ANTIGENICITY OF PNEUMOCOCCUS 333 



concentration of formalin must be raised to 0.5 per cent if enzy- 

 matic activity is to be destro} r ed. On the other hand, exposure of 

 the pneumococcal suspensions to heat, when properly carried out, 

 yields a vaccine of great stability and of high antigenic integrity. 

 Dubos observed that exposure to temperatures that rob the cell of 

 its ability to grow and multiply may fail to halt the action of the 

 protease of Pneumococcus. To devitalize the organisms, to prevent 

 autolysis, and to preserve the antigenic properties of pneumococci, 

 Dubos heated cultures or suspensions to 75° with the least pos- 

 sible delay and so obtained antigens of greater stability and im- 

 munizing power than could be produced by any other method. 



Confirming the claims of Barnes and White and of Dubos that 

 immediate heating of pneumococci for use as antigens is the best 

 method for insuring the stability of the preparation, O'Meara and 

 Brown, 1031 during the course of an investigation on the readiness 

 with which pneumococci disintegrate, reported that, in spite of a 

 large number of preservatives tried, no agent except heat was 

 found that would completely arrest autolysis of pneumococcal cul- 

 tures. 



Day 307 used formol, or antiformin, as a killing agent, but aban- 

 doned its use since it weakened the immunizing properties of the 

 cultures. Truche 141920 favored an alcohol-ether mixture for the 

 routine preparation of antigens in the production of equine anti- 

 pneumococcic serum, and claimed that cultures so treated, dried 

 in a vacuum, and suspended in salt solution, were tolerated better 

 by horses than were heat-killed cultures. 



There have been reported many other methods for rendering 

 pneumococci harmless for immunizing purposes. As early as 1902, 

 Sergent 1206 treated suspensions of agar cultures of pneumococci 

 with crystal violet. Although the dye in the concentration used 

 failed to kill the cocci it effected a partial attenuation, so that rab- 

 bits surviving the intravenous or intraperitoneal injection of the 

 treated cultures were able six to eight days later to resist increased 

 doses of the same cultures that proved fatal in animals which had 



