The Cytological Interpretation of the Feulgen Reaction BY EDGAR STEDMAN AND ELLEN STEDMAN

The chemical test for deoxyribosenucleic acid discovered by Feulgen (1914) and applied by Feulgen & Rossenbeck (1924) under the name of nucleal stain to histological sections has served many useful purposes in the hands both of its originator and of subsequent workers. It now finds much favour as a staining process for cytological preparations containing dividing cells, partly, no doubt, because of the particularly clear pictures of the chromosomes which result from its use, but mainly because of the belief that it specifically stains those nuclear structures which contain nucleic acid. Thus, because the chromosomes are the only structures in dividing cells which become stained by this method, it is assumed that these organs contain the whole of the nucleic acid of the nucleus, an assumption which is sometimes expressed by the statement that nucleic acid does not occur elsewhere in the nucleus than in the chromosomes. As an aid to studies of the morphology of chromosomes, nucleal staining may be unobjectionable, but when it is employed for ascertaining the distribution of nucleic acid within tho nucleus it becomes open to serious criticism. For such a use is based on the assumption that the nucleic acid 'stains' in 8itu, an assumption which ignores the well known fact that the violet dye produced when isolated nucleic acid is submitted to Feulgen's reaction (hydrolysis with dilute hydrochloric acid and treatment ofthe neutralized product with Schiff's reagent) is soluble in water. Unless nucleic acid in the nucleus behaves differently from isolated nucleic acid, it follows that those nuclear structures which become stained by Feulgen's process do so because they are composed of nondiffusible material which adsorbs or otherwise combines with the diffusible, violet dye, and that consequently nucleal staining can provide no certain information regarding the distribution of nucleic acid in the nucleus of the living cell. Evidence of an indirect nature supporting these conclusions has been adduced elsewhere (Stedman & Stedman, 1943, 1947). Direct proof is now provided that the hydrolysis of isolated cell nuclei under conditions used in nucleal staining gives rise to diffusible products which react with Schiff's reagent to form a soluble violet dye.