114 FIXATION 



fixation is very prolonged. Frozen sections are generally used, and 

 fixation of lipids (as opposed to their preservation) is therefore 

 unnecessary. 



It has been known for many years that there is a gradual loss of 

 lipids if tissues are left in solutions of formaldehyde for a long 

 time.^^^' ^^^' ^^^' ^^ Cholesterol, triglycerides, and cerebrosides 

 appear to remain unaffected, but certain phospholipids begin to 

 disappear. Lecithins are well retained, but phosphatidyl serine, 

 phosphatidyl ethanolamine, and sphingomyelins become reduced 

 in amount. There appear to be two factors in the loss of phospho- 

 lipids. On one hand there is thought to be a splitting oflF of glycero- 

 phosphoric acid, the fatty acids remaining in the tissues; on the 

 other there may be a direct removal of lipid in colloidal solu- 

 tion. ^^^' ^^ In this connexion it is to be remarked that phosphatidyl 

 serine dissolves in 4% aqueous formaldehyde to form a clear 

 colloidal solution.^^ 



Phospholipids have a strong tendency to take up water and 

 extend their surface by growing outwards in worm-like 'myelin 

 forms'. This can easily be seen by smearing some lecithin across 

 the bottom of a cavity-slide and watching through the microscope 

 on the addition of water (fig. 15, a). The outgrowth of myelin 

 forms from the lipid constituents of cells would be damaging 

 morphologically and would also favour gradual solution. It was 

 shown by Leathes ^^^ that calcium ions have a remarkable effect in 

 preventing these outgrowths (fig. 15, b). The idea of adding cal- 

 cium chloride to formaldehyde solution therefore suggested it- 

 self. ^^ The salt has a double function: it checks the distortion and 

 solution of certain lipids, and it also improves fixation in the same 

 way as sodium chloride or other indifferent salts. 



After treatment with formaldehyde, phospholipids are less 

 soluble in lipid solvents and therefore less extractable by these 

 from tissues.^^^'^^^'^^ Experiments on this subject may be carried 

 out as follows. ^^ Phospholipids are incorporated in elder-pith, and 

 the latter then placed in formaldehyde solution; frozen sections 

 are cut and treated with various lipid solvents, and then with one 

 of the colouring- agents for lipids (p. 299). In a series of experi- 

 ments of this sort, with formaldehyde at 4% in 1% (anhydrous) 

 calcium chloride solution as fixative, lecithins, 'cephalins', and 

 sphingomyelins were all rendered insoluble even in boiling ethanol 

 followed by boiling ether, and also in parafiin wax at 60° C pre- 

 ceded and followed by xylene. This shows that formaldehyde is 



