Methods for Testing Stains 321 



la Heidenhain's hematoxylin, Zenker's fixed material is used and embedded in 

 paraflBn. The paraffin is removed in the usual manner. Sections are mordanted in 

 1.5-4% aqueous NH4Fe(S04)2 -121120 (violet crystals) for 30 minutes to 3 hours, 

 rinsed in tap water, stained 1-3 hours in 0.5% solution of hematoxylin in distilled 

 water, ripened 4-5 weeks, rinsed again in tap water and diflFerentiated in the above 

 iron alum solution, controlling the difiFerentiation by microscopic examination. 

 They are then washed in running water about 5-10 minutes, and are counter- 

 stained in a 0.1% strength of eosin Y in 25% alcohol for 2-5 minutes, dehydrated, 

 cleared and mounted in balsam. By this technic, the nuclei should be black and the 

 cytoplasmic structures, pink. 



In testing its behavior as a constituent of Wright's stain the formula given on 

 page ID3-4 of Staining Procedures is employed and the compound stain, after dis- 

 solving in methyl alcohol, is applied to blood smears for one to three minutes with- 

 out diluting and is then diluted on the slide in distilled water with two to six minutes 

 application of the diluted stain. The smear is washed in distilled water and dried 

 and examined under the microscope. In judging a good Wright's stain, special 

 attention is given to the question of whether the red cells show a desired yellow 

 pink or whether all the granules of the various leucocytes are differentially stained. 



The eosin-methylene-blue medium is prepared as follows: 1 g. peptone, 0.2 g. 

 K2HPO4, 13^ g. agar and 100 ml. distilled water. Dissolve by heat and add 5 ml. of 

 20% aqueous lactose, 2 ml. of 2% aqueous eosin Y and 2 ml. of 0.5% aqueous methy- 

 lene blue. After sterilizing, pour into Petri dishes and inoculate with Escherichia 

 coll and Aerobacter aerogenes and allow to incubate for 24 hours. A good sample 

 should show red growth with a strong metallic sheen for the former organism, pink 

 growth without metallic sheen for the latter and no reddening of the medium except 

 where growth has occurred. 



Ethyl Eosin, C. I. No. 770 



Identification: Ethyl eosin is the potassium (or sodium) 'salt of the ethyl ester of 

 tetrabromofluorescein, C22HuOoBr4K (or Na). The method of identification is as 

 follows: Dissolve 50 mg. in 250 ml. of 50% alcohol. Dilute 5 ml. of this solution and 

 2 ml. of 1% Na2C03 solution to 200 ml. with 50% alcohol. R^ad in Beckman spec- 

 trophotometer. Absorption maximum 531-533 mn; ratio P-15/P-|-15 is from 1.27 

 to 1.62. 



Method of Analysis: Spectrophotometric. Percent dye = D-peak X 130. D-peak 

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



For certification, samples must contain not less than 78% anhydrous dye. 



Biological Tests: Ethyl eosin is tested for the demonstration of Negri bodies in 

 the central nervous system of rabid animals. Pieces of the hippocampus major, 

 3-5 mm. in thickness, are placed between squares of ordinary writing paper (cut 

 end next to the paper) and are immersed in acetone 23^-6 hours. The paper is re- 

 moved and the fixed tissue is placed in fresh paraffin at 58-60° C. for 4 hours or 

 overnight. Sections are cut 5 /x thick, floated onto glass slides and fixed by gentle 

 heat over Bunsen burner, and placed in oven for 45 minutes at 58-60° C. They are 

 washed in two changes of xylene and passed through two changes of absolute alco- 



