STAINING OF PARAFFIN SECTIONS 2S3 



color. This is deteriniuecl by obser\'ing the progress of the staining 

 under the microscope at short intervals during the procedure. It is 

 not advisable to stain too deeply, since later removal is never alto- 

 gether satisfactory, although it is possible. In the following method 

 we will employ Harris's hematoxylin as a nuclear stain and eosin as 

 a cytoplasmic stain. 



After preparing the nuclear and cytoplasmic stains, it is necessary 

 to assemble a series of jars just large enough to hold the slides and 

 in sufficient numbers to contain all the reagents needed. Shell 

 vials, a little over 1 inch in diameter and about 4 inches long, are 

 useful for the beginner. The Coplin jar is a more useful vessel, 

 since it can accommodate at least fi^'e slides at one time. Fourteen 

 of these jars will be foimd adequate. The fluid in each should 

 extend about 2 inches from the bottom, so that the upper third of 

 the slide will be free of fluid. 



Following is a list of jars and the fluid in each: (1) Xylol; (2) 

 aniline oil; (3) 95 per cent alcohol; (4) 70 per cent alcohol; (5) 50 per 

 cent alcohol; (6) distilled or tap water; (7) hematoxylin stain (one- 

 half stock solution, one-half water); (8) water; (9) 50 per cent 

 alcohol; (10) 70 per cent alcohol; (11) eosin in 70 per cent alcohol; 

 (12) 95 per cent alcohol; (13) aniline oil; (14) xylol. 



When using paraffin sections the paraffin must be removed before 

 proceeding, since paraffin does not mix with either alcohol or water, 

 and the dyes are dissolved in the latter. The slide is placed in the 

 xylol jar for about three minutes, during which the paraffin will 

 dissolve. After this step the sections should never be permitted 

 to remain out of the fluids until permanently prepared. After the 

 xylol, the slide is drained of free xylol and placed in the aniline oil 

 jar for two minutes, then drained and placed in the 95 per cent alco- 

 hol for two minutes, and similarly in the 70 per cent, 50 per cent, 

 and water for two minutes each. 



The slide is placed in the hematoxylin stain for three to five 

 minutes, then rinsed in water and examined under the microscope. 

 The section should have a rich purple or blue appearance. Micro- 

 scopic examination should show a purplish chromatin network in 

 the nuclei of the preparation. If the general appearance is a light 

 lavender, the section is imderstained. It should then be placed in 

 the stain for a moment or so more and reexamined. If the section 

 appears dark purple it is probably overstained. In progressive 

 staining it is desirable to stain until just the right effect has been 

 produced and then to stop further staining by placing the section 



