320 Biological Stains 



Decolor ization Test: To 100 ml. of a 0.01% solution of the dye add 2 ml. of A^ NaOH. 

 The color should turn red immediately, fading after 10 minutes to a straw color, 

 and after 20 minutes should become almost colorless. (Some good samples become 

 colorless in 10 minutes.) Addition of a few drops of normal acid should restore the 

 blue color almost instantly. 



Method of Analysis: Dissolve 200 mg. of dye in 175 ml. of distilled water, add 

 25 ml. of pH 4.5 acetate buffer, heat to boiling and titrate with 0.05 N TiCla. The 

 end point will vary, depending on the sample, from yellow to dark yellow-green, 

 and is generally easy to detect. Because of the uncertain composition of anilin blue, 

 no attempt is made to determine the dye content. Specifications have been made 

 which place the minimum TiCls consumption per gram of dye at 12.0 ml. of 0.1 N 

 solution. 



Biological Tests: Anilin blue is tested in Mallory's connective tissue stain on ani- 

 mal tissue fixed in Zenker's fluid and embedded in celloidin or paraflBn. The pro- 

 cedure followed is the one given under acid fuchsin, p. 319. With a good sample, the 

 collagen fibrils, reticulum, amyloid, and mucus stain blue. 



THE FLUORAN DERIVATIVES 



The fluoran derivatives are readily reduced by titanous chloride and excellent 

 checks are obtained in duplicate reductions, but due to the heterogeneous character 

 of some of the commercial samples of the halogenated dyes of this group, large 

 errors in the indicated dye content are encountered. It has been found that the 

 dye content of these samples may be determined more accurately and conveniently 

 by the conversion of the dye into the form of the insoluble color acid. 



Certain members of this group are used as biological stains, but as there is con- 

 siderable difference in the purpose for which the various members of the group are 

 employed, different biological tests are necessary for each of the dyes listed. The 

 biological tests employed at the present time are given for all these dyes, with 

 the single exception of fluorescein. 



The following methods, employed for the standardization of the fluorans, include 

 (1) the identification or qualitative examination; (2) the quantitative analysis; and 

 (3) the biological tests for each individual dye. 



EosiN Yellowish, C. I. No. 768 



Identification: Eosin yellowish (eosin Y) is the sodimn salt of tetrabromofluores- 

 cein, C2oH605Br4Na2. The following method of identification is employed: Dissolve 

 50 mg. of dye in 250 ml. of distilled water. Dilute 5 ml. of this solution and 2 ml. of 

 1% Na2C03 solution to 200 ml. with distilled water. Read in Beckman spectro- 

 photometer. Absorption maximum 515-517 myu; ratio P— 15/P+15 is from 1.21 

 to 1.77. 



Method of Analysis: Spectrophotometric. Percent dye = D-peak X 147. D-peak 

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



Por certification, samples must contain not less than 80% anhydrous dye. 



Biological Tests: Eosin Y is tested for three biological purposes : as a counterstain 

 against hematoxylin, as a constituent of Wright's stain, and in the eosin-methylene- 

 blue medium. 



