210 UADlATlU.N JJlOLUCiV 



3. LOCALIZATION OF SUBSTANCES IN CELLS 



It imisl not l)c' supposed that an awareness of the difficulties just suin- 

 mari/ed lias operated to inhibit the development of a chemical cytology. 

 (Juite the contrary has been the case; from the earliest days of cytology 

 there have been attempts to supplement the morphological descriptions 

 of cells by some idea of the chemical composition. 



A few substances such as hemoglobin or chlorophyll are visible in the 

 living cell because of the natural color, but in most cases a substance can 

 become visible only because of a color reaction carried out on a micro- 

 scopic slide. To be useful cytochemically such a test or stain must ful- 

 fill certain criteria: 



1. It must be possible to carry out the test under conditions which will 

 not seriously distort the cell morphology, a requirement which excludes 

 a great many of the color reactions of analytical chemistry. 



2. The specificity of the reaction must be known from data obtained 

 in vitro. 



3. The reaction must be one which will proceed without interference 

 in the presence of large amounts of proteins, and often in the presence of 

 nucleic acids or lipids. 



3-1. PRESERVATION OF INTRACELLULAR SUBSTANCES IN SITU 



Lison (193G) has considered critically many of these so-called "histo- 

 chemical" techniques; additional ones are briefly mentioned by Glick 

 (1949). The microchemical tests for inorganic ions, such as ferric, 

 chloride, and phosphate, and for smaller organic molecules, such as uric 

 acid and oxalic acid, are for the most part closely analogous to those of 

 microchemistry and cause the appearance on the slide of a colored pre- 

 cipitate or crystals only when the reacting group is in solution. For 

 cytological studies this means that the group will not react Avhen it is a 

 part of a large molecule, such as that of protein, but only after it has been 

 split off. These tests can undoubtedly be interpreted as an indication 

 that the reacting substance is present in the section of tissue, but this is 

 but a poor imitation of the precise conclusions which are possible from the 

 methods of analysis of tissue masses (Hogeboom, 1951) or from refine- 

 ment of microchemical methods to reach down to the level of a single 

 cell (Xorberg, 1942). For cytological studies the intracellular localiza- 

 tion is most important, and there are good reasons to (juestion the validity 

 of methods of microscopical demonstration of small diffusible chemical 



detoetable by absorption. Tlic |)ossiI)iliti('s have not been widely explored, but a 

 fluoiosceiit Scliiff reatfciit lias hccii dcNcloiM'd and found to dcnioustrate marked 

 aldfliyde (jjlasnial?) reaction in cells where the test api)eared couipletely negative by 

 ab.sorption contrast technique (L. Ornstein, unpublished). Fluorescent cytological 

 techniques also offer one possibility of avoiding the distributional error (p. 235). 



