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) Picked Plaque 10^0
• 2x Top Agar
• Agar plate
• Arthrobacter

Procedure:

1. Set up an Aseptic zone.
2. Add 0.5 ml Arthrobacter and 10^0 lysate to a Centrifuge tube 10 min for infection.
3. Add 2 ml of LB Broth, 22.5 ul Calcium Chloride (aq) to a tube and repeat to another tube.
4. Add the infected lysate to their respective tubes.
5. Add 2.5 ml of 2x Top Agar, pipette up and down then decant the solution to an agar plate and repeat.
6. Wait for 10 min to solidify (slightly shooked during) and place into the incubator.

Observations, Results & Data:

The plaque assay plates showed small clearings that are about 0.1 ~0.2 mm in diameter and the have tiny bumps on

the clearings.  The large clearing on plate 10^-1 at the top left corner in the picture above is a bubble formed when the top agar was solidifying.

Interpretations & Conclusions:

The potential plaques on the plates could be small plaques or cracks in the top agar layer, while the bump observed makes the clearings likely a break in the agar since that a bump may be the result of cracks, there is no way to completely rule out the possibility of both.

Next Step:

If the plaque assay shows plaques, the sample may have potential plaques, then the experiment can keep focus on the current sample.