A new method of titrating the infectivity of egg-adapted influenza virus suspensions is described.
For this purpose a plastic tray has been made, drilled with one hundred conical cups. In each cup a small piece of chorio-allantoic membrane from a chick embryo is suspended in a buffered salt solution. To these surviving tissue suspensions virus dilutions are added, and the tray is put in an incubator and rocked mechanically for 60 hr.
In those cups in which the inoculum has been sufficiently large, the membrane becomes infected by the virus which multiplies within the cells and, bursting out, builds up by infection of other cells a high concentration of virus in the suspending fluid. The virus multiplication is detected by removing the pieces of tissue and adding to the cups a drop of washed chicken erythrocytes. The presence of virus in the cup is shown by the pattern of the sedimented erythrocytes, and virus multiplication is inferred because the original seed virus is too dilute to be detectable by haemagglutination.
The method has been compared with in ovo titration. As a means of detecting egg-adapted infective virus, the tissue suspension is between 20 and 80 times less sensitive; as a titration system, however, approximately one cup is equivalent to one egg, so that by using sufficient replicates accurate assays of infective virus can be made.
The technique is readily adaptable to the titration of serum-neutralizing antibodies.
In addition to haemagglutination, the infectivity end-point of the titration can also be estimated by a specific complement fixation test, and it is suggested that, with modifications, the new technique may be applicable to the titration of a large number of other viruses.