1889.] On a pure Fermentation of Mannite and Glycerin. 



349 



The sterile liquid thus obtained was then carefully inoculated with 

 a minute quantity of a pure culture of the organism and placed in an 

 incubator, the temperature of which was maintained at 38 — 40° 0. 



The fermentation commences in the course of a few days and con- 

 tinues for several weeks. 



Three separate fermentations of mannitol were thus carried on. 



In each case, before commencing the chemical examination of the 

 products, the contents of the flask were submitted to microscopic 

 examination and to plate-cultivation with gelatine-peptone ; in each 

 case the growth was found to have remained pure, only the charac- 

 teristic colonies of the organism itself making their appearance on the 

 plates. 



'Fermentation of Mannitol No. 1. 



Two litres of mannitol solution, prepared as above described, were 

 duly sterilised and then carefully inoculated with a minute trace of 

 a pure cultivation of the organism. The liquid was then placed in 

 an incubator the temperature of which was maintained at 38 — 40° C, 



The fermentation commenced on the third day and continued 

 with more or less activity for about one month, but the liquid re- 

 mained in the incubator for upwards of three months ; at the close of 

 this period it was submitted to plate-cultivation as above indicated, 

 and the chemical examination then proceeded with. 



Separation of Alcohols. — The fermented liquid, which still contained 

 a quantity of undecomposed carbonate of lime, was distilled down to 

 about one-third of its bulk and until the distillate gave only the 

 faintest indications with Lieben's highly sensitive iodoform reaction 

 for alcohol. The residue in the distilling flask was set aside to be 

 examined for acids (see below), whilst the distillate, which would 

 contain any alcohols, was redistilled over and over again so as to get 

 rid of the greater part of the water. When the volume of liquid 

 amounted to only about 50 c.c. a careful determination of the specific 

 gravity was made, and this indicated the presence of 11*415 grams 

 of absolute alcohol. Finally the liquid was dehydrated with fused 

 carbonate of potash, and on then distilling it passed over at 

 79 — 80° C, showing it to be pure ethyl alcohol. 



Separation of Volatile Acids. — The residue remaining in the flask, 

 after distilling off the alcohol as above, would contain any acids in 

 the condition of calcium salts ; in order to liberate these acids a 

 calculated quantity of hydrochloric acid was added, sufficient exactly 

 to decompose the carbonate of lime originally added to the liquid. 

 On then repeatedly distilling with water the volatile acids passed over 

 and were converted into barium salts for analysis. 



The hydrochloric acid was not, however, added all at once, but 

 in several distinct portions, and after each addition the liquid was 



