284 TECHNIQUE 



in water. If overstained, the excess can be extracted by placing 

 the sHde in 70 per cent alcohol to which a drop or so of hydrochloric 

 acid has been added. However, this is not satisfactory with this 

 type of stain. We will suppose that the correct staining effect 

 has been attained without extraction. After rinsing in running 

 tap water for fifteen minutes the slide is placed in 50 per cent 

 alcohol for a minute or so, then in 70 per cent alcohol for two 

 minutes. It is next placed in the cytoplasmic stain, such as 70 per 

 cent alcohol and eosin, for thirty to fifty seconds. It should be 

 removed and rinsed in 95 per cent alcohol and examined for the 

 cytoplasmic stain effect which should not be too deep in color. 

 Then the slide is placed in aniline oil for two minutes, drained, and 

 placed in xylol for two minutes, and placed in the first xylol jar 

 for two minutes more. The purpose of the two xylol baths is to 

 prevent carrying over any aniline which would stain the final 

 preparation. The student should have at hand a bottle of Canada 

 balsam, or gum damar, dissolved in xylol to a relatively thick 

 viscous consistency but thin enough to flow easily. Clean and 

 thoroughly dry cover-glasses should also be ready. A pair of fine 

 forceps will also be needed. The slide is then taken from the 

 second xylol jar, the undersurface wiped dry, placed on filter paper 

 on the table and 1 or 2 drops of the balsam placed over the section. 

 A cover-glass is lifted with the forceps and carefully lowered over 

 the section. It is well to let one edge of the cover-glass rest at the 

 side of the slide and then to let it fall gently by its own weight over 

 the section, thus preventing air bubbles from collecting under the 

 cover. After the section has thus been covered, it should be placed 

 on the warming table or in a warm oven for some hours in order that 

 the xylol in the balsam mixture may evaporate and thus harden the 

 gum. The hard gum has the same optical refraction as the slide 

 and cover, and so allows light to pass through without refraction 

 and also seals the cover to the slide. 



After the gum has hardened the slide can be cleaned by di])i)ing 

 in toluol, draining, and permitting to dry. However, the cover 

 should not be rubbed until the gum has thoroughly hardened. 



Regressive Staining. In this technique the sections are far over- 

 stainetl at first and then excess stain is extracted until just the 

 right effect is produced. An excellent example of a well-tried stain 

 for tiiis ])urj)()se is the so-called Heideiihain's henuitoxylin. It is 

 highly recommended for differentiation of mitotic figures and is, 

 therefore, indicated for studies in cell division, spermatogenesis, 

 and oogenesis. Two solutions are used, a mordant and the stain. 



