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Week 8 - pHstat Experiments

The bulk of this week was spent gathering data from the pHstat. Since the experiment last Friday went well, Rachel decided to start a pH gradient experiment. For this kind of experiment, the computer generates a sine curve that has an amplitude and frequency that we set. The experiment is supposed to reproduce the natural pH cycle present in the ocean due to changing carbon dioxide levels throughout the day. When the sun is out and the phytoplankton are performing photosynthesis, the carbon dioxide decreases, so the pH decreases. At night carbon dioxide starts building up, so the pH increases. This cycle can be represented by one complete sine wave per day so that’s what the frequency was set to. The amplitude was set at 0.5 and the wave was to be centered at a pH of 8. We used a culture of Isochrysis and ran the experiment for three days so we could see longer term affects. I used the spectrofluorometer method to measure the intracellular pH at various time points. It appears that the intracellular pH is changing with respect to the external pH, which is very promising results. We will have to wait to get the results back from another lab about the dissolved inorganic carbon levels to see what is really going on, but I am excited to already be getting such great data.

I also tried to make another calibration curve for Alexandrium with the flow cytometer, but it failed again, just like last week. The samples looked weird when I made them, there was white stuff clumping around the cells when I spun them and my buffers looked cloudy, so I checked out my buffers under the microscope. I found that they were all contaminated with what appeared to be bacteria. I filtered them and will probably try to make another curve next week with my now decontaminated buffers.