302 DYEING 



An aqueous solution of Nile blue contains these substances : — 



cation of the dye (blue) ; 



anion of the dye (sulphate) ; 



Nile red (red or rose, maximum absorption at about 500 m/x, 



fluorescent) ; 

 imino-base of Nile blue (orange-yellow, maximum absorption 



at about 482 m/x, not fluorescent). 



XX^N^X^X 



HsC^^ 



Imino-hase of Nile blue ^^'' ^"^ 



The proportions in which these substances occur vary according 

 to the concentration at which the Nile blue was made up. Nile red 

 is abundant in strong solutions. The amount can be increased by 

 boiling the solution with a little sulphuric acid.^^^ The imino-base 

 of Nile blue is relatively more abundant in dilute than in strong 

 solutions. Like Nile red, it is soluble in toluene and in aqueous 

 solutions of Nile blue. 



When a section is exposed to a solution of Nile blue, the Nile 

 red present in it dissolves in all the liquid lipid components without 

 distinction. Two substances are present in the solution, how^ever, 

 that are capable of reacting with acidic tissue-constituents; 

 namely, the cation of Nile blue and the orange-yellow base. 

 Either or both of these can react with acidic lipids (fatty acids) or 

 lipids capable of acting as acids (phospholipids). Since the cation 

 dyes blue and the salts of the base are of the same colour, we do not 

 know, in any particular case, which of the tw^o has been responsible 

 for colouring these particular lipids blue. (The colour is sufficiently 

 intense to mask the Nile red dissolved in them.) On one hand the 

 lipid-soluble base would permeate lipids much more readily than 

 the cation; on the other, there is plenty of cation but very little 

 base in strong solutions of the dye. It seems certain, however, that 

 cations can colour fatty acids, for basic fuchsine (rosaniline 

 chloride) reacts with oleic acid to form rosaniline oleate and hydro- 

 chloric acid.^^- 



Beyond these reactions, Nile blue naturally acts like any other 

 basic dye in colouring all acidic tissue-constituents, such as chro- 

 matin and RNA. The orthochromatic colour is of course blue, but 



