358 PATHOGENIC MICROORGANISMS 



For the isolation of pneumococci from mixed cultures or from mate- 

 rial containing other species, such as sputum, surface smears of the 

 material are made upon plates of neutral glucose-agar, glucose-serum- 

 agar, or blood agar. According to the number of bacteria present in 

 the infected material, it may be smeared directly upon the plate, or 

 diluted with sterile broth before planting. After incubation for twenty- 

 four hours, the pneumococcus colonies are easily differentiated from all 

 but those of streptococcus. With practice, however, they may be dis- 

 tinguished from these also, by their smoother edges and greater trans- 

 parency and flatness. 



For the isolation of pneumococci from sputum, the sputum may be 

 washed by gently rinsing in successive watch glasses containing salt 

 solution, then rubbed up in a glass tube or mortar with a little broth 

 and injected into a white mouse, either into the base of the tail or into 

 the peritoneum, taking great care not to puncture the liver. If virulent 

 pneumococci are present, death will occur within 24 hours or there- 

 abouts. Pneumococci will be found in pure culture in the heart's 

 blood, and in large amounts in the peritoneal exudate. 



Resistance. On artificial media, the viability of the pneumococcus 

 is not great. Cultures upon agar or bouillon should be transplanted 

 every third or fourth day, if the cultures are kept within an incubator. 

 In all media in which rapid acid formation takes place, such as glu- 

 cose media, the death of cultures may occur more rapidly. In media 

 containing albumin and of a proper reaction, preservation for one or 

 even two weeks is possible. The longer the particular race has been 

 kept upon artificial media, the more profuse is its growth, and the 

 greater its viability, both qualities going hand in hand with diminish- 

 ing parasitism. The length of life may be much increased by preserva- 

 tion at low temperature, in the dark, and by the exclusion of air. In 

 calcium carbonate broth and kept in the ice-chest, cultures may often 

 remain alive for months. 



Neufeld has succeeded in keeping pneumococci alive and virulent, 

 by taking out the spleens of mice dead of pneumococcus infection and 

 preserving them in a Petri dish in a dessicator, in the dark and cold. In 

 this way, the organisms can be cultivated from the spleen, and will be 

 found virulent for longer periods than in culture media. 



In sputum the viability of pneumococci seems to exceed that ob- 

 served in culture. The studies of Guarnieri, 1 Bordoni-Uffreduzzi, 2 and 



1 Guarnieri, Att. della R. Acad. Med. di Roma, iv, 1888. 



2 BordonirUffreduzzi, Arch. p. 1. sc. med., xv, 1891. 



