Rational:

Picking a plaque and performing a serial dilution, as well as perform plaque assays to isolate the bacteriophage

Procedure:

• First, created an aseptic zone ensure no bacteria would contaminate the plates
• Picked a plaque from the 9/12 plaque assay, and added it into 100μL Phage Buffer
  • Tube labeled as 10^0 dilution
• Added 90μL of PB into two more micro-centrifuge tubes
  • For one tube, added 10μL of 10^0 into it, creating the 10^-1 dilution
  • For the second tube, added μL of 10^-1 into it, creating the 10^-2 dilution
• Then added 10μL of each dilution to 0.5 mL of arthrobacter
• Obtained a 50 mL conical vial and added 20mL LB Broth, 22.5 μL CaCl2, and 25 mL 2X TA
  • Created enough TA for 10 plates; three individual plates for the members in the group, and one more for the control plate
• Immediately mixed the TA with the 10^0, 10^-1, and 10^-2 dilutions, and then plated
• Let the plates sit for 15 minutes, and then incubated all 10 plates

Observations:

9/12 Plaque assay; can see the formed plaques

The 9/12 spot test

The 10^0, 10^-1, and 10^-2 plates

• Justin was the only member in the team to have plaques in the spot test
• For the plaque assay, there were many plaques, possibly indicating that the bacteriophage are lytic

Questions on the board:

1. Although all members of group 4 had plaques on their plaque assays, only Justin had plaques on his spot test. This may have resulted from the fact that bacteriophage can go through two “reproduction” cycles: the lytic and lysogenic cycle(s). Justin’s bacteriophage may be lytic, and immediately puts the bacteria through lysis, while his group members’ bacteriophage maybe lysogenic. This could explain the spot test because the soil they pulled from were in the same general area, and they all have bacteriophage in their soil, as confirmed by the plaque assay.
2. 

   Lathan would need 40.2μL of 10^0 to web his plate

    

Next Steps:

Repeat the picking of plaque from the 10^0 plate if plaques appear, as well as run the plaque assays again. If plaques do not appear, pick another plaque from the 9/12 plaque assay and repeat.

Conclusion:

The lab proceeded without problem since Lathan explained the procedure of serial dilution and the process in the video lecture. Every member in my team worked together to finish the lab as swiftly as possible, while ensuring no contamination occurred.