Title: Titer Calculations

Date: 7 November 2018

Rationale: After the dilutions were run, the titer of the flooded lysate can now be calculated. The titer must be at the 10^8 level or above in order to be considered adequate or else another plate will be flooded in order to try and increase the titer and amplify the lysate.

Results/Calculations:

• The first picture is the plaque assay of the 10^-1 lysate, which did not yield countable plaques
• The second picture is the plaque assay of the 10^-2 lysate, which yielded many plaques but too many to reasonably count.
• The third picture is the plaque assay is the plaque assay of the 10^-4 lysate, which yielded 27 countable plaques. *NOTE: the top agar did not set correctly on many of the plates (including plates shown and not shown) and the radius of the plate was taken into account when a webbed plate calculation was done.)
• The fourth picture is the clean top agar control plate.

Titer: (27 pfu/10 uL)*(1000uL/1 mL) = 2.7 x 10^7 (the titer is a medium titer and is not considered high enough, therefore another webbed plate will be done and flooded.)

Webbed plate calculation: Aplate/Aplaque = plaques needed to web –> 1225*pi(short radius from top agar shift used)/(0.8)mm^2*pi = 1914 plaques to completely web a plate

1914/2.7×10^7 = .00070889 mL to web a plate or .070889 microliters to web a plate (7.09 of 10^-2 lysate was used in order to more accurately measure this amount)

Procedure: Under an aseptic zone, 3 plaque assays + a control will be made in order to ensure a webbed plate. One plate will be done with the calculated web amount, another plate will be done with twice the calculated amount, and a third plate will be done with three times the calculated amount.

The following recipe was used for the plaque assays:

• 8 mL LB Broth
• 10 mL 2x Top Agar
• 90 microliters CaCl2

~ 4.5 mL transferred into a test tube containing 0.5 mL Arthrobacter + 7.09 uL/14.18 uL/2.127 uL(*) lysate. The plates were left to cool and then placed into the incubator to grow.

(*)Ran out of 10^-2 lysate so 2.127 of 10^-1 lysate was used instead to maintain the integrity of the concentration.

Conclusions: The titer from the previous experiment was too low to be considered acceptable, so the webbed plate will then be flooded in an attempt to amplify the titer of the lysate and hopefully achieve a titer of 10^8 or greater.