372 
LEWIS KNUDSON 
with the Hberation of some nitric acid. If the sucrose is present, therefore, 
in the nutrient solution, a considerable portion may be inverted. The 
period of sterilization seems to be a factor in this acidification, for in some of 
the preliminary experiments no such inversion occurred when the entire 
nutrient solution was sterilized at one time. 
Throughout the experiments here reported the following method was 
adopted. The nutrient solution was made up in two portions. Portion A 
Fig. I. See text for explanation. 
includes all the salts except calcium nitrate. Portion B includes calcium 
nitrate only. To solution A, which is slightly alkaline, the sucrose was 
added. The solutions were originally made up double strength so that 
equal quantities of A and B would give the desired concentration of the 
sucrose and salts necessary for the nutrient solution. The method of pro- 
cedure is as follows: When iioo cc. of the culture solution is desired, 
550 cc. of each solution (A and B) is accurately measured out. Solution 
A is placed in the culture flask and this is provided with a cotton stopper 
with a central tube. Through this tube is inserted the stem of a 9-centimeter 
funnel, and the funnel and neck of the flask are then covered with cotton 
to prevent any contamination after sterilization. Solution B is placed in a 
liter flask stoppered with cotton and the stopper and neck are also enclosed 
