Isolation Cultivation 291 



alcoholic solution of dye in 95 c.c. of distilled water). The 

 stain is applied to the fixed specimen and heated until it 

 begins to steam, when the stain is washed off in a 20 per 

 cent, solution of crystals of sulphate of copper. The prepara- 

 tion is then dried and mounted in balsam. 



Hiss finds this stain a useful aid in differentiating the 

 pneumococcus from the streptococcus with which it is 

 easily confounded if the capsules are not distinct, and to 

 which it is probably closely related. 



Isolation. When desired for purposes of study, the 

 pneumococcus may be obtained by inoculating rabbits with 

 pneumonic sputum and recovering the organisms from the 

 heart's blood, or it may be secured from the rusty sputum 

 of pneumonia by the method employed by Kitasato for 

 securing tubercle bacilli from sputum: A mouthful of fresh 

 sputum is secured, washed in several changes of sterile 

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

 carefully separated, and a minute portion from the center 

 transferred to an appropriate culture medium. 



Buerger,* in conducting a research upon pneumococcus 

 and allied organisms with reference to their occurrence in the 

 human mouth, under the auspices of the Rockefeller Insti- 

 tute, used a 2 per cent, glucose-agar of a neutral, or, at most, 

 0.5 per cent, phenolphthalein acid titre. 



"The medium was usually made from meat infusion and contained 

 1.5 to 2 per cent, peptone and 2.5 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 fol- 

 lows: A swab taken from the mouth was thoroughly shaken in a 

 tube of neutral bouillon. From this primary tube dilutions in bouil- 

 lon 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 col- 

 onies. 



Cultivation. The organism grows upon all the culture 



media except 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 be- 



*"Jour. Exp. Med.,"Aug. 25, 1905, vn, No. 5. 



