Rationale:

The goal for today was to analyze the results of the previously performed plaque assay and either perform a serial dilution if there were plaques present or perform another plaque assay.

Results of 09/24/18

• Contamination of the group control plate yet again. Plaque assay was also negative.
• It was also discovered that the lack of plaques and contamination could be due to the apparent contamination of the Arthobacter cultures in the lab. There were no plaques present because there was no Arthrobacter present to infect. This meant that a new plaque assay had to be performed with an older culture of Arthobacter to truly determine if the soil sample is negative.
• LB Broth was clearly contaminated, contained a precipitant very similar to the past 2 contaminated plaque assays.
• 

  Contaminated LB Broth

  

  Uncontaminated LB Broth

  

  Contamination

  

  Empty Plaque Assay

   

Materials:

• 2.5-mL 2X TA
• 400-μL Arthobacter (Lab was low on uncontaminated Arthro so we had to lower amount used)
• 2.1-mL LB Broth
• 22.5-μL Calcium Chloride
• Enriched Lysate

Procedure:

• Established aseptic zone.
• Aliquoted 2.1-mL LB broth into a conical vial.
• Added 22.5-μL of calcium chloride to the LB broth.
• After, added 2.5-mL of 2X TA to control top agar and plated immediately. Left to solidify and set in the incubator.
• Combined 10-μL of lysate with the 400-μL of Arthobacter and left to infect for 15 minutes.
• After time had passed, aliquoted 2.5-mL of 2X TA to broth mixture, added infected lysate, and plated immediately.
• Left plate to solidify and put in the incubator until the next lab.

Results/Data:

• Previous plaque assay had been negative, however careful measures were taken to ensure the experiment was performed aseptically. Additional burners were added and almost all equipment was wiped with 70% ethanol before use.
• LB Broth also was examined before use and was completely clear before the experiment. If the broth is contaminated next week, there is contamination elsewhere.
• 

  Uncontaminated LB Broth

   

Conclusions:

• If the plaque assay returns negative with no contamination, we will know that there is no phage present in the soil sample taken. Recently it’s been very difficult to tell due to the influx in contamination across the lab. Still unsure as to what is causing it as extra measures are taken to ensure that the experiment is performed aseptically.
• Arthrobacter was contaminated, so using this guaranteed culture that was grown at the beginning of the year should yield legitimate results.

Next Steps:

• Analyze the plaque assay’s results and either perform a serial dilution and pick a plaque, or find a new soil sample.
• Finally find some phage in the lysate.