THE NATURE OF TOXIN 105 



1 liter. The addition of cresols or other similar preservatives 

 to the cultures renders the toxin filtrates unfit for examination, 

 and the germ residues must be freed from it before accurate 

 examination of the fats can be made. The cresols pass unchanged 

 into the solvents along with the fatty acids, making their pmi- 

 fication difficult and wasteful. A trace of cresol renders an 

 iodin determination fallacious because of the formation of addi- 

 tive compounds with the halogen in Hiibl's solution. 



Having determined approximately the composition of the 

 fatty acid complex derived from the germ bodies and toxin 

 broth, this complex was then tested against the serums of immu- 

 nized animals — it being the custom to check the tentative anal- 

 ysis in this manner. Knowing the complex to contain about 80 

 per cent of unsaturated fatty acid corresponding closely in its 

 various values to an oleic acid, and about 20 per cent of lower, 

 non-volatile saturated acids having a calculated M.W. of 250, 

 and melting point of 58°C., several trial artificial antigen com- 

 plexes were prepared, having approximately the same values, 

 from the purest obtainable fatty acids procured from sources 

 other than the germ bodies. These artificial antigens were: 

 No. 1, oleic acid 80 per cent, palmitic acid 16 per cent, myristic 

 acid 4 per cent. No. 2, oleic acid 83.3 per cent, pahnitic acid 

 16.7 per cent. No. 3, oleic acid 84 per cent, stearic acid 16 

 per cent. (The specimen of oleic acid used was the normal 

 acid, having an I. V of 87.5). They were prepared for testing 

 by combining the Na salts of the acids in the above proportions 

 in alcoholic solution in such weights that 1 cc. contained 2 mgm. 

 of the complex, and to each 5 parts of the solution there was 

 added 4 parts of a 1 per cent alcoholic solution of cholesterol, 

 the latter being employed to furnish an adsorption surface for 

 the antigen proper. 



Experiments in complement fixation were then made with the 

 serums of rabbits that had been immunized, some with washed 

 C. diphtheriae germ bodies, others with toxin. Three separate 

 tests were made with the serums of two groups of animals, using 

 the sheep-rabbit hemolytic system, fresh guinea-pig comple- 

 ment, and a control antigen suspension in salt solution of thor- 

 oughly washed diphtheria bacilli from a twenty-four hour broth 



