38 Virus Inoculation-Plaque Assay
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Comments
The multiplicity of infection (MOl) is important in quantitative experiments of virus inoculation.
- Titrate the virus in the inoculum by plaque assay or other means.
The plaque forming units (PFU) can be calculated using the following formula (Bryan, 1957):
PFU = 0.69 x TCID so
Count the density of the cell population to be infected
Calculate the number of virus particles to be infected/cell; for example, if you inoculate 10 6 cells with 10 7 virus particles, the MOl is
10
Subprotocol 7
Plaque Assay Method
The following procedure is based on the methods of Hink and Straus
(1979).
Materials
- Centrifuge and centrifuge tubes
- Magnetic stirrer and magnetic bar
- Dissecting microscope
- Screw-capped bottle
- Autoclave
- Disposable tissue culture dishes (60 mm diameter and 15 mm depth;
e.g., Lux; Microbiological Associates)
- Culture medium
- 0.1 M 2-{N-morpholino) ethansulfonic acid
- Methylcellulose (4,000 centipoises)
Procedure
Equipment
Solutions
Chemicals
1. Dispense 2.0 ml culture medium into sterile disposable tissue culture Cell
petri dishes.
mono layers
2. Add 1.0 ml cell suspension containing between 1.2 and 1.5 x 10 6 cells
at log phase of growth to each dish.
3. Distribute the cells evenly throughout the dish by rotating it in a swirling motion.
4. Incubate the culture at 28°C for 2 h to allow formation of a monolayer
of cells.
5. Decant the medium from the dishes.