Growth Conditions
Temperature: 33°C
Recommendations for Infection: Dilute virus with PBS prior to inoculation. While candling the eggs, draw a pencil line around the air sac. Using an 18.5 gauge needle, poke a hole into the air sac. Using a 1.0 mL syringe fitted with a 22.5 gauge needle, insert the needle through the hole and into the allantoic cavity. Inject 0.2 mL of diluted inoculum into the allantoic cavity. Seal the hole with nail polish or sterile tape. Incubate eggs at 33°C 2 days, checking embryo viability every 12-18 hours and discard non-viable eggs. To harvest allantoic fluid, refrigerate eggs for at least 2 hours post incubation. Use sterile scissors to cut away the shell around the air sack. Aspirate the allantoic fluid with a syringe or a pipette. Avoid capturing blood or yolk in the allantoic fluid.
Incubation: 2 days with humidity
