214 HAROLD C. ROBINSON AND LEO F, RETTGER 



from the negative tryptophane tests, no indol could be detected 

 in coh cultures even in 2 per cent decolorized opsine. There was 

 always good growth, and the peptone water controls of the same 

 organisms were strongly indol-positive. Tests were made after 

 three and five days of incubation. 



Unfiltered cultures of B. coli in 1 per cent opsine gave the biuret 

 and the Hopkins and Cole protein tests after only two days of 

 incubation. It is interesting to note that in some old B. coli 

 cultures which had been kept in the laboratory for about two 

 months, the clear supernatant fluid gave a negative biuret test, 

 while the sediment was positive. This would seem to indicate 

 that in cultures of B. coli the protein tests were due solely to the 

 bacterial protein of the cells, and that no soluble protein was 

 formed in the medium, as Uschinsky claimed to be the case 

 with cultures of B. diphtheriae in his protein-free medium. 



In comparisons of the growths in 1 per cent opsine bouillon 

 before and after decolorization with charcoal there were rather 

 striking irregularities, especially with the diphtheria group. 

 Although, in general, growth was a little slower and scantier in 

 the decolorized medium, one or two strains seemed consistently 

 to thrive better in it. B. diphtheriae (J!^o. 8) was slow in devel- 

 opment in the decolorized medium, and did not form a film as it 

 did in the undecolorized fluid. It was found that the decoloriza- 

 tion resulted in only a very slight increase in the acidity of the 

 opsine, (about +0.1), and a very small decrease in the Sorensen 

 formalin titration for monamino acids. Animal charcoal which 

 had been washed free of chlorides was used. On the whole, 

 then, the undecolorized medium was rather more favorable to 

 growth, although when agar was used the difference was very 

 slight. It is probable that the charcoal removed something else 

 from the medium besides the coloring matter, but no definite 

 conclusion as to the nature of this material was reached. It was 

 shown not to include the monamino acids. 



In order to determine whether opsine could be improved as a 

 culture medium by the addition of certain inorganic salts and 

 carbo-hydrates the following substances were added to 2 per cent 

 opsine and to fresh beef infusion + 1 per cent opsine. 



