140 Bulletin 79 



the cell sap, and its freezing in the intercellular spaces, or (2) by 

 the condensation of the cell sap as a result of the water being 

 removed. The second and third problems, are, therefore, answered. 



EFFECTS OF COLD ON THE PROTOPLASM 



The only other cause that might result in the death of the 

 plant or parts of the plant is the inability of the protoplasm to 

 withstand temperatures below a certain point. It is not to be 

 presumed that the protoplasm would be killed as a result of the 

 destruction of the enzymes of the cell, since these are able to with- 

 stand low temperatures uninjured. 



To determine this, pieces of stems of the various species and 

 varieties of spineless cacti were subjected to a killing temperature 

 of — 20° C. ( — 4° F.). To do this, a large-sized ice-cream freezer 

 containing the ordinary freezing mixture of ice and salt was used. 

 This gave a temperature of — 20°C.( — 4°F.). The cactus stems were 

 allowed to remain at this temperature for 48 hours, and the ice 

 and salt mixture was replenished from time to time. When removed 

 from the freezer the stems were frozen hard, and upon thawing gradu- 

 ally it was evident that they had been killed, since they had lost 

 their turgor, and the water in the stems was not absorbed by the 

 protoplasm. Other tests also indicated that the protoplasm was 

 dead. 



Along with this experiment small pieces of the cactus stems were 

 fitted carefully about bulbs of thermometers which were inserted 

 in large test tubes, the ends of which were closed with cotton bat- 

 ting. These test tubes were placed in a mixture of ice and salt and 

 allowed to remain until the temperature reached ^20° C. (—4° F.). 

 This length of time, as before noted, varied with different species. 

 When this temperature had been reached, the thermometers with the 

 pieces of cacti attached were removed, and the protoplasm was 

 studied. The pieces of cacti were frozen hard. After thawing 

 gradually there was every indication that the protoplasm was dead. 

 The cells had lost their turgor, and the protoplasm did not absorb 

 water. Microscopic study showed that the cells were plasmolyzed 

 and the protoplasm had collected toward the centers of the cells in 

 shapeless masses. The nuclei were shrunken, and the chloroplasts had 

 lost, in part, their roundish shape. This p'" '■"iplasm stained immedi- 

 ately with a solution of eosin in water, which condition, will not obtain 

 with living protoplasm. The cells had lost their former outline 

 and the intercellular spaces were greatly enlarged. This latter. 



