THE DIFFERENTIAL ACTION OF DYES 233 



An interesting though compHcated example of rehance on 

 density for differential colouring is provided by Taenzer's method 

 for showing elastic fibres. (The method is usually called Unna's, 

 but Unna himself ^^^ attributed it to his pupil.) The dye used is 

 orcein (p. 185), dissolved in strongly acid alcohol. Differentiation 

 is carried out in alcohol, usually strongly acidified. The brownish- 

 red colour is better retained by elastic fibres than by other tissue- 

 constituents. The explanation appears to be as follows. *^'^ 



Orcein is used in alcoholic solution because it is scarcely soluble 

 in water. It has no particular chemical affinity for elastin. It is an 

 amphoteric dye (p. 190). In neutral solution (40% ethanol), being 

 now itself acidic, it colours collagen more strongly than elastin. 

 On the more acid side of its iso-electric point (about pH 5-7), it is a 

 feebly basic dye. It now no longer colours collagen strongly, on 

 account of the mutual repulsion of the positive charges, but is 

 taken up by various negatively charged objects. It diffuses easily if 

 dissolved in 70% ethanol, and is thus able to enter tissue-constitu- 

 ents that would have been impermeable to it in weaker ethanol. 

 Elastic fibres, the matrix of cartilage, acidic mucus, and chromatin 

 are coloured rather strongly, other tissue-constituents feebly. 

 Orcein is not sufficiently basic to have a particularly strong affinity 

 for the three last-mentioned substances, which are more electro- 

 negative than elastin. When the section is subsequently put in 

 acidified ethanol, the dye is easily extracted because it is extremely 

 soluble, and it would eventually be washed out everywhere. 

 Density rather than electric charge now^ controls events. Since 

 there is a lot of matter in the elastic fibres, a lot of dye is held by 

 them ; and when the somewhat less dense constituents (chromatin, 

 etc.) have lost all visible remnants of it, enough still remains in the 

 elastic fibres to show them clearly. 



If this explanation be correct, the result is achieved partly be- 

 cause elastin is even denser than chromatin, partly because orcein 

 is too feeble a basic dye (at the pH at which it is used) to allow the 

 final result to be controlled primarily by electric charges. 



The dyeing of elastic fibres by orcein is probably not controlled 

 only by their density: permeability is likely to play a part. 



Carminic acid, another amphoteric dye, behaves rather similarly 

 to orcein; but much more of it is taken up by the chromatin, pre- 

 sumably because the dye is more strongly basic at the pH of the 

 solution. During the differentiation in acid alcohol the dye is 

 therefore retained as long by chromatin as by elastin. 



