Guilliermond - Atkinson — 142 — Cytoplasm 



The other dyes which stain vacuoles, i.e., cresyl blue, Nile blue, 

 naphthylene blue and naphthylamine blue, behave a little differ- 

 ently. Between slide and cover glass, if the solution is too strong, 

 they may, at the same time that they accumulate in the vacuoles, 

 stain the cytoplasm and the nucleus with a diffuse color. This 

 diffuse color occurs, furthermore, only in the phases which precede 

 the death of the cells. At all events, with these dyes as with 

 neutral red, the vacuoles destain and the staining of the cytoplasm 

 and nucleus becomes accentuated as soon as death occurs. The 

 coloration of the vacuoles by these dyes, as is the case for neutral 

 red, is therefore possible only as long as the cell is living. It may 

 be added that if the dyes are used in weak concentrations, in the 

 fungi especially, they may be reduced in the cells and one sees the 

 coloration disappear under the cover slip and reappear if the latter 

 is raised for purposes of aeration. 



We have also been able to germinate wheat grains in a medium 

 to which these dyes had been added. Nile blue proved slightly 

 more toxic than neutral red and neutral violet ; cresyl blue is very 

 appreciably more toxic than Nile blue ; naphthylene blue and naph- 

 thyalmine blue are extremely toxic. These dyes are accumulated 

 exclusively in the vacuoles of root cells, just as is neutral red, and 

 brilliantly stain the vacuoles of meristematic cells, of root hairs 

 and of cells of the root cap. The staining persists as long as the 

 cells are in a living condition. 



These dyes are more toxic for fungi. They do not generally 

 accumulate in the vacuoles but are reduced by the Saprolegniaceae 

 and by other fungi when cultivated in their presence. 



In collaboration with Gautheret, some of the experiments on 

 Saccharomyces cerevisiae that we carried out with neutral red 

 were repeated, this time using Nile blue and cresyl blue. The 

 experiments showed that these dyes behave like neutral red but 

 have a much more complex action on the cells. These two dyes, 

 Nile blue especially, may stain the cytoplasm. At a high pU, for 

 example, Nile blue is at first retained exclusively by the cyto- 

 plasm which it stains diffusely. Then, at the end of a certain 

 time, the dye is taken up by the vacuoles and from there excreted 

 into the medium. When there is insufficient aeration, as is usually 

 the case, the dye, held at first by the cytoplasm, is reduced by the 

 cytoplasm to its leuco-derivative, in which form it goes into the 

 vacuole and finally, still in its colorless state, is excreted into the 

 external medium where it is re-oxidized in the presence of air. 



Cresyl blue is only weakly retained by the cytoplasm and accu- 

 mulates especially in the vacuole, but it may be reduced, as is Nile 

 blue, before being excreted into the medium where it is re-oxidized. 



Investigation of these dyes was carried further and the accumu- 

 lation of neutral red, Nile blue, and cresyl blue by yeast cells was 

 studied at different pB. values. The method consists of sowing a 

 known quantity of yeast (S. cerevisiae) in Schoen's medium (min- 

 eral salts, asparagine, glucose) to which 0.005% of the dye has 

 been added at pU values scaling from 3-10. At the end of twenty 



