INTRODUCTION TO FIXATION 25 



duced them. It is usual to use the surname alone: thus 'Bouin' 

 means Bouin's fluid. 



Chemical fixation is generally achieved by putting an organism 

 or part of it in a fluid. In micro-anatomy and gross histology it is 

 sometimes necessary to fix a large volume of tissue in a single 

 piece. The fixative may be injected into a blood-vessel and thereby 

 reach all depths in the piece at almost exactly the same mo- 

 ment. This results in an evenness of fixation that cannot be 

 achieved in any other way with large pieces. The total volume of 

 fixative that can be held in the blood-vessels is, however, small in 

 comparison with that of the tissue; the fluid that has been injected 

 has to diflPuse through their walls before it can exert any effect on 

 cells other than those of the blood-vessels; and many cells lie a 

 long way (on the microscopical scale) from any blood-vessel, even 

 a capillary. Perfusion should therefore not be used as a general 

 rule. In cytology it is seldom necessary or desirable. The single 

 cell that it is desired to study can easily be obtained by cutting out 

 a piece of tissue a millimetre or so in diameter. If this be simply 

 thrown into a much larger volume of fixative, the latter will reach 

 all parts without much delay. The rate of penetration of fixatives is 

 discussed in detail below (pp. 37, 67, 150). 



Vapours are occasionally used instead of fluids, but most fixa- 

 tives are not volatile, and the method is applicable only to very 

 minute pieces. The advantage gained is that nothing is dissolved 

 out of the tissue. 



It is possible to fix in two stages, by taking preliminary action to 

 stop autolysis and then applying a fixative fluid. Autolysis may be 

 stopped almost instantaneously by placing tissues in some harm- 

 less, non-fixative fiuid maintained at a very low temperature. 

 Isopentane chilled to below —160° C is suitable. Ice-crystals form 

 in the tissues, but if the temperature is low enough they are very 

 small, and surprisingly little damage is done. In the process of 

 'freezing- thawing', the tissue that has been chilled is simply 

 allowed to thaw in an ordinary fixative at room-temperature or 

 thereabouts.*^^ In 'freezing-substitution' an organic fixative fluid 

 replaces the ice directly, without any melting of the ice-crystals. 

 Ethanol *^^ or methanol ^^^ may be used. The alcohol is chilled to 

 — 40° C or lower before the tissue is transferred to it from the iso- 

 pentane, and then allowed gradually to warm up. It acts on the 

 protein in the total absence of fluid water. These methods have 

 not been sufliciently used to allow their value to be judged with 



