530 Messrs. Twort and Ingram. Isolating and [Nov. 7,. 



by distilled water, but that it is soluble in a glycerine saline solution,, 

 although from the above it is clear that some of the essential substance 

 remained in the residue. 



A further series of experiments was made, using ethyl alcohol as our 

 solvent. Two grammes of dry timothy grass bacillus were powdered, 

 placed in a Soxhlet apparatus with 100 c.c. of absolute alcohol, and 

 extracted for three hours. The residue was dried in an incubator, and the- 

 alcohol evaporated to dryness, leaving a dark yellowish sticky mass. The 

 extract and residue were then weighed separately, and it was found that 

 the original weight of the bacilli was reduced from 2 grm. to about 

 1-25 grm., the difference being represented by the extract. Media were- 

 prepared with the extract and residue thus obtained, the tubercle bacillus 

 of our original medium being replaced by 1 per cent, of the extract or 

 residue. Other batches of these media were thus prepared, some of which 

 contained only \ or -|- per cent, of the extract or residue. Tubes from each 

 batch were inoculated from young growths of Johne's bacillus, and incubated 

 at 39° to 40° C. 



Good growth was obtained on all the media containing the extracts, but,, 

 as a rule, there was none on the residues. 



These experiments prove that the substance in the timothy -grass bacillus 

 essential for the growth of Johne's bacillus is extracted by hot ethyl alcohol. 

 As is well known, if this hot alcoholic extract is allowed to cool, a white 

 flocculent precipitate forms, 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 cold chloroform, leaving an insoluble liquid portion which floats on the 

 surface of the chloroform, but is soluble in water. 



Media prepared with any one of these different parts of the alcoholic 

 extract give positive results with Johne's bacillus, the best being that 

 which is insoluble in chloroform. 



So far these extracts have not been purified, and it is possible that the 

 essential substance contained in each portion is identical. 



In considering pseudo-tuberculous enteritis from an hygienic standpoint 

 some important factors have to be considered. The disease is widely 

 distributed, and is easily conveyed from one animal to another, probably 

 by means of contaminated food, such as grass. In the early stages the 

 symptoms are slight and indefinite, and an early diagnosis is impossible. 

 The affected animals lose considerable weight, and with milch cows the 

 quantity of milk given is greatly diminished. In view of these facts it is 

 clear that a reliable diagnostic vaccine would be of great economic value, 



