92 JOHNE^S DISEASE 



ourselves, we have thought it advisable to quote at 

 some length in order that the reader may fully 

 appreciate our respective opinions. 



Nature of the ** Essential Substance."— Since the 



publication of our experiments with media containing 

 acid-fast bacilli, or extracts obtained from the bacilli, 

 we have attempted to isolate the substance — the 

 "essential substance" — contained in these bacilli and 

 extracts, in the hope that we might be able to throw 

 some light on its chemical nature. To carry out our 

 experiments we obtained large quantities of B. phlei 

 by growing the bacillus on glycerine-peptone-beef 

 broth for fourteen days at 2>7'^ C. ; the cultures were 

 then filtered, and the growth washed and dried. We 

 prepared the alcoholic extract as before, and from 

 this we attempted by means of various solvents to 

 separate the " essential substance." In the first place, 

 the wax was obtained in a fairly pure state by 

 repeatedly dissolving in hot alcohol, precipitating by 

 cooling, and filtering. The wax, when added to media, 

 gave negative results, and need not be considered 

 further. 



The two portions of extract soluble in cold alcohol, 

 when evaporated to dryness, were extracted with such 

 solvents as ether, petroleum ether, acetone, and methyl 

 alcohol, but with very unsatisfactory results, the 

 essential substance being mixed up with most of the 

 portions obtained. The experiments took a consider- 

 able time to carry out, as the only way in which we 

 could test for the substance was by preparing media 

 containing the separate portions, and by testing the 

 growth of Johne's bacillus on the various batches 

 of media. Usually it was necessary to incubate the 

 cultures lor at least two months before we could 

 be certain that no growth of Johne's bacillus had 



