac and actually graph my data. The results seemed to be a good representation of the data and suggested that we had been successful. Next I worked on extracting DNA (using the soils kit and DNA extraction protocol) for the last six samples collected on the most recent research cruise. This took all day, but I really enjoyed doing it! After the DNA was extracted and isolated, I used the nanodrop equipment in lab to measure the concentration of DNA in each sample and diluted each out accordingly. Surprisingly, each sample contained a lot of DNA; anywhere from 20-50ng/ul. By the way, I do understand that 20 nanograms is never considered a large number, but when dealing with DNA, this is exciting! Each was diluted to ~10ng and then subsequently diluted to a 1 in 10 and a 1in 50 dilution. This part took forever, mostly because trying to label tiny Epindorfs with a sharpie permanent marker takes ninja-like concentration and focus! After this labeling and diluting was all done, it was time to get ready for PCR! The 1 in 10 diluted samples were chosed to be placed in the machine, and a master mix was made up and the process was initiated. The products were collected and run through a gel. Fortunately, every sample worked and looked good in the snapshot we took.

The next experiment consisted of testing potential Mn-oxidizing medium plates to see if the organisms were truly oxidizing manganese. By smearing isolated colonies on Whatman paper and putting 2ul of LBB on each isolate, I could visualize the results. When each smear turned blue, I could conclude that Mn oxidization was occurring. Most of the plated samples yielded positive results! The positive plates were then isolated again to make sure that the results were truly correct.

Overall, that was week three in the lab. The only other exciting events were a lab meeting with Brad Tebo (who is my senior scientist and department chair I believe) and the NSF site visit which seem relatively painless, and was very interesting to be a part of. It rare that I get the opportunity to see the administration and funding side of research and I was glad to have the opportunity. Oh and did I mention that the interns I got free lunch at the site visit? It was awesome ;) Lets hope that this week can be even better...