TITERING OF BACTERIAL VIRUSES
Professor of Biology and Chemistry
University of Cincinnati Clermont College,
Batavia OH 45103
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aliquot of phage over
a prewarmed plate.
Related protocols: 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.
|sterile capped 16x150 mm test tubes
sterile capped 13x100 mm tubes
sterile pipettes, 0.1 mL and 1.0 mL
hot block, 45°C
37°C incubatormelted 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:|
|Equipment for dilutions: 10 uL phage stock into 10 mL
||2. Prepare a dilution of the phage such that there are about 103 particles per mL (usually a dilution of 106: 10 uL into 10.0 mL, repeat a second time).|
|Add 0.1 mL down the side The bacteria are on top:
||3. Set up seeded top agar:
a. Pipet about 2 mL of melted agar into sterile capped 13x100 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.|
|6. When gelled (1-2 minutes), invert plates, incubate ON at 37°C.|
|THE NEXT DAY:|
|7. Count all plaques, record data, calculate phage particles/mL in the original culture.|
To do the calculation of number of phage particles per mL, apply the following formula:
phage/mL = (number plaques/plate) x (1/mL plated) x dilution factor
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.
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