Chemical Fixation 



In general, chemical fixation of cells and tissues is carried out by im- 

 mersion in reagents which have been found by experience to give images 

 resembling those of the living cell and, in most cases, known to combine 

 with specific chemical groups of cell constituents. The choice of a suitable 

 fixative, both with respect to the kinds and amounts of chemical re- 

 agents, is dictated by the purpose for which the fixative is to be used. 

 For example, the investigator interested primarily in studying chromo- 

 some morphology frequently uses acidic fixatives (Carnoy's — 3 parts 

 absolute ethyl alcohol : 1 part glacial acetic acid; Bouin's — 5 parts satu- 

 rated aqueous picric acid : 5 parts 40 per cent formaldehyde (for- 

 malin) : 1 part glacial acetic acid), which leave the chromosomes in a 

 highly precipitated form suitable for staining and microscopic study, but 

 do not necessarily preserve the chemical organization of the cell. On the 

 other hand, the investigator concerned with the study of enzyme activhy 

 in the cell selects fixatives (acetone, formaldehyde) which produce min- 

 imal denaturation and solubilization of proteins and, at the same time, 

 preserve cell structures sufficiently well to permit identification of sites 

 of chemical activity. The development in recent years of methods of 

 freezing and dehydrating tissues which do not involve their immersion 

 in reactive chemical agents, has proved to be of particular value in solv- 

 ing the problem of preservation of chemical groups and enzyme systems 

 in cells. 



Individual components of most commonly used fixatives do have 

 known chemical effects and they are used because of these known effects. 

 In general, however, particular combinations of fixing agents are based 

 more on experience than on specific knowledge of effect. It is obviously 

 somewhat difficult to determine whether one has achieved good fixation 

 or not in any given case. Certain information concerning the organiza- 

 tion of protoplasm, however, does give a basis for deciding what kinds 

 of chemical agents may be most suitable for a particular study. 



It is generally agreed that the essential effect of all fixatives on living 

 protoplasm is the separation of its solid phase from its liquid phase 

 (Gersh, 1959). During the fixation process, the solid phase may separate 

 out in the form of fibrils, granules, nets, or vacuoles depending on the 

 kind of fixative employed. While it is recognized that fixatives produce 

 changes in the physical and chemical organization of protoplasm, the 

 precise mechanisms operating to produce these changes are, for the most 

 part, poorly understood. According to Wolman (1955), the basis of liv- 

 ing protoplasm is protein, which exists mostly in the form of long-chain 

 molecules interconnected by cross linkages (Figure 11-1). Specific 



210 / CHAPTER 11 



