THE MITOTIC CYCLE 



To calibrate a cytochemical method independently of bulk analyses, 

 it is necessary to establish that there is some constant stoichometrical 

 relationship in the amount of dye absorbed or liberated by a given 

 quantity of DNA. It might be expected that for this purpose the Feulgen 

 reaction could only be used empirically, for so many factors influence 

 the density of the staining. However, by measuring the rate at which the 

 constituents of deoxyribonucleoproteins are removed from frog cartilage 

 nuclei during Feulgen hydrolysis, Di Stefano^^ claims to have shown 

 that the optimal point in this procedure corresponds with the removal 

 of half the bases, and that this result can serve as a basis for micro- 

 colorimetric estimations. Di Stefano's assumptions have been criticized 

 by Leuchtenberger^' and found not to be valid in practice by Ris 

 and MiRSKY.*" These authors have calibrated their measurements on 

 Feulgen-stained nuclei from their previous chemical estimations for 

 isolated nuclei of the same kind, Lison and Pasteels calibrate their 

 Feulgen preparations by including a fragment of rat testis tissue to serve 

 as a standard. This is fixed, sectioned and stained at the same time as 

 the material on which microphotometric measurements are to be made. 

 The results are expressed in relative terms. Swift^^ has also made rela- 

 tive measurements of the DNA content of nuclei in a range of tissues by 

 microcolorimetry of Feulgen-stained material. 



The reaction of DNA with methyl green has been used as a basis 

 for such estimations (Kurnick,^^ *2 Kurnick and Mirsky^^). In the 

 well known method of Unna for distinguishing between RNA and 

 DNA, methyl green is used to stain the latter component. Kurnick has 

 shown that a positive reaction with methyl green is given by highly 

 polymerized nucleic acids, irrespective of the nature of the constituent 

 pentose ; and that DNA is sufficiently greater in molecular weight than 

 RNA to make the reaction specific for the former. After treatment of 

 a section with hot water, the Feulgen-positive material no longer 

 stains in methyl green (Pollister and Leuchtenberger**) for there 

 has then been some depolymerization. Kurnick^^ found that after 

 removal of histones by cin hydrochloric acid one molecule of dye 

 reacts with lo atoms of phosphorus in highly polymerized DNA, a 

 figure which he subsequently revised to 13. Kurnick's figures for the 

 DNA content of mammalian nuclei come within the range of those 

 obtained by other means, though his value for the chick erythrocyte is 

 low. 



In comparing somatic nuclei of the same species from diflferent 

 tissues, for example liver and erythrocyte nuclei, Ris and Mirsky found 

 that a close approximation to the same value for DNA content was 

 obtained only when the DNA was evenly dispersed throughout the 

 nucleus. This condition is attained by isolation of the nuclei in 30 per 

 cent sucrose, followed by fixation in formalin; but some DNA is said 



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