The preferred method of growing influenza A viruses is by the inoculation of specific pathogen free
(SPF) embryonated chicken eggs, or specific antibody negative (SAN) eggs. The supernatant fluids of
faeces or tissue suspensions obtained through clarification by centrifugation at 1000 g are inoculated
into the allantoic sac of three to five embryonated SPF or SAN chicken eggs of 9–11 days’ incubation.
The eggs are incubated at 37°C (range 35–39°C) for 2–7 days. Eggs containing dead or dying
embryos as they arise, and all eggs remaining at the end of the incubation period, should first be
chilled to 4°C for 4 hours or overnight, and the allantoic fluids should then be recovered and tested with
a screening test (such as haemagglutination [HA] test), influenza A type-specific test (such as agar gel
immunodiffusion test [AGID] or solid-phase antigen-capture enzyme-linked immunosorbent assays
[ELISA]) or influenza A subtype-specific test (such as haemagglutination inhibition [HI] and
neuraminidase inhibition [NI] tests) or a molecular test to detect influenza A specific nucleic acid
signatures (such as real-time reverse transcription polymerase chain reaction [RT-PCR] test) as
described later (see Section B.3.2). Detection of HA activity, in bacteria-free amnio-allantoic fluids