Learning Module - Experiment 2B

Growth of bacteriophage, titering, and transduction.

Bacteria must express the maltose receptor:

Phage binding to maltose receptor

Under poor growth conditions, the lysogenic pathway is followed:

Lysogenic pathway under poor growth conditions

Under good growth conditions, the lytic pathway is favored:

Lytic pathway under good growth conditions

Progeny phage are released from lysed bacteria:

Release of progeny phage from lysed bacteria

PLAQUE GROWTH

Plaques enlarge by successive rounds of infection and lysis:

Growth of bacteriophage lamda

The extent of plaque formation depends on the Multiplicity of Infection (M.O.I.):

Plaque formation depends on Multiplicity of Infection (MOI)

TRANSDUCTION

Conditions are chosen to promote lysogeny:

  • Adsorb phage to DM700 at lower temperature (30? C)
  • Grow bacteria on poor media (Minimal A glucose)
  • Incubate plates at lower temperature (30? C)

DM700 cannot grow on media lacking cysteine

Grow transductants under selective conditions, in our case we select for the ability to grow on media lacking cysteine

DATA TABULATION

Count the number of PFUs (or CFUs) on a series of plates:

Calculating the number of PFUs (or CFUs)

Calculate the number of PFU (or CFU) per mL of phage stock

For example: 37 plaques were observed.

Hence, (37 plaques / 0.1 ml) x 10^6 dilution = 3.7 x 10^8 PFU / mL of original phage stock.