GROWTH OF BACTERIA IN PROTEIN-FREE PRODUCTS 211 



in water, yielding a brown but perfectly clear solution which is 

 easily and completely decolorized by filtration through animal 

 charcoal. The following tests were made with the decolorized 

 solution. 



Different modifications of the biuret method for the presence of 

 proteins gave negative results, thus bearing out the contention 

 of Dalimier and Lancereaux that the product is protein-free. 

 The bromine water test for tryptophane was always negative. 

 This observation is opposed to that of the previous authors, 

 who claimed that tryptophane is present. Millon's Reagent 

 gave a very weak reaction. No tyrosin crystals could be identi- 

 fied upon slow evaporation of a strong solution of the opsine, but 

 leucine crystals were very numerous. 



In the Sorensen formol titration, 5 cc. of 1 per cent opsine solu- 

 tion required 3.5 cc. of N/20 NaOH to neutralize it after the addi- 

 tion of neutral formalin. In other words, 1 gram of opsine con- 

 tained 49 mgm. of nitrogen as monamino acid. A 1 per cent so- 

 lution of opsine has a reaction with phenolphthalein of about -I- 1. 



The first experiments were conducted with the opsine media 

 as prepared by Dalimier and Lancereaux. Since opsine is so 

 readily soluble, the different media were prepared with ease. 

 For opsine bouillon, opsine and sodium chloride were added to 

 the water and the mixture heated for a few minutes, or until 

 solution was complete. The reaction was then adjusted, and 

 the medium boiled over a flame for a few minutes and filtered. 

 In the preparation of agar, bouillon was first made as described, 

 and the desired amount of agar added. The agar medium was 

 always tubed without filtering. Witte's powdered agar was 

 employed. All of the media were sterilized for fifteen minutes 

 at 12 pounds of extra steam pressure. 



Inoculations were made from twenty-four hour slant agar 

 cultures, and the growths recorded as follows: 



— No growth 



=1= Very scanty 



-[- Scanty 



-|- =fc Sparse 



+ + . Moderate 



+ -l-± Abundant 



4--{--f Luxuriant growth 



