Measuring phage

1. MEASURING PHAGE TITER
DR. S. SIVASANKARA NARAYANI., M.SC., M.PHIL., PH.D., MRSB (UK)
26-09-2021
DR.SS

2. INTRO
Phage Titering is a procedure used to quantify the density of plaque-forming units (PFU, analogous to
a bacterial culture's colony-forming units) in a given lysate.
Plaque : When plated with a lawn of susceptible cells, a bacteriophage will form a circular area of
reduced turbidity called a plaque
Plaque assay : The process of determining phage concentration by dilution and plating with susceptible
cells is called titering or the plaque assay
plaque-forming unit (PFU): A bacteriophage capable of productively infecting a cell is called a plaque-
forming unit (PFU).
26-09-2021
DR.SS

3.  A bacteriophage stock is diluted in the following manner:
0.1 mL of the phage stock is diluted into 9.9 mL of dilution buffer (making a total volume in this
first dilution tube of 10.0 mL).
From this first dilution tube, 0.1 mL is withdrawn and diluted into a second tube containing 9.9
mL of dilution buffer.
From the second dilution tube, 0.1 mL is taken and diluted into a third tube containing 9.9 mL of
dilution buffer. From this third tube, 1.0 mL is withdrawn and added to 9.0 mL in a fourth tube.
Finally, 0.1 mL is withdrawn from the fourth dilution tube and plated with 0.2 mL of susceptible
cells in melted top agar. After incubating the plate overnight, 180 plaques are counted. What is
the titer of the bacteriophage stock?
• The series of dilutions described in this problem can be written so that each dilution step is represented as a
fraction. To obtain the overall dilution factor, multiply all fractions.
0.1mL 0.1mL 0.1mL 1.0 mL
X X X X 0.1mL
10mL 10mL 10mL 10mL 26-09-2021
DR.SS

4. Expressing this series of fractions in scientific notation yields the following overall dilution
(1x 10-2) x (1x 10-2) x (1x 10-2 ) x (1x 10-1) x (1x 10-1mL) = 1x 10-8mL
To obtain the concentration of phage in the stock suspension, divide the number of plaques counted by
the dilution factor.
The number of plaques counted
concentration of phage =
dilution factor.
180PFU
=
1x10-8 mL
= 1.8 x 1010 PFU/mL
Therefore, the bacteriophage stock has a titer of 1.8 x 1010 PFU/mL 26-09-2021
DR.SS

5. REFERENCE
• Calculations for molecular biology and Biotechnology – A Guide
to mathematics in the laboratory, Second Edition – Frank. H.
Stephenson
26-09-2021
DR.SS
sivasan91@gmail.com

6. 26-09-2021
DR.SS