230 DYEING 



A good example of a difference in chemical affinity among dyes 

 is seen in the colouring of cellulose cell-walls. Most acid dyes 

 have no affinity for these: they act chiefly by the linking of their 

 anions to the amino- and other basic groups of proteins. As we 

 have seen, however (p. 199), Congo red and some other dyes are 

 able to act in quite a different way by forming hydrogen bonds with 

 the hydroxyl groups of cellulose. 



Certain basic dyes used in pairs exhibit small differences in 

 affinity that can result in very noticeable differences in effect. The 

 most familiar example is the mixture of methyl green (triaryl- 

 methane) and pyronine G (xanthene). This was introduced by 

 Pappenheim,^^*' ^^^ who showed that the basiphil cytoplasm of 

 certain leucocytes could be coloured red with pyronine, while 

 chromatin became green or bluish green. Thus two basic dyes 

 coloured two basiphil substances differently. The method was 

 improved by Unna,^^^ whose formula included phenol. There is no 

 full and universally accepted explanation of the results obtained 

 with Unna's mixture and its modern variants, but the following 

 facts are relevant. 



It will be remembered that if a basic dye be used at various 

 levels of pH, certain tissue-constituents show themselves capable 

 of combination with it even in very acid solution, while others are 

 more sensitive to acidity and react with the dye only at somewhat 

 higher pH (p. 194). Now if two basic dyes differed somewhat in 

 their capacity to bind themselves to objects at a particular pH, it 

 should be possible for a mixture of them to give differential 

 colouring. 



If the usual mixture of the two dyes be used at pH 1-5, the 

 pyronine predominates everywhere over the methyl green; the 

 converse is true at pH 9-3. At intermediate pH both dyes act, but 

 not equally on all tissue-constituents.^^^' ^^^ One sees now how 

 Unna hit on the use of phenol by empirical experiments, for it 

 gives the weak acidity that allows both dyes to act, and to act 

 differentially. It is usual nowadays to buffer at pH 4-7 or 4-8.^^' ^^^ 



At the appropriate pH, chromatin is coloured mainly by the 

 methyl green and appears green, blue-green, or blue, while nucleoli 

 and basiphil cytoplasm are red with pyronine. It is usually RNA 

 that binds the red dye, but this must be confirmed by failure to 

 colour after the use of ribonuclease. (For a very convenient source 

 of this enzyme, see Bradbury.^') Similarly, reliance must not be 

 placed on methyl green as an indicator of the presence of DNA, for 



