Date: Monday, September 17th, 2018

Title: Plaque Assay Setup and Soil Sample C Collection

Rationale: The purpose of today’s lab is to set up a plaque assay with the lysate from Soil Sample B as well as gather more soil if the plaque assay yields negative results.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Plaque Assay Procedure:

1. An aseptic zone was set up.
2.  Spot tests were evaluated and found to have yielded negative results
3. 10 microliters of the Soil Sample B enriched lysate was added to a culture tube with 5 mL ATC 21022.
4. Culture tube was set aside for 15 minutes so phage could infect arthro.
5. Agar was made with the following formula:
   1. 8 mL LB broth
   2. 10 mL 2x TA
   3. 90 microliters 1M CaCl2
6. 4.5 mL of the top agar solution was added to the culture tube.
7. TA solution with arthro and phage was added to agar plate, left to harden for 10 minutes, inverted and incubated.

Soil Sample C:

• Tree Circumference: 380 cm
• Small Canopy Diameter: 1606.5 cm
• Large Canopy Diameter: 1976 cm
• Average Canopy Diameter: 1791.25 cm
• Tree Height: 13.68 m
• Tree Coordinates: 31°32’59” N 97°6’57” W

Observations: Controls were all contaminated, but individual spot tests were negative for plaques and contamination. It’s unclear how the controls were contaminated. The top agar wasn’t contaminated or the individual plates would have also had contamination.

Results: This experiment yielded a plaque assay to be evaluated for possible plaques. This also yielded a new soil sample to be experimented on if the plaque assay from Soil Sample B yields negative results.

Next Steps: The next step is to evaluate the results of the plaque assay. If negative, the next step is to work with Soil Sample C and try for phage with it. If positive, the next step is serial dilutions and plaque assays.

Conclusion: From the results of testing on this sample and the soil meta data, it could become evident in what type of environment phages tend to reside, and whether there is a difference between soil around live oaks or red oaks.

In addition to the experiments, there were also two guiding questions asked of the class to consider:

1. Group 4 all had plaques on their plaque assays. Justin had the most and well defined plaque (but all 3 got plaque). They each did a spot test in addition to their plaque assays, but only Justin had a plaque on his spot test. What do you think is going on?

• The likely reason is simply that the sample Justin used had the highest titer, resulting in better defined, clear spots. It’s also possible that he was more careful during his procedure, leading to less outside influences.

2. Lathan checked a purified lysate by a plaque assay using 10 microliters of 10^-3               lysate. He got 14 plaques. How many microliters of Lathan’s lysate should he add             web a plate (8 cm in diameter) if his average plaque diameter is 1mm?

• 14 plaques divided by 10 microliters = 1.4 x 10^3 pfu per mL 10^-3 lysate. The plate radius = 40 mm, and the plaque radius = .5 mm. The area of the plate over the area of the plaque = 6.4 x 10^3 pfu to web. 6.3 x 10^3 pfu divided by 1.4 x 10^6 pfu per mL = 4.6 x 10^-3 mL 10° lysate to web = 4.6 microliters 10° lysate to web.