CYTOCHEMICAL TECH^^[QUES FOR NUCLEIC ACIDS 69 



give a positive Feulgen reaction. Naturally ocurring aldehydes produce a 

 pink color without acid hydrolysis. These include lipids, which may remain 

 in tissues after formalin or osmic acid fixation, constituents in elastic fibers, 

 and certain materials in plant cell walls, particularly those of xylem ele- 

 ments.^" In inadequately washed tissues formaldehyde or paraldehydes 

 may remain, to give a positive reaction. Some fixatives, particularly those 

 containing chromic acid or other oxidizing agents, partially oxidize glyco- 

 gen, mucin, starch, cellulose, and other polysaccharides, so that these give 

 a pink color with Schiff's reagent. Also where sections are inadequately 

 rinsed in the SO2-HCI baths after staining, they may turn a more or less 

 uniform pink in water or alcohol. All of these false reactions are readily 

 distinguished from DNA staining since they produce a colored compound 

 without previous hydrolysis, and their extent can be estimated by com- 

 paring hydrolyzed slides with unhydrolyzed controls. In some cases mate- 

 rials that produce a pink color in unhydrolyzed control sections are re- 

 moved by the Feulgen hydrolysis. Since the Schiff reagent is acid, it slowly 

 hydrolyzes DXA, so that previously unhydrolyzed tissues may be stained 

 if allowed to remain in the reagent for long periods (6 hours or more). 



Because interfering substances are quite common, for all new tissues to 

 be investigated unhydrolyzed controls should be run, particularly if quan- 

 titative techniques are to be used. Feulgen-positive material should also be 

 investigated with acid or enzyme extraction techniques where its DNA 

 nature is doubtful. Such investigations would clarify, for example, the 

 nature of the cytoplasmic granules in brain tissue reported by Liang^^ 

 and Chu.^2 Also, one should never conclude on the basis of a negative 

 Feulgen reaction, that DNA is "absent" from a nucleus. Even where all 

 steps have been properly followed, no perceptible color can be seen in 

 some nuclei, for example, in many mature oocytes. The DNA in such nuclei 

 is obviously too dilute to produce a visible reaction. 



3. Localization 

 A few investigators, particularly Stedman and Stedman,^^-'^ have sug- 

 gested that DNA is made readily diffusible by hydrolysis, and may pro- 

 duce, on contact with the fuchsin-sulfurous acid, a soluble dye that is 

 adsorbed by various cell structures. The locahzation of dye given by the 

 Feulgen reaction is thus thought to have little or no relation to the actual 

 distribution of DNA. A great many people have argued against this sug- 



so B. B. Hillary, Botan. Gaz. 101, 276 (1939). 



SI H. M. Liang, Anai. Record 95, 511 (1947). 



«C. H. Chu, Science 106, 70 (1947). 



"E. Stedman and Ellen Stedman, Xature 152, 267, 503 (1943). 



9^E. Stedman and Ellen Stedman, Symposia Soc. Exptl. Biol. 1, 232 (1947). 



" E. Stedman and Ellen Stedman, Biochem. J. 47, 508 (1950). 



