NATURE OF THE "ESSENTIAL SUBSTANCE" 101 



interesting, not only because it supports our theory, 

 but also because it shows what a small quantity of the 

 essential substance is sufficient to stimulate the growth 

 of Johne's bacillus. 



More recently we have attempted to obtain a pure 

 acid from the barium and sodium salts, but without 

 success, the chief difficulty being to obtain a sufficient 

 quantity of the barium salt. In view, however, of the 

 positive results obtained with the barium compound, 

 we now directed our attention to known organic acids. 



In the first place it will be remembered that from 

 our experiments with B. phlei we came to the con- 

 clusion that the essential substance might be formed 

 by this bacillus from certain alcohols and sugars, so as 

 substitutes for the essential substance we now tested 

 acids that can be formed from these substances. The 

 following is a list of acids investigated : Glycerine, 

 tartronic, gluconic, saccharic, mucic, fumaric, malic, 

 aconitic, glycolic, glyoxylic, succinic. Glyceric alde- 

 hyde and glycerose were also tested. 



Each of these substances was made up into media, 

 and as a basis the same glycerine-saline-egg mixture 

 was used as in the previous experiments. Each acid 

 was tested in percentages varying from o'oi to I'o, 

 and in several series of media the acid was previously 

 neutralized with sodium hydrate or baryta water. 

 Tubes of each batch of medium were inoculated with 

 Johne's bacillus, and after incubation for some weeks 

 at 37° C, a slight growth was obtained on certain of 

 the tubes containing glycerinic acid, and to a less 

 extent on those containing tartronic acid, and on those 

 containing gluconic acid. 



In all these cases about i per cent, of acid was 

 present, but it had been almost completely neutralized 

 with NaOH before making: into medium. We also 



