Rationale:

Repeating the last plaque assay due to contaminated arthrobacter. Will be performing another purifcation plaque assay from the previous 10^0 lysate from 9/24 (Kept in refrigerator)

Procedure:

• Created an aseptic zone to prevent the possibility of bacterial contamination
• Retrieved 10^0 lysate from refrigeration and created a 10^-1 and 10^-2 dilution
  • Created by adding 90μL PB into microcentrifuge vial and adding 10μL of the previous dilution
• Added the dilutions of lysate into separate 400μL vials of arthrobacter into infect
• Obtained a 50mL vial and added in 16.8 mL LB Broth, 180μL 1M CaCl2, and 20 mL 2XTA (Enough for 9 plates)
• Immediately pipetted 4.5 mL TA mixture into the arthrobacter vial(s), shook to mix, and plated
• Let sit for 15 minutes and then incubated

Observations:

• The control TA plate sat for less than 15 minutes
• Surprising that the arthrobacter was the one to be contaminated, rather than the LB Broth or TA
• Lathan explained that the bacteria in our sample most likely are not arthrobacteria anymore since there were no plaques, and rather were another strain of bacteria

10^0 plate with contamination. Interesting pattern created

10^-1 contaminated plaque assay

10^-2 exhibiting contamination

The control TA with contamination

Conclusion/Next Steps:

It was unfortunate that there as contamination since after this plaque assay, the lysate would’ve been able to be used to create a titer and web a plate. It has shifted lab time one session back. The next steps would be to analyze the results from this plaque assay and hopefully be able to create/calculate a titer and web a plate