ELECTRON MICROSCOPY 1737 



pH 4.5, not at pH >7. Nagai suggested that this selective silver deposition is the result 

 of migration and adsorption of silver atoms on the chloroplast surface, rather than 

 evidence of the presence of a reducing agent directly in the latter. In a second paper, 

 Nagai (1951) noted that several plant substances — particularly ascorbic acid and di- 

 oxyphenylalanine (to a lesser extent, also tannings and flavone derivatives) — can reduce 

 silver nitrate under the conditions of the Molisch test. 



Later (1952) Nagai observed the Molisch reaction in many marine and fresh water 

 algae, and concluded that the only appropriate reducing compound found in all of them 

 is ascorbic acid. Algae which showed a negative Molisch test also gave no evidence 

 of ascorbic acid in chromatographic separation. In sorrel and similar "oxalate plants" 

 the Molisch test gave no clear positive results, despite the presence of ascorbate, but this 

 could be attributed to the interference of oxalic acid. 



The effect of light was considered by Nagai in still another paper (19522). He found 

 that the Molisch test was negative in etiolated seedlings, but became positive after 2 

 hours exposure to light, simultaneously with the visible formation of chlorophyll. Nagai 

 suggested that the photochemical effect consists in attracting to the chloroplast the 

 silver formed elsewhere by a non-photochemical reaction of ascorbic acid with silver 

 nitrate, and consequent formation of localized black deposits instead of a diffuse, plas- 

 matic precipitate. This seems a much less plausible explanation than sensitized photo- 

 chemical reduction of silver ions (or photochemical reduction of chlorophyll by ascor- 

 bic acid, followed by re-oxidation of reduced chlorophyll by silver ions). 



Hagene and Goas had noted (1945) that the disintegration of chloroplast struc- 

 ture (e. g., by heat) is accompanied by a cessation of fluorescence, and inquired (1949) 

 whether the capacity of chloroplasts for the reduction of silver nitrate shows a paral- 

 lelism with their fluorescence. They found this to be true, but not under all condi- 

 tions: fluorescence is preserved, e. g., after freezing chloroplasts in liquid air, while the 

 reducing capacity disappears. No general parallelism exists between the capacity for 

 silver nitrate reduction and for photosynthesis. It is not clear whether in these experi- 

 ments the photochemical component of the silver nitrate reduction was considered rather 

 than the — perhaps quite unrelated — thermochemical reduction. 



Metzner (1952') observed the distribution of silver deposits in sections from the 

 lower surface of leaves of Agapanthus umbellatus and cotyledons of Impatiens parviflora. 

 Blackening was found to occur in the cytoplasm as well as in plastides and nuclei. 

 Within the chloroplasts, the reaction occurred preferentially in the grana and at the 

 surface (perhaps at a membrane). The chloroplast reaction becomes predominant at 

 pH 3, that in the cytoplasm at pH 7-8. Both reactions occurred in the dark; light ac- 

 celerated the reduction. The action spectrum of AgNOs reduction by (initially) living 

 Agapanthus cells showed peaks in the red and in the violet, indicating sensitization by 

 chlorophyll and suggesting that direct photochemical decomposition of unstable silver 

 salts (precipitated in the cells) does not contribute much to the blackening. In cells 

 killed by several minutes immersion into silver nitrate solution in the dark, the action 

 spectrum (for silver deposition in a following light period) was quite different, showing 

 no peaks in the chlorophyll bands — except for the reaction in the grana. Cells killed 

 by lead acetate still reduced AgNO,,, but only in the plastides; cells killed by brief boiling 

 were completely inactive. Metzner (1952^) tried to sensitize the photochemical silver 

 nitrate reduction in chloroplasts by vital staining with Rhodamin B, but the action 

 spectrum of stained cells stiU showed the "green gap" characteristic of sensitization by 

 chlorophyll. 



Thomas, Post and Vertregt (1954) first used the electron microscope to 

 study the distribution of silver in chloroplasts and their fragments. They 



