76 The Preparation of Microscope Slides 



be transferred to either 70 per cent or 90 per cent alcohol in which they 

 should remain until no further color comes away. Specimens so treated 

 never show the fine needlelike crystals after mercuric fixation, but there 

 is no reasonable explanation of why this should be so. 



Tissues fixed in picric acid naturally are a bright yellow. All this 

 bright yellow color cannot be removed, as some of it is due to the 

 formation of complexes with the proteins. However, there are two meth- 

 ods by which much more of the yellow color can be removed than by 

 washing in alcohol alone. The first is to add a few grains of lithium car - 

 bonate to the 70 per cent alcohol in which the specimen is being washed . 

 The lithium salt appears to free some of the bound picric acid. Another 

 method, which is more troublesome but much more satisfactory, is to 

 transfer the specimen to: 



This liberates almost all the bound picric acid and is much t he bes t 

 a ftertreatment for picric acid-stained ma terials that has appeared in the 

 literature. It might be explained that the objection to the retention of 

 bound picric acid is that it interferes seriously with some forms of 

 staining. 



Treating Hard Materials. It so happens that some materials, even after 

 fixation, are so hard that they cannot possibly be cut into sections. This 

 hardening is caused either by the presence of calcium in the form of 

 bone or calcareous plates or by the presence of chitin. The removal of 

 bony or calcareous material is not as easy as it sounds, for if one were 

 merely to hang the material in an acid mixture there would be a great 

 hydrolysis of the protein. Therefore, it is necessary to have in the solu- 

 tion, besides acid, something that will prevent hyd rolysis and swelling. 

 The most commonly used reagent for this purpose is phloroglucinol. 

 The following is a typical formula: 



Note: The phloroglucinol is dissolved in the warm acid, cooled, added 

 to the water, and the alcohol then mixed in. The acid should be heated 



