Research Question:

…………To find out how the presence of bacteriophages in the soil of oak trees would affect the pathological process of Oak Wilt Disease and if the variation in oak species has a correlation with the presence of bacteriophages.

Rationale:

…………To test if sample Soil A has the presence of bacteriophages, a spotting test will be administered to Soil A Direct Isolation & Enrichment. By adding drops of lysate from sample Soil A to a bacterial lawn on the agar plates, we can determine if bacteriophages are present in the lysate or not. A Plaque Assay also helps us determine if there is the presence of bacteriophages, by adding Arthrobacter directly to the lysate, we can tell the existence of bacteriophages by checking the presence of plaques on the agar plate.

Experimental Procedure for Plaque Assay for Soil A:

……………………. 1. Set up an Aseptic zone(Sprayed with Ci-Decon, wiped dry, then sprayed 70% …………………………Ethanol and let it evaporate)  on the workbench,  prepare:

…………………………………………………….(1) Enrichment Lysate

…………………………………………………….(2) LB Broth

…………………………………………………….(3) 2x TA

…………………………………………………….(4) 1M CaCl2

…………………………………………………….(5) 0.5 ml Arthrobacter

…………………………………………………….(6) 50ml conical tubes

…………………………………………………….(7) Micropipettes & Serological Tubes

…………………………………………………….(8) Phage Buffer

………………………2. Checking the results of Spotting Test for sample Soil A

………………………3. Add 10µL of your lysate to a cultural tube w/0.5mL Arthrobacter, sit for 15 min.

………………………4. Add 8 ml LB Broth, 90 ul CaCl2 (aq) to a 50 ml conical tube.

………………………5. Add 10 ml 2x TA to the 50 ml conical tube, pipette to mix properly, take out 1 ml ………………………….and drop on the assigned section on the control plate.

………………………6. Take 4.5 ml of Top Agar from the 50 ml conical tube and pipette inside the ………………………….cultural tube to mix with lysate+Arthrobacter and add to a new agar plate, ………………………….slightly swirl to cover the plate evenly, set still for 10min before place in ………………………….incubator top-side down.

Observations, Results & Data:

……………………..

………………From the Spotting test, the TA Control appears to be uncontaminated as the gel still remain its original transparent yellow color. The Spotting plate is presented with several spots, proved to not be bubbles since the positions are inconsistent with the sketches drawn before. I hypothesize those may be either potential plaques or contamination An Interesting point worth noting is the “spots” are more prevalent in the Direct Isolation section, and not in the Enrichment section, which I originally presumed to have a higher probability to generate plaques due to theoretical higher concentrations of bacteriophage than Direct Isolation.

Interpretations & Conclusions:

……………….If I presume that all previous experiments for the Spotting test are not contaminated, then I would deduct the so-called “spots” might not be plaques due to the locations of the spots, more on the Direct Isolation section than the Enrichment section. The plate itself could potentially be contaminated before they were used since other unused plates also presented with similar conditions as described by the TA’s. I would wait for the Plaque Assay to yield its results to determine if sample Soil A contains bacteriophages or not.

Next Step:

………………If the Plaque Assay yields promising results I would continue trying to isolate the bacteriophages in sample Soil A, otherwise, I will search for new samples.