128 INFECTION 



tuberculoma or the tissue changes incident to tuberculosis, differing, 

 however, from true tubercle in that they do not contain living bacilli 

 and consequently are not infectious. When cultures of diphtheria 

 bacilli are filtered and the residue washed, it is found that extracts of the 

 bacterial substances or the bodies of the dead bacilli themselves are quite 

 free from the typical toxin; but the bacterial substances or the proteins 

 isolated from them, when injected into the subcutaneous tissues of 

 animals, are found to produce a strong inflammatory reaction and necro- 

 sis of the tissue-cells. 



Bacterial Split Proteins. These have been studied extensively by 

 Vaughan and his coworkers, who have ascribed to them the chief role 

 in and a very important relation to the processes of infection and im- 

 munity. 



Massive cultures of colon, typhoid, pneumonia, and diphtheria microorganisms 

 are grown in special large tanks containing agar; anthrax is grown in Roux flasks, 

 and tubercle bacilli in glycerin beef-tea cultures. After removal of the growths the 

 bacterial cellular substances are washed once or twice with sterile salt solution by 

 decantation, and then repeatedly washed with alcohol, beginning with 50 per cent, 

 and increasing the strength to 95 per cent. The substance is then placed in large 

 Soxhelet's flasks and extracted first for one or two days with absolute alcohol, and 

 then for three or four days with ether. These extractions should be thorough in 

 order to remove all traces of fats and waxes. 



After extraction the cellular substance is ground, first in porcelain, then in agate 

 mortars, and passed through the finest meshed sieves to remove bits of agar. The 

 person grinding the cellular substance should wear a mask in order to protect him- 

 self against poisoning. Vaughan reports that, despite this precaution, several workers 

 have been acutely poisoned, especially with the typhoid bacillus. Of course, there 

 is no danger of infection, as the bacteria are killed during the treatment. If the 

 finely ground cellular substance, in the form of an impalpable powder, is kept in wide- 

 mouthed bottles in a dark place, it will retain its toxicity for years. This powder 

 constitutes the bacterial protein substance, which may be split up by various means. 

 Vaughan found digestion with 2 per cent, caustic soda in absolute alcohol especially 

 satisfactory for extracting the poisonous group from bacterial or any other protein. 



A weighed portion of the protein, prepared as above, is placed in a flask, cov- 

 ered with from fifteen to twenty-five times its weight of absolute alcohol in which 

 2 per cent, of sodium hydroxid has been dissolved. The flask, fitted with a reflux 

 condenser, is heated on the water-bath for one hour, where it is allowed to cool and 

 the insoluble portion collected on a filter. After thorough draining the insoluble 

 part is returned to the flask and the extraction repeated. It has been found that 

 three extractions are necessary in order to split off all the poisonous group. The 

 temperature of these extractions is 78 C., the temperature of boiling absolute alco- 

 hol. By this method the protein is split into two portions, one of which is soluble in 

 absolute alcohol and is poisonous, while the other is insoluble in absolute alcohol and 

 is not poisonous (Vaughan). 



Nature of Bacterial Proteins. Vaughan and his coworkers regard 

 bacteria as essentially parti culate, specific proteins. He has not been 

 able to demonstrate the presence of cellulose and carbohydrates; fats 



