The drying process subsequent to embedding and sectioning consists 

 of removal of ice from the frozen tissues in a vacuum at temperatures 

 (—40 to — 60° C) sufficiently low to prevent changes in ice crystal 

 structure. The principal of the freeze dryer is shown in Figure 11-3. 

 It consists essentially of ( 1 ) a vacuum or drying chamber in which the 

 specimen is suspended, and (2) a cold bath which serves to keep the 

 drying chamber at a low temperature. Under vacuum, water vapor is re- 

 moved from the specimen onto the walls of the cold drying tube. Re- 

 moval of water can be enhanced by placing a chemical desiccant such as 

 phosphorus pentoxide on the bottom of the drying tube. Following re- 

 moval of water, the specimen is allowed to reach room temperature and 

 prepared for embedding by standard techniques. 



Freezing-Substitution 



This method involves placing the specimen after quick freezing in one 

 of a number of reagents (e.g., ethanol, methanol, butanol, acetone) 

 maintained at low temperature (—20 to —60° C). These reagents serve 

 to dissolve out the ice crystals in the specimen and to replace them with 

 the reagent. Freezing-substitution is useful for preparing smears and im- 

 prints of tissues prior to cytochemical staining, since they can be in- 

 cubated in staining solutions immediately following solvent substitution 

 at low temperatures without prior drying or embedding. 



The rapid cooling of tissues is superior to chemical fixation because 

 (1) postmortem changes are minimal, (2) chemical substances, par- 

 ticularly proteins, are precipitated but not made soluble, (3) solidifica- 

 tion of tissues as the result of freezing increases their viscosity and 

 reduces the extent of diffusion of substances, (4) displacement and dis- 

 tortion of cell structures are reduced, and (5) little or no extraction of 

 soluble components occurs. 



STAINING 



The stains most often employed in routine cytological studies are solu- 

 tions of organic aromatic dyes which may be acid (anionic) or basic 

 (cationic) in character depending on the particular molecular groups 

 they contain. For a compound to function as a dye it must contain 

 ( 1 ) an auxochrome group which is responsible for the electrolytic dis- 

 sociation of the compound, and (2) a chromatophore group which 

 imparts to the compound the property of color. 



Dyes are classified as acidic or basic depending on whether the auxo- 

 chrome they contain forms a salt with an acid (basic dye) or a base 



SURVEY OF CYTOLOGICAL TECHNIQUES / 215 



