Phage Spot Dilution Plating

 

                                   

 

This protocol is used for "titering" a phage, meaning that we can learn how many plaque forming units (PFU) are contained within a sample.  This procedure requires the mathematical understanding of dilutions and dilution factors, in addition to good laboratory technique with a pipettman.  In the plate on the left (note-this only required 1 plate per sample!), the sample was at a high concentration, since plaques were evident even at the 10-11 dilution.  The plate on the right, however, only had plaques at the 10-7 dilution.  This student actually used each plate for 2 separate phage samples, and didn't do a very good job of spreading the smeg around the plate before spot dilution plating.  Still, the experiment worked. 

Objective:  To learn how many plaque-forming units of phage are within a liquid sample that you have prepared.

Materials:  7h9 plates, Pipettman, 200 uL Pipettman tips, Sharpie Magic Marker, 37C incubator, Test tubes (SIZE) 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.)  Spot plating of the phage dilutions onto a   M. smegmatis lawn.  In the above, when expecting 109 phage in 100 uL, we can expect to have 108 phage in a 10 uL sample.  It is important to remember this logic, because 10 (not 100) uL of the diluted phage will be placed on top of M. smegmatis. The bacterial lawn will allow the virus to grow, lysing the bacteria and forming plaques allowing for the indication of how concentrated the phage is. 

A.)   Draw a grid on the back of your plate (petri-dish) with a marker, and label each portion of the plate with a number identifying the dilution to be applied to it in the media on the opposite side. 

B.)    Plate 100 uL of M. smegmatis culture onto the surface of a 7h9 plate.  Several plates can be done at a time.  Allow the plate to dry for 20 minutes, leaving the lid of the plate on so that the plate does not become contaminated.

C.)    Plate 10 uL of each phage dilution onto a separate section of the plate.  Allow the plate to dry for 20 minutes, leaving the lid of the plate on so that the plate does not become contaminated. 

3.)     Grow the plate in an incubator for 24-48 hours in order to determine the titer.  The plaques will only be apparent if virus was present in the dilution applied, and virus will be more or less identifiable.         

4.)    Observe the plaques.  At what dilution were plaques no longer visible?  Be sure to record your results!