Division 4 Virus isolation in embryonated fowls' eggs

The clarified supernatent fluid should be inoculated in 0,2 ml amounts into the allantoic cavity of each of a minimum of four embryonated, fowls' eggs which have been incubated for 8 to 11 days. Ideally, these eggs should be obtained from a specific pathogen free flock, but when this is impracticable it is acceptable to use eggs obtained from a flock shown to be free of antibodies to Newcastle disease virus. The inoculated eggs are held at 37 ^oC and candled daily. Eggs with dead or dying embryos as they arise, and all remaining eggs four days after inoculation, should be chilled to 4 ^oC and the allantoic-amniotic fluids tested for haemagglutination activity.