Rationale:

The purpose of today’s experiment was to flood and web a plate using the calculated lysate from titer calculations.

Results from 11/05/18

• All of the plates were positive for plaques up to the 10^-2. There was also contamination present on the top agar control. It was decided that it was best to continue with the amplification process by webbing and flooding a plate due to the time constraints present.
  

  10^0 Plaque Assay 11/05/18

  

  10^-1 Plaque Assay 11/05/18

  

  10^-2 Plaque Assay 11/05/18

  

  Contaminated Top Agar 11/05/18

   

Materials: 

• Phage Buffer for flooding and Plaque Picking
• Diluted Lysate
• Agar Plates
• Calcium Chloride
• Arthrobacter
• LB Broth
• 2X TA

Procedure:

1. Established an aseptic zone.
2. Calculated titer of lysate using the equation #pfu/volume of lysate in µL * (10^3 µL/mL) *10^0. 10^0 dilution was used as it had the most clear plaques and offered the most consistent phage morphology.
3. Once titer was calculated, 10 plaques were measured for the radius, and the plate’s radius was measured to determine how many plaques were needed to web a plate using the equation #Plaques Needed = Area of Plate / Area of Plaques
4. Using the # of Plaques Needed to web a plate, the equation: Volume of Lysate (mL) = (# of plaques needed to web / titer)
5. Once the volume was calculated, it was decided that the 10^0 plaque assay from 11/05/18 had to be flooded, and a plaque assay had to be performed from the remaining 60 µL of that lysate.
6. To flood the plate, 8-mL of phage buffer was added to the top of the agar and the plate was closed and left on a shaker to shake for an hour.
7. A plaque assay was then run with the 60 µL lysate.
8. 2.0 mL of LB broth was added to one conical vial and 2.5 mL of LB broth was added to another.
9. 22.5 µL of Calcium chloride was added to both conical vials.
10. The remaining 60 µL of lysate was combined with 500 µL of Arthrobacter and left to infect for 15 minutes.
11. After approximately 15 minutes, the infected lysate was added to the conical vial with 2.0 mL of LB broth in it as the other is our top agar control.
12. Then 2.5 mL of 2X TA was added to both conical vials and plated immediately.
13. The plates were then left to solidify.
14. After solid, plates were flipped and inserted into the incubator.
15. After the 1 hour, the flooded plate was removed from the shaker and the new lysate was gathered and filtered through a 0.22 micron syringe filter to create a new filtered lysate.
16. A plaque assay procedure identical to the previous one was then run, using 30 µL of lysate instead of 60 µL.

Results and Data:

• The calculated titer of the lysate was 1.5*10^4 pfu/mL
• The Average radius of plaques gathered was 0.05 cm and the radius of the plates was 4.25 cm.
• The calculated area of the plaques were 0.007854 cm^2 with the area of the plate being 56.745 cm^2.
• The number of plaques needed to web the plate was 7.225*10^3 plaques, which meant that a volume of 481 µL of lysate would be needed.

Conclusions:

Due to the extremely low titer of lysate, an extremely high volume of lysate was needed to lyse a plate. There was not enough of the diluted lysate to web this plate, so it was decided to flood the 10^0 plate and gather a high volume of lysate from that, and run another plaque assay with the remaining 60 µL of lysate. Once the plaque assay is complete, that plate is to be flooded as well and the lysates are to be combined and the titer will be calculated from the two and the amplification process will continue.

Next Steps:

The next steps are to flood both plates and calculate the new lysate required to web a plate. Have to continue working towards a high titer of lysate.