Rationale: 

The goal of today’s lab was to analyze the previous plaque assay for any possible indicators of phage present and perform a serial dilution if phage was found. If plaques were negative, a new soil sample was to be obtained.

Results of 09/26/18

• Plaque assays were negative with no clear indicators of phage presence.
• LB Broth was contaminated, as well as our control plate, with only a small spot of Artho still alive.
• 

  Empty Plaque Assay

  

  Contaminated LB broth

  

  Contaminated Plate

Materials

• 2.5-mL 2X TA
• 500-μL Arthobacter 
• 2.0-mL LB Broth
• 22.5-μL Calcium Chloride
• Enriched Lysate

Procedure

• Obtained new soil sample from possible Burr Oak outside of North Village.
• Returned to lab and established an aseptic zone to perform plaque assay.
• Aliquot 2.0-mL LB broth into vial.
• Added 22.5-μL of calcium chloride to vial.
• Combine 0.5-mL of Arthobacter with 10-μL of enriched lysate and left to infect.
• After 15 minutes of infection, 2.5-mL of 2X TA was added to the conical vial containing the LB broth.
• Added the infected lysate to the top agar solution and plated immediately, left to solidify,
• Once plate was solidified, plate was left in the incubator for 48 hours.

Results/Observations:

• The tree that the soil was gathered from was an extremely large burr oak in an empty field. The only other tree was an equally as large pecan tree right next to the oak. The oak tree seemed healthy, with no visual indications of poor health. What was very interesting was the differing soil consistencies surrounding the tree. 3 separate samples were gathered: one was clay-like, the other was less wet and very dark, and the third was almost completely dry and pebbly.
• 
• Plaque Assay and LB broth were performed with seemingly no errors to the aseptic zone. LB broth was clear during use and showed no indication of contamination.

Analysis/Conclusions:

• The contamination of the plaque and LB broth was most likely caused to an error in the aseptic technique used during experimental procedure. It could be possible that a pipette tip touched a contaminated surface, or procedures were performed too far from the flame, possibly introducing contamination to the plaque assay.
• The different soil types are indicators of varying soil compositions surrounding the sample tree, it could be possible that these varying compositions give way to different environments for phage and Arthobacter.

Next Steps:

• The next steps are to check the plaque assays after the 48 hours have passed to see if there are any plaques present. If plaques are present, I will pick a plaque and perform a serial dilution to get a high titer of phage and begin purification. If there are no plaques present, then the new soil gathered will be cleaned and enriched for a new round of plaque assays.