How to count cells with a phage infection

There are three types of bacterial cells during a phage infection:
  • Infected cells - they are going to die and release more phage particles
  • Uninfected cells - they are lucky and have not yet encountered a phage particle
  • Resistant cells that are never infected by this particular phage
    The other datum of importance is the number of phage particles. This may be several times the numbers of cells because one infected cell can produce from 5 to 200 phage particles. This multiplier is characteristic of each particular system. We cannot count dead cells or the debris from a cell that has lysed by any sort of plating on Petri dishes, but this material shows up with an electron microscope when we use it to count phage particles.

    Our reagents are excess phage and antiserum to the phage. We have two kinds of Petri dishes prepared. One has just the nutrient medium with agar, and the other has nutrient medium, agar, and it has been lightly inoculated with the bacteria at a concentration that will grow everywhere on the Petri dish. The experiments are:

    1. Dilute a sample to a concentration that will give some colonies on the Petri dish but not too many. Mix with antiserum to tie up and make harmless the phage. Add to Petri dishes with nutrient agar.
    2. Dilute as in 1, but this will usually be not so dilute because there are fewer of the cells that we expect to grow. Add a large excess of phage to kill those cells that are susceptible but were not yet infected. Add to Petri dishes with nutrient agar.
    3. Dilute as above, but usually much more dilution to get the desired concentration. No treatment, so there are phage particles as a source of infection and cells that are already infected. Add to Petri dishes that already are inoculated with bacteria. These would grow to make the plate cloudy, but those that get infected do not grow very long and release more phage in their location. No growth means a clear area on the Petri dish.
    The Java applet shows what happens, but the colors are just to help you. The colonies would be exactly the same color, and you would have to substract to get the numbers of each type of cells.

    You should easily puzzle out the numbers of resistant cells and uninfected cells if you were given the dilution factors. However, the experiment with the lawn of bacteria gave a number that was the total of phage particles and infected cells. Please design an experiment to distinguish between them without using electron microscope data.