Phage Discovery Guide: Plaque Assay Protocol

For POLS Labs

Plaque assays with bacteriophage involve overlaying “bottom agar” with “top agar” that contains bacteriophage-infected host bacteria.

This video will show you the technique for doing this with a single tube of infected bacteria.  In practice, you will likely set up infections of several tubes of bacteria using serially diluted bacteriophage sample. The purpose of plaque assays using serially-diluted bacteriophage is typically done to calculate the titer of your bacteriophage sample.  “Titer” refers to the number of infectious virions (aka plaque-forming units or pfu) per volume of viral stock.  Virus titers are most commonly described as “pfu/mL”.

Alternatively, you may use this assay in an attempt to create “webbed” plates, where the bacteriophage are diluted to 30,000 plaques per plate (give or take an order of magnitude).  This method requires that you have already determined the titer of your virus (described in the previous paragraph).  Webbed plates are those that have been infected with enough bacteriophage to end up with 90-95% of the bacteria within the top agar to be killed by the following day.  The number of plaques per plate will depend on the size of the plaques and the length of time the plates are incubated.  Rather than determining the titer of your bacteriophage, this variation of a plaque assay should provide you with the highest viral recovery possible from a single plate.

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