Methods have been described by which the number of elementary bodies present in a suspension can be estimated. It has been shown that by means of replicate counts, in which the Petroff-Hausser chamber was used, a high degree of accuracy can be attained. By means of the Gates densitometer, the number of elementary bodies in a suspension can be determined with a coefficient of variation of about 3.0 per cent. A method has been described by which the accuracy of estimation of the infectious titer of a suspension can be increased without greatly enlarging the number of animals employed. This consists of selecting as the end-point that dilution of virus which on intradermal inoculation in a rabbit would lead theoretically to an equal number of positive and negative results. The statistical advantages of this method have been confirmed by the experiences of other laboratories.
By the application of the methods described, there was shown to be a direct correlation between the number of elementary bodies and the number of infectious units of virus present in a given suspension. At the mean of the distribution this ratio is as the logarithms 9.62 to 8.0. To extrapolate this curve, in order to determine the number of elementary bodies present in a single infectious unit, while tempting, is probably not justifiable. It must likewise be remembered that the data given apply to a particular strain of vaccine virus, and that the number of infectious units has been determined by intradermal inoculation of rabbits. It appears also that this method may be of value in studies of the virulence of different strains of vaccine virus, since by its application one may determine not only the infectious liter of a suspension, but its content of elementary bodies.
In the agglutination reaction it was found that optimum titers of serum were obtained when the test antigen contained from 2.0 x 109 to 1.05 x 1010 elementary bodies per cc. Approximately 1.95 x 108 particles per cc. of suspension were required for the production of visible agglutination.