Plaque assay of Phage PDF

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Plaque assay of Phage...

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Worksheet 7: Plaque Assay of Phage Titer 1. From the results below, calculate the PFU/mL of the original T4 bacteriophage sample that was used. Remember to show all calculations and use scientific notation when writing your answer. Assume the plaque assay was conducted using the same protocol as Exercise 6-4. (1.0 pt)

From Initial Phage Dilution Tube Labeled

# of plaques

10-2

10-3

10-4

10-5

10-6

10-7

10-8

TNTC

TNTC

TNTC

417

39

19

0

TNTC: Too Numerous To Count PFU/mL = (# of plaques) / (Dilution factor) (mL of phage added to E. coli) PFU/mL = 39 / (1 x 10^-6) (0.1) PFU/mL = 3.9 x 10^8 PFU/mL

2. Michael, the absent-minded research technician, is attempting to do a plaque assay but has run into a problem as he forgot to make any soft agar for the assay! Instead of soft agar Michael decides to use “regular” agar that contains double the amount of agar, the ingredient that is used as the solidifying agent in microbiological media. Would you expect Michael’s plaque assay to work? What would you expect to see and why? (1.0 pt) If the agar is too thick, the bacteriophage would not be able to move at all and infect more cells. So no plaque will be observed. But we would expect to see very very small plaque when soft agar is used instead, because the soft agar will allow bacteriohage to move around and infect more bacterial cells.

3. Describe how plaques are formed, be specific. (1.0 pt) The phage that infected a bacterial cell will replicate and destroy the cell releasing new phage that can go on to infect cells that are in direct contact with the cells that have lysed. All bacterial cells that have been destroyed produce an area of clearing on the plate. The formation of a PLAQUE, the area of clearing on the bacterial lawn, is the result of a bacteriophage infecting and destroying bacterial cells....