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Week 3: Beginning the Experiment

Monday morning found me working with one of the pH buffers again. Eventually, Matt told me to titrate the buffer with a 0.25 basic solution and this solved the problem. I also remade the 20 U/ml HRP solution that is used in the H2O2 assay. I made up more liquid Lept media and transferred my bacteria from plates to this medium. I left the bacteria to shake overnight. I spent the afternoon preparing 3 L of minimal media that would be utilized in my actual experiment.

Tuesday was a big day as I began collecting data for my experiment. I finished making up the minimal media and split it out amongst 6 containers. I transferred all my bacteria into one tube and then inoculated 4 of the containers with 1 mL of bacteria. I did not inoculate the fifth container since this was to be my control for the experiment.  I covered two of the inoculated containers with aluminum foil and left the other two open to light. This will allow me to compare manganese production and oxidation in light versus dark environments. I spent the rest of the day collecting data at time 0. This consisted of me running the H2O2 assay and LBB assay and then creating calibration curves for them. My H2Ocalibration curve was better than any of my previous attempts.

On Wednesday, I collected data for time 1 and therefore this was my first time running the whole experiment through in its entirety. I took approximately 40 mL out of each of my samples (the light and dark containers) and collected data on several different variables—including manganese oxides, growth, hydrogen peroxide, etc. Several of the samples and reagents had to be read immediately since they are time sensitive. This included the manganese (III) and hydrogen peroxide assays. Since this was my first day running the experiment, I mostly focused on learning the steps that I need to take in order to be most efficient. I spent the rest of the day cleaning equipment and setting up for my next data collection.

On Thursday, I spent the morning setting up for my next time point and then ran the experiment again. I set up the experiment by myself and then had Matt look it over. I realized that there were several steps that I had overlooked! Therefore, I believe it will be helpful to create a protocol that I can refer to. In the afternoon, I began analyzing my data and creating graphs. Since Friday was a holiday, this was my last day of work for the week.