72 



HEWSON SWIFT 



■4.0 



2.0 



>thymu$ 

 'liver 



minute* 10 20 30 



Hydro I y sis time 



Fig. 5. Effect of hydrolysis time on the amount of Feulgen dye bound per nucleus. 

 Tissues fixed in acetic alcohol and sectioned at 20 ai. Each point represents the mean 

 of 12 tetraploid class interphase nuclei from mouse liver and thymus, measured at 

 560 m/i. Dye expressed in arbitrary units. Density readings at 30 minutes are too low 

 to be accurate, but are below 0.6. 



A'' HCl at 60° C. As hydrolysis time is increased, the intensity increases to 

 a maximum (about 12 minutes for acetic alcohol-fixed mouse tissues) and 

 then declines. The increase represents formation of aldehydes, and is asso- 

 ciated with removal of purines from the tissue.^* The subsequent decrease 

 involves actual loss of DNA from the tissues and possibly also chemical 

 breakdown. Ely and Ross** reported that material capable of giving a 

 positive Feulgen reaction was extracted from a suspension of isolated thy- 

 mus nuclei. The actual extent of DNA removal was not estimated. When 

 fixatives that contain chromic acid or bichromate are used (e.g., 

 Flemming's, Navashin's, Sanfelice) the intensity reaches its maximum 

 somewhat later, and a long plateau occurs before DNA is lost. Measure- 

 ments of Flemming-fixed frog cartilage nuclei showed maximum intensity 

 from 20 to 50 minutes in A^ HCl at 60° C'^^ Such fixation, if prolonged, 

 enables hydrolysis to proceed to a maximum before DNA removal, which 

 must be inhibited through interaction with the heavy metal. The intensity 

 obtained with chromic acid fixatives in general is no greater than with 

 acetic alcohol, however, so that one might conclude that fixation in fluids 

 without chromic acid enables hydrolysis to be near maximal before appre- 

 ciable DNA is removed. 



Maximum hydrolysis time may be different for different species. For 

 example, Hillary^" found hydrolysis in the alga Spirogyra needed to be 



'"8 E. D. DeLamater, H. Mescon, and J. D. Barger, J. Invest. Dermatol. 14, 133 (1950). 

 !•" H. S. DiStefano, Chro77iosoma 3, 282 (1948). 



