CYTOCHEMICAL TECHNIQUES FOR NUCLEIC ACIDS 73 



longer than for most plants (15 to 20 minutes), and McMaster^o^ found 

 acetic alcohol-fixed sea urchin eggs stained maximally after only 8 minutes, 

 instead of the usual 12 minutes for most vertebrate nuclei. Where Feulgen 

 intensity is to be compared between species, these differences may be of 

 considerable importance. A difference in optimal hydrolysis time for differ- 

 ent tissues of the same animal has also been described, although Feulgen 

 intensity was determined only by eye.^*^^ When such tissues are measured 

 by a microphotometer, and the amount of dye per nucleus computed, there 

 is not significant difference between the hydrolysis curves (Fig. 5). Thus, 

 although hydrolysis maxima may differ between species, there appears to 

 be almost no evidence of a difference between tissues of the same species. 

 Sperm nuclei, however, are an exception. Formalin-fixed mouse sperm 

 nuclei start to degenerate after about 20 minutes of hydrolysis, although 

 other nuclei, including the spermatocytes, remain intact. In the annelid 

 Sahellaria, acetic alcohol-fixed sperm show appreciable disintegration even 

 after 12 minutes,!"^ although other testis nuclei do not. This change is 

 probably influenced by the alteration during sperm formation in basic pro- 

 tein to which the DNA is bound. One other exception studied by Hillary^" 

 is found in plant nuclei with high. tannin content. Hydrolysis time curves, 

 even in acetic alcohol-fixed tissues, are altered to the chromic acid type, so 

 that such nuclei would not be optimally hydrolyzed at 12 minutes, and thus 

 would be paler than in surrounding cells. Such nuclei are often noticeably 

 brownish, for example, in fern epidermis. In addition, Patau and Srini- 

 vasachar"" have reported rather unpredictable changes in amounts of dye 

 bound by mitotic stages in onion, after various hydrolysis or posthydroly- 

 sis treatments; for example, certain treatments were found to affect pro- 

 phase nuclei more than metaphases, and others had the opposite effect. 



The shape of the Feulgen dye absorption curve shifts during hydrolysis, 

 as shown in Fig. 4. The reason for this is not altogether clear. In general, 

 nuclei on one slide show curves all roughly of one shape, regardless of tissue, 

 but in some cases differences in distributions of the two components within 

 nuclei have been found."' Such variations in the color produced by the 

 Feulgen reaction obviously should be considered where quantitative de- 

 terminations are made, particularly where the two-wavelength method of 

 measuring is employed. 



6. Variations in the Feulgen Reagent 



Preparations of fuchsin-sulfonic acid recommended by different authors 

 show wide variation in dye, pH, and SO2 content. The effect of these vari- 



108 R. McMaster, Ph.D. Thesis, Columbia University, New York, 1953. 



"9 M. Alfert and H. Swift, Exptl. Cell Research 5, 455 (1953). 



1'" D. Srinivasachar, Ph.D. Thesis, University of Wisconsin, 1953. 



1" L. Ornstein, /. Lab. Invest. 1, 85 (in discussion of paper by J. F. Scott) (1952). 



