THE MODE OF ACTION OF VITAL DYES 287 



For an example of the practical use of sodium sulphoxylate/ 

 formaldehyde in the reduction of methylene blue for vital studies, 

 see Smith. 4'^ 



The penetration of leucobases has been investigated especially 

 by Harris and Peters, ^^^ who used methylene blue. The leucobase 

 is, of course, colourless (non-chromophoric) and non-ionic (p. 163). 

 It is about 25 times as soluble in chloroform as in water. Ionized 

 methylene blue scarcely penetrates, except w^here cell surfaces have 



\/\N=^\^ H 



Methylene blue Leucobase of methylene blue 



been damaged. When the dye is used in this form, it is probable 

 that part of it is reduced in the vicinity of the cells and enters as 

 the leucobase. For some reason that has not been explained, 

 acidity makes the cell-membrane more permeable to the leuco- 

 base; but if strongly acid solutions are used, hydrogen ions are 



H 



The cation derived from the leucobase of methylene blue, in strongly acid solution 



added to one or both of the dimethylamino-groups, and new, 

 ionized forms of methylene blue are thus produced, which cannot 

 readily penetrate. As the pH is lowered from alkalinity, the in- 

 creased permeability of the cell-membrane increases the uptake of 

 methylene blue until pH5 is reached ; from this degree of acidity 

 onwards the uptake is less, because there is less leucobase. 



If ciliates are put in solutions of the leucobases of vital dyes, a 

 most surprising appearance is given, for the animals swim about 

 actively with coloured macronuclei. It is as though a fixed and 

 stained preparation had come alive. Thionine, brilliant cresyl blue, 

 and several other dyes are suitable. *^^ When a metachromatic dye 

 such as thionine is used, the whole organism is at first blue, but 

 the cytoplasm becomes violet and then raspberry-coloured, while 

 the macronucleus remains blue. It is rather strange that the use of 

 the leucobase makes it easier to colour the macronucleus, for 

 lipid-solubility would not be thought likely to have this effect; but 

 perhaps the cause is simply the abundance of the colouring agent in 



