76 Dr. A. Harden and Mrs. D. Norris. Production of [Jan. 25, 



two weeks, and four weeks. A different strain of the organism was used in 

 Experiment 1 from that used in Experiments 2 and 3. 



The glycerol added, and the amount unfermented, were estimated by 

 means of the method of Zeisel and Fanto (10), as described below. 



Estimation of the Residual Glycerol. — 100 c.c. of the liquid in which the 

 organism had grown were evaporated at a low temperature under reduced 

 pressure, in order to ensure the removal of all alcohol. The residue was 

 then taken up in a small quantity of water, the volume made up to 100 c.c. 

 with water, and 5 c.c. of this solution taken for the estimation. 



Search for Acetylrnethylcaroinol. — A portion of the culture medium was 

 distilled and the distillate tested for reducing power with Fehling's 

 solution. In every case the distillate was found to be non-reducing and the 

 absence of acetylmethylcarbinol was further confirmed by trying the Voges 

 and Proskauer reaction, which was invariably negative. It was also found 

 impossible to prepare any osazone. 



Detection of 2.3-Butylenc Glycol. — This substance was detected and estimated 

 as previously described, the estimation being made on 300 c.c. of the culture 

 medium in which the organism had been grown. In every experiment this 

 glycol was found to be present, the other products being ethyl alcohol, formic, 

 lactic, and succinic acids, carbon dioxide, and hydrogen. 



The diacetylphenylosazone prepared from the oxidation product of the 

 butylene glycol was analysed and gave the following results : — 



01372 grm. substance gave 0-3656 grm. C0 2 and 0-0876 grm. H 2 0. 

 C = 72 - 6 per cent. ; H = 7 per cent. 



0-0950 grm. substance gave 17 - 5 c.c. 1ST at 21° C and 765'5 mm. 1ST = 21-12 

 per cent. 



Ci 6 H 18 N 4 requires C = 72'2 per cent., H = 6-8 per cent., N = 21-05 per 

 cent. 



The lactic acid produced was also characterised by the preparation and 

 analysis of the zinc salt and by Fletcher and Hopkins' reaction (11). 



0-1500 grm. of the zinc salt dried at 105° C. gave 0-0499 grm. ZnO. 



ZnO = 33-28 per cent. 

 (C 3 H 5 03)2Zn requires ZnO = 33-4 per cent. 



The analysis of the calcium salt prepared from the succinic acid formed 

 gave the following results : — 



0-0550 grm. substance gave - 0202 grm. CaO. CaO = 36 - 7 per cent. 



(GtlLtO^Ca requires CaO = 35 - 9 per cent. The pure acid was also isolated 

 in this case and gave M.P. 183-4° C. 



The percentage of these substances on the weight of glycerol fermented is 

 shown in the following table, Columns 1, 2, and 3. Columns 4 and 5 



