Week 4: Experiment One Measurements

This week marked my first full week running through my experiment. Matt was gone Monday and therefore it was up to me to make sure that I measured my daily time point correctly. My cultures had grown up over the weekend and therefore there were lots of bacteria in both of the light cultures. The dark cultures exhibited slower growth. I spent Monday running my time point (consisting of collecting samples and adding reagents to test for manganese (III), hydrogen peroxide, manganese oxides, total dissolved manganese, pyoverdine and optical density), cleaning up equipment/acid washing and setting up for the next time point.

On Tuesday, I spent the morning collecting my daily time point. As I mentioned above, the daily time point consists of collecting samples that will allow me to analyze manganese oxidation over time by observing the changing amounts of manganese (III), manganese oxides and total dissolved manganese within my cultures. I also mentioned that I measure optical density, which is a measure of the growth of bacteria within my cultures. In the afternoon, I measured samples that I had collected from the past time points and tested for the presence of manganese (III). I had added LBB reagent to these samples when I collected them and therefore all I had to do was pipette small volumes from each sample into a well plate and measure the absorbance on the spectrophotometer. Since I had already created an LBB calibration curve, I was able to use this curve to calculate concentration of manganese (III) from the given absorbance values. I ended the day by streaking a new plate with P. Putida bacteria and left this to grow up overnight.

I spent Wednesday collecting my daily time point in the morning. I attended the welcome lunch that OHSU put on for all of the summer interns. This was a great opportunity to hear what other interns are studying this summer. There was a wide-range of projects that encompass many areas of research. In the afternoon, I made up 20 ml of Lept media and inoculated it with P. Putida GB 1 that I transferred from the plate that I had streaked the previous day.

Thursday was a busy day. I was able to get my time point done early in the morning and then went to the solar simulator with Amelia, another intern in the Tebo lab. She had a few samples that she needed to place under the solar simulator and since I went with her, Matt had me place a few different manganese (III) ligand complexes under the solar simulator. We then took absorbance values of these samples and compared them to the values of the original samples before solar simulation. After heading back to the lab, I finished acid washing and tidying up before making up 50 ml of minimal media. I made up this media and then inoculated it with 1 ml of bacteria that had grown up overnight in the Lept media. Before leaving for the day, I made up 2 L of minimal media and left it to be autoclaved in the morning.

I began Friday by autoclaving the minimal media that I made on Thursday. While this was autoclaving, I measured my time point. After allowing my minimal media to cool, I finished adding the necessary components and inoculated four of the samples with the P. Putida that I had grown up. These were to be my light and dark cultures for the second round of culture experiments. I did not inoculate bacteria into one sample, as this was to serve as my control. I measured time 0 for Experiment 2 and then spent the rest of the afternoon measuring outstanding samples from past time points. In particular, I measured the amount of total dissolved manganese in my samples. To do this, I added formal B reagent to my samples in 48 well plates and also made up a set of standards to use in creating my calibration curve. I measured the absorbance on the spectrophotometer. This ended my fourth week!