Simplified Technic for Inoculating into Amniotic Sac of Chick Embryos. ∗

Burnet¹ has reported the isolation of influenza virus direct from throat washings, by inoculation into the amniotic sac of chick embryos and subsequent passages with a suspension of the ground tracheas removed from the embryos following a suitable period of incubation.

The technic employed by Burnet involves cutting a window in the egg shell over the embryo, puncturing the shell membrane and transferring the air sac to between the shell and the chorioallantoic membranes beneath the opening. The cut portion of the shell, including the attached shell membrane, is then removed exposing the chorioallantois beneath the window in the shell. A small slit is made in the chorioallantois, and by means of a fine forceps the underlying amniotic membrane is grasped and drawn through the opening in the chorioallantois. Inoculation is made into the amniotic sac by puncturing the exposed membrane with a capillary pipette. The membrane is then released and allowed to withdraw through the opening in the chorioallantois. Finally, the window in sealed. (6) No further enlargement in the opening in the shell is required for removal of the embryo.

Experimental. We have found the following technic to be somewhat simpler than the one described by Burnet.

During transillumination of the egg to determine whether the embryo is alive, the border of the air sac is marked upon the shell with a pencil. The shell under the circular pencil mark is then cut through with a dental drill, leaving the shell membrane intact. Alcohol is applied, the exposed shell membrane is cut through with sterile scissors, and the shell cap over the air sac is removed, thus exposing the diverted layer of the shell membrane which covers the chorioallantois.