34 S. S. COHEN 



at all. For example, the ultraviolet absorption of the mitotic apparatus 

 suggests the presence of 2 to 3 % protein-bomid nucleic acid. However, this 

 seems rather to be due to the presence of firmly bound mononucleotide, 

 perhaps adenosine triphosphate (ATP), in this material (Mazia, 1957). 



The reactions of cell structures with chemical reagents to give colored 

 products has long been known, as in the use of dyes. Structures which became 

 colored by binding basic dyes, such as rosaniline, hematoxylin, or azure A 

 applied in acid solutions, were termed basophilic. This property reflects the 

 presence of acidic groups and has been particularly important in the develop- 

 ment of knowledge of the structural and functional relations of the nucleic 

 acids. The earHer embryological studies of Brachet exemphfy such a use of 

 this teclmique. 



Wide use has been made of differential staining techniques, as in the use 

 of a mixture of methyl green and pyronine for the study of the nucleic acids. 

 Brachet (1953) and Kurnick (1955) have discussed various problems asso- 

 ciated with the use of these dyes. Methyl green reacts with highly polymerized 

 DNA, as it exists in chromosomes; its reactivity has been observed to be a 

 fmiction of the degree of polymerization of the nucleic acid (Kurnick, 1954). 

 Pjn^onine reacts with relatively low molecular weight nucleates, such as 

 commercially obtainable E.NA. It was originally thought that this reaction 

 was specific for RNA; however, Kurnick (1951) has shown that pyronine will 

 also react with depolymerized DNA. In a well-controlled preparation 

 (Kurnick, 1955), chromosomes are clear green, and nucleoli are bright red. 

 RNA -containing areas of the cytoplasm also stam red. 



The Feulgen reaction has provided the most important nuclear stain for 

 the characterization of DNA (Di Stefano, 1948; Swift, 1955). The reaction 

 has also been used successfully on microorganisms to reveal structures which 

 are comparable to the chromatin of higher cells, in that both types of struc- 

 ture contain DNA. The Feulgen stain is a modified Schiff reaction, given 

 generally by aldehydes; it mvolves the absorption of SO 2 from a decolorized 

 fuchsin-SOg mixture to give a red fuchsin stain. Cellular constituents contain 

 few compounds besides DNA capable of mterfering with the specificity of 

 the reaction. Fatty aldehydes may be removed by alcohol extraction after a 

 gentle acid hydrolysis of acetal phosphatides. The hydrolysis also removes 

 purines from DNA, liberating the active carbonyl group in the aldehydo 

 sugar, 2-deox}Tibose, which is then capable of binding SO 2. The specificity of 

 the stain depends on the nmch greater tendency of 2-deoxysugars to exist 

 in the straight chain aldehydo form than do furanoses, such as the ribose of 

 RNA, or the pyranoses present in many polysaccharides. 



The Schiff reaction has also been modified for the detection of polysacchar- 

 ides in the cell. Prior incubation with periodate cleaves the glycol configura- 

 tion to two aldehyde groups, which are then also detectable by fuchsin-SOg. 



