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By precipitation with acetone at 4°C. a refined diphtheria toxoid was obtained as a dry powder, readily soluble in aqueous solutions. The powder itself appears to be stable. When dissolved in half the original volume of physiological salt solution, the toxoid remained stable, in the cold room, for a period of 7 months. Only toxoids should be used which have been completely detoxified by the treatment with formalin. The formalin is immediately and completely removed by precipitation with acetone. Filtration through a Mandler filter or the addition of a preservative did not appear to have any deleterious effect. Incubating at 39°C. and freezing for 20 hours did not impair its activities. The flocculating value of the purified toxoid containing phenol was almost completely destroyed by freezing, while when merthiolate was the preservative, it was unchanged. Approximately 62 per cent of the nitrogen was eliminated in the process with approximately 15 per cent loss in flocculating value. The flocculating values were nearly double those of the original material and thus corresponded with the concentration, which was onehalf the volume. By this method, the antigenic activity of a toxoid below standard may be increased to a satisfactory standard. The antigenic activity of the refined product in the immunization of guinea pigs may in general parallel its flocculating values but the relation between the results of these tests may not be proportional nor constant. As an immunizing agent in the guinea pig, the refined toxoid possesses an antigenic activity that is equal to, or greater than, that of the unconcentrated toxoid.

An earlier study having established that (1), as soon as the detoxification of diphtheria toxoid is complete, the formalin can be eliminated by precipitation with acetone without apparent injury to the toxoid, further experiments were undertaken to determine the most suitable concentration of formalin and period of exposure when the preparation was incubated at from 36 to 37°C. The preparation of diphtheria toxoid by the methods of Glenny (2) and of Ramon (3) involves prolonged incubation at elevated temperatures and with minimal quantities of formalin, from 0.3 to 0.4 per cent, so carefully adjusted that, when detoxification has reached the desired stage, changes affecting the antigenic action will not take place. There are obvious advantages in avoiding this complicated procedure and in adopting one that would eliminate the formalin as soon as the product was satisfactorily detoxified.

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