Equal mixtures of influenza A (PR8) and B (Lee) viruses, based on predetermined ID50 values of the individual preparations, were titrated in closely spaced steps near the 50 per cent infectivity end-point. Typing of the hemagglutinins found in the allantoic fluids after incubation of the eggs for 72 hours showed an approximately equal distribution between types A and B, when less than 2 ID50 of the mixed seed had been injected. With larger inocula influenza A became dominant because it reproduces at a faster rate than the influenza B virus. While this result agreed with expectations, it was found that passage of the allantoic fluids revealing influenza A agglutinins in mixture with anti-A serum, and of B agglutinins with anti-B, yielded the heterologous virus in many instances, even when only one-half of an ID50 of mixed seed had been administered in the original titration. "Negative" fluids obtained from embryos injected with as little as one-eighth of an ID50 upon passage yielded on occasion virus of one or the other type.
Similar closely spaced titrations near the 50 per cent end-point of single strains (PR8) indicated that if hemagglutinins were found after incubation of 72 hours they were of high titer, as a rule. However, in some embryos hemagglutinins became detectable only between the 3rd and 4th days of incubation. In addition, negative allantoic fluids removed from embryos 72 hours after injection yielded on occasion virus on passage, yet no hemagglutinins were found in some of these eggs after an additional incubation period of 24 hours, or a total of 96 hours.
None of the possible explanations for these various observations, which have been discussed in detail, is completely satisfactory. However, the data indicate that in infectivity titrations the ID50 end-point obtained at 72 hours, or even after 96 hours, does not reflect the total amount of virus present in the material titrated. The data also denote that separation of variants, or mutants or "genetic recombinants" by the limiting dilution technic, although possible, does not represent an absolutely safe procedure.