42 FIXATION 



material. As fixatives he also used the vapour of osmium tetroxide 

 and the heat of steam. 



Tvi^o of Hardy's figures are shown in fig. 4, c, d. He found that 

 weak protein solutions generally showed separate microscopical 

 particles after fixatives had acted, while stronger ones resulted in 

 the appearance of a sponge or net. As the figures show, the net was 

 thickened at the nodal points. The size of the meshes varied with 

 the fixative used. The vapour of osmium tetroxide (a non- 

 coagulant fixative, as we have seen) was alone in not producing a 

 microscopically visible structure. 



Hardy's main contribution was his discovery that there is no 

 Grundsiihstanz. The whole of the coagulum consists of granules or 

 spongework, with water in between. This was evident when suffici- 

 ently thin sections were cut, for there was nothing in the meshes of 

 the net that could be demonstrated even by saturated solutions of 

 dyes. In thicker sections the out-of-focus appearance of other 

 parts of the net gave the misleading appearance of an interstitial 

 substance, but Hardy realized that in finished microscopical 

 preparations the meshes were occupied only by Canada balsam or 

 other mounting medium. Thus, as he said, the essence of fixation 

 by coagulant fixatives is the separation of fluid from solid, of 

 water from protein ; and the solids, if sufficiently abundant, hang 

 together to form a network (or in some cases a honeycomb-like 

 structure). As a result, water can often be squeezed by hand from 

 a fixed protein, though a pressure of 400 lb to the square inch will 

 not separate it from an unfixed protein gel. When water has been 

 separated from protein, it can be replaced by other fluids. While 

 the water is still present, there is usually little change of volume ; 

 but when the replacement occurs, there is usually shrinkage. 

 Similarly a silicic acid gel shrinks w^hen its water is replaced by 

 another fluid. 



It has already been remarked (p. 22) that the network pro- 

 duced by the action of coagulant fixatives has the same refractive 

 index as dry protein, and that is why mounting media having about 

 the same r.i. as dry protein give such glassy transparency to 

 microscopical preparations of the tissues of organisms. 



Hardy considered that osmium tetroxide in aqueous solution 

 produced a network in egg-white, but the effect was more prob- 

 ably due to the water of the solution acting in the absence of salt 

 on globulin. 



It might be thought that the structure produced in protein sols 



