45 



Luyet and Gibbs (1937) repeated the experiments of 

 IMolisch, using the ei)idermis of the onion. They followed 

 this author's procedure in all details except that their 

 material was vitally stained with neutral red before being- 

 cooled, and that their low temperature chamber was set 

 to about - 10°. With the higher cooling velocity furnished 

 by this lower temperature, all the cells were observed to 

 freeze in 5 to 6 minutes. Sudden flashes during freezing 

 indicated that the individual cells were subcooled and that 

 they congealed at once. After thawing, they were all dead, 

 as shown by the release of the stain. 



Zacharowa (1926), in a study of the effect of hydrogen 

 ion concentration on cold resistance, put to freeze epi- 

 dermal sections of red cahhage in test tubes immersed in 

 freezing mixtures and containing a little quantity of either 

 distilled water or a weak solution of sodium hydroxide 

 (0.01 to 0.001 N) or sulphuric acid (0.0001 to 0.00001 N). 

 The material, seeded with an ice particle, was left to freeze 

 and cool for one hour. It was then thawed and its vitality 

 was tested by plasmolysis. Half of the cells were found 

 alive after exposure to - 3.9° and one-fourth after expo- 

 sure to -5.75°, in distilled water. The sections in the 

 sodium hydroxide solution were slightly more resistant, 

 and the sections in the sulphuric acid solution less resis- 

 tant than those in distilled water. 



Chambers and Hale (1932) followed under the micro- 

 scope the gradual freezing of single epidermal cells in 

 precisely known temperature conditions. Epidermal 

 strips of the red onion were placed, together with the tip 

 of a thermo-needle, in a hanging drop of liquid paraffin 

 and the temperature of the whole system was slowly low- 

 ered, in a cooling room, while inoculation with an ice- 

 tipped pipette prevented subcooling and subsequent too 

 rapid freezing. When the temperature reached -7° to 

 - 10°, ice was formed between the cellulose wall and the 

 protoplast but the latter was usually still capable of 

 plasmolysis. At - 10°, the cytoplasm and nucleus disinte- 

 grated, but the tonoplast still resisted. These phenomena 



