480 LEWIS DAVIS 



place of the beef has not demonstrated any increase in toxico- 

 genicity or shown any other advantages to warrant its use. Beef 

 from various sources, including both cold storage and fresh prod- 

 ucts, has been employed in the infusion with equally satisfac- 

 tory results. The increased acidity at times encountered with 

 cold storage meat has not been found to require any special treat- 

 ment. Preliminary fermentation of the infusion with a culture 

 of Bad. coli, as first recommended by Smith (1899) to remove any 

 fermentable carbohydrates is, at best, an unsatisfactory proce- 

 dure, and in our experience, entirely superfluous. The formation, 

 by the colon bacillus, of decomposition products which may give 

 trouble on injection of the final diphtheria toxin, must also be 

 taken into consideration. 



Allowing the beef to infuse over night is not very practical with 

 large scale preparation. Equally satisfactory results are ob- 

 tained by adding twice the quantity of water to the minced beef 

 and bringing to a boil in the steam kettle in the course of about 

 an hour and a half. The resultant infusion liquor is then ob- 

 tained by the use of a suitable press. 



Fat must be eliminated from beef infusion, as even traces of 

 fat have a decided inhibitoiy action on the production of diph- 

 theria toxin. This has been demonstrated on numerous occa- 

 sions when, out of the same lot of broth, those flasks which showed 

 particles of fat gave final toxin of which the L+ dose was greater 

 than 0.5 cc, while the strength in the remaining flasks was 0.25 

 cc. or less. We are probably dealing here with a surface tension 

 phenomenon, similar to that discussed by Larson, Cantwell and 

 Hartzell (1919) in their recent paper on the influence of the sur- 

 face tension of media on the growth of bacteria. It seems quite 

 likely that the fat depresses the surface tension of the bouillon 

 thus forcing Corynebact. diphtheriae to grow beneath the surface 

 with resultant diminution in the formation of pellicle and toxin. 

 Experimentation with differing concentrations of peptone 

 from 0.5 per cent to 4 per cent has shown that the most potent 

 toxin requires a peptone content around 2 per cent. Amounts 

 up to 4 per cent may be satisfactorily employed, but with no ad- 

 vantage over the smaller concentrations. The peptone used in 



