CULTURAL REQUIREMENTS OF BACTERIA 327 



Many confirmatory experiments of the same nature have 

 shown that within rather wide limits, the amount of charcoal 

 used and the duration of boihng are without appreciable influence 

 on the result, and as a standard method of producing the decol- 

 orized infusion, 2 per cent of charcoal and fifteen minutes boiling 

 have been adopted. 



The most obvious interpretation of the experiment is that the 

 charcoal treatment removes certain substances, perhaps amino 

 acids or polypeptides which occur also in commercial peptone, 

 from the infusion. If polypeptides are concerned, they would 

 probably be hydrolyzed by boihng with strong acid, and the 

 resulting hydrolysate would not have the property of reactivat- 

 ing the decolorized infusion, while if one or more amino acids are 

 responsible for the phenomenon, they would perhaps withstand 

 acid hydrolysis. 



Reactivation of decolorized infusion loith a sulphuric acid 

 hydrolysate of casein 



A quantity of commercial casein was hydrolyzed by boiUng 

 for eighteen hours with a mixture of six times its weight of water 

 and three times its weight of concentrated H2SO4. The result- 

 ing solution was freed from H2SO4 by Ba (OH) 2, the precipitate 

 washed with water, and the filtrate and washings combined and 

 concentrated. A quantity of the resulting hydrolysate equiva- 

 lent to 0.5 gram of the original casein was used in the preparation 

 of media as follows: 



J . , fDecolorized infusion 25 cc. 



\Glucose-salt solution 25 cc. 



Lot 2 Same plus casein hydrolysate 0.5 gm. 



[Water 25 cc. 



Lot 3 < Glucose-salt solution 25 cc. 



[Casein hydrolysate 0.5 gm. 



