Wrapping up D. sonorensis characterization

 Introduction:

Through this past week, my team and I have been collecting the data needed to complete a full poster surrounding our project. We performed a catalase test on D. sonorensis to wrap up the biochemical tests. In our preparation, certain tests had to be redone to ensure our information was presentable. While the time spent actively performing lab procedure was minimal this week, this blog will outline the methods use as we extrapolate the final biochemical information we could surrounding D. sonorensis and it's plaque. 

Methods:

Performing a catalase test on D. sonorensis:

1. Prepare a one-way streak plate of D. sonorensis and allow it to incubate for 24-48 hours.

2. Add 2 drops of 3% Hydrogen peroxide to a large colony on the culture dish.

3. Observe for the formation of oxygen gas bubbles. 

Observing homogenization differences of D. sonorensis when suspended in TGY versus R2B:

1. Prepare two Eppendorf tubes- one with 3ul R2B, the other with 3ul TGY.

2. Inoculate D. sonorensis culture into both Eppendorf tubes. Repeat inoculations until sufficient amount of culture is visible in either tube. 

3. Centrifuge tubes for 30 seconds or until culture is nested at the bottom of the tube.

4. Vortex both Eppendorf tubes

5. Observe for homogenization of culture

Results:

D. sonorensis is positive for the catalase enzyme. After the addition of the 3% Hydrogen peroxide, a reaction occurred where visible oxygen bubbles had formed within the colony. When suspended in TGY, D. sonorensis did not fully homogenize into the broth. The plaque structure was not compromised after the vortex, and remained slightly separate from the broth. This differs from R2B, which post vortex, D. sonorensis had fully homogenized. 

Discussion: 

This past week, not much hand-on work was done, as we placed our efforts on perfecting and fine tuning our poster presentation. We were able to swiftly perform the catalase test after hesitation from not having the controls picked out. D. sonorensis was positive for the catalase enzyme. The primary function of this enzyme in bacteria is to decompose hydrogen peroxide to prevent oxidative stress. Hydrogen peroxide is a byproduct of various metabolic processes within the cell, and the catalase enzyme is important in breaking the molecule into oxygen and water, which protects the cell from damage.

We had performed the vortex procedure once prior, but not enough culture was inoculated into the Eppendorf tubes before the vortex. In images, there were no visible culture in either tubes, which provided zero valuable comparative basis of homogenization between the different nutrient broths. In a repeated trial, I had to inoculate multiple times to ensure enough culture was present to yield and effective before-and-after. This brought in a greater risk of contamination to the tubes, but it was unimportant-as we were observing for morphological changes. In this second trial, I had ensured a clear layer of culture was found in the tubes with the centrifuge before I suspended the broths. We had to be quick about taking images after suspending, so we were able to make out the homogenization differences. In the image with the nutrient dense TGY media, the broth was opaque, and upon further observation, one can see the plaque still retained, but spread out due to the vortex. This aids our idea that the more nutrient dense media will give a more resilient plaque, since when compared to a less nutritious R2B, the plaque was more discernable. The R2B Eppendorf tube fully homogenized upon suspension. The less nutrient dense media showed a compromised plaque upon forces such as that of the vortex. These data points aid our story of how the plaque can be made strong or weak, dependent on the nutrient density of the media. 

Next week in lab will involve a summary of all projects worked on in the lab. We will analyze what more needs to be studied, and what feels redundant. From there, I hope to find inspiration for future projects, or find an existing one to join. 

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