Titration of Herpes Simplex Virus in One-Day Eggs by an Improved Inoculation Method and Its Application to the Serum Neutralization Test

A new and simple technique for the yolk sac inoculation of 1-day eggs is described. Since the nonspecific death rate within one week following the inoculation was fairly constant regardless of the diluent used or of the season of experiment, averaging less than 20% in one week, a formula for the LD50 calculation was standardized by adopting 0.2 as a correction factor for nonspecific deaths. The sensitivity of the new titration for egg-adapted herpes virus appeared to be about equal to that of other yolk sac titration methods employing older eggs, but the 1-day egg titration required only one week as compared with 10–12 days needed with older eggs. The standard deviation of titers using ten 1-day eggs per dilution was approximately 0.3–0.4 log10. When compared with the pock counting technique, the titer was approximately one log10 lower, and serial 1-day egg passages of the virus did not alter the ratio of PFU (pock-forming units) to OIU (1-day egg infective units). However, the 1-day egg technique was thought to be of special value in the serum neutralization test because of the technical ease, quick determination of titer and constancy of result, when a constant virus-varying serum system was utilized, the virus-serum mixture was incubated at 37°C for 1 hr and five eggs were used per dilution.