Rationale:

Performed another plaque assay on the lysate to further purify the bacteriophage

Procedure:

• Created an aseptic zone to minimize chances of bacterial contamination
• Picked a plaque from the 9/21 plaque assay (10^0 dilution) and pipetted into 100μL of PB
• Created a 10^-1 dilution by adding 10μL of lysate (10^0) to 90μL of PB
• Created a 10^-2 dilution by adding 10μL of lysate (10^-1) to 9 μL of PB
• Obtained a 50 mL conical vial and added in 18mL LB Broth and 202.5μL CaCl2 in (Calculated for 9 plates)
• Added 10μL of lysate (All three) to separate 0.5μL arthrobacter vials (Allow to infect)
• Added in 22.5 mL 2XTA to the conical vial, and immediately pipetted 4.5 mL of the TA into the bacteriophage+arthrobacter vial and plated (For all three)
• Let the plates sit for 15 minutes and then incubated

Observations:

Not as many plaques on the 10^-1, but still a considerable amount

Significant amount of plaque on the 10^0

10^-2 plaque assay with only a few plaques

The group TA control was contaminated yet again, possibly indicating something is contaminated in either the LB Broth or TA

Conclusion/Next Steps:

The lab group was able to finish this procedure without problems or using up too much time since we are repeating procedures. Next lab period, will be looking for countable plaques on the 10^0 plate to create a webbed plate