54 S. S. COHEN 



importance of studying these integrated structural and enzymatic relations 

 (Warburg, in 1913; Quastel, in 1930; Peters, in 1937), it can be seen that the 

 most important impetus and guidance in this field has come from anatomists, 

 cyiiologists, embryologists, and virologists. 



2. On Cytoplasmic Staining 



Two types of basophilia, important in the differentiation of cytoplasmic 

 structure, have been distinguished. In the common test for basophilia, a 

 basic dye reacts with and is absorbed by the acidic substance organized in 

 visible or submicroscopic particulates. With most basic dyes, the peak of 

 absorption is shifted only shghtly toward longer wave lengths and the cell is 

 stained with a color comparable to the color of the dye in solution. The 

 existence of submicroscopic particles containing RNA in the apparently 

 clear cytoplasm of cells particularly active in protein synthesis is revealed 

 by a strong basophiha of this type, which can be removed by pretreatment 

 with ribonuclease. This type of basophiha usually parallels the observed 

 ultraviolet absorption of this region of the cell.^ 



Earlier, Paul Ehrlich had described metachromasy as a special staining 

 reaction of certain dyes, such as thionine or toluidine blue. In these instances, 

 the color of the adsorbed dye is quite different from the color of the dye 

 solution itself. The relatively rare compounds that are stained in this way 

 are polymeric sulfuric acid esters such as agar, heparin, chondroitin sulfuric 

 acid, mucin, and the metaphosphates, which may exist in cells as volutin 

 granules. Other acidic polymers, such as the ubiquitous nucleic acids, wdll 

 not shift the color of boimd toluidine blue from blue to pmk or purple, as will 

 agar. The shift in color and in the absorption spectrum was shown by 

 MichaeHs (1950) to be a function of the dimerization and polymerization of 

 this dye, an aggregation which was readily reversible. The shift is also a 

 function of the nature of the absorbent and it is believed by some workers 

 (Walton and Ricketts, 1954) that the extent of metachxomasia is mainly 

 dependent on the solubihty characteristics of the dye-substrate complex. 

 Nevertheless, it is still not clear why the nucleic acids should not influence 

 the dye, yielding the spectrum of the monomer, whereas a substance such as 

 agar does influence the spectrum, as though the dye were highly polymerized. 



^ In a remarkable monograph (Hedberg, 1953) it is reported on the basis of ultra- 

 violet absorption measurements that the RNA content of the human ovarian oocyte is 

 less than 0.1 %, being too low to be determined by this method. Despite the apparently 

 negligible concentration of RNA, it should be noted that the oocyte accomplishes an 

 enormous increase of protein unaccompanied by any significant increment in RNA 

 content. Hedberg poses the question of whether this protein has been synthesized by the 

 oocyte or by the surroundmg follicular epithelium. 



Similarly, one may note the report (Bhargava, 1957) of the absence of RNA in bull 

 sperm, which are capable of incorporating radioactive amino acids into protein. 



