The timing of phage release

You have now determined the generation time of T4 phage in E. coli grown under standard conditions.

So what is happening during this time? What are the dynamics of phage assembly? 

To answer these questions, we have to increase the sophistication of our analysis.  We will use centrifugation to separate phage associated with bacterial cells from those free in solution.

From an overnight culture of E. coli take 0.5 ml and add it to 4.5 ml of fresh LB.

Set up 8 of these secondary cultures, and grow them up until A = ~0.5. (~109 cells/ml).  

We are now going to do a "time course" experiment.  In this type of experiment, we examine how a particular property of they system changes over time.  

In this particular experiment, we are going to measure how many phage are present in the supernatant after we have removed the bacteria and all phage bound to or within the bacterial cells. 

We add 106 phage/ml to each culture.  The phage are added at different times, but we will harvest the cultures all together. 

The cultures are placed in the centrifuge and spun at 13,000g for 1 minute.


The pellets at the bottom of the tubes contain all of the intact bacteria; free phage are left in the solution.

We remove the supernatants and store the pellets frozen at -80 °C; we will use the pellets later in the second part of the experiment.

Phage Release

Now, we will take our supernatants, each derived from a particular culture at a particular time after the addition of phage. 

For each time point, we will measure the concentration of PFU.

Save your graph of phage release for your lab report.

When are phage assembled within the infected cell?   To determine if, and when, the pelleted bacteria contain active phage, we need to break the bacterial cells open.

Take the frozen pellets from the first part of your experiment.  To each pellet, add 1 ml of resuspension buffer  and resuspend the cells.

  Resuspension buffer:
50mM Tris-HCl (a buffer)
5mM EDTA (a chelator)
500mM NaCl pH 8.0

Now add 15 µL chloroform and 50 µL of a 1% solution of the denaturing detergent sodium dodecyl sulphate (SDS) and mix the solution.

This combination of chemicals breaks open the bacterial cells and releases their contents, it will not, however, disrupt intact phage.

Take a sample of each supernatant and dilute it 1:100 in LB; this dilutes the chloroform/SDS sufficiently so they will not kill the bacteria you use in the plaque assay.


  Questions to answer
  • Why did the phage disappear from the supernatant and where did they go?
  • When did phage reappear in the supernatant? 
  • When did PFU appear in the pellet fraction and how did this time relate to the appearance of phage in the supernatant?
  • Make a model of phage infection, assembly and release that fits your data.

Use Wikipedia | revised 11-Jun-2008