ii8 



water, 50 c.c. ; 95% alcohol, 50C.C. A good counter-stain with hematoxylin 

 or carmine. Stain in hematoxylin or carmine, rinse in water (or alcohol if Hcl. 

 carmine is used), and stain with picric alcohol 10-30 seconds ; wash off with 

 67% alcohol, etc. Since picric acid tends to wash out the nuclear stain, it is 

 best to over-stain somewhat with the nuclear dye and regulate carefully the 

 time of staining with the picric alcohol. 



I 97. Picro-fuchsin. Formulas: (a) General stain, — \% aqueous solu- 

 tion of fuchsin acid, 10 c. c. ; saturated aqueous solution of picric acid, 75 c.c. ; 

 distilled water, 25 c.c. (b) For nervous tissue, — i%aqueous solution of fuchsin 

 acid, 15 c.c. ; saturated aqueous solution of picric acid, 50 c.c. ; distilled water, 

 50 c.c. This is a valuable counter-stain to hematoxylin, especially serviceable 

 in the differentiation of white connective tissue fibers. The nuclei are a pur- 

 plish brown (hematoxylin stain), the connective tissue red, cell bodies and 

 muscle yellow-orange. In special cases the relative amount of fuchsin acid 

 may be decreased or increased, thus giving a preponderance to the yellow or 

 red in the general stain. 



Stain well with hematoxylin, rinse in water, and stain with the picro-fuch- 

 sin 15-30 seconds ; wash away the excess of stain with distilled water or 67% 

 alcohol. Picro-fuchsin will gradually wash out the hematoxylin, therefore 

 stain strongly with hematoxylin and regulate carefully the time of staining 

 with picro-fuchsin. Picro-fuchsin is quite sensitive to alkalies, so that tap-water 

 (unless slightly acidulated) should not be used for washing out, and the mount- 

 ing medium should be slightly acid or neutral, not alkaline. 



\ 98. Heidenhain's Iron Hematoxylin. Three steps are necessary, (a) 

 mordanting, (b) staining, (c) differentiating. 



1. Mordant sections 1-2 hours in a \% aqueous solution of ferric alum 

 (iron-ammonium-persulphate), rinse in water a few (5-10) minutes, and 



2. Stain for 1-3 hours in a )i% aqueous solution of hematoxylin (form- 

 ula : 16% alcoholic solution of hematoxylin 3 c. c, water 97 c. c, chloral hy- 

 drate 2 grams), rinse in water, and 



3. Differentiate by dipping the slide into the mordant for a few seconds 

 and then rinsing in tap-water, repeating the operation until the correct differ- 

 entiation has been attained, as ascertained by examination under the micro- 

 scope. The chromatin (nucleus) should be a deep blue or blue-black, the 

 cytoplasm ( cell-body ) gray or light blue. After differentiating it is necessary 

 to wash the section well in running water for about 20 minutes to ensure the 

 complete removal of the ferric alum. The above times are for tissue fixed in 

 Flemming's or Hermann's fluid ; shorter times may be given for tissue fixed 

 by most other methods. 



A counter-stain is ordinarily not needed, but, if desired, the sections may 

 be stained for (O seconds to 2-3 minutes with a y^ saturated aqueous solution of 

 orange G. 



This is a valuable stain in all cytological work, and for all muscular tissue. 



