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S. S. Brody and M. Brody 



to determine the effects of freezing on these cells. Studies were made of the 

 morphological state of the photosynthetic apparatus, the photochemical 

 activity of the chloroplasts, and the state of pigment aggregation. 



1. Morphology: The structure of the chloroplast lamellae of Chlorella 

 pyrenoidosa and Porphyridium cruentum was examined before and after 

 freezing with the aid of an electron microscope. The algae, after being 

 cooled to 77 K, were allowed to warm to room temperature, and were then 

 immediately fixed with osmium tetroxide. The material was prepared with 

 procedures similar to those given by Brody and Vatter (14). 



The ultrastructure of the frozen Chlorella cells showed the following 

 changes: extensive vacuolization of the cell cytoplasm, some damage to the 

 nucleus (which probably accounts for subsequent failure of reproduction) and 

 a small degree of vacuolization between the lamellae. Perhaps this last 

 effect is related to the decreased activity of the frozen cells in carrying out 

 Hill reaction. 



2. Photochemical Activity : Hill reaction was measured with whole cells 

 using quinone as the hydrogen acceptor. Manometric techniques were 

 essentially similar to those described by Ehrmantraut and Rabinowitch (Z3) . 

 Two aliquots of cells were prepared; one was kept as a control, the other 

 was cooled to 77°K, held at this temperature for about 10 minutes, and then 

 thawed. Great care was taken to keep the cells in the dark. Hill reaction 

 activity of the two aliquots was measured manometrically, each vessel con- 

 tained 100 mm of Chlorella pyrenoidosa, Z mg of quinone, 0. 05% KCL, and 

 1/20 M phosphate buffer (pH 6. 5). (The quinone was freshly purified by 

 steam distillation from ION sulfuric acid, and later sublimated in vacuum. ) 

 The samples were flushed with nitrogen and broughtto 20°C. Illumination 

 was provided by a tungsten lamp. 



It was found that cells which have been frozen and thawed still show Hill 

 reaction but with a lowered efficiency. Figure 3 represents a portion of an 

 experiment in which the production of oxygen by Chlorella cells has been 

 determined. Essentially similar results were obtained by Horowitz (33), 

 who used Chlorella, and 2, 6 dichlorophenolindophenol as Hill oxidant. 



The major effects of freezing upon the Hill reaction are a lowering of yield 

 by a factor of about 5, and an acceleration in loss of activity. But, since 

 oxygen production as a function of light intensity and time of illumination is 

 quite normal, it would seenn that the photochemical apparatus is still 

 relatively intact after freezing. 



