unsaturated lipids, which renders them insoluble. Osmium tetroxide pro- 

 duces a similar effect on unsaturated lipids, the osmium being reduced 

 during the fixation process to black oxides. This blackening of osmium 

 occurs in such structures as the Golgi complex, and has been utilized 

 for many years as a method for revealing this structure in the cytoplasm 

 of certain cells. 



Freezing-Drying 



Because of the physical and chemical effects of fixation by immersion 



in chemical reagents, such treatment is generally not suitable for most 



cytochemical studies in which the aim is to detect particular substances 



(e.g., enzymes) and to determine the location of these substances in the 



Top plate y'Trap at -TS^C 



To vacuum pump 



Bottom plate ^Specimen at - 40°C 



Figure 11-3. Diagram Showing the Basic Principles of the Freeze Dryer 

 in Cross-Section. (From Bell, L. G. E., 1952. "The Application of Freezing 

 and Drying Techniques in Cytology," Intern. Rev. CytoL, 1, Fig. 1, p. 46.) 



cell. One method which, in most respects, is at least a considerable im- 

 provement involves: (1) rapid cooling and freezing of tissues by im- 

 mersion in a cold bath, and (2) their dehydration by vacuum desiccation 

 at low temperature to prepare them for subsequent embedding, section- 

 ing, and staining. 



In the freezing technique, the initial cooling or quenching of the tissue 

 is usually carried out at temperatures in the range of —160 to —190° C. 

 This is most easily accomplished by plunging small pieces of tissue, cul- 

 ture cells on coverslips, or smears and imprints on slides into a quench- 

 ing solution cooled by liquid nitrogen. The solutions most frequently 

 used are isopentane, propane, or a mixture of the two. The most im- 

 portant aspect of the freezing process is the quick cooling of materials 

 through the temperature range —30 to —40° C. It is within this range 

 that the rate of ice crystal formation is most rapid. The use of liquid 

 nitrogen permits materials to be cooled rapidly through this critical 

 range to lower temperatures where only finer ice crystal formation occurs. 



214 / CHAPTER 11 



