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. After collecting the lysate it will be divided into two tubes, one for Direct Isolation and the other for Enrichment. Direct.Isolation allows us to test in future experiments if the sample contains bacteriophage and Enrichment allows us.to let potential existing bacteriophage to multiply by manually adding Arthrobacter, letting us get more conclusive results in further experiments.

Experimental Procedure for Soil Washing(continue)                                                                                      Direct Isolation & Enrichment of sample Soil A:

…………..50 ml conical tube Soil A has been in the fridge for 44.5 Hrs.

…………..Prepare:

………………..(1) One Tube-top Filter

………………..(2) One 15 ml conical tube

………………..(3) Vacuum Suction (In the Hood)

………………..(4) 0.5 ml of Arthrobacter

………………..(5) One plastic dropper

…………..1. Attach Vacuum Suction to the Tube-top Filter, slowly drop the liquid onto the filter ……………..paper(.22um).

…………..2. Filtered for 30 min.

…………..3. Split the collected lysate and add 7.5 ml to a 15 ml conical tube for Direct Isolation, the ………………original 50ml conical tube is for Enrichment, which has 11 ml.

…………..4. Place the Directed Isolation lysate in the fridge.

…………..5. Add 0.5 ml of Arthrobacter to the 50 ml conical tube for Enrichment and placed on ………………Shaker.

Observations:

• After Filtration, the lysate in both tubes (15 ml & 50 ml conical tube) present a yellow, transparent color, which can deduct the particles in the lysate are extremely small and invisible to the naked eye.

Results & Data:

………The lysate filtered has a clear yellow color with no debris when examined by the naked eye.

………The 50 ml conical tube Enrichment is labeled: JY5 Soil A Enrich 8.24.18.

………The 15 ml conical tube Direct Isolation is labeled: Jy5 Soil A Direct 8.24.18.

Interpretations, Conclusions:

……….The filter time in the washing procedure may be slightly faster if the liquid is filtered through a filter with a larger hole before than filtering directly with the Tube-top Filter.

Next Step:

………..In the next lab, on 8.27.18, a Spotting Test will be administered on sample Soil A to determine if there are bacteriophages present in the lysate in both Direct Isolation and Enrichment.

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

…………………………………………………….(1) Direct Isolation & 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

…………………………………………………….(9) Agar Plates