INTRACELLULAR TOXIN OF BACILLUS PYOCYANEUS. 165 



The intracellular toxin can not be extracted, at least not in appreciable 

 quantity, bv pliysiologic salt solntion. 100 mg. of the germ substance 

 was suspended in 50 c.c. of physiologic salt solution, placed in a sealed 

 tube, and vigorously agitated in a mechanical shaker for twelve hours, 

 at the end of which time the emulsion was filtered through heavy Swedish 

 filter paper, and the filtrate found to be without toxic effect. This is 

 found to be true, whether the germ substance was freshly taken from 

 agar cultures or had been previously extracted with alcohol and ether. 



When the germ substance was extracted with one per cent, of sulphuric 

 acid and this extract treated with absolute alcohol, a precipitate was 

 obtained. This precipitate, after having been washed until the washings 

 were free from acid, dried and injected into animals, proved to be toxic, 

 but much less so than the original germ substance. 



While no appreciable amount of toxin could be obtained from the cell 

 substance, a hemolytic body was extracted with physiologic salt solution ; 

 100 mg. of the germ substance which had been hardened by the action of 

 alcohol and ether, was suspended in 50 c.c. of physiologic salt solution 

 and agitated for twelve hours, after which it was filtered through heavy 

 Swedish paper. To three tubes containing each 10 c.c. of a 5 per cent, 

 solution of defibrinated blood, the following quantities of physiologic salt 

 solution extract of the germ substance were added: 



Tube 1 received 10 c.c. of the extract. 

 Tube 2 received 1 c.c. of the extract. 

 Tube 3 received 0.1 c.c. of the extract. 



These tubes were placed in the incubator for twenty-four hours, at 

 the end of which time complete hemolj'sis had occurred in Tube 1. In 

 Tube 1, about one-third of the blood had been dissolved, while in Tube 

 3 the hemolytic action was found to be very slight. 



The hemolytic substance was also extracted from the unhardened fresh 

 germ. Some of this germ substance was shaken with physiologic salt 

 solution for twelve hours and then passed through a Berkefeld filter; 

 13 c.c. of this filtrate was found to completely dissolve all the blood 

 corpuscles in 10 c.c. of a 5 per cent, blood solution in twenty-four hours 

 at incubator temperature. 



My conclusions may be stated as follows : 



1. Bacillus pyocyaneus does contain an intracellular toxin, 



2. This toxin can not be extracted from the cell by physiologic salt 

 solution. It is in part extracted by dilute sulphuric acid. 



3. Heat to 120 C. in the autoclave continued for thirty minutes only 

 slightly diminishes the toxicity of the cell substance. 



4. The cell substance of Bacillus pyocyaneus contains a hemolysin, 



