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Week 5: Experiment Two Measurements

Monday was my busiest day yet since I had two time points to measure. I measured time eight from experiment one in the morning and then acid washed equipment before measuring time one from experiment two. This took me quite awhile to accomplish and therefore I spent most of my day focusing on getting my two time points in. I spent the afternoon measuring the amount of manganese (III) present in outstanding samples. The samples already had LBB added to them so all I had to do was measure them. However, this process of measuring the samples takes awhile since the samples must be diluted until none of the typical LBB blue color is visible. Then the samples have to be run through the spectrophotometer one at a time. Amelia, another intern, helped me with this.

On Tuesday, I once again ran two time points since I needed to measure samples from both experiment one and experiment two. However, after collecting samples from experiment one, I terminated that experiment to make room for experiment three, which I planned to begin later in the week. I spent the afternoon measuring outstanding samples collected from previous time points. In particular, I focused on measuring the total dissolved manganese by running the formaldoxime assay. Since my samples were very concentrated the first time I ran this assay, I began by making up new standards with a greater concentration range. This will allow my calibration curve to more accurately determine the concentration of total dissolved manganese present in my samples. I ended the day by streaking a plate with a P. Putida GB 1 mutant and left it to grow overnight.

On Wednesday, I only had one time point to measure since I had terminated experiment one the day before. This was accomplished in the morning and therefore I had the afternoon to catch up on measuring all of my collected samples. It is amazing how fast unmeasured samples build up when you are measuring two time points a day! In order to measure the amount of manganese oxides present, I had to dilute several of the samples with more LBB reagent. I also spun down the samples so that all the cells and particulate matter would separate from the fluid that I wanted to sample from. I then ran my samples through the spectrophotometer. I then transferred the bacteria grown yesterday to Lept so that it could grow up overnight in a liquid medium.

Thursday began with me running out of the necessary A-red reagent that is required to run the hydrogen peroxide measurements. While Matt had placed an order, it hadn’t come in yet. Therefore, I had to reduce the number of hydrogen peroxide measurements made. I completed my time point despite this minor set back and also made up 50 ml of minimal media. I transferred bacteria from the Lept media into the 50 ml of minimal media. After lunch, I finished tidying up and then made 3 L of minimal media. This would be utilized in making up my cultures for experiment three.

Friday has proven to be one of my busiest days in the lab since I have weekly seminars and the lab group meetings. I finished my time point early in the morning (the A-red reagent had come in!) and also finished making the minimal media that I had made up and autoclaved the day before. I inoculated four of the five cultures with bacteria and once again covered two of the four with foil. This experimental set up was identical to the ones in past weeks except for the fact that I was now testing manganese oxidation in a P. Putida mutant rather than P. Putida wild type. I measured t=0 for experiment three and then spent the afternoon working on data analysis.