Research Question:

To find out how the presence of bacteriophages in the soil around red or white oak trees has a correlation with the health condition of oak trees.

Rationale:

A Plaque Assay helps us determine if there is a 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. Since 9/24 Plaque Assay was ruled invalid due to inactive arthrobacter.

Plaque Assay for Soil (9.17.18):

Materials:

• Micropipette
• Serological pipette
• Centrifuge tube(1.5ml)
• 50 ml conical tube
• LB Broth
• CaCl2(aq)
• Sample (9.17.18) Lysate
• 2x Top Agar
• Agar plate
• Arthrobacter

Procedure:

1. Set up an Aseptic zone.
2. Add 0.4 ml Arthrobacter and 10 ul Filtered Enriched Lysate to a Centrifuge tube 10 min for infection.
3. Add 2.1 ml of LB Broth, 22.5 ul Calcium Chloride (aq) to a 50 ml conical tube
4. Add the infected lysate to the 50 ml conical tube
5. Add 2.5 ml of 2x Top Agar to the 50 ml conical tube, pipette up and down then decant the solution to the agar plate
6. Wait for 10 min to solidify (slightly shooked during) and place into the incubator.

Observations, Results & Data:

Plaque Assay 2 result for sample (9.17.18) showed contamination on the control plate, which makes the result of this plaque assay invalid. The plaque assay plate also showed negative results, no plaques were visible on the plate.

Interpretations & Conclusions:

According to TA’s phage positive control over arthrobacter’s activity, the arthrobacter used on Monday seems to be inactive since there were no plaques formed, indicating there weren’t enough active arthrobacter cell count for the phages to infected and generate a plaque. So the contamination seen on the control plate may not be arthrobacter but some other species of bacterial colony.

Next Step:

Since the Plaque Assay run on Monday is invalid due to the inactive arthrobacter, today was a re-run, so if the result is negative the next step would be searching foe new samples, if positive, the next step would be the purification process.