Research Question Discussion:

After discussing with fellow BEARS in the SEA members, I have developed a prototype research question that expresses both something not too narrow and that can be applicable to a real-life situation. The dependent variable in this study is whether or not the Arthrobacterphage is present or the positive signs appear from either a spot test an/or the plaque assay. We considered many variables that could potentially affect the results of the lab. These variables included: soil pH, soil type, distance from water resource, temperature, weather, elevation, climate, and tree species. From here we were able to narrow down the factors. Since we will be working on Baylor University campus, the area for collecting samples is small enough that variables such as temperature, climate, elevation, and the weather should be relatively the same. As far as soil type, we don’t know if there is a sufficient amount of soil type samples to test. If we studied the tree height, it would be difficult to measure it accurately. Same as the distance away from a water source. After all, what exactly do we mean by water source? Is the water source a sprinkler, creek, or the Brazos River? The class felt like tree species would be a good variable to test. Specifically, the independent variable would be the different types of oak trees, so we could relate it to the real-world problem of oak wilt disease. The following question has two parts: one as a broad, generalized question and one as a specific applied real-life scenario. We wrote the question in this format so we can have a testable hypothesis. Based on the observation from the first spot test, finding a bacteriophage is not easy. So ideally, if we do find a sufficient amount of bacteriophage in the next few weeks, we can continue to answer the second part of the question, and thus, apply the scenario to a real-life situation. If not, we can simply drop the second part.

Purposed Prototype Research Question:

Does the presence of Arthobacterphage appear more dominant one oak tree species than the others? If so, in this species is there a correlation between the presence of Arthrobacterphage and the present of oak wilt fungus growth?

If this prototype research question is used, we will need to define what we mean by “oak wilt fungus growth.” Also, we will need to define where we will be collecting the sample.

As far as metadata, if we used the prototype research question above, we would need to identify the oak tree species.

Rationale:

Through washing the soil, the bacteriophages will become separated from the dirt and bacteria. The addition of the arthrobacter will allow for the determination of whether the bacteriophages present attack arthrobacter specifically or not.

Procedure:

1. Cleaned the counter area with CiDecan and wiped it dry. Then, cleaned with EtOH (70%) and allowed it to evaporate.
2. Added approximately 15 mL of soil sample (collected on Tuesday from Area 2) into a 50 mL conical vial and labeled it “KEA 8/21/18 Soil A.”
3. Through aseptic technique (performed the following over an EtOH (100%) lamp and being extremely cautious by not allowing the conical vial or cap to touch anything), poured the LB Broth into the 50 mL conical vial with about 15 mL of the soil sample to the 35 mL mark.
4. Used an analytical balance to measure the mass of the conical vial.
5. Mixed the LB Broth into the soil by hand-shaking for approximately 15 minutes.
6. The vial was then centrifuged at 3000xg for 5 minutes.
7. The supernatant was then pipetted into a 0.22 μm top vacuum filter to separate the lysate.
8. The lab counter was cleaned with CiDecan and EtOH (70%).

Observations:

• With the addition of the LB Broth, the conical vial weighted 53.18 grams.
• Before the conical vial was centrifuged, the hand-shaken soil and the LB Broth appeared to look like chocolate milk.
• After the conical vial was centrifuged, the supernatant appeared to be light colored and murky with a few dark particles floating at the top. The pellet had streaks of dark and light brown throughout. The following image shows the pellet in the conical vial.

• After approximately 20 minutes of filtering out the supernatant, only a little less than 10 mL of the lysate formed. The lysate was a light shade of yellow.

Next Steps:

On Friday (8/24), the supernatant will finish filtering. It will be spilt into the direct isolated lysate and the enriched isolated lysate. Next, 0.5mL of arthrobacter will be added to the enriched lysate. After finishing these steps, the enriched lysate should be prepared for Monday’s spot test.