322 Biological Stains 



hoi and two changes of 95%, then 70% alcohol to distilled water; then stained 2 

 minutes in 1% ethyl eosin in 95% ethyl alcohol, adjusted to pH 3.0 with N/10 HCl. 

 (If this solution fails to stain, about 1% acetic acid is added.) They are then washed 

 in distilled water, stained 30 seconds in a solution of 0.3 g. methylene blue in 30 ml. 

 95% ethyl alcohol and mixed with 100 ml. distilled water, adjusted to pH 5.6 by 

 adding 2 ml. of acetate-acetic acid (sodium acetate, 25 g.; glacial acetic acid, 5 ml.; 

 distilled water, 250 ml.) to 60 ml. of fluid, washed in distilled water and differen- 

 tiated in water acidulated with acetic acid until the sections become brownish red, 

 rinsed in distilled water, dehydrated, cleared in xylene and mounted in balsam. 

 By this technic, the nerve cells are stained blue and the Negri bodies terra cotta to 

 cardinal red. 



Eosin Bluish, C. I. No. 771 



Identification: Eosin bluish (eosin B) is the sodium salt of dibromodinitrofluo- 

 rescein, C2oH6N209Br2Na2. The method of identification is: Dissolve 50 mg. in 

 250 ml. of distilled water. Dilute 5 ml. of this solution and 2 ml. of 1% Na2C03 solu- 

 tion to 200 ml. with distilled water. Read in Beckman spectrophotometer. Absorp- 

 tion maximum 516-519 m/x; ratio P-15/P + 15 is from 1.00 to 1.10. 



Method of Analysis: Spectrophotometric. Percent dye = D-peak X 226. D-peak 

 (color density at peak) measured on dye solution described under "Identification". 



Samples of this stain should contain not less than 85% anhydrous dye. 



Biological Tests: Eosin B is tested as a counterstain for hematoxylin by the follow- 

 ing technic: Material fixed in either Bouin's fluid or Zenker-formol is embedded in 

 parafl5n and cut 10 /x in thickness. Sections are mounted on slides with Mayer's 

 albumen fixative. They are then run down to distilled water, stained for from 5 to 10 

 minutes in Mayer's hemalum, dipped once or twice in tap water, then holding them 

 in forceps dipped into the solution of eosin once, twice or three times, dehydrated 

 and mounted. 



The solution of eosin is 0.5% solution of the stain in 20% alcohol. If it overstains. 

 dilute with distilled water. In going quickly from Mayer's hemalum into the eosin a 

 precipitate is formed in the latter and the color is also precipitated into the tissue so 

 that it does not wash out readily. 



Eosin B is also tested in Mallory's phloxine-methylene-blue stain. Zenker-fixed 

 material is embedded in paraflBn. The paraffin is removed in the usual manner and 

 the material is stained 20 minutes or longer in a 2% aqueous eosin B (which is 

 substituted for the 5% phloxine). It is washed in distilled water; stained 30 minutes 

 in borax methylene blue (methylene blue, 1 g.; borax, 1 g.; distilled water, 100 ml.; 

 diluted to 10 times its volume with distilled water) by pouring the solution off and 

 on several times; washed in distilled water; differentiated and dehydrated in a dish 

 of 95% alcohol containing a few drops of 10% alcoholic collophonium (rosin). The 

 sections are kept in constant motion so that the decolorization is uniform. Staining 

 is controlled under the microscope; when the pink color has returned to the section 

 and the nuclei are still a deep blue, the dehydration is finished quickly with absolute 

 alcohol, sections are cleared and mounted in balsam. The cytoplasm should stain 

 pink in contrast to the blue of the nuclei. 



