Isolation 495 



of staining the capsules of the pneumococcus : The organism 

 is first cultivated upon ascites serum-agar to which i per 

 cent, of glucose is added. The drop containing the bacteria 

 to be stained is spread upon a cover-glass mixed with a 

 drop of serum or a drop of the fluid culture-medium, and 

 dried and fixed. A half-saturated aqueous solution of gen- 

 tian violet is applied for a few seconds and then washed 

 off in a 25 per cent, solution of carbonate of magnesium. 

 The preparation is then mounted in a drop of the latter 

 solution and examined. 



If it is desired to stain the capsules and preserve the 

 specimens permanently in balsam, Hiss employs a 5 or 10 

 per cent, solution of fuchsin or gentian violet (5 c.c. saturated 

 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. 



To demonstrate the organisms in sections of tissue either 

 Gram's or Weigert's methods may be used, with beautiful 

 results. 



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 J^lood, or it may be obtained from the rusty sputum 

 of pneumonia by the method employed by Kitasato for 

 securing tubercle bacilli from sputum: A mouthful of fresh 

 sputum is 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 titer. 



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



