122 FIXATION 



When sections that had been 5 min. in any of the antemedia 

 were brought down to water and bleached, it could easily be 

 shown that the lipid still remained in them, for it could be coloured 

 black by Sudan black. If, however, sections were left for 40 min. 

 in the antemedia, the ones treated with xylene or toluene were 

 proved to contain no lipid, while those that had been in benzene or 

 chloroform still contained it. 



Sections that had been 5 min. in the antemedia were brought 

 down to water, bleached with hydrogen peroxide, taken up to the 

 antemedia again, and left there for 30 min. On subsequent treat- 

 ment in the usual way with Sudan black, they were shown to 

 contain no lipid. This applied in all cases, whether the antemedium 

 was xylene, toluene, benzene, or chloroform. 



It is to be noted that so long as the lipid-sites were still blackened 

 by osmium, they contained lipid. The evidence suggests strongly 

 that the black substance is a compound of lipid with osmium. 

 This compound is resistant to solution by benzene and chloroform, 

 but dissolves slowly in xylene or toluene; when bleached by 

 hydrogen peroxide, it is soluble in any of the lipid solvents. 

 Xylene is capable of acting as an oxidizing agent,^-^ and the same 

 may perhaps apply to toluene. It is to be supposed that osmium 

 tetroxide reacts with the double bonds of lipids in much the same 

 way as it does with those of the substances discussed on pp. 61-2, 

 but no direct evidence on this subject is available. 



The possibility that osmium tetroxide sometimes simply 

 oxidizes lipids, without forming an additive compound, must be 

 kept in mind. Hofmann ^^^ believed that this was the way in which 

 it ordinarily reacted with lipids. He made a careful study of oxida- 

 tion by osmium tetroxide, and reached the conclusion that it can 

 act as an adjuvant to other oxidizers. Thus, certain substances 

 that cannot be oxidized by potassium chlorate alone, can be 

 oxidized if osmium tetroxide is present as well. The latter oxidizes 

 the substrate and is itself reduced: the chlorate re-oxidizes it, and 

 the process begins again. Wolman ^*^ suggests that osmium tetrox- 

 ide may act as an oxidative catalyst in this way in microtechnique, 

 when mixed with other oxidizers. 



In the presence of a strong oxidizer, such as chromium trioxide, 

 the mixed triglycerides of adipose tissue are generally blackened by 

 osmium tetroxide, while conjugated lipids as a rule are not. This 

 is useful as a rough pointer in preliminary histochemical work. 

 Since the fatty acid component of conjugated lipids is often highly 



I 



