June 3. 1918 Density of Cell Sap and Winter Hardiness in Grain 499 



Table II. — The effect on sap density of treating leaves of barley and wheat with toluene 



for different periods 



Exposure to toluene. 



Hours. 



14-15 



22.5-23 



37-5-38 



46-46.5 



61.5 



Freezing point of sap after treatment (°C.). 



Barley. 



Sample i. 



-1.29s 



-1-515 

 -I. 310 



'-1-725 

 -1-35° 



Sample 2. 



-o- 975 

 -I- 565 

 -1-355 



^■—T. 710 

 -I- 315 



Average. 



-I- 135 

 -1.540 



-I. 718 



Wheat. 



Sample i. 



■1.230 

 ■1-575 

 ■1-335 

 -1.320 



Sample 2. 



— I. 040 



— I. 670 

 -1-275 



-1-395 



Average. 



— I. 140 



-1.623 

 -1-305 

 -1-385 



o The freezing point of these samples was not determined until several hours after extraction. Molds 

 had then appeared on the sap, and this is perhaps responsible for the large depression. 



The results show a gradual increase in the depression of the freezing 

 point with exposure to chloroform and toluene up to about 23 hours. 



This would indicate that the plasma membrane had been rendered 

 thoroughly permeable at that time. There appeared to be no marked 

 difiference whether the leaves were kept on ice or at room temperature. 

 Since enzymic action would be least at the low temperature, it was thought 

 best to keep the samples on ice during treatment, and this was done in 

 most cases. 



EFFECT OF CHLOROFORM AND TOLUENE ON THE DEPRESSION OF THE 



FREEZING POINT 



Since chloroform and toluene are slightly soluble in water, it seemed 

 desirable to determine their eflfect on the freezing point of the sap as a 

 possible explanation for the variation in duplicate samples. Accord- 

 ingly, different amounts were added to distilled water, and the freezing 

 point of the solutions determined. The results are presented in 

 Table III. 



Table III. — Effect of chloroform and toluene on the freezing point of distilled water 



The solubiUty of chloroform and toluene appears to be too low to 

 affect the freezing point enough to explain the discrepancies observed 

 in dupHcate samples. The slight error due to variation in the amount 

 taken up by the sap could scarcely have been an important factor in 

 the results reported later in this paper. This particularly would be true 



