Cultivation 447 



water to free it from the bacteria of the mouth and pharynx, care- 

 fully separated, and a minute portion from the center transferred 

 to an appropriate culture-medium. 



Buerger,* in conducting a research upon pneumococcus and alUed 

 organisms with reference to their occurrence in the human mouth, 

 under the auspices of the Rockefeller Institute, used a 2 per cent, 

 glucose-agar of a neutral, or, at most, 0.5 per cent, phenolphthalein 

 acid titer. 



"The medium was usually made from meat infusion and contained 1.5 to 2 

 per cent, peptone and 2.4 per cent. agar. Stock plates of these media (serum- 

 agar and 2 per cent, glucose-serum-agar) were poured. The agar or glucose- 

 agar was melted in large tubes and allowed to cool down to a temperature below 

 the coagulation point of the serum. One-third volume of rich albuminous 

 ascitic fluid was added, and the resulting media poured into Petri plates. These 

 were tested by incubation and stored in the ice-chest ready for use. . . . 



"The plan finally adopted [for inoculating the plates] was as follows: A 

 swab taken from the mouth was thoroughly shaken in a tube of neutral bouillon. 

 From this primary tube, dilutions in bouillon with four, six, and eight loops 

 may be made. A small portion of the dilute mixture was poured at a point near 

 the periphery of the prepared plates. By a slight tilting motion the fluid was 

 carefully distributed over the whole surface of the plates. Care must be taken 

 to avoid an excess of fluid. It was found that plates made in this way gave 

 a sufficiently thick and discrete distribution of surface colonies." 



Cultivation.— The organism grows upon all the culture-media ex- 

 cept potato, but only between the temperature extremes of 24° and 

 42°C., the best development being at about 37°C. The growth is 

 always meager, probably because of the metabolic formation of 

 formic acid. The addition of alkali to the culture-medium favors 

 the growth of the pneumococcus by neutralizing this acid. . Hiss and 

 Zinsserf advise that the culture-media used for the pneumococcus 

 be made with 3 to 4 per cent, of peptone. 



Colonies. — The colonies which develop at 24°C. upon gelatin 

 plates (15 per cent, of gelatin should be used to prevent melting at 

 the temperature required) are described as small, round, circum- 

 scribed, finely granular white points which grow slowly, never attain 

 any considerable size, and do not liquefy the gelatin. 



If agar-agar be used instead of gelatin, and the plates kept at the 

 temperature of the body, the colonies appear transparent, delicate, 

 and dewdrop-like, scarcely visible to the naked eye, but under the 

 microscope appear distinctly granular, a dark center being sur- 

 rounded by a paler marginal zone. 



Upon the medium recommended by Buerger for isolating the 

 pneumococcus, the colonies appear in from eighteen to twenty-four 

 hours, the surface colonies being circular and disk-like. When 

 viewed from above, the surface appears glassy with a depressed 

 center. When viewed from the side or by transmitted light, they 

 appear as distinct milky rings with a transparent center. This 



* "Jour. Exp. Med.," Aug. 25, 1905, vil, No. $■ 

 t "Text-book of Bacteriology," 1910, p. 356. 



