38 Virus Inoculation-Plaque Assay
361
- Culture medium
Solutions
- Virus inoculum
Viruses
Procedure
1. Culture the cells to be infected in an appropriate culture vessel.
(ell culture
2. Culture the cells for 1 or 2 days.
1. Add several drops of inoculum to the culture, and continue the cul- Inoculation
ture.
2. Observe the appearance of any cytopathic effects.
1. To collect the virus liberated into the culture medium, harvest the me- Virus
dium when the cytopathic effects reach a maximum.
harvest
2. Centrifuge the medium at 150 g for 15 min.
3. Pass the supernatant through a 0.4- or 0.6-fim filter.
4. Store the filtrate at -80°C for use as an inoculum.
Results
Infection can be recognized by the appearance of cytopathic changes in
the inoculated cells. Virus occlusion bodies, as well as a suspension of
non-occluded virus, can be obtained using this method.
Subprotocol 6
Quantitative Inoculation (Infectivity and Multiplicity of Infection)
Infectivity is expressed as the median tissue culture infective dose
(TCIDso)'
Materials
- 96-Well tissue culture plate
- Pasteur pipettes
- Microplate shaker
- Culture media
- Virus inoculum
Procedure
Equipment
Solutions
Viruses
1. Harvest the cells, and prepare a cell suspension with culture medium Seeding
at a density of 2 x lOs cells/ml.
2. Dispense 100 fil cell suspension to each well of a 96-well culture plate.
361
- Culture medium
Solutions
- Virus inoculum
Viruses
Procedure
1. Culture the cells to be infected in an appropriate culture vessel.
(ell culture
2. Culture the cells for 1 or 2 days.
1. Add several drops of inoculum to the culture, and continue the cul- Inoculation
ture.
2. Observe the appearance of any cytopathic effects.
1. To collect the virus liberated into the culture medium, harvest the me- Virus
dium when the cytopathic effects reach a maximum.
harvest
2. Centrifuge the medium at 150 g for 15 min.
3. Pass the supernatant through a 0.4- or 0.6-fim filter.
4. Store the filtrate at -80°C for use as an inoculum.
Results
Infection can be recognized by the appearance of cytopathic changes in
the inoculated cells. Virus occlusion bodies, as well as a suspension of
non-occluded virus, can be obtained using this method.
Subprotocol 6
Quantitative Inoculation (Infectivity and Multiplicity of Infection)
Infectivity is expressed as the median tissue culture infective dose
(TCIDso)'
Materials
- 96-Well tissue culture plate
- Pasteur pipettes
- Microplate shaker
- Culture media
- Virus inoculum
Procedure
Equipment
Solutions
Viruses
1. Harvest the cells, and prepare a cell suspension with culture medium Seeding
at a density of 2 x lOs cells/ml.
2. Dispense 100 fil cell suspension to each well of a 96-well culture plate.