Growth Conditions
Plant cells 24-48 hours in advance and infect when cultures are 80-90% confluent. Process virus by freeze/ thawing it three times by vapor phase of liquid nitrogen and 37.0°C water bath (vortex mixing on high speed for 20 seconds between each freeze/ thaw), sonicating with 40% power for 30 seconds, and then a final vortex mixing on high speed for 30 seconds. Remove cell growth medium from the flask, and inoculate with a small volume of virus stock diluted in virus growth medium to provide a MOI of about 1-3. Adsorb 1-2 hours at 37.0°C in a humidified 5% CO2 atmosphere, rocking every 15-20 minutes to redistribute inoculum. End adsorption by adding virus growth medium.
Incubate infected culture for 1-3 days at 37.0°C in a humidified 5% CO2 atmosphere, until CPE are well advanced through 90% of the culture.
