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



C. DIPHTHERIAE. THE ANTIGEN 



The strain of organism used was the Park no. 8. The cul- 

 tures were prepared in a manner calculated to produce the 

 greatest luxuriance of growth and the maximum of toxin. For 

 these purposes there was used veal infusion to which were added 

 2 per cent pepton and 0.5 per cent NaCl. The pepton was for 

 the most part a ''proteose" pepton prepared by the Digestive 

 Ferments Company which was found to yield toxin of high 

 grade. The proteose broth was adjusted to a pH of 7.9 with 

 NaOH prior to autoclaving. The organisms were trained to 

 rapid pellicle fonxiation by frequent transplantations before the 

 final inoculations upon large surfaces of the nutrient broth as 

 recommended by Bunker- which were made upon shallow depths 

 of broth in Roux flasks laid on the flat and slightly inclined, 

 giving an area of 40 square inches per flask and a depth varying 

 from f to 1^ inches. Good pellicles covered the surfaces in 

 twelve hours, and the growths were maintained at 35°C. for 

 five to six days. It was essential that the medium should be as 

 nearly fat-free as possible, and accordingly every precaution 

 was taken in that regard. Careful siphonage and filtration 

 while the broth was strongly acid, that is before the addition 

 of alkali, with strict attention to cleanliness of vessels and glass- 

 ware insured a medium containing a negligible amount of fat. 



At the conclusion of the incubation period the toxic broth 

 was separated from the germ mass by filtration through paper. 

 The germ residues were then examined for fat content by meth- 

 ods described in earlier articles. The total moist residue, a 

 portion of which was kindly supplied by Dr. Clark of the labora- 

 tory of Parke, Davis and Company, used in the examination 

 weighed over 1 kgm. The fatty acid complex obtained there- 

 from was found to consist of approximately 80 per cent of unsat- 

 urated acids, and 20 per cent of saturated, nonvolatile acids. 



The toxin broth filtrates to the amount of 50 liters were also 

 examined and found to contam the same fat complex in quanti- 

 ties averaging 60 to 80 mgm. per liter, an amount equal to 

 about two-thirds of that obtained from the germ residue from. 



« Jour. Bact., 1919, 4, 4. 



