Scientific Question:

Does the presence of Arthrobacter phage vary in oak species? Is there one species where they are more dominant?

Rationale:

In today’s lab, the goal was to successfully perform a plaque assay assessment to test the presence of phage in soil samples. Although the results of the spot test yielded negative results for the presence of phage, plaque assays have the ability to yield different results.

Materials Used: 

• 10 μL Lysate
• PY Broth
• PY 2X TA
• 40% Dextrose
• 1M Calcium Chloride
• 0.5 mL of Arthrobacter host
• Agar plate
• Pipettes (Micropipettes and serological pipettes)

Procedure: 

1. I began this procedure by setting up an aseptic zone on my workbench.
2. Next, I gathered the necessary materials to create the top agar. Each group member grabbed their own plate for their plaque assay. I labeled mine with a sharpie and began to make the top agar.
3. Because the top agar made was supposed to be split 4 ways, the measurements for the top agar solution varied slightly from the spot test. For this procedure the top agar consisted of:
   • 8 mL of LB Broth
   • 10 mL of PY 2x Top Agar
   • 90 μL of Calcium Chloride
4. I added the 8 mL of LB broth into our 50 mL conical tube by using the serological pipette.
5. After the LB Broth was added, I used a 20-200 μl micropipette to transfer 90 μL of the calcium chloride to the conical tube.
6. Then I took the filtered lysate from the spot test procedure in the micro centrifuge tube and I used a 1-10 μL micropipette to transfer 10μL of the lysate to the vial containing the 0.5 mL of arthrobacter. I allowed this mixture to sit for approximately 15 minutes to let any possible phage present infect the bacterial hosts.
7. After 15 minutes had past, 5 mL of the top agar was extracted from the tube to add to the arthrobacter solution, the vial was mixed gently, and then plated immediately onto my plaque and let it sit for 15 minutes.
8. Unfortunately, while transferring the top agar to my partner’s vial, we noticed the vial had broken and was leaking my partner’s solution all over the table, so before advancing any further, an aseptic zone was reestablished. We then continued with the transfer of top agar to the remaining vials.
9. We then took the leftover agar and added it to the control plate, leaving the agar to solidify and add to the incubator for 48 hours.

Observations/Results/Data:

• The top agar this time looked very similar to the top agar created for the spot test. There was no contamination of the top agar as everything was performed aseptically.
• The color of the top agar stayed relatively dark yellow, very clear once poured and solidified.

Conclusions/Next Steps:

• The top agar has yet to be examined, but if the results for phage are positive there will be empty lawns throughout the plate where the phage has killed its bacterial host.
• If no spots are examined, then the soil sample will be confirmed negative for phage and I will have to collect more soil samples from different plots on campus.