Protocol 6.3: Collecting Phage Lysate

For POLS Lab

Once you have taken your bacteriophage sample through two rounds of purification, it is time to amplify your clonal virus population.  This process involves first determining the titer of your purified phage.  (We will use a spot test of serially-diluted samples for this.)  Then, using the titer information, we will perform full-plate plaque assays using diluted viruses that should create a “webbed plate”.

Once the plaques on the webbed plates have grown to an appropriate size, that is that they have killed most of the Mycobacterium smegmatis in the top agar, it is time to collect the bacteriophage.  This is the best time to collect the bacteriophage because the recoverable virus titer should be highest at this point.

The video clearly shows the procedure of collecting the plate lysate.  You will see that once the lysate is drawn up into the syringe, it is then sent through a sterile filter.  As you know, this process will allow virus to pass through the filter but not any bacteria.

The purified and amplified bacteriophage will need to be titered at this point.  Our goal is to collect lysate that has greater than 5 x 10^9 pfu/ml (plaque-forming units per milliliter).  This “high titer” bacteriophage is necessary for the characterization steps of this process.

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