Filtered vs. Unfiltered Medium with Pseudomonas putida - Week 7

I started this week by running my last time-point from last week’s experiment which was growing P. putida with and without light in minimal medium A with 100uM manganese and 0.37 uM  iron. Then Matt and I wanted to look at the differences on filtered and unfiltered medium with P. putida. We believe that if the P. putida produced catalase in the medium, we would see the concentration of hydrogen peroxide decrease over time on the time scale we set up: 14, 8, 4, 2, 1, 0 minutes. However, it did not came out as we expected. There were a lot of background noise from the spectrophotometer. After I wrapped up the last time-point from last week’s experiment, I run a formaldoxime assay to measure the total manganese on all the time-point sample we had collected from our experiment. I also run a leucoberbelin blue (LBB) assay. This was used to measure manganese oxide (MnO_x) or Mn IV. Our results on the LBB assay came out a bit off. Specially our LBB on filtrates because some of the P. putida passed through the filters and started making more MnO_x as we saved our sample for five days. And for the formaldixime assay and LBB assay on the filters, we need to make sure take out any solids and cells present in the medium because they can affect absorbance from the spectrophotometer. We need to repeat this experiment.

This week’s experiment was similar to the one from last week, except we compared filtered and unfiltered samples of P. putida in minimal medium with same additives at same concentrations. Right after the cultures were introduced to the Minimal medium, I run the first time-point. I did an amplex-red + hydrogen peroxide assay and saved eight other filtrate and filters, four for formaldoxime assay and four LBB assay. The result from the filtered and unfiltered medium were similar at our T₀. After about 24 hours, we started to see more obvious results and which showed as we expected. The fluorescence of hydrogen peroxide in the unfiltered medium was decreasing over time on the time scaled we set up like the experiment from last week: 14, 8, 4, 2, 1, 0 minutes. Higher fluorescences means higher hydrogen peroxide concentration. After a certain point the fluorescence became constant, which meant there was no more hydrogen present to react with the reagent. On the other hand, the filtered medium showed a fairly high constant fluorescence, which means the concentration of hydrogen peroxide was somewhat constant.  We believe if we allowed the culture to grow  in well-plate set up, the fluorescence would decrease to a point where the change would be constant.

On Friday, I went on a trip with the other CMOP interns and Vanessa to the Bonneville Lock and Dam. We had a tour on the powerhouse and on fish reservation management. There was a fishladder. And I thought fish would jump up the ladder as they move up, but actually most of the fish pass through a hole at the bottom. We saw the fish swimming upstream through a large glass window. The fish were forced to pass by a narrower passage where a person count all the different kind of fish that pass by. This was done to keep track of the  number of different fish species that has gone up stream of the Columbia River. It was a fun trip.

I still haven’t had time to run the formaldoxime assay and LBB on the filters. I have planned to do them on Monday afternoon.