Rationale:

The purpose of todays lab was to analyze the results of the plaque assays performed prior. If plaques were present, dilutions and plaque assays would be performed from picked plaques. If no plaques were found, final soil gathering and enrichment were to take place.

Plaque Assay Results from 10/08/18:

• Plate was littered with plaques.
• Top Agar control was uncontaminated, meaning there is a high possibility this is not some other organism such as in previous trials.
• 

  Group Control Plate

  

Materials:

• Micropipette to pick plaques
• Phage Buffer for Dilutions
• 2X TA
•  Arthobacter 
• LB Broth
• Calcium Chloride
• Enriched Lysate

Procedure:

1. Established an aseptic zone
2. Began by circling 6 plaques to pick.
3. To pick a plaque, used a micropipette tip to poke the top agar and lifted out (avoid bacterial lawn beneath) and inserted tip into 100-μL of phage buffer. Repeated for each plaque in separate containers. This was the 10^0 dilution
4. Chose the first plaque picked for dilutions and 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.
7. 2.0-mL of LB broth was added into 3 separate conical vials.
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.

Data:

• Plaque morphology was relatively difficult to gather. The plaques were very small, seemingly circular, and some had more jagged edges than the others. For the most part they were relatively the same size except for a few outliers.
• 
• Plaque assays were performed with no complications.

Conclusions:

• There is a clear indicator of phage presence, but it is impossible to determine which type and how much are present.
• The soil sample gathered is phage positive, being the first soil sample gathered to test positive.

Next Steps:

The next steps are to check the plaque assay results and determine the titer of phage. From this, amplification and purification will be needed to single out a specific phage from the possible multiple phages that could be present.