Rationale: Today, I will be checking the results of our spot tests conducted on 8/27, as well as start the procedure of a plaque assay. The objective of today is to correctly run a plaque assay.

Scientific Question: Does the presence of Arthrobacteriophage appear more dominant in one oak tree species compared to another? If so, in this species, is there a correlation between the presence of Arthrobacteriophage and the presence of Oak Wilt Fungus growth?

Procedure:

• First, we cleaned our lab table/area with CiDecon and 70% ethanol and placed an alcohol burner in the area to create an aseptic zone
• We then obtained three agar plates, one for each of us to run our plaque assay on, as well as one plate where four of our groups (Groups 5, 2, 1, and 6) would run our control
  • My plate was labeled “Plaque Assay”, along with my initials, the date, and “Enriched Lysate Soil A”
• We first measured out 22.5μL of 1M CaCl2 with the P200 micropipette [Min. 20μL and Max 200μL] (Labeled with a yellow sticker) and transferred it into our TA 50 mL conical vial
• We then added 2mL of LB Broth to out TA conical vial via a cartwheel pipette with a 5 mL tip
• This is the point in our experiment where we realized that we forgot to multiply the materials by 4 since there are three of us in our group (Plus the TA needed for our control), so we then multiplied our materials (Originally 0.5mL LB Broth, 2.5 mL 2X TA, 22.5mL 1MCaCl2) by four (My personal plaque assay plate, Kathryn’s, Emily’s, and our team’s control), resulting in our group obtaining 8mL LB Broth, 10mL 2X TA, and 90mL 1M CaCl2
• We then added another 67.5 μL of 1M CaCl2 to our TA conical vial via the P200 micropipette [Min. 20μL and Max 200μL] (Labeled with a yellow sticker)
• Similarly, we added another 6mL of LB Broth to our TA conical vial via a cartwheel pipette, with a 10 mL tip
• I then added 10μL of my enriched lysate to 0.5 mL of Arthrobacteria via the white pipette (The P10 pipette) [Min. 1μL and Max 10μL] and let it sit for 15 minutes
  • We do this to allow time for any bacteriophage in the lysate to infect the Arthrobacteria
• After waiting 15 minutes, we then added 10 mL of 2x TA to our TA vial with a cartwheel pipette with a 10 mL tip, and then immediately added 1mL to our control dish (Quadrant shared)
  • We have to immediately add it to the dish because as soon as the TA is added into the mixture, the solution will start to solidify
• I then added 5mL of the TA to my arthrobacteria + enriched lysate vial, and mixed it with the pipette (Cartwheel pipette with a 5 mL tip)
• I then added the mixture onto my plate, and let it sit for around 10 minutes to solidify
• The plate was then transferred to an incubator set at room temperature
• We cleaned up our work area with CiDecon and 70% ethanol

Results:

• So my spot test turned out negative, and our team’s control TA dish was contaminated. This was a minor setback, but with the start of the plaque assay, we are hoping that we will get positive results that our lysate(s) have bacteriophage

Observations:

• The procedure of the plaque assay was very similar to the spot test, however the only change in procedure was that the lysate and arthrobacter were introduced together first so if there are any bacteriophage in the lysate, it would be able to infect the lysate
• 

  The control plate for our four groups; notice the quadrants drawn on the bottom of the plate

  

  The plate for my plaque assay, prior to adding the TA or arthro + lysate mixture

  

  The negative results of the spot test

  

  The contaminated control plate of Group 2’s spot test

   

• Next Steps: We will be waiting for the results of the plaque assay. If it turns out negative, then we will need to obtain another sample soil different from where we got our first sample (Sample A). If positive, then I think a retesting of a spot test may be wise to double check the lysate.