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Week 2: Preparing Media and Growing Bacteria
The week began with the NSF annual site visit on Monday. This was an all day event that I attended with the other interns. It was really nice to hear a broad overview of what CMOP is all about and the research that is being performed throughout the program.
Tuesday found me back in the lab and proved to be a very busy day. I spent most of the day making media. I had never made media before and ran into a couple challenges along the way and therefore had to remake some of the media. I made two different kinds of media so that I could grow up my bacteria in a rich environment and then transfer them to an environment that would essentially starve them, allowing me to better study manganese oxidation. I ended the day by making different dilutions of manganese oxides with unknown concentrations. I transferred a small bit of sample to a well plate and mixed it with LBB reagent. Through use of the spectrophotometer and the calibration curve that I had created last week, I was able to use the absorbance values to determine the unknown concentrations. I thought this was really neat!
On Wednesday, I finished making up the Lept media. One component that I had to add to create this media required a pH of 7.8. My initial pH was 5.46 and therefore I used sodium hydroxide to titrate the solution to the correct pH. This took awhile since I wanted to make sure that I did not add too much sodium hydroxide, causing the pH to rise above 7.8. After adding all of the necessary components to create Lept media, I poured a number of plates. Before leaving, I made up 1M HCl and used it to refill several of our stock bottles.
I began Thursday by performing a bit of housekeeping—restocking our distilled water bottles and washing used equipment. Because the H2O2 assay had not given me a good calibration curve, I focused on making up my standards and performing this assay once again. I repeated this test several times using different concentrations and volumes. The calibrations curves were still not as good as we would have liked them to be. After this, I incubated my plates with P. Putida GB 1 and hoped that the bacteria would grow, as this would indicate that I made the media correctly! I spent the remainder of the day making manganese oxides and manganese (III) solutions with specific concentrations. These solutions would eventually be used for kinetics experiments.
On Friday, I saw that bacteria had grown on my plates! I then transferred the bacteria to another plate, which I would leave to grow over the weekend. Matt and I attempted to determine why the H2O2 assay was not producing a nice calibration curve. We decided that it would be best to start from scratch and remake the original solutions and buffers. I spent most of the day making the two buffers. I had a difficult time getting the two buffers to the correct pH and had to titrate them while using the pH probe. Just when I was nearly done, I spilled one of the buffers and had to start over making that solution! I spent the afternoon working with Modi, a volunteer who is working with Matt as well. We attempted to run kinetics experiments before heading home for the weekend.
