Related protocols: Preparation of Phage Stocks
Commonly Used Media for Phage Growth
Agar Overlay Technique
When an individual bacterial virus grows in a host suspended in a top agar lawn, its progeny infect and lyse the surrounding host cells. This causes the appearance of a hole or plaque in the otherwise homogenious bacterial lawn. Since a plaque represents a single virus, the number of viruses in the aliquot is equal to the number of plaques which appear.
EQUIPMENT: SUPPLIES:
sterile capped 16x150 mm test tubes
sterile capped 13x100 mm tubes
sterile pipettes (0.1, 1.0 mL)
hot block, 45C
vortex
Bunsen Burner
37C incubator
melted top agar, about 60C
phage culture to be titered
sensitive host bacteria
(such as E. coli B)
sterile dH2O in 10.0 mL repipet
pre-warmed LB agar plates
(or tryptone soy agar plates)
PROTOCOL:
THE PREVIOUS NIGHT:
1. Inoculate about 2-3 mL of nutrient broth or tryptone soy broth with a sensitive host (E.g: E. coli B). Grow ON at 37C in hot block.
THE DAY OF ASSAY:
2. Prepare a dilution of the phage such that there are about 103 particles per mL (usually a dilution of 106: 10 L into 10.0 mL, repeat a second time).
3. Set up seeded top agar:
a. Pipet about 2 mL of melted agar into sterile capped 13x100 mm tubes in 45C hot block.
b. Pipet about 0.1 mL host bacteria (E. coli B) into melted agar (down the side of the tube is OK).
4. Add phage: Pipet 10 or 100 L of 10-6 phage into the host-inoculated tube (deliver with care, just below surface of agar).
5. Vortex to mix.
6. Pour out and distribute over a pre-warmed agar plate, immediately tilt back and forth to evenly distribute before it begins to gel. Let set until gelled.
6. When gelled (1-2 minutes), invert plates, incubate ON at 37C.
THE NEXT DAY:
7. Count all plaques, record data, calculate phage particles/mL in the original culture. Apply the following formula:
For example, if you got 347 plaques when you plated out 0.1 mL of a 106 diluted phage suspension,
the titer is 3.47 x 109/mL.