PRIMARY fixatives: NON - CO AGUL ANTS 121 



There is no coagulation of nucleoprotein, nor is DNA pre- 

 cipitated from solution. ^^^ 



Reactions with lipids. As we have seen (p. 120), Schultze and 

 RudnefT *'*^ had already in 1865 recognized the capacity of fats 

 and other lipids to reduce osmium tetroxide. It was shown by 

 Altmann ^ that whereas oleic acid and olein are blackened by this 

 substance, palmitic and stearic acids and their triglycerides are 

 not. This led to the understanding that osmium tetroxide reacts 

 with the double bonds in lipids. Since lipids generally occur in 

 nature as mixtures, and some of the constituents of these mixtures 

 are usually to some degree unsaturated, most lipids as they occur 

 in organisms will sooner or later be darkened by the action of 

 osmium tetroxide. As a general rule, mixed triglycerides in the 

 form of storage-fat blacken with osmium tetroxide more easily than 

 conjugated lipids. 



When osmium tetroxide reacts with lipids, three possibilities 

 present themselves. The tetroxide may be reduced to the dark 

 lower oxide ; or it may combine with the lipid to form a dark com- 

 pound; or both may occur. If a compound is formed, alteration 

 in solubility is to be expected. It is well known that the sites of 

 storage fat are often black in paraffin sections mounted in Canada 

 balsam. Lipids are unlikely to survive such embedding and 

 mounting, unless profoundly altered. The possibility presents 

 itself, however, that the lipid has gone and only insoluble osmium 

 dioxide remains to mark its former sites. 



Comments on this subject are scattered through the literature. 

 Recently the Chinese cytologist Chou,^^^ working at Oxford, has 

 made a systematic study of the subject. The subcutaneous fat 

 of the mouse was used in his experiments. This was fixed in 

 1% osmium tetroxide or in Flemming's strong fluid, ^"^ which 

 contains osmium tetroxide at 0-4%. The fixed tissue was de- 

 hydrated with ethanol, left for 30 min. in an antemedium, and 

 embedded in paraffin. Sections were soaked for 5 min. in the same 

 fluid that had been used as antemedium, brought down to water, 

 and mounted in an aqueous medium. The antemedia used were 

 xylene, toluene, benzene, and chloroform. 



The fat-oites were in all cases black. If, however, the sections 

 were left in xylene or toluene for 40 instead of 5 min., the fat-sites 

 were colourless: the globules appeared empty. They remained 

 black, however, if benzene or chloroform was used for the same 

 period. 



