Iron Hematoxylin-stained Testis 229 



should be filtered immediately before use if the finest slides are required 

 because chromosome figures in a rat can be obscured by a very small 

 particle of dust. 



The slides are next taken from distilled water and placed in the mordant 

 solution. It matters little how long they remain in this solution although 

 the usual directions call for keeping them there overnight. This varies 

 with every type of tissue studied and is greatly dependent on tempera- 

 ture. If the solutions are heated to 50° C, with the understanding that this 

 will cause a swelling of the section and a general obscuring of the finer 

 details, the period may be shortened to as little as 10 min. T he finest stains , 

 however, are t hose obtained by leaving the sections in the mordant solu- 

 tion at room temperatures overnigh t. On removal from the mordant solu- 

 tion, the sections should be rinsed very briefly in distilled water; the pur- 

 pose of the rinse is to remove the surplus mordant from the surface of 

 the slide without extracting it from the tissues. The slides are then placed 

 in the hematoxylin solution where they should remain for approximately 

 the same period as they have been in the mordant; the length of time is 

 not important, although from 3 to 24 hr is the customary period. Sections 

 may be removed from time to time from the staining solution and ex- 

 amined with the naked eye. Successful preparations show the sections to 

 have been blackened completely, although a slight bluish tinge in the 

 black is permissible. If they have not become blackened completely in 24 

 hr, it is necessary only to place them, after a brief rinse, in the mordant 

 solution and leave them there for another 24 hr before returning them 

 to the stain. 



If, however, the sections are sufficiently blackened on removal from the 

 staining solution, they may be differentiated , that is, the hematoxylin stain 

 extracted from all portions of the sections except the chromosomes, which 

 are to be studied. This is done customarily with the same solution in 

 which they were mordanted, although, of course, a fresh solution or a 

 stronger solution may be used if desired. Differentiation at the commence- 

 ment of the process goes relatively slowly, so that all the slides, which are 

 carried in a glass rack, may be removed and placed in the 2.5 per cent 

 iron alum without any very great care. The actual time in which differ- 

 entiation takes place cannot be forecast since it depends on a large num- 

 ber of uncontrollable factors; it is never less than five minutes nor is it very 

 often more than a few hours. Sections, therefore, should be withdrawn 

 from the iron alum every 4 or 5 min and examined briefly under the low 

 power of a microscope. It is a matter of great convenience for the con- 

 trolling of differentiation of chromosomes in this type of preparation if an 

 ordinary student microscope ( of the type customarily used in laboratory ) 

 can be lifted with a glass plate over the stage, so that one can, without fear 

 of damage to the instrument, place a slide, wet with iron alum, on the 



