70 HEWSON SWIFT 



gestion.'"-^^-^^ Although the possibility that some slight alteration in DNA 

 localization may take place during hydrolysis is difficult to exclude en- 

 tirely, and definitely may occur with over-hydrolysis, this is very obviously 

 not an important factor in the usual Feulgen method. Dye distribution is 

 essentially unchanged whether the Feulgen reaction is carried out on whole 

 tissue blocks,^"" on very thin smears, or on cell homogenates. It is similar in 

 very early and mid-stages of hydrolysis. Prolonged washing after hydroly- 

 sis has little effect. Moreover, the distribution of stain, caused by adding 

 excess DNA to the hydrolysis bath, or hydrolyzed DNA to the staining 

 solution, where adsorption actually does occur, is completely different, 

 showing nucleolar and cytoplasmic coloring. 



4. Fixation 



The type of fixation used is important in determining the maximum in- 

 tensity obtainable. It also causes variations in the effects of hydrolysis, 

 and even in the color of the Feulgen complex itself (Fig. 4). Nuclei from 

 tissues fixed in neutral formalin are usually more intense than after acetic 

 alcohol. In addition, when a block of tissue is fixed, the peripheral nuclei, 

 with either acetic alcohol or formalin, are significantly darker than those 

 toward the center of the block, as demonstrated by photometric determi- 

 nations of the amount of dye per nucleus.^" This variation in intensity may 

 be as great as 30%. No such gradient is shown with fresh-frozen or frozen- 

 dried material. When a fresh piece of tissue is placed in fixative, the com- 

 position of the fixing fluid obviously varies as it penetrates the block. 

 Dilution of fixative occurs, and certain constituents penetrate more rapidly 

 than others. Also certain constituents soluble in the fixative are more 

 readily removed from the outer cells. In Table I are shown measurements 

 on nuclei from the same rat liver, fixed in the same fixative, and stained 

 together. A 25% difference was found between inner and outer nuclei. 

 When cells were homogenized in sucrose before fixation, centrifuged down, 

 and resuspended in fixative, the Feulgen values were still higher. From an 

 analysis of the absorption curves of these nuclei, one can conclude that the 

 difference is not caused by variation in dye distribution, but rather by 

 actual differences in the amounts of dye bound per nucleus. From measure- 

 ments on nuclei stained with the Millon reaction for tyrosine, ^^^ some 

 protein, possibly the globulin fraction, '°- appears to be partially extracted 



36 R. E. Stowell, Slain Technol. 20, 45 (1945). 



" R. E. Stowell, Stain Technol. 21, 137 (1946). 



58 J. Bracket, Symposia Soc. Exptl. Biol. 1, 207 (1947). 



33 C. H. Li and M. Stacey, Nature 163, 538 (1949). 

 ""> J. F. Lhotka and H. A. Davenport, Stain Technol. 22, 139 (1947). 

 '" E. M. Rasch and H. Swift, J. Histochem. and Cytochem. 1. .392 (1953). 

 '»2 W. R. Kirkham, Federation Proc. 11, 240 (1952). 



