360 CONNECTIVE TISSUES. 



mate solutions, as in the March! method. The bichromate, which is 

 itself an oxidising agent, acts in the various double linkages, and 

 prevents the osmic acid from being reduced except by the substances 

 having the strongest reducing power ; these are the true fats and mix- 

 tures of cholesterin and unsaturated fatty acids. 



Since the latter show double refraction in polarised light (see 

 below), while the true fats do not, it is possible to differentiate these 

 two groups of substances. 



The behaviour of mixtures of cholesterin and fatty acids is para- 

 doxical, since cholesterin alone and fatty acids alone do not blacken 

 with osmic acid after bichromate. The same paradoxical behaviour 

 is exhibited by these cholesterin fatty acid mixtures in their staining 

 reaction with hsematoxylin after mordanting with bichromate 

 (see next paragraph). One must assume that cholesterin is present 

 in these mixtures in a special physical state, in which it exhibits a 

 greater reducing power. 



The myelin sheath of normal nerves does not contain true fats 

 and gives, accordingly, no blackening with bichromate-osmic acid 

 treatment. In the early stages of degeneration globules of true fat 

 are formed, which stain black with bichromate-osmic acid and then 

 give the positive Marchi reaction. With other tissues rich in lipoids, 

 such as the adrenal cortex, no such clear distinction can be obtained 

 because the lipoids are present therein, not only together with true 

 fats, but also mixed with them, so that a globule of fatty material 

 frequently contains both groups of substances. 



(3) STAINING WITH HJEMATOXYLIN AFTER MORDANTING WITH 

 BICHROMATE. This method, which was introduced originally by 

 Weigert for the staining of the medullary sheath of nerves, is 

 applicable to all fatty substances. Like the preceding method, it 

 also depends on the presence of a double linkage in the molecule, 

 as pointed out by Thorpe. Substances having a double linkage 

 are oxidised by the bichromate solution and, in this process of 

 oxidation, a chromium compound is formed which is insoluble in 

 fat solvents and which at one stage of the oxidation has the property 

 of forming a dark blue lake with haematoxylin. If oxidation be 

 continued, however, hsematoxylin will again cease to stain. The 

 rate of oxidation with bichromate varies, as might be expected, 

 with the concentration of the solution used and with the temperature 

 at which it is carried out. Working under similar conditions, it is 

 found again that the different fatty substances vary in the readiness 

 with which they are oxidised by bichromate and, consequently, 

 reach the stage of staining with hsematoxylin after different periods 



