286 DYEING 



in the tube at the other end. Mechanical stirrers in the end-tubes 

 keep the dye-solution and sap (or other fluid) moving, while the 

 middle tube admits a stirrer to the lipid-solvent. After the lapse 

 of an hour the concentration of the dye that has diffused through 

 the lipid-solvent into the sap is determined colorimetrically. 



There is a general correspondence between the rapidity with 

 which a dye passes through a layer of chloroform and its ability to 

 enter cells of various kinds, but the relation is by no means 

 exact. ^^^' ^^ In the circumstances of the experiment the chloroform 

 contains a certain amount of water, and it has been pointed out ^^ 

 that passage through the layer of chloroform does not actually 

 prove the necessity for lipid-solubility. It has been held ^^ that 

 vital dyes may enter the living cell by either an aqueous or a lipid 

 path, and that the former is open to the cations of basic dyes. 

 Seki,^^^ however, found no correlation between the diffusibility 

 of basic dyes in aqueous media and their ability to enter living 

 cells. 



In his paper of 1887, in which he introduced methylene blue as 

 a vital colouring agent for nerve-fibres, Ehrlich ^^^ mentioned that 

 the dye tends to be reduced to its leucobase in the tissues and in 

 this state to diffuse easily out 0/ cells. Today, the ready diffusibility 

 of leucobases into cells is of greater interest. It is usual to reduce 

 methylene blue to its leucobase in order to make it enter axons 

 more easily. The reduction of dyes to leucobases for this purpose 

 may be done by adding sodium thiosulphate and acidifying. The 

 resultant sulphurous acid acts as reducer. Alternatively one may 

 use a compound of sodium sulphoxylate (NaHSOg) with formalde- 

 hyde. This substance (with two molecules of water) is sold as 

 white lumps or powder under various trade names (Rongalit, etc.). 

 It was used by Unna for the reduction of methylene blue long 

 jjgQ 515, 517 j|-g power to reduce dyes derives from its capacity, 

 when in solution, to take oxygen from water and thus convert 

 itself to the compound of acid sodium sulphite with formalde- 

 hyde : — 



NaHSOa.CHsO -f Hp = NaHSOa.CHgO + Hg 



The hydrogen formed by this reaction combines with the dye. 

 The commercial product contains some sodium sulphite (NagSOg), 

 which gives an alkaline reaction to the solution. Since the leucobase 

 is formed more slowly in alkaline solution, and also tends to be 

 precipitated, it is usual to acidify slightly with hydrochloric acid. 



