May 3i, 1924 Cell Sap Density and Environmental Conditions 
849 
TECHNIQUE OF DETERMINATION OF DENSITY OF SAP 
The importance of freezing the samples preliminary to extraction of the sap 
has been fully discussed by Dixon and Atkins (29), by Gortner and Harris (41), 
and by Gortner, Lawrence, and Harris (42). These studies indicate that this 
preliminary freezing is essential to increase the permeability of the tissues which 
yield a more representative sample of the solutes from the smaller as well as from 
the larger vacuoles. Sap from unfrozen tissues has in all cases resulted in lower 
concentrations than that from frozen tissue, because of the less complete extrac¬ 
tions of the former. 
Sap density determinations were made by means of the standard freezing- 
point depression method used by Cavara (16) as early as 1902-1905, later by 
Dixon and Atkins (29), and further developed by Harris, Gortner, and Lawrence 
(41, 4®, 46, 41), whereby the osmotic pressure of the sap is calculated from the 
observed depression of the freezing point. Freezing points were determined by 
means of the classical Beckmann thermometric method (6), which has been 
widely used in chemistry and biology. The thermometer was read to thou¬ 
sandths of degrees and the results were then expressed in freezing point lowering 
in degrees C. (A) and in atmospheres of osmotic pressure (P), after corrections 
for undercooling were made by using the formula suggested by Harris and Gortner, 
as follows: * 
A=A'—0.0125 mA, in which A=the true depression of the freezing point, 
A'=the observed depression of the freezing point, ju=the amount of under¬ 
cooling in degrees C., and a constant (0.0125) experimentally determined, accord¬ 
ing to the tables calculated by Harris and Gortner. As a part of the laboratory 
routine it was necessary to determine the freezing point of double distilled water 
at the beginning and the end of each day's work. Through the adoption of the 
simplified technique it became possible to make 50 freezing point determinations 
in one day, all of which were made by the writer. 
On several occasions during the course of these investigation^ the importance 
was forcibly brought out of exercising extreme precautions in the collecting and 
freezing of the samples and the extraction of the sap. Otherwise there can be 
no assurance that the results obtained will be dependable. Evaporation, espe¬ 
cially, must be most carefully watched. A striking example of this is afforded 
in the case of Orthocarpus tolmiei. Specimens of this species collected in an 
opening in the spruce-fir forest in Big Cottonwood Canyon on September 9, 1920, 
at an elevation of 8,850 feet and treated in the regular manner showed a freezing 
point depression of the sap of 1.76° C. or an osmotic pressure of 21.2 atmospheres. 
In marked contrast, the sap of leaves collected from the same plants at the same 
time but allowed to lie in the shade exposed to the air for 12 hours depressed the 
freezing point 2.67° (32.0 atmospheres). This difference was caused largely 
by the drying out of the leaves. With the water supply cut off and the evapo¬ 
ration of moisture from the stomata continuing, a material concentration of the 
remaining sap is the obvious result. 
Another striking example occurred in connection with two collections of coni¬ 
fers in the spruce-fir type of Ephraim Canyon on July 18 and 22. Both lots were 
collected and frozen in accordance with the prescribed methods, except that one 
lot on each date was allowed to stand subjected to hot summer temperatures on 
the laboratory table for six hours after it had been taken from the freezing mix¬ 
ture but before being taken out of the freezing tubes, while the sap of the other 
lot was extracted and tested immediately after the leaves had thawed out. The 
results of this comparison are given in Table I. 
