204 GEORGE H. HOGEBOOM AND WALTER C. SCHNEIDER 



histochemical methods with direct enzyme assays of cell fractions isolated 

 by centrifiigation and find that entirely different results are obtained by 

 the two procedures. The histochemical tests indicated that phosphatase 

 activity is confined mainly to the nucleus (see Chapter 18), whereas the 

 cell fractionation experiments demonstrated apparent localization in the 

 cytoplasm. A convincing explanation for this discrepancy was offered by 

 the latter investigators in the finding that cell nuclei have a strong affinity 

 for calcium and lead phosphates. Since these two metallic ions are used to 

 precipitate the inorganic phosphate liberated by alkaline and acid phos- 

 phatase, it is apparent that the histochemical methods are capable of 

 producing an artifact and are therefore probably not suitable for demon- 

 strating the intracellular distribution of the enzymes. It should be pointed 

 out that these data do not necessarily invalidate conclusions derived from 

 histochemical studies of the distribution of phosphatases among histo- 

 logically defined areas of various tissues.^^ 



Recent reports have indicated, however, that certain other histochemical 

 methods may provide accurate intracellular localization of enzymes. 

 Thus, Hoffmann et al?^ found that cytochrome oxidase, as demonstrated 

 by the Nadi reaction, is localized exclusively in the mitochondria of lymph- 

 oid and myeloid cells. Mitochondrial localization of succinic dehydro- 

 genase in cells was also demonstrated by Malaty and Bourne,^* who used 

 tetrazolium salts to give a colored reaction product. These findings have 

 confirmed results obtained by the cell fractionation technic (see below). 

 It would therefore appear that histochemical methods can provide reliable 

 information in some instances but that the data should be checked by in- 

 dependent means. It is, in addition, reassuring to note that, by the applica- 

 tion of both histochemical and cell fractionation methods, it has been 

 possible to obtain confirmatory results on the one hand and to detect 

 artifacts on the other. Although the possibility must be entertained that 

 the results of both methods may be false, their simultaneous use offers 

 considerable promise as a means of solving cytochemical problems. 



3. SUBMICROMETHODS 



The submicromethods^i-'" ^j-g theoretically capable of the quantitative 

 analysis of single cells and their structural components and in this respect 

 represent the ideal cytochemical tool. In practice, however, the results do 

 not indicate that this ideal has been achieved except in special instances, 

 because it has not as yet been possible to increase the sensitivity of enzymic 

 assays and chemical analyses sufficiently to permit the study of any but 



35 G. T. Hoffman, A. Rottino, and K. G. Stern, Blood 6, 1051 (1951). 



36 H. A. Malaty and G. H. Bourne, Nature 171, 295 (1953). 

 " K. Linderstr0m-Lang, Harvey Lectures 34, 214 (1938-39). 



