A bacterial virus, or bacteriophage, can infect a specific kind of bacterial cell. Like all viruses, the bacteriophage relies on the cellular machinery for energy production and for macromolecular synthesis. A lytic virus causes the host bacterial cell to burst or lyse and releases new mature phage particles. The lysis of the bacterial culture by bacteriophages can be seen as a loss of turbidity in a broth culture or as a zone of clearing called a plaque on an agar surface with otherwise confluent growth of bacteria. We will use the plaque assay to titer (enumerate) phages in a suspension. In this exercise, you will titer a suspension of the bacteriophage T4 using its host strain E. coli B.
In order to grow and replicate within a bacterial cell, the bacteriophage
must be able to bind to the surface of the potential host cell, to penetrate
into the cytoplasm and to use the host replication machinery to produce
new phages. One can demonstrate that a specific bacteriophage will not
grow in a host strain with which it is incompatible. Therefore bacteriophages
can be used as a tool in identification of bacterial strains. Bacteriophage
typing relies on the specificity of the host range of each
In this experiment, you will prepare lawns of bacterial cells using the agar overlay method. An agar overlay results in a more uniform lawn than that prepared by streaking and allows for easy visualization of plaques since the lawn is a very thin film over a clear bottom layer of agar. The bottom layer is necessary to prevent drying of the overlay during incubation.
1. Suspension of phage T4. .
2. Overnight cultures of E. coli B.
3. Four plates of bottom agar
4. One 9.9 ml sterile water blank; three 9.0 ml sterile water blanks
5. Sterile pipet tips and pipets
6. Four tubes of top agar, approximately 5 ml per tube. The medium should be melted and the tubes kept at 50oC in a water bath until ready to use.
1. Label 4 agar plates for the dilutions series for virus titering. You will be diluting the virus suspension to l0-5 and plating 0.1 ml (100 Ál) to give a l0-6 dilution on the last plate. The plates should be labeled l0-3 to l0-6.
2. Arrange the dilution tubes; the tube containing 9.9 ml sterile water is to be used first. You will be making one 1/100 dilution followed by three successive 1/10 dilutions (See Dilution Scheme link below). Label the tubes as indicated.
3. Use the micropipet and a sterile yellow tip to transfer 100 Ál of the virus suspension into the first 9.9 ml dilution tube. Discard the tip and mix carefully. Continue the dilution series transferring 1 ml of the l0-2 dilution to the next tube (l0-3) and so on. Remember to discard the tip into the appropriate container after each dilution step. Mix each tube thoroughly prior to removing a sample for the next dilution tube.
4. Each pair will need four tubes of top agar. Keep them at 50oC in the water bath. From this point on you must work quickly so that the agar overlay does not solidify until after it is poured onto the plate. Remove one tube from the water bath, add 0.2 ml (200 Ál) of E. coli B. Mix gently. Using the l0-2 dilution tube, remove 100 Ál of the phage suspension, mix gently and well, wipe off any water on the outside of the tube, then pour the contents onto the plate marked l0-3. Tip the plate quickly to spread the top agar over the entire surface. Put aside to cool. Using the same procedure, proceed to add, mix and plate the other dilutions. Remember to put each one on the appropriate plate. Always use a fresh pipet tip for each step. NEVER REMOVE A TUBE OF TOP AGAR FROM THE WATER BATH UNTIL YOU ARE SURE YOU ARE READY TO PROCESS THE BACTERIA AND PHAGE MIXTURE RAPIDLY.
5. Incubate all plates at 37oC for 24-48 hrs.