Titering of Bacterial Viruses
Preparation of Phage Stocks
Commonly Used Media for Phage Growth
Agar Overlay Technique
When an individual bacterial virus grows in a bacterial 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 homogeneous bacterial lawn. Since each plaque represents a single virus, the number of viruses in the aliquot added to the plate is equal to the number of plaques which appear.
Here is the experimental plan:
sterile capped 16×150 mm test tubes
sterile capped 13×100 mm tubes
sterile pipettes, 0.1 mL and 1.0 mL
hot block, 45°C
37°C incubator melted top agar, about 60°C
phage culture to be titered
sensitive host bacteria grown ON in TSB
(such as E. coli B)
sterile dH2O in 10.0 mL repipet
pre-warmed to 37°C tryptone soy agar plates
(or LB agar plates for lambda phage)
THE PREVIOUS NIGHT:
1. Inoculate about 3-4 mL (for thirty plates) of nutrient broth or tryptone soy broth with a sensitive host (E.g.: E. coli B). Grow ON at 37°C in hot block. (Aeration is not mandatory.)
THE DAY OF ASSAY:
2. Prepare a dilution of the phage such that there are about 10^3 particles per mL (usually a dilution of 10^6: 10 uL 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 13×100 mm tubes in 45°C hot block.
b. Pipet about 0.1 mL host bacteria (E. coli B) using a 1.0 mL pipet into melted agar (down the side of the tube is OK).
4. Add phage: Pipet 10 uL or 100 uL 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 sit undisturbed until gelled.
7. When gelled (1-2 minutes), invert plates, incubate ON at 37°C.
THE NEXT DAY:
8. Count all plaques, record data, calculate phage particles/mL in the original culture.
(Note that the plaques are round areas of clearing in the otherwise solid lawn of host bacteria (E. coli B in this instance