48 Biological Stains 



to a base in its action being known as its isoelectric point. It also 

 assumes that these compounds, although insoluble, act as though 

 they were electrolytes dissolved in any fluid in which they are 

 immersed. Hence, on this assumption, such a compound acts as a 

 base or as an acid in any staining solution according to whether 

 its isoelectric point is below or above the H-ion concentration of 

 that solution. The chemical theory therefore postulates that any 

 cellular element takes a basic dye if the H-ion concentration of the 

 staining solution is below its isoelectric point, an acid dye if it is 

 above that point. 



Now, exactly such a phenomenon as this is to be observed in 

 staining any tissue. With very acid staining solutions even the 

 nuclei take the acid dyes; if one employs successively a series of 

 solutions of decreasing H-ion concentration, the aflinity of the 

 nuclei for the acid dyes rapidly becomes less ; till at a fairly definite 

 reaction, usually in the region of pH 4, they lose their affinity for 

 acid dyes and take the basic dyes. In solutions near neutrality, 

 therefore, the nuclei take basic dyes, the cytoplasm the acid dyes. 

 Finally at a point considerably to the alkaline side of neutrality 

 even the cytoplasm takes the basic dyes. Such an observation is 

 interpreted to mean that the isoelectric point of the nuclei lies 

 considerably to the acid side of neutrality, that of the cytoplasm 

 considerably to the alkaline side. (This is merely another way 

 of making the statement, given two paragraphs above, that the 

 nuclei are acid in character, the cytoplasm basic.) Now assuming 

 that this interpretation is correct, it might be possible to determine 

 the isoelectric points of different parts of the cell by staining at 

 different reactions. Stearn and Stearn (1928), who were among 

 the leading exponents of the chemical theory in the days of the 

 most heated arguments on the subject, tried to determine in this 

 way the isoelectric points of different species of bacteria. They 

 concluded it to be the pH-value of the crossing point of the staining 

 curves of acid and basic dyes. This idea has been distinctly dis- 

 credited of recent years. For one thing, staining determinations 

 of any protein must be made on fixed tissue; and the isoelectric 

 point of a fixed protein may well be very different from that of the 

 fresh material. Also, as Singer (1952) points out in his review of 

 the factors which control staining, the position of the crossing 

 point of the staining curves is determined by various other factors, 

 and does not definitely indicate the isoelectric point. He con- 

 cludes, nevertheless, that although this point cannot be precisely 

 defined by pH staining characteristics, it is possible to compare 

 the curves of different proteins and to learn therefrom the relative 

 position of their isoelectric points. Such results show that there 

 is a definite tendency for cationic dyes to unite with anionic ele- 

 ments of the tissue, and vice versa. 



This is not by any means the whole of the chemical theory of 



