Rationale:

The purpose of today’s lab experiment was to analyze the plaque assays performed on 10/31/18 and continue with purifying the phage particles to get a consistent morphology. We are working towards obtaining high titer lysates.

Results 10/31/18:

• Plates were all plaque positive, but unfortunately the 10^-2 dilution had several air bubbles that had popped on the top agar, making the results inconclusive. Going to continue with the final round of dilutions to get a consistent plaque morphology and pick a plaque from the 10^-1 diluted plate.
• 

  10^-1 Dilution 10/31/18

  

  10^0 Dilution 10/31/18

  

  10^-2 Dilution 10/31/18

  

  TA Control 10/31/18

Materials:

• Phage Buffer
• Plaque to Pick
• LB Broth
• 2X TA
• Calcium Chloride

Procedure:

1. Established an aseptic zone.
2. To pick a plaque, used a micropipette tip to poke 1 plaque from the 10^-1 plaque assay (avoiding bacterial lawn beneath) and inserted tip into 100-μL of phage buffer to release phage particles.  This was the new 10^0 dilution.
3. Added 10-μL of the 10^0 dilution to 90-μL of phage buffer, making a 10^-1 dilution.
4. Made a 10^-2 dilution by repeating the dilution process with the 10^-1 dilution and adding it to 90-μL of phage buffer.
5. Once diluted, 3 separate plaque assays were run with each dilution.
6. 2.0-mL of LB broth was added into 3 separate conical vials and 2.5-mL was added to a fourth top agar control.
7. Next, 22.5-μL of calcium chloride was added to each of the conical vials.
8. 30-μL of each of the diluted lysates were combined with 3 separate 500-µL quantities of Arthrobacter and left to infect for approximately 15 minutes..
9. After about 15 minutes, the diluted lysates were combined with their respective LB Broth mixtures and 2.5-mL of 2X TA was added to each of the conical vials.
10. Swirled and plated top agars immediately.
11. Once solidified, they were added to the incubator until the next lab.
12. Calculated titer by counting plaque formation on the 10^-1 dilution from 10/31/18 and used the equation Titer = #pfu/volume of lysate in µL * (10^3 µL/mL) *10^1

Results and Data:

• The plaque assays were performed with no error during the experiment except for slight air bubble formation along the side of the plate.
• The calculated titer of the current lysate used to make the 10^1 dilution on 10/31/18 was 3.167*10^4 pfu/mL.

Conclusions:

• Overall, the calculated titer was on the lower end of the spectrum, which is consistent with the rest of the class. The phage concentration that was begun with was not very high initially as it required more than 10 µL of lysate to even show any plaque formation on any diluted plaque assays. Also, the titer is low due to the fact that there have been no webbing and flooding involved that could potentially give rise to high titer lysates as the lysate has not moved beyond the purification process.

Next Steps:

The next steps in this experiment are to analyze the results from this plaque assay, calculate a new titer, and move towards webbing and flooding a plate.