Plant Cells at Low Temperatures . 407 
B.S.P., leaves ; Pyrola rotundifolia , L., leaves ; Linnaea borealis , L. var. 
americana (Forbes), leaves ; Populus tremuloides , Michx., cortical tissues. 
During the winter some difficulty was experienced in collecting Pyrola and 
Linnaea from under the snow, thus limiting the number of observations that 
could be made with these plants. 
Methods. 
According to the observation of Dixon and Atkins ( 5 ), sap expressed 
by simple pressure from living tissues does not give a fair sample of the sap 
in the vacuoles of the living cell. In the researches of these authors the 
protoplasm of the living cell was rendered permeable to all the solutes by 
the application of extreme cold, liquid air being used for this purpose. 
After freezing by this means the protoplasm is killed, rendered quite perme- 
able, and the sap with all the solutes can be extracted by comparatively 
slight pressure. 
As liquid air was not obtainable for these experiments, liquid C 0 2 was 
used. This gives a temperature of — 72°C., which results in the instant 
freezing of the tissues. The material was then placed in a glass vessel, 
rapidly thawed, and on treatment in a small screw-press about 25 c.c. of 
sap could be obtained from a comparatively small amount of material of all 
the plant tissues investigated. Care was taken to ensure that all the C 0 2 
was evaporated before the extraction of the sap. The increased ease with 
which the sap is extracted by this method is striking. One sample of 
untreated cortex of Populus tremuloides gave only a few drops of sap on 
pressing ; an equivalent weight of cortex killed by freezing yielded 14 c.c. of 
sap with slight pressure. 
The A of the expressed sap was determined at once — usually within 
the hour — by the ordinary Beckman method, and the measurement of the 
electrical conductivity was then measured by the method of Kohlrausch. 
The estimation of sugars was made by treating the extracted sap with 
a minimum amount of basic lead acetate to precipitate the tannins. The 
excess of lead was then removed by sodium carbonate. The extract freed 
from tannins was tested with Benedict’s sodium citrate solution (6). All 
determinations were made in drops rather than in cubic centimetres, since 
the quantity of extract available would not permit of the latter method. 
The same burette was used throughout the investigation, thus ensuring 
a uniformity in the size of the drops. The results recorded in each case 
were the number of drops required to decolorize twenty-five drops of 
Benedict’s solution ; the relative reducing power of the extract before and 
after inversion being determined by comparison with that of a standard 
glucose solution. The relative amount of glucose, sucrose, and maltose were 
determined according to the methods described by Haas and Hill ( 7 ). 
