Rationale:

The purpose of today’s lab was to analyze the plaque assay results from the previous lab. If plaques are present, then the purification process will continue. If no plaques are present, then new serial dilutions will be formed to retest phage presence.

Results from 10/22/18:

• Plaque results were negative.
• Although results were negative, top agar control was uncontaminated, and is a clear indicator that there truly were no phage particles present.
• New serial dilutions will be run with new lysates.
• 

  10^0 PA 10/22/18

  

  10^-1 PA 10/22/18

  

  Top Agar Control 10/22/18

  

  10^-2 PA 10/22/18

   

   

Materials: 

• Phage Buffer
• 10 Circles Plaques to Pick
• LB Broth
• 2X TA
• Calcium Chloride
• Agar Plates

Procedure:

1. Established an aseptic zone.
2. Circled 10 new plaques to pick.
3. To pick a plaque, used a micropipette tip to poke 10 plaques separately the top agar and lifted out (avoid bacterial lawn beneath) and inserted tip into 100-μL of phage buffer.  This was the 10^0 dilution
4. Added 10-μL of the 10^0 dilution to 90-μL of phage buffer, making a 10^-1 dilution.
5. Made a 10^-2 dilution by repeating the dilution process with the 10^-1 dilution and adding it to 90-μL of phage buffer.
6. Once diluted, 3 separate plaque assays were run with each dilution as well as one top agar control.
7. 2.0-mL of LB broth was added into 3 separate conical vials and 2.5-mL added to control.
8. Next, 22.5-μL of calcium chloride was added to the conical vials.
9. 10-μL of each of the dilutions was combined with 3 separate 0.5 mL quantities of Arthrobacter and left to infect.
10. After about 15 minutes, the diluted lysates were combined with their LB Broth mixtures and 2.5-mL of 2X TA was added to the broths.
11. Swirled and plated top agars immediately.
12. Once solidified, they were added to the incubator until the next lab.

Results:

• Experiment went smoothly, pushed a little farther into the top agar to see if there was a possibility plaques were not being picked properly. Plates were plated without any issues as top agar did not solidify before plating.

Conclusions:

• The experiment was performed with good aseptic technique, and should yield results with no data skewing. A possibility for error is in the plaque picking as pushing deeper could possibly puncture the bacterial lawn below. There were no clear signs of puncturing, but it is a possibility.

Next Steps:

• The next steps are to analyze the plaque assay results for plaque presence. If plaques are present then purification will continue in hopes of obtaining a high titer lysate.