Previous Results:

• Webbed plates from last lab had numerous plaques, but were not webbed.

Objective:

• To correctly calculate the titers of the plates from last lab and decide which lysates should be increased for a webbed plate
• Plate a “10^0 #4” plaque assay and webbed plate

Procedure: Calculations

1. Aseptic Zone was created with CiDecon, 70% Ethanol, and an Ethanol burner
2. “10^0 #2” and “10^0 #3” plates were obtained and 5 plaques on each plate were measured using a dissecting scope. The diameters were averaged to prepare for titer calculations.
   1. “10^0 #2” avg plaque area = 2.66 mm^2
   2. “10^0#3” avg plaque area = 0.83 mm^2
3. Titers were calculated with the following equations:
   1. “10^0 #2” had 48 plaques on the plate
      1. (48 pfu/10 microliters) x (10^3 microliters/mL) x (10^0) = 4.8 x 10^3 mL = Low Titer
   2. “10^0 #3” had 15 plaques on the plate
      1. (15 pfu/10 microliters) x (10^3 microliters/mL) x (10^0) = 1.5 x 10^3 mL = Low Titer
4. The volume needed to web a plate for each lysate was calculated:
   1. “10^0 #2” = 2133.3 plaques/(4.8 x 10^3) mL = 440 microliters
   2. “10^0 #3” = 6836.7 plaques/(1.5 x 10^3) mL = 4560 microliters
5. The “10^0 #3” volume needed to web was too high, and therefore not plated. “10^0 #2” lysate was then left to create a webbed plate using a higher volume (in next procedure)

Procedure: “10^0 #4” Lysate and Webbed

1. Aseptic technique was still used
2. Overlay Agar was made in 50 mL tube using 6 mL LB broth, 7.5 mL 2x TA, and 67.5 microliters CaCl2
3. To make the “10^0 #4” plaque assay 10 microliters of lysate was added to 0.5 mL Arthrobacter and allowed to infect for 10 min
4. To make the webbed plate, 64 micro liters of “10^0 #2” lysate was added to 0.5 mL Arthro and infected for 10 min
5. 4.5 mL of Overlay Agar was added to each Arthro tube for the experimental tubes, leaving 5 mL of Overlay Agar in the 50 mL of the control plate
6. All three Agars were plated and left to harden for 15 min, then incubated for 48 hours

Results:

• The titers of both the “10^0 #2” and the “10^0 #3” plates were low titers and will need to be increased in order to move on in the procedure.

Next Steps:

• During next lab the webbed plate and “10^0 #4” plate will be examines for positive results. If the plate is webbed, then the next step in the procedure will occur. If not, then the titer of the “10^0 #4” plate will be calculated and a webbed plate will be created from that lysate.