86 Microscopic Histochemistry 



(b) the part of dye which is adsorbed in excess of the ion 

 exchange mechanism can be removed with alcohol/^^ or that 

 adsorption can actually be prevented by the use of deter- 

 gents incorporated in the staining solution. ^^^ 



Extreme caution is warranted in the interpretation of the 

 results of such experiments. First of all, the results depend 

 within wide limits on minor variations in technique ( the dye 

 used, its concentration, time of staining, washing, and differ- 

 entiation). Especially differentiation is a very delicate step. 

 While acid in an aqueous medium has a relatively moderate 

 effect on the staining by basic dyes, traces of it transferred 

 to the differentiating alcohol (either from the dye solution 

 or from the buffer used to wash out the excess dye ) may de- 

 colorize the section extensively during the first few seconds 

 of contact, even at pH values as high as 4-4.5. On the other 

 hand, tissues stained at a pH as low as, 1.2 or lower (and they 

 do stain with considerable intensity; even Gabbett's^^^ meth- 

 ylene blue, which contains 25 per cent sulfuric acid, will stain 

 nuclei), differentiated in a buffer of the same pH, and sub- 

 sequently washed thoroughly to remove all traces of acid 

 may retain a beautfful nuclear staining even after prolonged 

 differentiation in alcohol. ^^^ 



In summary, it may be said that, other things being equal, 

 substances possessing acidic groups are more likely to stain 

 with basic dyes, especially at pH levels below neutrality, than 

 substances without such groups. However, basophilia is the 

 result of many factors, most of which are very poorly under- 

 stood. It does not permit the drawing of even approximately 

 quantitative conclusions as to isoelectric points or the values 

 of dissociation constants. 



Extraction procedures 



Strictly speaking, "extraction" is a misnomer if applied to 

 the whole group of procedures to be described here, because 



130. Michaelis, L.: Cold Spring Harbor Symp. Quant. Biol., 12:131, 1947. 



131. Gabbett, H. S.: Lancet, 1887-1, p. 757. 



132. Gomori, G.: Unpublished. 



