CYTOCHEMICAL TECHNIQUES FOR NUCLEIC ACIDS 89 



Although methacrylate refractive index is adequate for some tissues, it 

 is not for others, and is not readily adjusted. 



The effect on absorption curves of the refractive index of the mounting 

 medium is shown in Figs. 7 and 8. When the same cell is measured in differ- 

 ent media, one of refractive index close to that of the specimen and the 

 other not, an estimate of the light loss due to scatter alone can be obtained. 

 The effect of anomalous dispersion is evident, since the proportion of light 

 lost by scatter is obviously greater in the region of maximum absorption, 

 although the swelling of the tissue in methacrylate may have added further 

 complications. In some cases the effect of anomalous dispersion is greater for 

 the protein component than for the nucleic acid (Fig. 8). Thus scatter 

 effects may easily lead to false interpretations of the nucleic acid-protein 

 ratio, depending upon the degree to which nucleic acid and protein indi- 

 vidually contribute to light loss. 



From such data it is evident that, if ultraviolet determinations are ever 

 going to provide cjuantitative estimates of nucleic acids in tissues, high 

 refractive index media should be employed. Where specimens are ade- 

 quately matched, however, cell details are not readily seen in visible light, 

 so that accurate centration of cell regions is difficult. Fluorescent screens 

 may be used for visualizing the sections, but these provide weak images at 

 best, and the intensities of ultraviolet light required may be harmful to the 

 specimens. Photographic scanning may be used but is tedious. An ultra- 

 violet-sensitive television camera'*^ or image intensifier would solve the 

 problem, but these are not as yet generally available. 



VIII. Conclusions 



Present cytochemical techniques by and large seem to be adequate for 

 the study of many problems concerning the role of nucleic acids in the cell. 

 None of the techniques discussed here, however, can be considered entirely 

 satisfactory, and a number of pitfalls confront the worker in cytochemical 

 research. These difficulties are avoided only by experience with the tech- 

 niques themselves, a certain skepticism of data rapidly obtained, and a 

 constant checking of specificity and stoichiometry wherever possible. Al- 

 though a list of steps to follow can be given for many cytochemical meth- 

 ods, for any new material modifications must almost always be made. 

 For example, nuclease enzymes may act rapidly, slowly, or not at all, 

 depending on the type of material and how it is fixed, and may even fail 

 to act specifically under some conditions. Thus a certain amount of em- 

 pirical manipulation must preface the use of almost any cytochemical 

 method. 



Improved cytochemical techniques are needed to extend the types of 



»«« G. K. Williams, Proc. Am. Assoc. Cancer Research 1, 60 (1953). 



