82 JOHNE'S DISEASE 



all the media containing the extracts, but as a rule 

 there were none on the residues. 



These experiments proved that the substance in the 

 timothy-grass bacillus and in allied bacilli that is 

 essential for the growth of primary cultures of Johne's 

 bacillus is extracted by hot ethyl alcohol. As is well 

 known, if this hot extract is allowed to cool, a yellowish 

 sticky sediment and a white flocculent precipitate of 

 wax, etc., form, and can be removed by filtration. The 

 clear-coloured filtrate, when evaporated to dryness, 

 leaves a thick oily residue, which becomes firmer on 

 cooling. Part of this residue is soluble in hot and in 

 cold chloroform, leaving an insoluble portion, part of 

 which floats on the surface of the chloroform. This can 

 be removed by filtration, and, unlike the chloroform 

 soluble portion, it is found to be readily soluble in water. 



Each of these portions was now tested in media, 

 and all gave positive results with Johne's bacillus, the 

 best being that which is insoluble in chloroform (see 

 Plate VI.). 



Our researches with regard to media for the cultiva- 

 tion of Johne's bacillus had reached this stage when 

 the results were communicated to the Royal Society 

 (November 5, 191 1), and, with some animal inoculation 

 experiments and the results of certain vaccine tests 

 to be discussed later, formed the subject of the Brown 

 Lectures given at the Royal College of Surgeons early 

 in January, 191 2. 



In May, 191 2, Halfdan Holth, working in the labora- 

 tories of Professor Jensen in Copenhagen — to whom, 

 early in 191 1, we had sent cultures of Johne's bacillus 

 — published an account of experiments with this 

 bacillus. (Results communicated March 21, 191 2.) 



In March, 191 1, from a natural case of the disease in 

 a Jersey cow which had reacted to avian tuberculin, 



