4 " Fixation (chap. 1) 



In some cases the fixative may have a mordanting (combining insolu- 

 bly) effect on the tissue, thereby bringing the two together in a staining 

 action and assisting in the attachment of dyes and proteins to each other. 



Tissues must be placed in fixatives as soon as possible after death. If, 

 however, delay is unavoidable, they should be placed in a refrigerator, 

 thus reducing autolysis and putrefaction to a minimum until the fixa- 

 tive can be applied. 



A single chemical seldom possesses all of the requisite qualities of a 

 good fixative; a fixing solution therefore is rarely composed of only one 

 chemical. A familiar exception is formalin. Other reliable fixatives con- 

 tain one or more chemicals to coagulate the proteins of the cells, and 

 one or more chemicals to render the proteins insoluble without actu- 

 ally coagulating them. Coagulant fixatives change the spongework of 

 proteins into meshes through which paraffin can easily pass, thus form- 

 ing a tissue of proper consistency for sectioning. In addition, the pro- 

 tein linkages are strengthened by coagulants against breaking down 

 during later procedures. Used alone, however, coagulants have disadvan- 

 tages in that they may form too coarse a network for the best cytological 

 detail. Also, coagulation tends to induce the formation of artificial 

 structures (artifacts). Noncoagulant fixatives produce fewer artifacts 

 but if used alone have the disadvantage of giving the tissue a poor con- 

 sistency for embedding. It therefore follows that the ideal fixing fluid is 

 a combination of one or more protein coagulants and one or more non- 

 coagulants. The most efficient solutions are of this nature, and combine 

 if possible all types of action. This excludes those designed for the fixa- 

 tion of some specific cell component, such as chromosomes, glycogen, 

 mitochondria. 



Because they contain ingredients which act upon each other, many 

 mixtures are most efficient when made up fresh. However, the individ- 

 ual parts usually can be made up as stock solutions and mixed together 

 immediately before use. Among the frequently used chemicals are form- 

 aldehyde, ethyl alcohol, acetic acid, picric acid, potassium dichromate, 

 mercuric chloride, chromic acid and osmium tetroxide. Since every 

 chemical possesses its own set of advantages and disadvantages, each so- 

 lution component should, whenever possible, compensate for a defect 

 in some other component. For example, in the case of the widely used 

 fixative, Bouin's solution: 1. Formaldehyde fixes the cytoplasm but in 

 such a manner that it retards paraffin penetration. It fixes chromatin 

 poorly and makes cytoplasm basiphilic. 2. Picric acid coagulates cyto- 

 plasm so that it admits paraffin, leaves the tissue soft, fixes chromatin 

 and makes the cytoplasm acidophilic, but it shrinks and makes chroma- 



