TUBERCUL OSIS. 323 



result. The marked disadvantage of abscess-formation 

 following the injections, however, remained. This fluid, 

 when filtered, possessed the properties of tuberculin. 



The mechanical fragmentation of the bacilli had been 

 used by Klebs in the studies of antiphthisin and tubercu- 

 locidin. Koch now used it with advantage in his studies, 

 and pulverized living, fresh, virulent, but perfectly dry 

 bacteria in an agate mortar, in order to liberate the ba- 

 cillary substance from its protecting envelope of fatty 

 acid. In the trituration only a very small quantity of the 

 bacteria could be handled at a time, and Koch seemed 

 thoroughly aware of the risk incurred from inhalation of 

 the finely pulverized bacillary mass. 



Having reduced the bacilli to fragments, they were 

 removed from the mortar in distilled water, and collected 

 by centrifugation, in a small glass tube, as a muddy re- 

 siduum at the bottom of an opalescent, clear fluid. For 

 convenience he named the clear fluid TO, the sediment 

 TR. TO was found to contain tuberculin. In order 

 to separate the essential poison of the bacteria as perfectly 

 as possible from the irritating tuberculin, the TR or 

 fragments were dried perfectly, triturated once more, 

 re-collected in fresh distilled water and re-centrifugated. 

 After the second centrifugation microscopic examination 

 showed that the bacillary fragments had not been resolved 

 into a uniform mass, for when TO was subjected to stain- 

 ing with carbol-fuchsin and methylene-blue it was found 

 to exhibit a blue reaction, while in TR a cloudy violet 

 reaction was obtained. 



The addition of 50 per cent, of glycerin had no effect 

 upon TO, but caused a cloudy white deposit to be thrown 

 down from TR. This last reaction showed that TR con- 

 tained fragments of the bacilli which are insoluble in 

 glycerin. 



Experiment showed that TR had decided immunizing 

 powers. Injected into tuberculous animals in too large 

 dose it produced a reaction, but its effects were entirely 

 independent of the reaction. Koch's aim in using this 



