Phage Dilution Plating

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These methods can be done for a variety of reasons: 1) to make a lot of phage for research, 2) to learn about the lytic and/or lysogenic nature of the phage at different concentrations, or 3) to determine how much phage a sample contains. 

Procedure:

If your objective is 1), you therefore want to make a "lacy plate":  be sure to record the unique dilution when your phage creates this type of plate.  Comparing to other plate morphologies, this appearance indicates that the greatest amount of phage is present. 

If your objective is 2), carefully observe the appearance of the plaques, including their size and turbidity, at different dilutions of phage.

If your objective is 3), work backwards from your dilution calculations when the number of plaques on the plate can be counted; recall that 100 uL of the dilution was plated. 

Materials:  7h9 plates, Pipettman, 200 uL Pipettman tips, Sharpie Magic Marker, 37 C incubator, Test tubes with metal caps, phage buffer. 

Materials Substitutions:  Plaque formation can be done without an incubator at room temperature, however this will take longer.  Any size test tube will work.  If a Pipettman and tips are not available, I recommend using Pipettes and larger volumes for the dilutions. 

Procedure:

1.)    Time for Math!  How 'far out' do you want to dilute?  If you suspect your sample has a high concentration of phage (1010 is about as concentrated as is possible), then dilute '11 fold,' since we expect no phages to plaque if we plated 10-11.

Here is an example:  I expect my phage to be 'High-Titer,' and contain 1010 PFU / mL. 

100 uL of this solution (109 expected therein) into 1 mL phage buffer, suspend and mix with a flick of the thumb, and remove 100 uL of this and place into a new tube with 1mL phage buffer (108 expected therein)...continue this process until 100 uL of solution removed from a test tube is expected to contain 0 virus.  Do one additional dilution after this. 

2.)  Top agar plating of diluted phage mixed with M. smegmatis.  When expecting 109 phage in 1000 uL, we can expect to have 108 phage in a 100 uL sample aliquot.  It is important to remember this logic, because 100 of the diluted phage will be plated with M. smegmatis. The bacteria will allow the virus to grow, lysing the bacteria and forming plaques. 

3.)     Grow the plates in an incubator for 24-48 hours in order to form plaques.  The plaques will only be apparent if virus was present in the dilutions plated.        

4.)    Observe the plaques.  At what dilution were plaques no longer visible?  At what concentration of phage is a lacy plate observed?  How long did it take to form plaques?  Be sure to record your results!

 5.)  If you want to extract the phage from a lacey plate, use the Phage Extraction Protocol