CYTOCHEMICAL TECHNIQUES FOR NUCLEIC ACIDS 87 



estimated, however, since nucleic acids and protein have no appreciable 

 absorption in this region (unless tyrosine phenolic groups are oxidized) and 

 most light loss may be considered nonspecific. In some cases Caspersson 

 and his associates have determined the degree of absorption in this area, 

 and then extrapolated the curve into the region of specific absorption.^** -^^^ 

 As pointed out by many authors,^-^'^-'-^**'^*^ this can only give an extremely 

 rough estimate of the degree of nonspecific light loss. The shape of the 

 scatter curve has been determined by analogy to theoretical systems which 

 obviously are only indirectly applicable. ^^^-^^^ Also, where light scatter is 

 produced by absorbing particles, the extent of light loss may be much in- 

 fluenced by anomalous dispersion (see below) so that the amount of scatter 

 is larger in the general region where the specific absorption is greater. For 

 these reasons most of the determinations of nucleic acids that have been 

 made on tissue sections, where marked scatter almost invariably occurs, 

 are best considered qualitative or at most semiquantitative. Techniques for 

 direct measurement of the scattered light have recently been described.^" 

 This should provide an independent estimate of nonspecific light loss and 

 may greatly simplify its determination. 



If ultraviolet absorption measurements on tissues are to be made quanti- 

 tative, it is obviously of great importance to reduce scatter to a minimum. 

 In most cases glycerin has been used as a mounting medium. This is of 

 much lower refractive index than tissues, and considerable scatter may 

 result from its use, although with frozen-dried material, the nucleoprotein 

 may swell slightly on contact with glycerin and reduce the scatter some- 

 what. ^^ In thin smears, made in acetic alcohol, scattering is also reduced^^ 

 (Fig. 7B,C), probably in part because a considerable amount of protein is 

 removed. Smears of isolated nuclei show only small amounts of scatter,^^*-^^* 

 probably also associated with protein loss. Scattering may be practically 

 ehminated where the refractive index of the mounting medium is matched 

 to that of the tissue nucleoproteins. As yet no high refractive index mount- 

 ing medium has been described that is adequate for ultraviolet studies. 

 Saturated or nearly saturated solutions of chloral hydrate^" or zinc chlo- 

 ride^^* in glycerin have been suggested and are of the proper refractive 

 index. Both of these media remove nucleic acids from sections after acetic 



152 H. Hyden, Acta Physiol. Scand. 6, Suppl. 17, 1 (1943). 



i"T. Caspersson, Kolloid-Z. 60, 151 (1932). 



"^ T. Caspersson, Kolloid-Z. 65, 162 (1933). 



1'^ C. Leuchtenberger, R. Leuchtenberger, C. Vendrely, and R. Vendrely, Exptl. 



Cell Research 3, 240 (1952). 

 1S6R. C. Mellors and J. Hlinka, Federation Proc. 12, 246 (1953). 

 1" A. Kohler, Z. wiss. Mikroskop. 21, 273 (1904). 

 15' H. Koenig, D. Schildkraut, and E. Galler, J. Histochem. and Cytochem. 1, 384 



(1953). 



