108 C. C. WARDEN, J. T. CONNELL AND L. E. HOLLY 



We assume there can be no question that diphtheria toxin is 

 diphtheria antigen since its injection into animals produces a 

 specific antitoxin which unites with no other known antigen. 

 The concensus of opinion is that toxin is not a protein and does 

 not require protein for its development.^ If we accept, then, as 

 one postulate the statement that toxin is specific antigen, and 

 as another that the specific fat complexes of cells constitute 

 their antigens, it follows that diphtheria toxin must be composed 

 of the diphtheria fat complex in one form or another, and it 

 should be possible to demonstrate the correctness or falsity of 

 our assumption by means of the artificial fat antigen in a certain 

 colloidal state. 



In taking up this work we planned to commence with a broth 

 menstruum known to yield good toxin, and to combine with it 

 varying proportions of the fat antigen in various forms. Accord- 

 ingly a standard broth identical with that used for actual toxin 

 production was adopted. Artificial antigen no. 2 was prepared 

 in proper proportions in the forms of the fatty acids, the alkaU 

 salts, Na and K, the ethyl esters, and the glyceryl and choles- 

 teryl esters. The ammonium salts were too unstable for use 

 since in presence of Na ions in excess the NH4 ions are replaced. 

 Cholesterol was omitted from these antigens. 



We regarded the broth as a highly complex colloidal fluid con- 

 taining proteoses existing as particles of varjdng colloidal dimen- 

 sions, as shown by ultrafiltration^ unknown organic matter 

 from the veal infusion, coloring matter, and various electrolytes 

 in addition to NaCl, all under the influence of a prunary pH of 

 7.9. We assumed that the diphtheria bacilli, trained to the 

 most rapid reproduction on the most favorable medium must 

 also die and disintegrate rapidly according to a general principle 

 of life and death, ^ Hberating their fat complex in an emulsified or 



< Jordan, Genl. Bact., 1918, p. 266; Guinochet, Arch, de Med. exp., 1892, 4, 

 487; Hadley, Jour. Infec. Dis., 1907, Suppl. 3, p. 95. 



6 Bechhold-Bullowa, Colloids in Biol, and Med., 1919, p. 99. 



« Vesilova, Russk. Vrach., 1915, 9, 205; Park and Williams, Pathol. Microorg., 

 1920, p. 343. As an adjunct to the "life and death" principle in accounting for 

 the death of microorganisms, in fluid cultures at a time when the quantity of 

 lysin or toxin is at a maximum, is the fact of the existence in the fluid of the 



