Chapter XIII 

 VITAL STAINING OF THE VACUOLES 



Colloidal substances in the vacuolar sap:- The existence of dyes, 

 called vital dyes (methylene blue, cresyl blue, Nile blue, neutral 

 red, etc.) has been known for some time. They have the property 

 of penetrating living cells and of coloring some of the cytoplasmic 

 inclusions. 



Pfeffer first showed that methylene blue used in a 1% solu- 

 tion penetrates the cells of various plants (Azolla, Lemna, Spiro- 

 gyra) but accumulates exclusively in the vacuole and does not color 

 the protoplasm. Methylene blue brings about dark blue precipi- 

 tates in the vacuoles as a result of the flocculation of the tannins 

 contained in the vacuolar sap, with which the stain forms a com- 

 plex. Pfeffer stressed the remarkable property which the vacuoles 

 possess, of accumulating methylene blue and of taking on very 

 rapidly a coloration more intense than that of the solution of the 

 dye itself. The dye becomes, therefore, more concentrated in the 

 vacuole than it was in the solution. In staining tadpoles, Pfeffer 

 showed that methylene blue accumulates in certain inclusions of 

 the cytoplasm. Since that time it has been demonstrated that other 

 dyes, such as cresyl blue, Nile blue and neutral red, also have the 

 property of penetrating living cells, and numerous investigations 

 on plant cells as well as on animal cells have determined that these 

 dyes accumulate exclusively in the inclusions which are not a part 

 of the living matter (secretion granules of animal cells, vacuoles 

 of plant cells) . 



In research on yeasts, we ourselves have demonstrated that the 

 vacuoles enclose granules, showing Brownian movement, which 

 have the property of being stained instantaneously by cresyl blue 

 and neutral red. These granules can be easily fixed with alco- 

 hol and formalin, after which they are stained deeply by basic 

 stains (anilin blue, anilin violet and haematein) which give them 

 a reddish tint. This has permitted us to identify them as meta- 

 chromatic corpuscles. These were cited first in the bacteria by 

 Babes and found later in the Cyanophyceae by Butschli, who gave 

 them the name of "red granules". In later research also, we 

 showed that vacuoles formed by hydration of aleurone grains (Fig. 

 84), when seeds germinate, accumulate neutral red and this dye 

 brings about the flocculation of the proteins which the vacuoles con- 

 tain in solution. But these are isolated observations. It remained 

 for P. A. Dangeard to establish the fact that the ability to accumu- 

 late vital dyes is a general property of vacuoles. 



Taking up our work on the metachromatic corpuscles in the 

 fungi and algae, Dangeard demonstrated that these bodies are 

 visible only rarely in living cells and when they are visible, they 



