196 DYEING 



sulphuric groups of certain mucosubstances later still. These facts 

 provide the explanation of Bethe's results. 



Seki ^^^ chose sheet gelatine, untreated by any fixative, as a 

 model for amphoteric tissue-constituents. The dyeing of gelatine 

 is represented diagrammatically in fig. 26. The iso-electric point of 

 this substance is about pH 4-7. Since gelatine is positively charged 

 at lower pH than this, it has a strong affinity for ordinary acid 

 dyes ('levelling' dyes, see p. 235) in strongly acid solution. In the 

 same way wool has a strong affinity for these dyes at low pH. The 

 affinity of gelatine or wool for ordinary acid dyes falls off rapidly as 

 the pH rises above the iso-electric point, but now% as the charge has 

 become negative, there is increasing affinity for typical basic dyes. 



The curves representing the uptake of acid and basic dyes by 

 proteins cross somewhere, and the place of crossing is at or near 

 the iso-electric point of the protein. It might be thought that dye- 

 ing would be impossible beyond the iso-electric point, so that the 

 curves for acid and basic dyes w^ould reach the base-line here, 

 instead of crossing. It must be remembered, however, that pro- 

 teins are not without electric charges at the iso-electric point: such 

 charges still exist, but the positive and negative ones balance one 

 another. 



Accurate figures for the amounts of a basic and an acid dye 

 taken up by thin fibrin films have been obtained photometrically 

 by Singer and Morrison. ^"^ Their results confirm the general 

 correctness of the data provided by Seki, who used an arbitrary 

 scale (+, +-f, etc.) to represent the intensity of dyeing as judged 

 visually. 



The facts just recorded explain the common custom of using 

 basic dyes in w^eakly acid solution. The pH is low enough to pre- 

 vent the amphoteric cytoplasm from taking the dye, but not low 

 enough to prevent the chromatin from being coloured strongly. 



The complication is considerable when an amphoteric substance 

 is treated wath an amphoteric dye. If the iso-electric points of both 

 were the same, there would be little affinity between them. If, 

 however, the iso-electric points are different, there will be a 

 narrow range in which the charge on the dye will be opposite to 

 that on the protein; dyeing will occur within and to some extent 

 also beyond this range (fig. 26). 



After basic dyes have acted, there is generally little tendency to 

 extraction by water at neutrality. Many acid dyes are less fast to 



