29 August 2018 ✷ Plaque Assay

Because no plaques were present in my spot test (negative spot test), only 1 plaque assay will be run as to practice running this type of test (using filtered enriched lysate).

Procedure:

• CiDecon and 70% Ethanol were used to wash the workspace and prepare the aseptic environment. An alcohol burner was lit to clean the air.
• The plate was made–see table below for volume and concentration values. The LB broth was added first (4x the amount was made because the lab group consisted of 3 people with the addition of a control plate). Next, we added the calcium chloride. 2 mL of the mixture was removed for the test plate and put in a separate vial; 2.5 mL of top agar was added last so it wouldn’t harden while we were adding the other components. The tube was inverted to mix the components and then pipetted onto the dish.
  

  component	volume	concentration
  2X Top Agar	2.5 mL	1.05X
  LB Broth	2.0 mL	–
  1M Calcium Chloride	22.5 µL	4.5 µM
  1000 µM*V=4500µL*4.5µM = 22.5 µL
  2X*2.5 mL=M*4.5mL = 1 M

• 10 µL of filtered enriched lysate was added to a tube of arthrobacter and allowed to sit for 15 minutes.
• The remaining 7.5 mL of TA was added to the shared vial and mixed; then 5 mL was transferred to a vial for Rachel and 5 mL was transferred to a vial for Lauren. After 15 minutes, the arthro mix was added to the TA vial, inverted to mix, and poured into the petri dish. After the plate solidified, it was inverted and placed in an incubator until 2 pm on Wednesday, September 5.

Observations, Results, Data:

• Because the last plate I made was contaminated, I took extra care to ensure ALL work was done aseptically.
• However, since the last test was negative, it is highly unlikely that the plaque assay performed today will produce any positive results.
• Of the 24 people in the class, 9 had positive spot tests.
• UPDATED: after sitting in an incubator for nearly a week, the assay yielded a negative result, meaning no phage was found. However, there didn’t seem to be any contamination, which is an improvement from last week’s experiment.

Interpretations, conclusion, next steps:

• Because the bacteria (and potentially a phage) are mixed before entering the plate, the chance of infection is higher on a plaque assay than on a spot test, thus this test may be more telling of the presence of a phage in a sample. However, a control plate is necessary to ensure the agar isn’t contaminated because the phage may cover the entire plate rather than just a small circle as in the spot test.
• Though the chances for my particular plate are low, there is still a possibility my plaque assay may yield a positive result (phage present).
• The negative result from the spot test will be used in answering the class’s overarching scientific question regarding the presence of phages in relation to the types of oak trees on Baylor’s campus.
• In the future, more soil will need to be collected from another tree in hopes of isolating a new phage to continue research on.