Chapter XIII —133— Vital Staining 



This work has demonstrated that the toxicity of the vital dyes 

 which stain the chondriosomes is approximately the same for 

 phanerogams as for fungi. The least toxic is Janus green. Ger- 

 mination of wheat seeds may be obtained in media to which 0.0005- 

 0.001% of Janus green has been added. The roots grow well in 

 solutions as high as 0.005% but at higher concentrations their 

 growth is inhibited and they soon die. The other dyes are much 

 more toxic. Wheat seeds will germinate in them, with the excep- 

 tion of methyl green and Victoria blue, only in solutions between 

 0.0002-0.0008%. The dye is never taken up by the chondriosomes 

 and leucoplasts but accumulates only in the vacuole (Janus green 

 generally in its rose form) . Elodea canadensis can be kept alive in a 

 0.0005% solution of Janus green or of methyl violet. At the be- 

 ginning, the dye is taken up only by the chondriosomes but after 

 a short while these elements lose their color and the dye accumu- 

 lates only in the vacuole (in its rose form in the case of Janus 

 green) . 



It has also been possible in this work to follow the reduction of 

 Janus green to its rose derivative as it occurs in the course of vital 

 staining. If a strip of epidermis from a bulb scale of Allium Cepa 

 is colored between slide and cover glass in a 0.0005-0.005% solu- 

 tion of Janus green, the dye is too dilute to produce a macroscopic- 

 ally visible staining of the epidermis, but a microscopic examination 

 of the preparation shows that Janus green has been taken up by 

 the chondriosomes and leucoplasts. After several minutes, these 

 elements lose their color and there is no longer any trace of the 

 dye to be seen in the cell. If the same experiment is repeated in 

 solutions of 0.01-0.02% of Janus green, the strip of epidermis is 

 stained green macroscopically but at the end of one or two hours, 

 it changes to rose. Janus green has therefore been reduced to its 

 rose derivative. This reduction is irreversible and the rose deriva- 

 tive can no longer by re-oxidation resume its green form. If air 

 is excluded by sealing the slide with paraffin, reduction is more 

 rapid and can be followed under the microscope in a single cell. 

 At the beginning, the dye stains only the chondriosomes and leuco- 

 plasts, the chondriosomes more intensely than the leucoplasts, and 

 accumulates at the same time in those vacuoles which contain oxy- 

 flavanol compounds. At the end of about half an hour in cells m 

 which the vacuole is not stained, the chondriosomes and leucoplasts 

 lose their green color at the same time that the cytoplasm and 

 nucleus take on a rose tint. In cells in which Janus green has 

 accumulated in the vacuole, reduction often begins there. The 

 vacuole changes from violet to rose, more accentuated than the 

 green stain that it first showed, while the cytoplasm and nucleus 

 generally remain colorless. In reality the chondriosomes and leuco- 

 plasts only partially destain, for these two categories of elements 

 show a pale rose tint. The cells which have reduced Janus green 

 to its rose derivative can remain alive without air and show cyto- 

 plasmic currents for 48 hours. With concentrations above 0.02% 

 of Janus green, reduction of the dye to its rose derivative is some- 



