no 



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



The alcoholic solutions of the antigen fatty acids, salts and 

 esters were sterile and of convenient strengths for the pipetting 

 of small amounts to the broth with the minimum of alcohol. 

 Control experiments showed that the addition of corresponding 

 quantities of alcohol alone gave rise to no precipitation or other 

 appreciable change. The antigens were added in weights vary- 

 ing from 1 mgm. per 100 cc. to 20 mgm. per 100 cc. in a maxi- 

 mum of 1 cc. of alcohol, without material change in the pH 

 either at once or with the passage of time. After inoculation 

 the flasks were placed, without stirring, save where emulsifica- 

 tion was done at once, in the incubator at 35° to 37°C. where 

 thej^ remained undisturbed until examined. The pH of the 

 broth was also made to vary from the standard so as to try the 

 effects of such concentrations as 7.38, 8.0, 8.1 and 8.33. Some 

 of the flasks were made alkaline to the lower figures by ammonia 

 added in a sterile manner after autoclaving and coohng. Sev- 

 eral flasks were incubated under lowered oxygen tension. When 

 a flask was removed from the incubator the pH of the contents 

 was taken, and the degree of clearness noted, as well as the 

 presence or absence of sediment and of faint scum of unemul- 

 sified fat upon the surface. As a rule 2 cc. quantities taken from 

 the center of the fluid were then injected subcutaneously into 

 guinea pigs averaging 275 grams weight. 



The results of these experiments are shown in tables 4 and 5. 



TABLE 4 



FATTY ACIDS 



8 mg. artificial diphtheria antigen added to broth surface. 



Incubation at 37°C., five to six days 



Same dosage K or Na salts of fatty acids added to broth 



surface. Incubation 37°C. five to six days 



Mastic emulsions; alkali salts; no incubation 



Number of pigs dying in 1 to 4 days .21 



Number of pigs dying in 5 to 14 days 50 



Number of pigs dying in 14 to 30 days 29 



