Blog #3: Mid-summer Updates and Changes in My Project

Hi everyone! Hope you had an enjoyable 4th of July.

The last couple weeks have been especially busy. During week #4 of my research, I created treatments and incubated them in microcosms (2-L carboys) at room temperature. As I collected all the necessary subsamples, I began making slides from the frozen cryo vials. I also pooled the triplicates, or three replicates, of my treatments onto 0.22 micron nitrocellulose filters to capture bacteria, after which I stored the filters in the -80 ºC freezer (which is extremely cold!).  I will use these filters later when I conduct t-RFLP (terminal restriction fragment length polymorphism) to assess changes in bacterial community composition. Lastly, I helped Professor Williamson prepare vials with sub-samples from each of my treatments so they could be analyzed for bacterial production via tritiated leucine assays. Since tritiated leucine is radioactive and I am not certified to handle such substances, Professor Williamson was primarily responsible for this part of the project.

There have been some changes to my project based on time constraints and other factors. First, Professor Williamson and I decided at the end of week #3 not to do the viral decay analysis using potassium cyanide. There are many reasons for this. When I did a trial run, I followed the protocol described by papers illustrating this method, but I did not obtain similar results, so I was not sure if I should apply this protocol to my experiment. My project focuses more on bacterial production and abundance as a result of viral and protozoan activity, so it is more important in this case to collect data on bacteria than to gather viral production data. A third reason I decided against this method is that with all the other sub-amples and assays required, there would be a lot of extra work and disturbance to the microcosms. I would be taking 50-mL subsamples from each of the microcosms three of the days, and then I would be taking additional subsamples from the 50 mL on intervals of one or two hours well into the evenings. This sampling schedule would be difficult to manage given the other subsamples I would be collecting simultaneously.

A second major change to my project is the reduction in the number of treatments. I eliminated the virus-enhanced treatment I had been planning to make, resulting in twelve microcosms (triplicates of four treatments: control, grazer-enhanced, grazer-reduced, and virus-reduced) instead of fifteen. I did this because when I looked at a slide from the viral concentrate under the epifluorescence microscope, I saw a drastic reduction of viruses instead of an increase in concentration, which rendered the viral concentrate essentially useless. One possible explanation for this is that the viruses might not have survived the tangential flow filtration process as well as expected. It would have been interesting to see whether a two- or three-fold increase in virus concentration would make a difference in their effect on bacteria, but with the treatments I still have, I can at least tackle the question of whether grazers or viruses play a larger role in microbial ecology in Lake Matoaka.

I will be posting more information on epifluorescence microscopy and observations for the bacterial production assays shortly.

Stay tuned!