961 
STUDIES IN AGGLUTINATION. . 
As soon as I had determined, by fermentation tests, that the fer- 
mentative properties of the fifteen types of dysentery bacilli were not of 
such a nature as to allow us always to separate them distinctly and that 
their separation, based upon their action upon alcohol-mannite, did not 
always conform to their division into the same groups if founded upon 
their agglutinative reactions, I undertook a careful comparative study 
of the agglutination reactions of all of these cultures toward animal 
immune serum. 
In most instances, although tests were made with the other strains, 
only the reactions obtained with the fifteen already referred to and 
tabulated will be given in the tables, excepting in particular instances. 
Rabbits in most cases were used for the preparation of the serum since 
they are the most convenient laboratory animals for immunization and 
because, according to the German authors, these animals do not produce 
secondary agglutinins, and therefore their serum is best suited for the 
differentiation of these organisms. Goats and horses were employed in 
only a few instances. 
As the organisms fermenting mannite are generally not very patho- 
genic for these animals, their immunization is a simple matter. Only 
two or three strains of the same type were used for immunization. The 
so-called non-acid types were as a rule toxie for rabbits and, unless great 
care was taken, the animals died from the introduction of either the 
dead or the living cultures, but among these types of bacilli, some strains 
were encountered which were not so toxic, even when they had but 
recently been isolated. The most satisfactory results in the production 
of sera were obtained from the administration of very small, primary, 
subcutaneous doses of living cultures, as advised by Hiss, allowing full 
recovery of the animals to take place before a second injection of an 
increased dose was given. 
The technique employed in the agglutination experiments consisted 
of the macroscopic test carried on in small test tubes. 
Suspensions of agar cultures, eighteen to twenty hours old, were made in 0.85 
per cent of sodium chloride solution, 8 cubie centimeters of saline solution being 
always employed for 8 milligrams of agar culture. The quantity of suspension of 
the cultures and serum in the tubes was always made up to 2 cubic centimeters, 
0.5 cubic centimeter of suspension (that is, 0.5 milligram of agar culture) being 
employed. Twenty-four hours was the limit of the test. The results determined 
by the use of the macroscopic method were as follows (see Tables IV, V, VI): 
