368 



SCiEl^CE. 



[N. S. Vol. XV. No. 375, 



COCCUS, and to Methods of Staining Cap- 

 sules: P. H. Hiss, M.D., College of Phy- 

 sicians and Surgeons, New York. 

 The author believes that up to the pres- 

 ent time it has not been demonstrated that 

 pneumococci and streptococci can at all 

 times be clearly differentiated from each 

 other. Well-marked capsules have been 

 found by various observers to occur on 

 organisms more reasonably classified as 

 streptococci than as pneumococci. On the 

 other hand, capsules may not be demon- 

 strable by the usual methods on pneumo- 

 cocci, especially when these organisms are 

 growing on artificial media. Pneumococ- 

 cus cultures may also show a predominance 

 of chains, while streptococci may occur in 

 pairs. The usual cultural characters and 

 reactions of these organisms are at the best 

 not diagnostic, and are subject to varia- 

 tions. 



Experiments by the author, with cul- 

 tures of pneumococci and streptococci from 

 many different sources indicatd well- 

 marked, constant differences between the 

 metabolic activities of pneumococci and 

 those of streptococci. These differences in 

 metabolism become apparent when the or- 

 ganisms are cultivated in the following 

 media: (1) A medium composed of ox 

 serum, one part; distilled water, two 

 parts; normal sodium hydroxid, 0.1 per 

 leent. (2) A medium composed of ox se- 

 rum, one part; distilled water, two parts, 

 and inulin, 1 per cent. These serum media 

 are not coagulated by boiling and are ster- 

 ilized at 100° C. Acid is formed in each of 

 these media by pneumococci when grown 

 at 37°C., and a solid yellowish-white coagu- 

 ium results. The coagulation is rapid in 

 the inulin medium, slower in the alkaline. 

 Streptococci do not form acid in these me- 

 dia, and no coagulation occurs. These 

 ifflfidia have, therefore, in all instances, 

 ■served to differentiate pneumococci from 

 streptococci. 



Other mono-, di- and poly-saccharids 

 were tested in media made in the same 

 manner as the inulin medium, but were 

 fermented and the media coagulated, by 

 various members of the streptococcus 

 group, as well as by pneumococci. Hence, 

 these carbohydrates are not of use in dif- 

 ferential tests. Special methods were de- 

 vised for demonstrating capsules on pneu- 

 mococci and streptococci. Chief among 

 these was to grow the organisms on ascitic 

 serum agar, preferably plus one per cent, 

 glucose. Spread the organisms on the 

 cover-glass by mixing with a drop of 

 serum, or a drop of one of the fluid serum 

 media. Dry in the air and fix by heat. 

 Then stain for a few seconds in a one half 

 saturated aqueous solution of gentian vio- 

 let. Wash off with a 0.25 per cent, solu- 

 tion of potassium carbonate, mount and 

 study in this solution. This is a good stain 

 for capsules of pneumococci in blood or 

 serum of infected animals. Pneumococcus 

 capsules may also be stained by the follow- 

 ing method and be mounted in balsam 

 without injury. A five per cent, or ten per 

 cent, solution of gentian violet or fuehsin 

 (500 sat. alcoholic sol.' gentian violet plus 

 95 c.c. distilled water) is used. This is 

 placed on the dried and fixed cover-glass 

 preparation and gently heated until steam 

 arises. The dye is washed off with a 

 twenty per cent, solution of copper sul- 

 phate (CuSO^ cryst.). The preparation is 

 then dried and mounted in balsam. By 

 these methods most streptococci were 

 found to have capsules. 



Well-marked examples of encapsulated 

 organisms, such probably as those which 

 have been described by some investigators, 

 and separated by them into new species 

 distinct from Streptococcus pyogenes, or 

 with no certainty differentiated from pneu- 

 mococci, have been examined and have been 

 found to correspond to Streptococcus pyo- 

 genes in the media mentioned in this paper. 



