Previous Results:

• The plate that was made in lab (10/8) was positive. It was not webbed, but had more plaques than previous plates, indicating a possible high titer.

Objective:

• To correctly calculate the titer of the 10^0 plate
• Collect the phage buffer (lysate) of both plates that were created during last lab to prepare for plating in a future lab

Procedure: Titer Calculation

1. Aseptic Zone was created with CiDecon, 70% Ethanol, and Ethanol burner
2. The 10^0 plate (made 10/8) was obtained and the number of plaques were counted. # plaques = 312
3. The titer of the lysate used was found with the following equation:
   1. (#pfu/volume used microL) x (10^3 microL/mL) x (dilution factor) = titer
   2. (312/10 microL) x (10^3 microL/mL) x (10^0) = 3.12 x 10^4 mL = low titer
4. To calculate the volume of lysate needed to web a plate, the diameter of 5 plaques were measured under the dissecting scope, then they were averaged
   • (1.5 mm + 2.0 mm + 2.0 mm + 2.1 mm + 1.9 mm) / 5 = 1.9 mm
5. The diameter of the plate was measured to be 85 mm
6. The areas of both the plaques and the plate were found
   • Plaque Area = 2.84 mm^2
   • Plate Area = 5674.5 mm^2
7. It was found that the number of plaques needed to web the plate would be 6920.12 plaques
   • 5674.5 mm^2 / 2.84 mm^2 = 1998.1 plaques
8. Then the volume of the lysate needed to web the plate was found using the following equation: (# of plaques) / (titer) = volume needed
   • 1998.1 plaques / (3.12 x 10^4 mL) = 0.064 mL = 64 microL

Procedure: Flooded Plates

1. The webbed plate and 10^0 plate (made 10/3) that were flooded 10/8 were obtained. The phage buffer of each plate was removed using 3 mL syringes
2. Phage buffer for each plate was filtered into separate 15 mL tubes for storage
3. Tubes were placed in the fridge to be plated during next lab

Results:

• The experiment was not completed, therefore there are no results to report.

Next Steps:

• During next lab two plates will be made, each plate containing one of the two 10^0 lysates that were created in lab 10/8. Then the titers of those plates will be calculated, hopefully high titers so the project can move forward.