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.  And dilution of picked plaque lysate can help us calculate the titer of the phages, and successfully culture more phages for further analysis.

Plaque Assay for Soil (10.1.18):

Materials:

• Micropipette
• Serological pipette
• Centrifuge tube(1.5ml)
• 50 ml conical tube
• LB Broth
• CaCl2(aq)
• Sample (10.1.18) Lysate
• 2x Top Agar
• Agar plate
• Arthrobacter

Procedure:

1. Set up an Aseptic zone.
2. Dilute the picked lysate to 10^-1 concentration.
3. Add 0.5 ml Arthrobacter and 10 ul original picked plaque lysate and 10^-1 lysate to a Centrifuge tube 10 min for infection.
4. Add 2 ml of LB Broth, 22.5 ul Calcium Chloride (aq) to a tube and repeat to another tube.
5. Add the infected lysate to their respective tubes.
6. Add 2.5 ml of 2x Top Agar, pipette up and down then decant the solution to an agar plate and repeat.
7. Wait for 10 min to solidify (slightly shooked during) and place into the incubator.

Observations, Results & Data:

The plate showed some disruption on the plates, no obvious plaques were discovered, but there was a small amount of liquid found on the plate.

Interpretations & Conclusions:

The plaque assay did not show obvious plaques, but the liquid and the disruption found on the plate might suggest that phages might lysed the bacteria, so no individual plaque was found, the new plaque assay would again confirm if there is an existence of phage.

Next Step:

Check the results of the plaque assay plates, discuss with the group then decide the next step.