A CRITIQUE OF CYTOCHEMICAL METHODS 213 



made with the advantage of artificial contrast which is introduced by 

 .staining or developing a metallic precipitate in c(>ll c()mpf)ncnts.'' One 

 of the oldest of these techniques is l)asic staining, which involves the use 

 of dye salts which, upon dissociation, carry the ctjlor, the chromophore, 

 in the cation and which therefore form colored salts with the anions of 

 strongly acid substances within the cell (Fig. 6-2C). It was early recog- 

 nized (e.g., Mathews, 1898) that, if basic dyes were applied in acid solu- 

 tion (after appropriate fixation), this property of cell substances, which 

 is included in the general term "basophilia" of tissues, constituted in 

 effect a test for strongly acidic substances. Specifically, in animal cells 

 these include the relatively uncommon sulfuric acid esters of polysac- 

 charides and the phosphoproteins plus the universal cell constituents 

 nucleic acids, w^hich are orthophosphoric esters of nucleosides. There 

 were many early cytochemical researches based on the supposition that 

 basophilia indicated the intracellular distribution of nucleic acid. One 

 outstanding example is the so-called "chromidial hypothesis," which as 

 applied to metazoan cells held that the basophilia of cytoplasmic struc- 

 tures was evidence of their origin from the nuclear chromatin (see Wilson, 

 1925, pp. 700^.). However, most cytologists used basic staining so as to 

 achieve maximum contrast for morphological studies (e.g., iron hema- 

 toxylin, applied to material which had been fixed in reagents containing 

 chromic acid). Such technique departed widely from the strict criteria 

 laid down by Alathews for specific staining by salt formation between 

 basic dyes and nucleic acids, and undoubtedly the increased contrast was 

 to a large extent due to adsorption of dye rather than chemical staining 

 (Pollister, 1952a). This distinction was rarely appreciated, however, 

 either by cytologists themselves or by others interested in the chemistry 

 of the cell, and as a result there developed a widespread distrust of 

 attaching any chemical significance whatsoever to the basophilic reac- 

 tion. This was not dissipated even when van Herwerden (1913, 1914) 

 developed the nuclease technique for identification of intracellular 

 nucleic acid basophilia. The modern use of basophilia for localization 

 of nucleic acids (Mazia and Jaeger, 1939; Brachet, 1942; PoUister, 1950; 

 Kaufmann et al., 1951) stems directly from van Herwerden's work 

 but rests on a much firmer biochemical basis since it is now known 

 that: 



1. There are actually two nucleic acids, the pentose type — ribose 

 (RNA), plasmonucleic acid — found in the cytoplasm, nucleolus, and to 

 some extent in chromatin, and the deoxypentose type — deoxyribose 

 (DNA), chromonucleic acid — which is normally restricted to chromatin 

 of the nucleus (Davidson, 1950). 



■^ The development of phase contrast microscopy fsee Fip;. 6-2.4 and Bennett et al., 

 1951) has nearly freed cytologists from the necessity of introducing artificial contrast 

 by these methods. 



