INTRODUCTION TO VITAL COLOURING 277 



leucocytes. He also saw the latter ingest coloured particles on a 

 microscopical slide. 



Nowadays it is usual to make one or more subcutaneous in- 

 jections and then to kill the animal, generally after the lapse of 

 several days, and fix the tissues with a suitable fixative (often 

 formaldehyde or mercuric chloride or Zenker). ^^^ Sections are 

 prepared and stained with a dye that contrasts in colour with the 

 vital colouring agent. Particles of carmine and other insoluble pig- 

 ments naturally remain in position, but it is a remarkable and con- 

 venient fact that the soluble disazo dyes used in this kind of work 

 do not quickly dissolve out of the cells during the after-treatment, 

 presumably because their escape is impeded by the coagulation 

 of proteins round them. As a result, it is usual to make observa- 

 tions on sections mounted in Canada balsam, although the uptake 

 of the coloured substance was an active, vital function of the 

 cells. 



It is not perfectly clear whether the disazo acid dyes sometimes 

 flocculate outside the cells and are then engulfed, or whether they 

 always penetrate in a finely dispersed state and are then aggregated 

 into microscopically visible particles within the cells. It seems 

 likely that the fate of such acid dyes as can penetrate cells depends 

 on their ability to flocculate. Those that exist as small molecules or 

 ions with little tendency to clump together are not used in vital 

 studies of this kind. They diflPuse out of cells as easily as they 

 diffuse in. Those that exist as large molecules or ions — notably the 

 disazo and trisazo -^ dyes — have a tendency to clump; these are 

 captured if they enter certain cells.^*^'^^- In some cases, especially 

 in excretory as opposed to phagocytic cells, it seems certain that 

 aggregation occurs after absorption. In the kidney tubules of 

 vertebrates, droplets containing such dyes first appear in the region 

 of what is commonly called the Golgi apparatus, and subsequently 

 spread into the ground cytoplasm.^^^ 



Whether insoluble particles or soluble dyes are used, it is char- 

 acteristic of this kind of colouring that pre- existent objects in cells 

 do not take up the colour. Thus, even if the colouring agent used is 

 a dye, it does not act as a dye in these circumstances, since a pre- 

 requisite for dyeing is something that can be dyed. Ranvier w^as 

 perfectly logical in using vital colouring agents of this kind while 

 claiming that cells could only be dyed when dead. The soluble 

 colouring agents used in this kind of work may give a slight, 

 diffuse colour to cellular or intercellular matter, ^^^ but the reaction 



