A CUlTUjUE OF CYTOCHEMICAL METHODS 217 



purine bases from nucleic acid, changed the thymus nucleic acid so that it 

 gave a positive Schiff reaction for aldehydes — a restoration of color to a 

 reduced leukofuchsin, and the}^ demonstrated that this reaction could he 

 carried out not onl}^ in vitro but also on tissue sections in which it colored 

 brilliantly the chromatin of the cell nuclei. For this reason they called it 

 the " nucleal reaction. " C'ytologists were quick to try this new technique, 

 and in a few years the reaction had been demonstrated to be positive on 

 the tissues of a wide variety of animals and plants (Milovidov, 1936). 

 This is an ideal (lualitative chemical reaction, highly specific (Fig. 6-2F) 

 for the unique substance of chromatin which Levene et al. (1930) even- 

 tually showed to be deoxypentonucleic acid (DNA), not a hexose polynu- 

 cleotide. The intense color (Table 6-2) is one of its important character- 

 istics. The Feulgen reaction at once demonstrated that the major 

 basophilic component of chromatin and chromosomes of both plants and 

 animals is always DNA and that the cytoplasm never contains this in 

 detectable amount. This was eventually fully confirmed by the analysis 

 of isolated nuclei and cytoplasm (Feulgen et al., 1937; Hogeboom et al., 

 1948). It thus became quite clear that, whatever the nature of the 

 basophilic substance in the cytoplasm, it was certainly not the same as the 

 nucleinic acid of Miescher and Altmann. When, therefore, as a result of 

 the earlier discoveries of pentoses and purine bases in the cytoplasm of 

 developing eggs, Brachet (1942) was led to reintroduce on a wide scale 

 van Herwerden's (1914) long-forgotten nuclease technique and conclude 

 therefrom that the Feulgen-negative basophilic substance of the cyto- 

 plasm was a pentose nucleic acid, it was not seriously questioned; like- 

 wise, no objections were raised when Caspersson and Schultz (1939) 

 stressed the fact that the ultraviolet absorption spectrum of these same 

 basophilic areas must be due to pentose nucleic acid because these parts 

 of the cell were Feulgen negative. Like basophilia, the Feulgen reaction 

 is not directly dependent on a physical property of nucleic acid but on a 

 chemical reaction which the acid can give after removal of the purines 

 from a part of the nucleotides. (From the results of Stacey et al, 1946, 

 it appears that the deoxypentose undergoes considerable intramolecular 

 change to become a substance which is capable of recolorizing the Schiff 

 reagent.) It must be understood that the very useful specificity, which 

 rarely has been seriously cjuestioned, resides in the release of reactive 

 groups as a result of the hydrolytic process, and it is customary to stain 

 simultaneously a control slide which has not been hydrolyzed. 



3-3. PROTEIN STAINING AND TESTS 



Although the bulk of the solid matter of the cell is protein, the prospect 

 for fruitful cytochemical protein analysis is by no means as bright as for 

 nucleic acids. It is certain that this protein mass must be a very hetero- 

 geneous mixture, including countless intracellular enzymes and other pro- 



