Studies in Fat Metabolism of Timothy Grass Bacillus. 273 



formed and the total protein nitrogen at the point of maximum growth will 

 be discussed later. 



Experiment 5. — Growth on, and Utilisation of Acetic Acid. 



Eoux bottles containing the usual inorganic salts, with 0'5 per cent, acetic 

 acid as sodium acetate, were steam sterilised and sown in the usual way. 



Estimation of the Acetic Acid. — The contents of one Eoux bottle (150 c.c. 

 of original medium) were made up exactly to 250 c.c, filtered through a 

 dry filter into a dry flask, and the acetic acid then estimated by distillation 

 of 20 c.c. acidified with phosphoric acid. The usual method of estimating 

 acetic acid by steam distillation was found unsatisfactory. The amount of 

 distillate which must be collected before the acetic acid has completely come 

 over is at least 2,000 c.c. ; this contains large and variable quantities of 

 carbon dioxide, which must be separately estimated by collecting the dis- 

 tillate in excess standard baryta, neutralising the excess with standard 

 hydrochloric acid, filtering off and separately estimating the barium car- 

 bonate. The whole process takes between three and four hours, and gives 

 moreover very variable and unreliable results. Instead, a modified procedure 

 was adopted. 



The distillation of the acetic acid was carried out in vacuo. The apparatus 

 used was identical with that described by Van Slyke (Joe. cit.) for the estima- 

 tion of ammonia, except that in place of the second smaller distilling flask, 

 a thick- walled filtering flask was found more convenient ; 10 c.c. of diluted 

 medium (or an amount containing from 0'06 to - l grm. of acetic acid) was- 

 placed in a Claissen flask of 300-400 c.c. capacity, fitted with a capillary 

 tube in the first neck, and a tap in the second neck. The liquid was- 

 acidified with four or five drops of saturated phosphoric acid. An equal 

 volume of 95 per cent, alcohol (previously distilled from caustic soda) was- 

 added. An exact volume (about 25 c.c.) of 0'1N baryta was run from a 

 burette into the distilling flask and the filtering flask, and the capillary 

 tube was adjusted to dip beneath the surface of the liquid. The contents 

 of the Claissen flask were then distilled in vacuo (10-15 mm.), and the 

 distillate collected in the baryta in the cooled receiver, any escape of acetic 

 acid being prevented by the baryta in the filtering flask. The distillation 

 was continued until only about 1 c.c. of liquid remained in the Claissen 

 flask. The temperature during the last five minutes should be at least 

 60°. When the distillation was complete, air was admitted by the capillary 

 and by opening the tap in the second neck of the Claissen flask ; 20 c.c. 

 of 50 per cent, alcohol were then poured into the flask and the distillation 

 repeated as before, care being taken that the temperature of the water-bath 



