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 search for possible bacteriophages in the soil, this experiment takes Soil Sample A and go through the Washing process to collect potential bacteriophage-containing lysate for further analysis. By using LB Broth, centrifuge and Tube-top filters with vacuum suction, potential bacteriophage lysate can be collected.

Experimental Procedure for Soil Washing of sample 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) Sample Soil A

……………………….(2) One 15 ml Conical Tube

……………………….(3) One 50 ml Conical Tube

……………………….(4) LB Broth

……………………….(5) Tube-top Filter

……………..2. Place sample Soil A to the 15 ml mark in the 50ml Conical Tube.

……………..3. Pour LB Broth to the 35 ml mark in the 50 ml Conical Tube Soil A.

……………………….(1) 50 ml Conical Tube Soil A weight: 51.20 g

……………..4. Vortex & Shake with Hand for 15 min to mobilize the soil.

……………..5. Set the 50 ml Conical Tube Soil A still for 15 min.

……………..6. To balance the weight in order to centrifuge,

…………………50 ml Conical Tube Soil A was added with water.

……………………….(1) 50 ml Conical Tube Soil A weight: 52.55 g

……………..7. Centrifuge at 3000 G for 5 min.

……………..8. Set the 50 ml Conical Tube Soil A still for 30 min.

……………..9. Filter the supernatant from the 50 ml Conical Tube Soil A with 1 microliter filter …………………paper.

……………10. Stored the Supernatant filtered from the filter paper into a new 50 ml Conical Tube …………………and stored in fridge.

……………11. Washing Procedure of sample Soil A is to be continued on 8.24.18.

……………12. Workbench was sterilized with Ci-Decon and 70% Ethanol for clean up.

Experiment Results and Observations:

…Observations:

• The Soil Sample has a brown color and a moist texture.
• After adding LB Broth and mobilizing the soil, the liquid is opaque and has a muddy color (On the left)
• After the 50 ml conical tube was centrifuged there are sediments at the bottom of the tube, as the sketch shows (On the Right) the sediments lays slightly to the side, which assume is due to the position of the tube when it was in the centrifuge. The supernatant has a slightly translucent yellow color with a couple of floating debris at the surface of the liquid.

• Going through a filter paper with a larger hole results in a faster filter time, yet the liquid still presents a translucent yellow color, indicating there are still particles of soil present in the liquid.

…Results:

………Due to the time limitations of lab hours and equipment, the washing procedure of sample Soil A has to be postponed to 8.24.18.

………The 50 ml conical tube is labeled: JY5 Soil A 8.22.18.

Interpretations, conclusions, and next steps:

…..Interpretations and Conclusions:

……….When putting soil samples in the 50 ml conical tubes in the future, make sure sizable debris such as parts of grass and sticks are picked out so after centrifuge there won’t be any debris floating in the liquid causing.difficulties to conduct the washing procedure.

…..Next Steps:

……….In the next lab, on 8.24.18, I will be finishing sample Soil A’s Washing procedure, splitting the volume of the lysate in half into a 15 ml conical tube for Direct Isolation and 50 ml conical tube for Enrichment.

……….For the coming experiment, I will need:

……………………………………………………………(1) One Tune-top Filter

……………………………………………………………(2) One 15 ml Conical Tube

……………………………………………………………(3) 0.5 ml of Arthrobacter

……………………………………………………………(4) One plastic dropper